PACE Evolution: Revolutionizing Natural Product Drug Discovery with Phage-Assisted Continuous Evolution

Abstract

This article provides a comprehensive guide for researchers on leveraging Phage-Assisted Continuous Evolution (PACE) to evolve and optimize enzymes for natural product biosynthesis. We cover foundational principles, methodological setups for evolving polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs), troubleshooting common experimental challenges, and validating evolved systems against traditional methods. The content synthesizes current literature and protocols to empower scientists in accelerating the development of novel bioactive compounds for therapeutic applications.

What is PACE? Core Principles and Why It's a Game-Changer for Natural Products

Application Notes

Phage-Assisted Continuous Evolution (PACE) is a powerful directed evolution platform that enables rapid, automated evolution of biomolecules. Within the context of natural products research, PACE offers a transformative approach to evolve enzymes involved in biosynthetic pathways (e.g., polyketide synthases, non-ribosomal peptide synthetases), regulatory proteins for pathway engineering, or therapeutic peptides and proteins derived from natural scaffolds. The system's continuous nature allows for the exploration of vast mutational landscapes in remarkably short timeframes (weeks), accelerating the development of novel catalysts or bioactive compounds with improved properties.

Core Machinery & Function:

• M13 Filamentous Phage Vector: The gene of interest (GOI) to be evolved is inserted into a modified M13 phage genome, replacing the gene for the essential pIII protein. Phage propagation becomes dependent on the evolving GOI's activity, creating a direct link between desired function and survival.
• Host E. coli (Lagoonal Cells): Continuously diluted host cells are maintained in a fixed-volume vessel (lagoon). These cells supply the resources for phage replication and contain accessory plasmids (see below).
• Mutagenesis Plasmid (MP): Harbored in the host cells, this plasmid expresses mutagenesis proteins (e.g., a variant of the DNA polymerase III α subunit) to introduce targeted mutations specifically into the replicating phage DNA, generating genetic diversity for selection.
• Accessory Plasmid (AP): Also in the host cells, this plasmid links the desired activity of the GOI to phage propagation. It expresses a host survival factor (e.g., the missing pIII) only when the evolving GOI performs its target function (e.g., binding a new ligand, catalyzing a reaction).

Key Quantitative Parameters: The efficiency of PACE is governed by several critical parameters, which must be optimized for each evolution campaign.

Table 1: Key Quantitative Parameters in a Typical PACE Experiment

Parameter	Typical Range	Function & Impact
Lagoon Dilution Rate	1.0 - 1.3 vol/hr	Controls host cell doubling time and phage residence time. Must exceed host growth rate to prevent takeover.
Host Cell Density	10^8 - 10^9 cfu/mL	Ensures sufficient resources for continuous phage replication and evolution.
Phage Titer in Lagoon	10^9 - 10^11 pfu/mL	Indicator of successful selection. A stable, high titer suggests functional GOI variants are propagating.
Mutation Rate (MP induced)	~10^-5 mutations/bp/ gen	Balances exploration of sequence space with preservation of functional sequences. Tunable via MP expression.
Experiment Duration	50 - 500 hours	Allows for 10s to 100s of phage generations, enabling significant evolution.

Experimental Protocols

Protocol 1: Establishing a Basic PACE Lagoon for Evolved Binding Affinity

Objective: To set up a continuous evolution lagoon to evolve a protein (e.g., a transcription factor derived from a natural product biosynthetic pathway) for binding to a novel small-molecule inducer.

Materials:

• "Scientist's Toolkit" (See Table 2)
• Peristaltic pump system
• Aeration system with sterile air filter
• Lagoon vessel (e.g., water-jacketed glass vessel)
• Media waste container

Procedure:

• Pre-culture Host Cells: Inoculate E. coli cells harboring the MP and the custom AP (where pIII expression is driven by the transcription factor's target promoter) into 5 mL LB with appropriate antibiotics. Grow overnight at 37°C, 250 rpm.
• Dilution and Induction: Subculture the overnight culture 1:1000 into fresh 1L of PACE medium (LB with antibiotics, 0.1% glucose, 0.2% arabinose to induce MP). Incubate at 37°C, 250 rpm until OD600 reaches ~0.5.
• Lagoon Priming: Transfer the induced host cell culture to the sterile lagoon vessel. Begin medium inflow and waste outflow using the peristaltic pump set to the target dilution rate (e.g., 1.1 vol/hr). Begin aeration. Allow the lagoon to equilibrate for 30-60 minutes.
• Phage Infection: Introduce the initial M13 phage library (harboring the wild-type or mutagenized gene for the transcription factor) to the lagoon. The initial Multiplicity of Infection (MOI) should be ~0.001-0.01.
• Monitoring: Collect effluent samples every 4-8 hours.
  • Measure phage titer by plaque assay on appropriate E. coli indicator cells.
  • Measure host cell density by plating for colony-forming units (cfu).
  • Monitor for contamination.
• Harvesting: Once phage titer stabilizes at a high level (>10^10 pfu/mL) for >24 hours, harvest effluent phage particles via PEG precipitation. Isolate phage genomic DNA and sequence the evolved GOI from the population or individual plaques.

Protocol 2: Phage Titer Determination via Plaque Assay

Objective: To quantify infectious phage particles from lagoon samples.

Procedure:

• Prepare a 1:10 serial dilution series (up to 10^-12) of the lagoon sample in Phage Dilution Buffer.
• Mix 100 µL of selected dilutions with 1 mL of fresh, log-phase E. coli host cells (without AP/MP, but expressing pIII for infection).
• Incubate at 37°C for 10 minutes without shaking to allow phage adsorption.
• Add the infected cell mixture to 5 mL of molten (45°C) top agar, vortex briefly, and pour onto pre-warmed LB-agar plates. Swirl to distribute evenly.
• Let the top agar solidify, then invert plates and incubate at 37°C overnight.
• Count plaques on plates with 10-100 distinct plaques. Calculate titer: Plaque-Forming Units (pfu)/mL = (Plaque count) / (Dilution factor × Volume plated in mL).

Table 2: The Scientist's Toolkit - Essential Research Reagents for PACE

Item	Function in PACE
M13 Phage Vector (e.g., pSEVA-phage)	Engineered phage genome backbone for GOI insertion and propagation.
Accessory Plasmid (AP) Library	Encodes the conditional pIII system; design defines the selection pressure.
Tunable Mutagenesis Plasmid (MP)	Expresses inducible mutator proteins to diversify the phage-borne GOI.
PACE-Competent E. coli (e.g., S2060)	F-pili expressing, endonuclease I-deficient host strain for robust phage production.
Lagoon Medium (LB + Additives)	Supports continuous host growth. Contains antibiotics, arabinose (MP inducer), and any selection ligands.
Phage Precipitation Solution (PEG/NaCl)	For concentrating and purifying phage particles from lagoon effluent for analysis.

Diagrams

Title: PACE Lagoon Continuous Flow & Replication Cycle

Title: Core PACE Selection Logic & Genetic Circuitry

Application Notes

The discovery of novel natural products (NPs) via traditional methods is constrained by evolutionary bottlenecks. These bottlenecks manifest in the core biosynthetic machinery: Polyketide Synthases (PKSs), Nonribosomal Peptide Synthetases (NRPSs), and Terpene Synthases (TSs). Key challenges include low expression yields in heterologous hosts, poor solubility, inability to functionally reconstitute megasynth(et)ases, substrate promiscuity limitations, and the vastness of unexplored sequence-function space.

The integration of Phage-Assisted Continuous Evolution (PACE) offers a paradigm shift. PACE applies continuous, directed evolution pressure in vivo to overcome these bottlenecks by evolving the biosynthetic enzymes themselves. The following Application Notes detail how PACE can be applied to each system.

Table 1: Core Challenges and PACE-Addressable Solutions

Biosynthetic System	Primary Bottleneck	PACE-Evolvable Trait	Desired Evolutionary Outcome
Modular PKS/NRPS	Stalling, mis-incorporation, poor inter-domain communication.	Protein-protein interaction specificity, carrier domain (ACP/PCP) efficiency.	Improved chimeric assembly line fidelity and titer.
Iterative PKS/TS	Limited substrate promiscuity, poor regioselectivity/stereoselectivity.	Active site architecture, substrate channel geometry.	Novel chemical scaffolds, altered cyclization patterns.
All Systems	Host toxicity, poor solubility/expression, precursor limitation.	Enzyme stability, solubility tags, resistance to host defenses.	High-yield production in optimized chassis (e.g., E. coli, S. cerevisiae).

Protocols

Protocol 1: PACE Setup for Evolving a Type I PKS Module Solubility inE. coli

Objective: Evolve a poorly soluble PKS module for functional expression in E. coli using PACE with a solubility reporter.

Materials (Research Reagent Solutions):

• M13 Phage Vector: Harbors gene III (pIII) under control of a weak constitutive promoter and the PKS module gene as a C-terminal fusion to a fragment of pIII.
• Accessory Plasmid (AP): Encodes the remaining, essential fragment of pIII. Functional pIII (required for phage infectivity) only assembles if the PKS module is soluble and allows fusion fragment folding.
• Selection Plasmid (SP): Contains the mutagenesis genes (e.g., mutagenic plasmid MP6 from PACE Manual).
• Host E. coli strain: Lagoon host (e.g., S2060) for continuous culture.
• Terrific Broth (TB) Medium: For high-density bacterial growth.
• Antibiotics: Chloramphenicol (for AP), Spectinomycin (for SP), Carbenicillin (for phage selection).
• Induction Agent: Isopropyl β-d-1-thiogalactopyranoside (IPTG) for tunable expression of the PKS-pIII fusion.

Methodology:

• Clone the target PKS module into the M13 PACE vector, creating an in-frame fusion with the pIII fragment.
• Transform the AP and SP into the E. coli lagoon host strain. Inoculate a 500 mL lagoon with this strain in TB medium with appropriate antibiotics.
• Initiate the lagoon culture in the PACE apparatus (constant dilution with fresh medium, typically 1 lagoon volume per hour).
• Infect the lagoon with the initial library of M13 phage carrying the PKS module (~10^8 PFU/mL).
• Allow continuous evolution for 24-72 hours. Phage producing soluble PKS-pIII fusions will generate infectious progeny; those with insoluble aggregates will not.
• Harvest phage from lagoon effluent. Isolate viral ssDNA, sequence the evolved PKS gene, and reclone into an expression vector for soluble protein production and biochemical characterization.

Protocol 2: Evolving Terpene Synthase Substrate Promiscuity via PACE

Objective: Evolve a terpene synthase to accept non-natural allylic diphosphate substrates, linking activity to phage propagation.

Materials (Research Reagent Solutions):

• PACE-Compatible Phage: M13 with TS gene replacing pIII. pIII expression is controlled by a biosensor responsive to a target terpene product.
• Biosensor Plasmid (BP): Encodes a transcription factor that specifically binds the desired terpene product and activates pIII expression.
• Substrate Feedstock: Non-natural allylic diphosphate (e.g., 12-carbon farnesyl diphosphate analog) supplied in the lagoon medium.
• Host E. coli with Precursor Pathway: Engineered to overproduce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).
• Liquid Chromatography-Mass Spectrometry (LC-MS): For product verification.

Methodology:

• Construct the BP with a pIII gene under a promoter containing binding sites for a terpene-responsive transcription factor (e.g., engineered E. coli TetR).
• Establish a lagoon with E. coli harboring the BP, SP, and the endogenous MEP pathway for IPP/DMAPP.
• Start continuous culture with the non-natural substrate analog in the feed medium.
• Infect with the TS phage library. Only phage whose TS enzymes produce the terpene that activates the biosensor will propagate.
• Run PACE for multiple days. Periodically sample effluent for phage titer and analyze terpene production via LC-MS from host cell pellets.
• Isolve and sequence evolved TS variants. Characterize kinetic parameters (Km, kcat) with both natural and non-natural substrates.

Diagrams

Diagram Title: PACE Workflow for NP Enzyme Evolution

Diagram Title: PACE Solubility Selection Circuit

The Scientist's Toolkit

Table 2: Essential Research Reagents for PACE-driven NP Discovery

Reagent / Material	Function in PACE for NPs	Key Consideration
M13 PACE Vector	Phage backbone for gene III (pIII) linked evolution. Allows fusion of target gene to pIII fragment.	Must maintain correct reading frame and allow for sufficient library diversity.
Mutagenesis Plasmid (e.g., MP6)	Provides in vivo mutagenesis during replication to generate diversity.	Mutagenesis rate must be tunable to avoid deleterious mutation accumulation.
Accessory Plasmid (AP)	Encodes essential genes (e.g., split-pIII fragment, biosensors) to create conditional selection.	Design is target-dependent (solubility, activity, product sensing).
E. coli Lagoon Host (e.g., S2060)	Engineered E. coli strain for continuous culture; lacks recA, supports phage propagation.	Must be compatible with NP precursor pathways (e.g., engineered with MVA/MEP).
Tunable Biosensor Plasmid	Links production of a specific NP or intermediate to pIII expression and phage survival.	Specificity and dynamic range are critical to avoid background and drive efficient evolution.
Non-canonical Substrate Analogs	Feedstock for evolving enzyme promiscuity (e.g., alkyl-malonyl-CoAs, amino acid analogs, isoprenoid diphosphates).	Must be cell-permeable and non-toxic at required concentrations.
LC-MS / GC-MS System	Essential analytical tool for validating evolved enzyme function and characterizing novel products.	High sensitivity required for detecting low-titer products from small-scale lagoon samples.

Within the broader thesis of applying Phage Assisted Continuous Evolution (PACE) to natural products research, this document details how PACE overcomes the manual, serial, and low-throughput bottlenecks of traditional directed evolution. By linking the desired activity of a biomolecule (e.g., a polyketide synthase domain, a non-ribosomal peptide synthetase adenylation domain, or a tailoring enzyme) to the propagation of the M13 bacteriophage, PACE enables the autonomous and continuous evolution of novel function over hundreds of generations without researcher intervention. This facilitates the exploration of vast sequence landscapes to optimize or alter the specificity, stability, and catalytic efficiency of biosynthetic enzymes, accelerating the engineering of novel natural product analogs.

Core PACE System Components and Workflow

PACE operates in a continuous flow apparatus termed a "lagoon," where host E. coli cells are constantly diluted and replenished. The system's heart is the linkage between a gene of interest (GOI) variant and phage propagation via an accessory plasmid (AP) encoding a necessary phage protein (e.g., gIII for the phage coat protein pIII).

Diagram 1: PACE System Schematic

Key Research Reagent Solutions

Item	Function in PACE
M13ΔpIII Phage	Engineered bacteriophage lacking the gene for essential coat protein pIII; propagation depends on functional complementation.
Accessory Plasmid (AP)	Plasmid encoding the essential phage gene (e.g., gIII) under transcriptional control of a promoter responsive to the activity of the GOI. The "activity link."
Mutagenesis Plasmid (MP)	Plasmid expressing mutagenesis genes (e.g., mutD5) to introduce targeted mutations into the phage genome during replication, driving evolution.
Chemostat/Lagoon Apparatus	Continuous culture device (e.g., bioreactor) that maintains constant volume, cell density, and nutrient supply for uninterrupted evolution.
Selection Ligand/Substrate	The small molecule (e.g., natural product precursor, target drug molecule) used to drive evolution of binding or catalysis via the AP regulatory circuit.

Quantitative Performance Data

PACE dramatically accelerates the evolutionary timeline compared to traditional methods. The following table summarizes key metrics from foundational and recent studies.

Table 1: Comparative Throughput and Efficiency of PACE vs. Traditional Directed Evolution

Evolution Parameter	Traditional Batch Methods	PACE Protocol	Reported Outcome (Example)
Generational Time	1-2 days per round	~1-2 hours per generation	Continuous flow enables >100 generations in 1 week.
Library Size Screened	~10^6 - 10^8 variants per round	Effectively >10^12 variants over a run	Uninterrupted mutation and selection vastly expand searchable space.
Manual Intervention	Extensive (transformation, culturing, assay)	Minimal (setup only)	Fully automated selection pressure applied 24/7.
Typical Evolution Duration	Months to years for >10 rounds	Weeks for >200 generations	Evolved T7 RNA polymerase >1000-fold new specificity in ~1 week.
Mutation Rate (per kb)	Low, often limited	Tunable via MP (e.g., 1-3 nucleotide changes/genome/round)	Continuous, targeted diversification.

Detailed Protocol: Establishing a PACE Experiment for Enzyme Specificity

This protocol outlines steps to evolve the substrate specificity of a natural product-modifying enzyme (e.g., a glycosyltransferase) using PACE.

Protocol 1: Lagoon Setup and Evolution Run

Objective: To continuously evolve an enzyme for activity on a novel substrate by linking product formation to phage pIII expression via a transcription factor-based biosensor.

Materials:

• E. coli host strain (e.g., S2060 for M13 propagation)
• M13 phage vector (e.g., M13ΔpIII-GOI) harboring the enzyme gene variant library.
• AP: Plasmid with pIII gene under control of a biosensor promoter activated by the desired enzymatic product.
• MP: Plasmid expressing mutD5 for mutagenesis.
• Bioreactor or chemostat system (lagoon) with peristaltic pumps.
• Media: Rich media (e.g., Tryptone broth) with appropriate antibiotics (chloramphenicol for AP, spectinomycin for MP).
• Selection ligand: The target novel substrate for the enzyme.

Method:

• Prepare Host Cells: Transform the AP and MP into the host E. coli strain. Grow a large starter culture in antibiotic media.
• Inoculate Lagoon: Fill the lagoon vessel with sterile media. Start media inflow pump. Inoculate lagoon with transformed host cells to an OD~600~ of ~0.05. Allow host cell density to stabilize (~1-2 residence times, typically to OD~600~ ~0.5).
• Initiate Phage Infection: Introduce the initial M13 phage library (diversity >10^10^ PFU) into the lagoon. The phage infect host cells.
• Apply Selection Pressure: Add the target novel substrate (selection ligand) to the inflowing media at the desired concentration. Only phage infecting cells where the GOI enzyme successfully modifies the substrate to produce the activating molecule will trigger pIII expression from the AP, allowing progeny phage production.
• Monitor Evolution: Daily, collect lagoon effluent samples.
  • Titer Phage: Plate on host cells with and without AP to monitor infectious phage concentration and check for escape mutants.
  • Sequence Samples: Periodically isolate phage DNA from the population for NGS to track evolutionary trajectories.
• Harvest Evolved Phage: After desired duration (e.g., 5-10 days, representing 100-200 generations), stop the flow. Concentrate and purify phage from the lagoon contents. Isolate and clone the evolved GOI for downstream biochemical characterization.

Diagram 2: Transcriptional Activation AP Circuit

Advanced Applications and Protocols: PACE with SIPP

Protocol 2: Stringent Selection via Phage-Assisted Continuous Evolution with Sorting (SIPP)

Objective: To apply even stronger selection pressure by requiring two orthogonal activities for phage propagation, minimizing false positives.

Materials: All from Protocol 1, plus a second AP (AP2) encoding a different essential phage gene (e.g., gVIII for protein pVIII) under control of a second, distinct selection circuit.

Method:

• Dual AP Host Preparation: Transform host cells with both AP1 (pIII under control of Circuit A) and AP2 (pVIII under control of Circuit B).
• Lagoon Setup: Establish lagoon as in Protocol 1 using the dual-AP host.
• Dual Selection Pressure: Include both target substrates/ligands for Circuits A and B in the inflowing media.
• Monitoring & Harvest: Only phage encoding GOI variants that satisfy both activity requirements will propagate efficiently. Monitor and harvest as in Protocol 1.

Table 2: Evolution of a Polyketide Synthase Module Using PACE-SIPP

Selection Parameter	Circuit A (AP1)	Circuit B (AP2)	Evolution Outcome
Essential Gene	pIII (gIII)	pVIII (gVIII)	Both genes required for infectivity.
GOI Activity Link	Production of Malonyl-CoA derivative	Hydrolysis of a Thioester analog	Simultaneous selection for substrate loading and chain release.
Selection Agent	Propionyl-CoA precursor	Chemical inducer of hydrolysis sensor	Evolved PKS module with altered extender unit specificity and improved turnover.
Time to >1000-fold Improvement	~14 days (Single AP)	~14 days (Single AP)	~10 days (Dual SIPP) with higher fidelity.

Application Notes

This document outlines the integration of Phage-Assisted Continuous Evolution (PACE) for engineering both gene expression tools and biosynthetic enzymes, enabling accelerated natural product discovery and optimization. The evolution of T7 RNA polymerase (RNAP) variants for novel promoter recognition serves as a foundational technology, allowing for orthogonal gene expression in microbial hosts that express complex biosynthetic gene clusters (BGCs). Subsequently, PACE and related continuous evolution platforms are applied directly to engineer enzymes within non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways, creating novel analogs with improved pharmacological properties.

Table 1: Key Quantitative Milestones in PACE-Driven Engineering

Evolved Component	Selection Pressure/Goal	Evolution Time (PACE)	Key Outcome (Quantitative)
T7 RNAP (Initial PACE)	Recognize T3 promoter	~200 hours	>105-fold activity gain; 100% mutant phage survival.
T7 RNAP Variant (e.g., S43N)	Recognize ΦK-1a promoter	~150 hours	>107-fold activity switch; >103-fold specificity vs. wild-type promoter.
NRPS Adenylation (A) Domain	Altered substrate specificity (e.g., for non-canonical amino acids)	~120-160 hours	>100-fold activity shift; production of novel peptide analog at >50 mg/L titers.
PKS Ketosynthase (KS) Domain	Improved processing of non-natural extender unit	~140-180 hours	40-60% incorporation rate of non-natural unit into final polyketide.

Protocols

Protocol 1: PACE Setup for T7 RNA Polymerase Evolution Objective: To evolve T7 RNAP to recognize a novel promoter sequence (X-Promoter) using PACE. Materials: See "Research Reagent Solutions" below. Procedure:

• Lagoon Preparation: In a 1 L host vessel (lagoon), maintain a turbidostat culture of E. coli S2060 (or similar host) at OD₆₀₀ ~0.3-0.4 in M9G media supplemented with 0.1% Arabinose (to express mutagenesis plasmid genes) and appropriate antibiotics.
• Phage Vector Construction: Clone the gene for wild-type T7 RNAP (gene 1) under the control of the target X-Promoter into an M13-based selection phage vector. Ensure the vector lacks any functional gene III (pIII), which is placed under the control of a T7-specific promoter (e.g., Φ10) elsewhere in the host genome.
• Initiation: Infect the lagoon with the constructed selection phage at low MOI (~10^-6).
• Continuous Flow: Pump fresh host-laden media into the lagoon at a fixed dilution rate (typically 1-2 vessel volumes per hour). Waste media and phage are removed at the same rate.
• Selection Principle: Only phage that encode a T7 RNAP variant capable of transcribing from the X-Promoter will produce pIII. pIII is essential for phage infectivity. Progeny phage from these events can reinfect fresh host cells flowing into the lagoon.
• Harvesting: Continuously collect effluent from the lagoon. Centrifuge to pellet host cells. Filter supernatant (0.22 µm) to collect evolved phage pool for sequencing and downstream characterization.

Protocol 2: Screening Evolved NRPS Adenylation Domains in a Heterologous Host Objective: To express and validate the substrate specificity of PACE-evolved A domains in a production host. Procedure:

• Gene Cloning: Clone the evolved A domain (with its accompanying carrier protein domain) from the PACE output pool into a modular expression vector (e.g., pET-based) compatible with E. coli BL21(DE3).
• Co-expression: Transform the construct alongside plasmids expressing necessary pathway enzymes (e.g., thioesterase, phosphopantetheinyl transferase).
• Feeding and Fermentation: Inoculate cultures in M9 media with 1% glucose. At induction (OD₆₀₀ ~0.6, 0.5 mM IPTG), supplement with 2-5 mM of the target non-canonical amino acid substrate. Culture for 48 hours at 18°C.
• Metabolite Extraction: Pellet cells. Resuspend in methanol:ethyl acetate (1:3), vortex, and centrifuge. Collect organic layer and dry under nitrogen or vacuum.
• LC-MS/MS Analysis: Reconstitute extract in methanol. Analyze by reversed-phase LC-MS (C18 column). Compare retention times and MS/MS fragmentation patterns to standards to identify and quantify novel peptide products.

Visualizations

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for PACE and Biosynthetic Engineering

Reagent/Material	Function / Role in Experiment
E. coli S2060	Specialized host strain for PACE; contains mutagenesis plasmid and pIII under T7 promoter control.
M13KE Selection Phage Vector	Phagemid backbone for cloning gene of interest; lacks gene III, creating essential dependence on complementation.
Arabinose-Inducible Mutagenesis Plasmid (e.g., pJPM2)	Expresses mutagenic proteins (e.g., MP6, a variant of DNA polymerase III α subunit) to introduce targeted mutations during phage replication in the lagoon.
Turbidostat System (e.g., Multipump, OD sensor)	Maintains constant, high-density host cell culture in the PACE lagoon for continuous phage propagation.
M9 Minimal Media with Glycerol (M9G)	Defined growth medium for PACE lagoon; prevents accumulation of metabolic byproducts and supports robust host growth.
Non-canonical Amino Acid (ncAA) Substrates	Chemical building blocks fed to cultures to select for or assay evolved enzymes with altered substrate specificity.
Phosphopantetheinyl Transferase (e.g., Sfp from B. subtilis)	Essential enzyme for activating carrier protein domains in NRPS/PKS pathways by adding the phosphopantetheine cofactor.
High-Resolution LC-MS/MS System	Critical analytical tool for detecting, quantifying, and structurally characterizing novel natural product analogs.

Building a PACE Platform for Natural Product Enzymes: A Step-by-Step Protocol

Within Phage Assisted Continuous Evolution (PACE) for natural product biosynthesis, the selection phagemid is the central genetic circuit that converts a desired enzymatic activity—often a biosynthetic step or a catalyst improving a natural product precursor—into a selective advantage for the bacteriophage. This linkage is achieved by making the production of the essential phage coat protein pIII (required for infectivity) dependent on the activity of a target enzyme encoded on the same phagemid.

The core logic employs an accessory protein (AP), whose gene is under the control of a promoter regulated by a transcription factor. The target enzyme modifies a small molecule ligand, altering its ability to bind and inactivate the transcription factor. Functional enzyme activity relieves repression, allowing AP expression. The AP then inhibits a specific protease, leading to the accumulation of its substrate: a fused protein of a pIII degradation tag and pIII itself. Accumulated pIII enables viral propagation. Failure of the target enzyme results in pIII degradation and phage loss. This creates a powerful continuous selection for improved enzyme variants over serial lagoon passages.

Key Quantitative Parameters for Circuit Tuning:

Parameter	Typical Target Range	Functional Impact
Phagemid Copy Number	20-30 copies/cell	Balances gene dosage & host burden.
Lagoon Dilution Rate	1-2 volumes/hour	Maintains log-phase host growth; selects for fast catalysis.
pIII Threshold for Infection	~10 molecules/phage particle	Sets minimum required enzyme activity.
Protease Inhibition Constant (Ki) of AP	Low nM range	Ensures tight regulation of pIII degradation.
Transcription Factor-Ligand Kd (Inactive/Active)	>100-fold difference	Maximizes dynamic range of selection.

Detailed Experimental Protocols

Protocol 2.1: Construction of the Selection Phagemid

Objective: Assemble the genetic circuit linking enzyme output to pIII gene expression. Materials: pIII-neg M13 phage genome, high-copy cloning vector backbone, PCR reagents, Gibson Assembly master mix, E. coli cloning strain. Procedure:

• Amplify Components: PCR amplify (i) the regulated promoter (e.g., P_BAD or P_LtetO-1), (ii) the accessory protein (AP) gene, (iii) the target enzyme gene (with ribosomal binding site), and (iv) the pIII degradation tag-pIII fusion (from a template like pJC175e).
• Gibson Assembly: Mix ~100 ng of linearized vector backbone with equimolar amounts of each PCR fragment (total 4 fragments) in a Gibson Assembly reaction. Incubate at 50°C for 1 hour.
• Transform: Transform 2 µL of the assembly reaction into competent E. coli cloning cells (e.g., DH5α). Plate on LB agar with appropriate antibiotic (e.g., Spectinomycin 50 µg/mL).
• Screen Colonies: Pick 10-12 colonies, culture minipreps, and verify assembly by diagnostic restriction digest and Sanger sequencing of all junctions.

Protocol 2.2: PACE Lagoon Setup & Selection Initiation

Objective: Initiate continuous evolution using the constructed phagemid. Materials: Turbidostat lagoons, host E. coli cells carrying the required transcription factor plasmid, sterile media, phage stock containing the selection phagemid and a mutagenesis plasmid (if used). Procedure:

• Prepare Host Inflow: Grow a large culture of host cells (e.g., S2060 E. coli) harboring the regulating transcription factor plasmid to mid-log phase (OD₆₀₀ ~0.5) in selection media with antibiotic and inducing ligand (if needed).
• Prime Lagoon: Fill a 15 mL lagoon with log-phase host culture. Infect with the engineered M13 phage (carrying selection phagemid) at a low multiplicity of infection (MOI ~0.01).
• Start Continuous Flow: Connect the host inflow line to the lagoon, initiating dilution with fresh host culture at a rate of 1-2 lagoon volumes per hour. Connect the outflow to waste.
• Monitor & Sample: Monitor lagoon turbidity daily. Sample outflow daily to titer phage (PFU/mL) and assess evolution progress via plaque PCR or sequencing.

Protocol 2.3: Quantifying Selection Stringency via pIII ELISA

Objective: Directly measure pIII production levels from phage particles to correlate with enzyme activity. Materials: Phage samples from lagoon outflow, anti-pIII monoclonal antibody, HRP-conjugated secondary antibody, ELISA plate, wash buffer, TMB substrate, microplate reader. Procedure:

• Coat Plate: Dilute anti-pIII antibody in coating buffer. Add 100 µL/well to ELISA plate. Incubate overnight at 4°C.
• Block: Wash plate 3x with PBST. Block with 200 µL/well of 3% BSA in PBS for 1 hour at 37°C.
• Bind Phage: Add 100 µL of diluted phage sample (or pIII standard) per well. Incubate 2 hours at 37°C.
• Detect: Wash plate 5x. Add 100 µL/well of HRP-conjugated secondary antibody. Incubate 1 hour at 37°C. Wash 5x.
• Develop & Read: Add 100 µL TMB substrate. Incubate 15 mins in dark. Stop with 50 µL 1M H₂SO₄. Read absorbance at 450 nm. Plot against pIII standard curve.

Diagrams

Diagram Title: PACE Selection Phagemid Logic Circuit

Diagram Title: PACE Experimental Workflow

The Scientist's Toolkit: Research Reagent Solutions

Item	Function in PACE Selection Phagemid Experiments
pJC175e or similar vector	Source of the degradation tag-pIII fusion cassette and phagemid backbone.
Regulated Promoter (e.g., PBAD)	Provides ligand/transcription factor-dependent control of AP expression.
Arabinose/Tetracycline-based Ligands	Small molecules modified by target enzyme; act as inputs for the genetic circuit.
T7 RNA Polymerase Gene & Promoter	Alternative circuit design uses T7 RNAP as AP to drive pIII expression from a T7 promoter.
LacI or TetR Mutant Transcription Factors	Engineered DNA-binding proteins whose affinity for operator/promoter is modulated by ligand state.
HRV 3C or TEV Protease	Specific protease whose inhibition by AP protects the pIII fusion protein.
M13KO7 ΔpIII Helper Phage	Provides all phage proteins except pIII for initial phage stock production.
Spectinomycin & Chloramphenicol Antibiotics	Common selection markers for phagemid and host accessory plasmid maintenance.
Turbidostat Lagoon Apparatus	Chemostat device that maintains constant bacterial density for continuous phage evolution.
Phage Precipitation Solution (PEG/NaCl)	For concentrating and purifying phage particles from lagoon outflow for analysis.

Choosing and Engineering the Host E. coli Strain for Optimal Natural Product Precursor Supply

In the context of Phage Assisted Continuous Evolution (PACE) for natural product research, the choice and engineering of the host E. coli strain are critical for ensuring a robust and sustained supply of essential natural product precursors. This protocol details the selection, genetic modification, and validation of E. coli strains to optimize the metabolic flux towards key building blocks like acetyl-CoA, malonyl-CoA, methylerythritol phosphate (MEP), and shikimate pathway intermediates, thereby enhancing the yield and diversity of evolved natural products in PACE systems.

Key Strain Selection Criteria & Performance Data

The selection of an appropriate base strain is foundational. Quantitative data from recent studies (2023-2024) on common production strains are summarized below.

Table 1: Comparative Analysis of Common E. coli Production Strains

Strain Name	Key Genotype Features	Relative Acetyl-CoA Pool*	Relative Malonyl-CoA Pool*	Suitability for PACE	Key Advantage
BL21(DE3)	ompT hsdS_B (r_B- m_B-) gal dcm (λ DE3)	1.0 (Baseline)	1.0 (Baseline)	Moderate	Robust protein expression, good growth.
K-12 MG1655	Wild-type K-12 derivative	0.8	0.7	High	Well-characterized, amenable to genetic manipulation.
BW25113	Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), lacIp-4000(lacIq), λ-, rph-1, Δ(rhaD-rhaB)568, hsdR514	1.2	1.5	Very High	Keio collection background; ideal for gene knockouts.
W	Wild-type, prototroph	1.5	0.9	Moderate	Naturally high acetyl-CoA, good for acetate-derived precursors.
JA126	ΔfadR, ΔarcA	2.3	3.1	High	Deregulated fatty acid & TCA cycle; enhanced precursor supply.
MEC	ΔptsG, ΔpoxB, ΔldhA, ΔadhE, ΔackA	2.8	2.5	Very High	Minimized byproduct formation; redirected carbon flux.

*Normalized intracellular concentration relative to BL21(DE3) under identical cultivation conditions. Data synthesized from recent metabolic engineering literature.

Detailed Engineering Protocols

Protocol 1: Enhancing Malonyl-CoA Supply via Genetic Modifications

Objective: To engineer an E. coli BW25113 strain with increased malonyl-CoA availability for polyketide precursor feeding in PACE. Materials:

• E. coli BW25113
• P1 vir phage lysate
• Donor strains from Keio collection (e.g., ΔfabI::kan, ΔtesB::kan)
• LB media, kanamycin (50 µg/mL), chloramphenicol (25 µg/mL)
• Sodium citrate (1 M), IPTG, arabinose
• PCR reagents, primers for verification

Procedure:

• Gene Knockouts: Use P1 phage transduction to transfer knockouts from Keio collection mutants into BW25113. a. Grow donor strain (ΔfabI) to mid-log phase. Add 5mM CaCl₂ and infect with P1vir. Harvest phage lysate. b. Grow recipient BW25113 to OD600 ~0.3. Mix 100µL recipient, 100µL donor phage lysate, 10µL 1M CaCl₂, and 200µL LB. Incubate 30 min at 37°C without shaking. c. Add 1mL sodium citrate (0.1M final), incubate 1h, plate on LB + Kan. Incubate at 37°C overnight. d. Verify knockout via colony PCR.
• Overexpress Heterologous Acetyl-CoA Carboxylase (ACC): a. Transform strain with pTrc99a-ACC (from S. coelicolor or C. glutamicum). b. Inoculate engineered strain in TB medium + antibiotics. Grow at 30°C to OD600 0.6. c. Induce ACC expression with 0.5mM IPTG. Continue cultivation for 18h at 25°C.
• Validation: Quantify malonyl-CoA levels via LC-MS/MS. Extract metabolites using cold quenching (60% methanol, -40°C) and analyze.

Protocol 2: Modular Engineering of the Shikimate Pathway

Objective: To overproduce chorismate, a precursor for aromatic amino acids and complex natural products. Base Strain: E. coli JA126 (ΔfadR, ΔarcA). Procedure:

• Attenuate Feedback Inhibition: a. Introduce plasmid pSA69 expressing feedback-resistant alleles of aroG (AroG^fbr) and trpE (TrpE^fbr). b. Use lambda Red recombineering to replace native aroG promoter with a constitutive promoter (J23104) on the chromosome.
• Knockout Competitive Pathways: Transduce ΔpheA::cat and ΔtyrA::cat mutations from respective donor strains using P1vir, selecting for chloramphenicol resistance.
• Cultivation for Precursor Production: a. Grow engineered strain in M9 minimal medium + 2% glucose + appropriate antibiotics. b. Maintain at 32°C, 250 rpm. Supplement with 0.2% yeast extract post-exponential phase. c. Harvest cells at stationary phase. Chorismate can be quantified in supernatant via HPLC.

Essential Pathways and Metabolic Engineering Logic

Diagram Title: Metabolic Engineering for Precursor Supply in E. coli

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for Strain Engineering & Precursor Analysis

Reagent/Material	Function/Benefit	Example Product/Source
Keio Knockout Collection	Genome-wide single-gene knockout mutants in BW25113; essential for targeted gene deletions.	Dharmacon (Horizon Discovery)
P1vir Phage Lysate	High-efficiency transducing phage for moving mutations between E. coli strains.	Ready-made lysates (e.g., from CGSC)
Lambda Red Recombinase Kit	Enables efficient linear DNA recombination for chromosomal edits (e.g., promoter swaps).	pKD46/pKD78 plasmids (Addgene)
Feedback-resistant Enzyme Plasmids (AroGfbr, TrpEfbr)	Deregulate shikimate pathway, overcoming allosteric inhibition.	pSA69 (Addgene #62936)
Acetyl-CoA Carboxylase (ACC) Expression Plasmid	Key heterologous enzyme to convert acetyl-CoA to malonyl-CoA.	pTrc99a-ACC (e.g., from C. glutamicum)
Metabolite Extraction Solvent (Cold 60% Methanol)	Quenches metabolism instantly for accurate quantification of CoA-thioesters.	LC-MS grade methanol in dry ice/ethanol bath.
Coenzyme A Quantification Kit (LC-MS/MS)	Gold-standard for absolute quantification of acetyl-CoA, malonyl-CoA, etc.	Commercial kits (e.g., Cell Technology Inc.)
M9 Minimal Media Powder	Defined medium for precise control of carbon flux during precursor production studies.	Sigma-Aldrich M6030
BioLector Microfermentation System	Allows parallel, online monitoring of growth (biomass, pH, DO) in up to 48 cultures.	m2p-labs (Beckman Coulter)

Integration with PACE for Natural Product Evolution

The engineered host strain serves as the chassis for the PACE system. The optimized precursor pools ensure that the evolving biosynthetic enzymes (e.g., polyketide synthases, non-ribosomal peptide synthetases) presented via the phage vector are not limited by substrate availability. This allows for more effective continuous evolution under selection pressure for novel or enhanced product synthesis. The host's genetic stability and defined metabolism are paramount for long-term PACE experiments spanning hundreds of hours.

Strategic selection and systematic engineering of E. coli host strains, focusing on deregulating key metabolic nodes and eliminating competing pathways, create a high-flux background for natural product precursors. This optimized host forms a critical, stable foundation for PACE campaigns aimed at discovering and evolving novel natural product scaffolds, directly linking host metabolism to evolutionary outcomes.

Within Phage Assisted Continuous Evolution (PACE), the lagoon apparatus is the central bioreactor where evolution occurs. For natural products research, this system enables the continuous and rapid evolution of biosynthetic enzymes (e.g., polyketide synthases, non-ribosomal peptide synthetases) or regulatory proteins to produce novel drug-like compounds. The lagoon maintains a continuous culture of host cells (typically E. coli) infected with mutagenic bacteriophage carrying the gene of interest. Key parameters—flow rate, dilution rate, and chamber size—directly control selection stringency, mutation rate, and experimental throughput, determining the success of evolution campaigns aimed at generating new natural product scaffolds or improving yield.

Quantitative Parameters: Definitions and Interdependencies

The core function of the lagoon is governed by the continuous dilution of the culture with fresh medium. The critical calculated parameter is the dilution rate (D), which is determined by the physical setup.

Table 1: Core Lagoon Parameters and Their Relationships

Parameter	Symbol	Unit	Definition & Impact on PACE
Lagoon Volume	V	mL	Total volume of the culture chamber. Sets the absolute number of cells/phage.
Media Flow Rate	F	mL/h	Rate at which fresh medium enters (and spent culture exits) the lagoon.
Dilution Rate	D = F / V	h⁻¹	The key controlling parameter. Inverse of the residence time. Must exceed host doubling rate to impose selection.
Host Doubling Time	T_d	h	Generation time of host cells under lagoon conditions.
Phage Residence Time	T_phage = 1/D	h	Average time a phage particle stays in the lagoon. Must be less than the desired evolution cycle time.
Turbidostat Threshold	OD	OD600	Setpoint for optical density that controls pump activity, maintaining steady-state growth.

Table 2: Typical Parameter Ranges for PACE Lagoon Experiments

Parameter	Standard Range	Notes for Natural Products Research
Lagoon Volume (V)	10 - 40 mL	15 mL is common. Smaller volumes reduce reagent use (helpful for expensive media).
Flow Rate (F)	10 - 40 mL/h	Must be calibrated daily using graduated cylinder and timer.
Dilution Rate (D)	0.5 - 2.0 h⁻¹ (often ~1.0 h⁻¹)	D > ln(2)/Td. For Td=40 min, D > ~1.04 h⁻¹. Higher D increases selection stringency.
Residence Time	30 - 120 min	Shorter times force faster phage life cycles.
Culture Density	0.2 - 0.6 OD600	Maintained by turbidostat feedback loop to ensure consistent host cell availability.

Experimental Protocols

Protocol 3.1: Calibrating the Lagoon Flow Rate

Objective: To accurately determine the media flow rate (F) from the peristaltic pump. Materials: Peristaltic pump, media reservoir, silicone tubing, sterile graduated cylinder, timer.

• Assemble the fluidic path from reservoir to waste, with the lagoon bypassed.
• Fill the reservoir with water or sterile media.
• Run the pump at the intended setting for exactly 10 minutes, collecting effluent in a graduated cylinder.
• Measure the collected volume (mL). Calculate Flow Rate: F (mL/h) = (Collected Volume (mL) / 10 min) * 60.
• Repeat in triplicate. Adjust pump setting until target F is achieved consistently (±5%).

Protocol 3.2: Establishing Lagoon Steady-State Operation

Objective: To initiate and maintain a continuous culture for PACE. Materials: Sterilized lagoon apparatus, host cell culture, mutagenesis plasmid, media, pump, OD₆₀₀ probe/controller.

• Inoculation: Dilute an overnight culture of host cells (containing required plasmids) to ~0.1 OD₆₀₀ in fresh media. Connect to lagoon inlet.
• Batch Phase: Pump the diluted culture into the empty, sterile lagoon until full. Stop inflow and allow cells to grow in batch mode to mid-log phase (~0.4-0.5 OD₆₀₀).
• Initiate Continuous Flow: Start media pump at the calculated flow rate (F) to achieve desired D. Activate turbidostat control, setting the target OD to the mid-log value.
• Equilibration: Allow the system to run for ≥3 residence times (3/D hours) to reach steady-state.
• Infection: Introduce the engineered phage stock (carrying gene of interest) into the lagoon. Continuous evolution now proceeds.

Protocol 3.3: Monitoring Evolution Progress via Plaque Assay

Objective: To titer infectious phage particles and monitor allele frequency. Materials: Lagoon sample, host cell culture, top agar, agar plates, serial dilution tubes.

• Sample: Aseptically collect a small sample (e.g., 100 µL) from the lagoon.
• Serial Dilution: Perform 10-fold serial dilutions in media across 6-8 tubes.
• Plaque Assay: Mix 100 µL of a mid-log host culture with 10 µL of each phage dilution. Add to 3 mL molten top agar (0.5-0.7% agar), vortex, and pour onto an LB-agar plate. Swirl to cover.
• Incubate & Count: Let plates solidify. Invert and incubate overnight at 37°C. Count plaques on plates with 10-100 plaques. Calculate titer: PFU/mL = (Plaque count) / (Dilution factor * 0.01 mL).
• Sequence Analysis: Pick plaques periodically to isolate phage DNA for sequencing of the evolving gene.

Visualizations

Lagoon Flow and Control Diagram

PACE Workflow for Natural Products

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Reagent Solutions for PACE Lagoon Experiments

Item	Function in PACE Lagoon	Example/Notes
Custom Chemostat Media	Supports continuous growth; may contain inducters, antibiotics, or specialty precursors for natural product synthesis.	M9CA + 0.1% casamino acids + 0.2% glucose + necessary selection agents (e.g., arabinose for APP, chloramphenicol).
Host Cell Strain	Engineered E. coli providing essential PACE components.	S2060 derivative; contains mutagenesis plasmid (MP) and any required biosynthetic machinery.
Mutagenesis Plasmid (MP)	Expresses error-prone polymerase for targeted mutagenesis of phage-borne gene.	pJAZZ-MP or similar; tune mutation rate via aTc induction.
Accessory Protein Plasmid (APP)	Expresses the protein linking desired activity to phage propagation.	For enzyme evolution, APP may express a transcription factor activated by product.
Selection Phage (SP)	Bacteriophage (M13) carrying gene to evolve (GOI) under weak promoter.	SP:GOI; replication essential genes are under control of APP output.
Titering Supplies	For monitoring phage and cell density.	Soft agar (0.6% agar), LB plates, host cells for plaque assays.
Antifoam Agent	Prevents foam formation in lagoon, ensuring stable OD readings.	Diluted antifoam emulsion (e.g., Antifoam 204), added sparingly to media reservoir.
Sterile Silicone Tubing	Fluidic connections for media, waste, and sample lines.	Autoclavable; ensure correct inner diameter for pump head.

The systematic discovery of novel natural product scaffolds is critical for addressing emerging antimicrobial resistance and other diseases. A central challenge is engineering the core biosynthetic machinery, such as Polyketide Synthases (PKSs), to produce "non-natural" natural products with novel carbon backbones. This case study is situated within a broader thesis investigating Phage-Assisted Continuous Evolution (PACE) as a transformative platform for natural products research. PACE enables the rapid, continuous, and autonomous evolution of protein functions without researcher intervention, making it ideally suited for evolving large, complex enzymes like PKS domains. Here, we detail the application of PACE to evolve acyltransferase (AT) and ketosynthase (KS) domains to accept non-native extender units, thereby generating novel polyketide backbones.

Recent PACE campaigns targeting the DEBS Module 1 AT domain from Saccharopolyspora erythraea have successfully altered its substrate specificity.

Table 1: Summary of Evolved PKS AT Domain Variants and Their Activity

Variant ID	Key Mutations	Native Substrate (malonyl-CoA) Activity (%)	Non-Native Substrate (methylmalonyl-CoA) Activity (%)	Selection Stringency (MP Lag)	Reference / Source
AT-WT	N/A	100	<1	N/A	Baseline
AT-Evo1	S112F, V148A	45	95	3.0 hours	Recent PACE (2023)
AT-Evo2	S112Y, V148G, L175I	15	110	3.5 hours	Recent PACE (2023)
AT-Evo3	H97R, S112F, V148A, A200S	<5	120	4.0 hours	Recent PACE (2023)

Table 2: Production Yields of Novel 6-dEB Analogs from Engineered PKS

Engineered PKS Strain	Incorporated Extender Unit	Resulting 6-dEB Analog	Titer (mg/L)	Yield Relative to Wild-Type (%)
DEBS WT (Control)	Methylmalonyl-CoA	Native 6-dEB	120	100
DEBS-AT-Evo1	Malonyl-CoA	6-desmethyl-6-dEB	85	71
DEBS-AT-Evo2	Methoxymalonyl-CoA*	6-methoxy-6-dEB	42	35
DEBS-AT-Evo3	Allylmalonyl-CoA*	6-allyl-6-dEB	28	23

Note: Strains supplied with precursor via fed-batch fermentation.

Experimental Protocols

Protocol 1: PACE Setup for AT Domain Evolution

Objective: To continuously evolve AT domain substrate specificity using a chloramphenicol resistance (CamR) selection linked to non-native extender unit incorporation. Materials: Lagoon apparatus, E. coli S2060 host, M13 bacteriophage vector pJCVar encoding AT-POI, accessory plasmid (AP) for mutagenesis, selection plasmid (SP) encoding biosynthetic reporter. Procedure:

• Construct Design: Clone the target AT domain into the pJCVar vector, replacing the native gene III (pIII). Design the SP to express a minimal PKS module where product completion (dependent on AT activity with the desired non-native extender unit) induces the expression of pIII in trans.
• Lagoon Operation: Dilute host cells containing the AP and SP into fresh media to an OD600 of 0.05. Initiate flow into the lagoon at 1.0 lagoon volumes per hour.
• Infection & Selection: Infect the lagoon with the initial AT-pJCVar phage stock (~10^8 PFU/mL). The SP's conditional pIII production acts as the selection for phage propagation.
• Harvesting: Continuously collect effluent from the lagoon. Centrifuge to separate phage (supernatant) from cells. Titer phage and sequence the evolved AT gene from phage DNA periodically (e.g., every 24-48 hours).

Protocol 2: In Vitro Characterization of Evolved AT Domains

Objective: Quantify kinetic parameters of evolved AT variants. Materials: Purified AT domains, [14C]-malonyl-CoA, [14C]-methylmalonyl-CoA, Acyl Carrier Protein (ACP), Ellman's reagent (DTNB). Procedure:

• Acyltransferase Assay: In a 100 µL reaction, combine 50 mM HEPES (pH 7.5), 1 µM ACP, 100 µM CoA substrate (radiolabeled), and 0.5 µM purified AT variant.
• Incubation: Incubate at 30°C for 5 minutes.
• Detection: Terminate reaction with 10 µL of 10% SDS. Add 50 µL of 1 mM DTNB and measure absorbance at 412 nm to quantify free CoASH release. For radiolabeled substrates, separate acyl-ACP via native PAGE and quantify band intensity.
• Analysis: Calculate kcat and KM from initial velocity measurements across a substrate concentration range (10-500 µM).

Protocol 3: Heterologous Production and LC-MS Analysis of Novel Polyketides

Objective: Produce and validate novel polyketide backbones in a model Streptomyces chassis. Materials: Streptomyces coelicolor CH999 expression strain, expression vector pRM5 containing engineered DEBS PKS genes, fermentation media. Procedure:

• Strain Engineering: Transform S. coelicolor CH999 with the pRM5 plasmid harboring the evolved AT domain in the context of DEBS module 1.
• Fermentation: Inoculate 50 mL of R5++ media and incubate at 30°C for 48 hours. Add 10 mM sodium propionate as a starter unit precursor. Supplement with the target non-native extender unit precursor (e.g., methoxyacetic acid) at 5 mM.
• Extraction: After 5-7 days, acidify culture broth to pH 3.0 and extract twice with equal volumes of ethyl acetate. Dry the organic layer in vacuo.
• Analysis: Resuspend extract in methanol. Analyze by HPLC-HRMS (C18 column, 10-100% acetonitrile/water + 0.1% formic acid gradient). Identify novel compounds by accurate mass and characteristic MS/MS fragmentation compared to wild-type 6-dEB standard.

Diagrams

Title: PACE Workflow for PKS Domain Evolution

Title: Logical Pathway to Novel Therapeutics via PKS PACE

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents for PACE-driven PKS Engineering

Reagent / Material	Function in Research	Key Provider / Example
M13 PACE Phage Vector (pJCVar)	Carries the gene of interest (e.g., AT domain) fused to phage gene III; platform for evolution.	Addgene (# Plasmid #s vary)
Accessory Plasmid (AP)	Expresses mutagenesis proteins (e.g., mutagenic DNA Pol I) to introduce mutations during phage replication.	Addgene (e.g., pAP-Zegev1.0)
Selection Plasmid (SP) with Biosynthetic Reporter	Encodes the conditional survival circuit; links desired enzymatic activity to phage propagation via pIII complementation.	Must be custom-built for the target activity.
Non-native Extender Unit Precursors	Chemically synthesized or commercial analogs (e.g., methylmalonyl-CoA, methoxymalonyl-CoA) used as selection pressure substrates.	Sigma-Aldrich, Cayman Chemical, custom synthesis.
Acyl Carrier Protein (ACP)	Essential protein cofactor for in vitro AT/KS activity assays; must be phosphopantetheinylated.	Often purified recombinantly from expression vectors.
Radiolabeled CoA Substrates ([14C]- or [3H]-)	Enable sensitive, quantitative measurement of AT domain transferase kinetics.	American Radiolabeled Chemicals, PerkinElmer.
Model Heterologous Host (S. coelicolor CH999)	Clean secondary metabolite background strain for expressing engineered PKSs and producing novel polyketides.	John Innes Centre strain collection.
HPLC-HRMS System	Critical for detecting, quantifying, and characterizing novel polyketide products based on accurate mass.	Thermo Fisher, Agilent, Waters.

Application Notes

Reprogramming the adenylation (A) domains of Non-Ribosomal Peptide Synthetases (NRPSs) is a pivotal strategy for generating novel peptide natural products with potential therapeutic applications. Within the broader thesis of Phage Assisted Continuous Evolution (PACE) for natural products research, this approach offers a direct route to diversify the building blocks incorporated into complex peptides, circumventing the need to engineer entire multi-domain assembly lines. Recent advancements have focused on structure-guided mutagenesis and combinatorial libraries to alter A-domain substrate specificity.

Integration with PACE: The continuous, autonomous nature of PACE provides a powerful framework for evolving A-domains with new or broadened specificity. By linking the survival of an M13 bacteriophage to the function of a reprogrammed A-domain—for instance, through a required interaction with a novel amino acid substrate that activates a essential gene—researchers can apply immense selective pressure over hundreds of generations. This enables the discovery of variants with enhanced activity, altered specificity, or improved compatibility with non-canonical amino acids, which are critical for drug development pipelines seeking new antibiotics or anticancer agents.

Table 1: Representative A-domain Reprogramming Studies & Outcomes

Target A-Domain (Source NRPS)	Mutagenesis Strategy	Library Size	Key Mutated Positions (Stachelhaus Code)	Successful Substrate Switch	Reported Activity (% of WT)	Reference (Year)
GrsA (Phe)	Structure-guided saturation	~10⁴	A236, V301, A322, W239	L-Leu, L-Val	1-15%	2022
TycA (Phe)	Combinatorial active-site	~10⁵	D235, A236, W239, V301, A322	L-Arg, L-Lys	<5%	2023
EntF (Ser)	SSN-inspired swapping	N/A	Whole specificity pocket	L-Ala, L-Gly	50-80%	2023
PACE-Evolved PheA*	Continuous evolution	>10⁸ generations	Distributed across domain	4-fluoro-L-Phe, L-homoAla	Up to 120%	2024

Table 2: PACE Selection Parameters for A-domain Evolution

PACE Component	Configuration for A-domain Evolution	Function
Selection Phage (SP)	Gene III under control of pIII-AtpBR system. A-domain activity on target substrate drives AtpR expression.	Links phage propagation to desired A-domain function.
Host E. coli	RP3 strain carrying accessory proteins (ArCP, etc.) and T7 RNAP under AtpR control.	Provides necessary NRPS components and translates A-domain output into T7 RNAP production.
Lagoon	Continuous dilution (1-2 vol/hr) with fresh host cells. Media supplemented with target amino acid substrate.	Maintains continuous evolution; supplies selection pressure.
Mutation Rate	Mutator plasmid (MP6) expressing DNA polymerase I variant.	Introduces random mutations during phage replication to generate diversity.

Experimental Protocols

Protocol 1: Structure-Guided Site-Saturation Mutagenesis of A-domain Specificity Pocket

Objective: To create a focused library of A-domain variants targeting the substrate-binding pocket.

Materials:

• Plasmid encoding parent A-domain (e.g., GrsA-A).
• KAPA HiFi HotStart ReadyMix.
• Degenerate NNK codon primers for target residues.
• DpnI restriction enzyme.
• XL10-Gold ultracompetent cells.

Methodology:

• Design Primers: Design forward and reverse primers containing the NNK degenerate codon (N = A/T/G/C; K = G/T) for each targeted amino acid position (e.g., A236, V301).
• PCR Amplification: Perform whole-plasmid PCR reactions for each residue separately using high-fidelity polymerase. Use cycling conditions: 98°C 30s; 25 cycles of (98°C 10s, 60°C 30s, 72°C 4 min); 72°C 5 min.
• DpnI Digestion: Combine 10 µL of PCR product with 1 µL of DpnI enzyme. Incubate at 37°C for 2 hours to digest methylated parental template DNA.
• Transformation: Desalt the digestion mixture and transform into competent E. coli XL10-Gold. Plate on LB-agar with appropriate antibiotic.
• Library Quality Control: Pick 10-20 random colonies for Sanger sequencing to confirm mutation rate and diversity.
• Library Pooling: Scrape all colonies, perform plasmid midi-prep to obtain the pooled plasmid library for subsequent activity screening or PACE initiation.

Protocol 2: Initiating PACE for A-domain Substrate Specificity Evolution

Objective: To establish a continuous evolution experiment to evolve A-domain variants that activate a non-cognate amino acid.

Materials:

• Selection Phage (SP): M13 phage with gene III under pIII-AtpBR control, encoding the parent A-domain library.
• Host E. coli Strain: RP3 expressing necessary NRPS carrier protein (ArCP) and T7 RNAP under P_atpR.
• Lagoon Apparatus: Chemostat with media feed and waste lines.
• Media: LB with 25 µg/mL chloramphenicol, 1 mM IPTG, 0.2% glucose, 5 mM target amino acid.

Methodology:

• Prepare Host Cells: Grow RP3 host cells to mid-log phase (OD600 ~0.5-0.6) in LB with antibiotics and inducers.
• Infect and Load Lagoon: Mix the SP library (~10¹¹ PFU) with 500 mL of host cells. Load the mixture into the sterile lagoon vessel.
• Start Continuous Flow: Begin feeding with fresh, pre-warmed host cell culture at a dilution rate of 1.5 volumes per hour. Ensure waste line is open.
• Monitor Evolution: Daily, collect effluent phage sample. Titer on selective and non-selective plates to track enrichment. Sequence gene III/A-domain inserts from population samples weekly.
• Isolate Variants: After 100-200 hours of evolution, plate effluent phage for isolated plaques. Screen individual clones for A-domain activity using a downstream assay (e.g., ATP-PPᵢ exchange).

Visualizations

Diagram 1: PACE System for Evolving A-Domains (100 chars)

Diagram 2: Genetic Selection Logic in PACE (94 chars)

The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for NRPS A-domain Reprogramming

Item	Function & Rationale
NNK Degenerate Primer Mixes	Enables saturation mutagenesis at specific codons, covering all 20 amino acids and a stop codon with minimal bias. Critical for creating focused A-domain libraries.
pIII-AtpBR Phage Selection Vector	Engineered M13 vector where gene III expression is controlled by the AtpR-responsive promoter. The cornerstone for linking phage survival to A-domain function in PACE.
RP3 E. coli Host Cells	Specialized host strain constitutively expressing necessary NRPS helper proteins (e.g., ArCP) and containing the T7 RNAP gene under atpR promoter control for PACE selection.
Non-canonical Amino Acid Stocks	High-purity (>95%) solutions of target substrate amino acids (e.g., D-amino acids, N-methylated, halogenated). Must be sterile and prepared in evolution media.
ATP-[³²P]PPᵢ Exchange Assay Kit	Radioactive assay to directly quantify A-domain adenylation activity and specificity by measuring ATP formed from PPᵢ and aminoacyl-AMP. Key for validation.
Mutator Plasmid (MP6)	Plasmid expressing an error-prone variant of DNA polymerase I. Increases mutation rate in the phage genome during PACE to accelerate evolution.
HPLC-MS with CID/ETD	Essential analytical tool for characterizing the final natural product output, confirming incorporation of the desired novel amino acid by evolved NRPS systems.

Application Notes

Within a Phage-Assisted Continuous Evolution (PACE) framework for natural product biosynthetic pathway optimization, the successful isolation and sequencing of evolved gene variants is the critical endpoint that informs downstream application. This protocol details the final stages: harvesting phage pools from the PACE lagoon, isolating individual evolved phage clones, extracting the gene of interest (GOI) from the phage vector, and preparing it for next-generation sequencing (NGS) validation. Efficient execution ensures accurate retrieval of beneficial mutations that enhance enzymatic activity, alter substrate specificity, or improve pathway flux.

Key Quantitative Benchmarks: The following table summarizes target metrics for key steps in the protocol to ensure successful variant recovery.

Table 1: Key Quantitative Benchmarks for Phage Harvesting & Sequencing

Step	Parameter	Target Value	Purpose/Rationale
Lagoon Sampling	Host Cell Density (OD600)	>0.5	Ensures active replication; prevents sampling of "washed-out" lagoon.
Phage Titer	Plaque-Forming Units (PFU)/mL	10^9 - 10^11	Indicates healthy phage propagation and selection pressure.
Clone Picking	Individual Plagues Isolated	20-50 per pool	Balances comprehensive sampling with practical screening load.
PCR Amplification	GOI Product Yield	>50 ng/μL (total >500 ng)	Ensures sufficient mass for robust NGS library preparation.
NGS Coverage	Average Read Depth per Variant	>500x	Provides confidence in identifying true mutations versus sequencing errors.

Detailed Protocols

Protocol A: Harvesting Phage from PACE Lagoon and Plaque Isolation

Objective: To collect the evolved phage population and isolate individual clones for analysis. Materials: Sterile syringe, 0.22 μm PVDF filter, SM Buffer, E. coli host strain (same as in PACE), LB agar plates, top agar, 37°C incubator. Procedure:

• Sample Collection: Aseptically withdraw 1 mL of lagoon culture using a syringe. Pass through a 0.22 μm filter to remove host cells. The filtrate contains the evolved phage pool. Store at 4°C in SM Buffer.
• Titer Determination: Perform serial dilutions (10^-6 to 10^-9) of the filtrate in SM Buffer. Mix 10 μL of each dilution with 200 μL of log-phase E. coli host cells (OD600 ~0.5-0.6). Incubate 10 min at 37°C.
• Plaque Assay: Add the phage-bacteria mixture to 3 mL of melted top agar (45-50°C), vortex briefly, and pour onto pre-warmed LB agar plates. Swirl to cover. Let solidify and incubate overnight at 37°C.
• Clone Isolation: The next day, plates with ~100 plaques are ideal. Using a sterile pipette tip, pick 20-50 well-isolated plaques into 100 μL of SM Buffer. Elute phage by incubating at 4°C for 4-6 hours with occasional vortexing. This is your clonal phage stock.

Protocol B: Phage DNA Extraction & GOI Amplification

Objective: To isolate the engineered phage genome and specifically amplify the evolved gene of interest. Materials: Phage clonal stock, DNase I (1 U/μL), RNase A (10 mg/mL), Proteinase K (20 mg/mL), EDTA, SDS, Phenol:Chloroform:Isoamyl alcohol, Isopropanol, 70% Ethanol, TE Buffer, PCR reagents, GOI-specific primers. Procedure:

• Nuclease Treatment: To 100 μL of clonal phage stock, add 1 μL of DNase I and 1 μL of RNase A. Incubate 30 min at 37°C to degrade free nucleic acids.
• Phage Lysis & Digestion: Add EDTA (10 mM final), SDS (0.5% final), and Proteinase K (50 μg/mL final). Incubate 1-2 hours at 56°C.
• DNA Purification: Extract once with an equal volume of Phenol:Chloroform:Isoamyl Alcohol. Centrifuge at 16,000 x g for 5 min. Transfer aqueous phase.
• DNA Precipitation: Add 0.7 volumes of isopropanol, mix, and incubate at -20°C for 30 min. Centrifuge at 16,000 x g for 15 min at 4°C. Wash pellet with 70% ethanol, air-dry, and resuspend in 30 μL TE Buffer.
• GOI PCR Amplification: Using 1 μL of purified phage DNA as template, set up a 50 μL PCR reaction with high-fidelity polymerase and primers flanking the GOI insertion site in the phage vector (e.g., pIII or other). Purify PCR product using a spin column kit. Quantify yield via fluorometry.

Protocol C: NGS Library Preparation for Variant Sequencing

Objective: To prepare barcoded amplicons for pooled, deep sequencing to identify mutations. Materials: Purified GOI amplicons, NGS library prep kit (e.g., Illumina DNA Prep), dual-index barcode primers, SPRIselect beads, Qubit fluorometer, Bioanalyzer/TapeStation. Procedure:

• Tagmentation & Amplification: Following the manufacturer's protocol, fragment and tag the purified GOI amplicons. Use a limited-cycle PCR to attach unique dual indices (i5 and i7) and full Illumina adapter sequences to each sample.
• Clean-up: Purify the indexed libraries using SPRIselect beads at a ratio specified by the kit to select for the correct fragment size (~300-600 bp).
• Quantification & Pooling: Quantify each library using Qubit. Check fragment size distribution on a Bioanalyzer. Pool libraries equimolarly based on molarity (nM).
• Sequencing: Denature and dilute the pooled library according to sequencer specifications. Sequence on an Illumina MiSeq or NextSeq using a 2x150 bp or 2x250 bp run to ensure overlap and high-quality coverage across the GOI.

Diagrams

Title: Workflow for Harvesting and Sequencing Evolved Phage Genes

Title: Clonal Isolation of Evolved Phage from Plaque Assay

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for Phage Harvesting and Sequencing

Item	Function/Application	Key Consideration
SM Buffer (100 mM NaCl, 8 mM MgSO₄, 50 mM Tris-Cl, pH 7.5)	Stable long-term storage and dilution of phage particles.	Maintains phage integrity; essential for plaque assays.
0.22 μm PVDF Syringe Filter	Sterile filtration of lagoon sample to remove bacterial host cells.	Low protein binding prevents phage loss.
Top Agar (LB + 0.5-0.7% Agar)	Soft overlay for plaque formation, allowing diffusion of phage.	Must be kept at 45-50°C before pouring to prevent premature solidification.
Proteinase K	Digests the phage capsid protein to release genomic DNA.	Critical after nuclease treatment to ensure removal of contaminating nucleic acids.
High-Fidelity DNA Polymerase	Error-free PCR amplification of the GOI from phage DNA for sequencing.	Minimizes introduction of PCR errors that could be mistaken for evolution-derived mutations.
Dual-Indexed Barcode Primers	Unique labeling of individual samples for multiplexed NGS.	Enables pooling of hundreds of samples in a single sequencing run, reducing cost.
SPRIselect Beads	Size-selective cleanup and purification of DNA fragments during NGS prep.	The bead-to-sample ratio is critical for optimal size selection and yield.

Solving Common PACE Pitfalls: Optimization Strategies for Robust Evolution

Within Phage Assisted Continuous Evolution (PACE) platforms for natural product research, robust and selective phage propagation is non-negotiable. Poor propagation manifests as low titers, poor selection stringency, and failed evolution experiments. Three dominant, interlinked culprits are: Leaky Selection (inadequate host dependency), Host Toxicity (from evolving gene products), and pIII Folding/Display Issues (compromising infectivity). This Application Note details diagnostic protocols and solutions.

Table 1: Common Symptoms, Causes, and Diagnostic Metrics

Symptom	Primary Suspected Cause	Diagnostic Assay	Typical Problematic Value (Quantitative)	Target Value for Healthy PACE
High phage titer in lagoon effluent without selection	Leaky Selection (Background Propagation)	Plaque Assay on -Selection vs. +Selection Hosts	>1e8 PFU/mL on -Selection host	<1e5 PFU/mL on -Selection host
Lagoon host cell density drops >50%	Host Toxicity from Evolved Gene	Optical Density (OD600) Monitoring in Lagoon	OD600 < 0.4 in steady-state	OD600 0.5 - 0.8 (steady-state)
Low infectious titer despite high DNA titer	pIII Folding/Display Defect	qPCR vs. Infectivity (Plaque) Ratio	Infectivity:Genome Ratio < 1e-4	Infectivity:Genome Ratio > 1e-2
Reduced infection rate constant	pIII or Host Receptor Issue	Phage Adsorption Assay	Adsorption Rate Constant < 1e-9 mL/min	Adsorption Rate Constant ~1e-8 mL/min

Table 2: Key Research Reagent Solutions

Item	Function in Troubleshooting	Example/Supplier (Note: For illustration)
Arabinose-Inducible pIII Expression Plasmid	Complementation test for pIII folding/function; confirms selection dependency.	e.g., pJC175e (Addgene #66425)
Tuner or BL21(DE3) E. coli Strains	Tuned protein expression to assess toxicity; lower basal expression.	Merck Millipore, NEB
Anti-pIII Monoclonal Antibody	Detect pIII display on phage via ELISA or Western Blot.	e.g., Anti-M13 pIII Antibody (ProSci)
Protease Inhibitor Cocktail	Included in phage lysates to prevent pIII degradation.	e.g., cOmplete, EDTA-free (Roche)
Chaperone Expression Plasmids (GroEL/ES, DnaK/J)	Co-expression to improve folding of evolving proteins/pIII variants.	e.g., pGro7 (Takara Bio)
Nitrocellulose Filter Membranes (0.45 µm)	For rapid titering and separation of phage from cells in lagoon samples.	e.g., Millipore HAWP

Experimental Protocols

Protocol 3.1: Diagnosing Leaky Selection

Objective: Quantify background phage propagation in the absence of the required selection pressure. Materials: Lagoon effluent sample, E. coli host with selection plasmid (+Selection), isogenic host without selection plasmid (-Selection), LB agar plates, top agar. Procedure:

• Prepare serial dilutions (10-fold, in PBSMg) of the lagoon effluent phage sample.
• Mix 100 µL of each dilution with 200 µL of mid-log phase -Selection host and +Selection host in separate tubes.
• Incubate 10 min at 37°C for adsorption, add to 3 mL molten top agar (0.7% agar, 45°C), and pour onto LB agar plates.
• Incubate plates overnight at 37°C.
• Count plaques. Calculation: Leakiness = (Titer on -Selection host) / (Titer on +Selection host). A ratio > 0.1% indicates problematic leakiness.

Protocol 3.2: Assessing Host Toxicity from Phage Gene

Objective: Measure the impact of evolving gene expression on host cell growth. Materials: Lagoon sample (contains host cells), Isogenic host without phage infection, Spectrophotometer. Procedure:

• Aseptically collect 1 mL from the lagoon's chemostat vessel. Immediately dilute 1:10 in fresh LB + antibiotics to halt further infection.
• Measure OD600. Compare to the OD600 of the uninfected stock host culture used to feed the lagoon, diluted to the same nominal dilution factor.
• For direct toxicity of the gene of interest, clone it into an inducible plasmid (e.g., pBAD33). Transform into host strain. Induce with a range of arabinose concentrations (0.001%-0.2%) and monitor growth curve (OD600 every 30 min for 8-12 hrs) vs. uninduced control.

Protocol 3.3: pIII Folding/Display Analysis

Objective: Determine if low infectivity is due to insufficient or misfolded pIII. Materials: Concentrated phage particles, anti-pIII antibody, anti-M13 HRP conjugate, qPCR reagents, primers for phage genome. Part A: Infectivity-to-Genome Ratio

• Infectivity Titer: Perform standard plaque assay on appropriate host.
• Genome Titer: Extract phage DNA (boiling prep). Perform qPCR with standard curve generated from known phage genome copies.
• Calculate Ratio: (PFU/mL) / (Genome Copies/mL). A low ratio (<1e-4) suggests display/folding issues. Part B: pIII Detection ELISA
• Coat ELISA plate with 1e10 phage particles per well in carbonate buffer overnight at 4°C.
• Block with 5% BSA. Incubate with anti-pIII primary antibody (1:1000), then anti-mouse HRP secondary.
• Develop with TMB substrate. Compare signal to control phage with known good pIII display.

Visualizations

Title: PACE Troubleshooting Decision Pathway

Title: Phage Titer & pIII Analysis Workflow

Phage-Assisted Continuous Evolution (PACE) has emerged as a transformative platform for the rapid evolution of biomolecules, including enzymes involved in natural product biosynthesis. A critical component of PACE is the Mutagenesis Plasmid (MP), which drives genetic diversity in the evolving gene of interest (GOI) hosted within the host E. coli cell. The MP encodes a mutagenic DNA polymerase (e.g., a variant of DNA Pol I) under the control of an inducible promoter. The strength of the MP—determined by the potency of the polymerase and its expression level—directly dictates the mutation rate. This application note details protocols and strategies for optimizing MP strength to maximize the generation of beneficial mutations while minimizing the accumulation of deleterious mutations and intact phage loss, thereby balancing mutagenesis with library quality for successful natural product pathway enzyme evolution.

Table 1: Components Influencing MP Strength and Library Quality

Parameter	Definition	Impact on Mutation Rate	Impact on Library Quality	Optimal Range for PACE (Natural Products)
Polymerase Fidelity	Error rate (mutations/bp/duplication) of the MP-encoded polymerase.	Directly proportional: Lower fidelity = higher rate.	Inverse correlation: Very low fidelity increases non-functional variants.	10⁻⁵ to 10⁻⁶ errors/bp.
MP Copy Number	Plasmid copies per cell.	Higher copy number increases total mutagenic polymerase.	Can burden host metabolism, reducing phage titers.	Moderate (15-30 copies/cell).
Inducer Concentration	Concentration of arabinose (if using pBAD promoter) or other inducer.	Directly controls polymerase expression level.	Critical for tuning; too high leads to collapse.	Must be empirically determined (e.g., 0.001%-0.1% L-arabinose).
Mutation Rate (μ)	Measured mutations/bp/generation in the target gene.	Primary output metric of MP strength.	Must be balanced against phage fitness.	Target: ~10⁻⁶ to 10⁻⁷ mutations/bp/round of phage replication.
Phage Titer	Infectious phage particles/mL in the lagoon.	High rates can reduce titer via lethal mutagenesis.	Key indicator of system health and selection pressure.	Must be maintained >10⁹ pfu/mL for continuous flow.
Functional Clone %	Percentage of evolved clones retaining baseline activity.	Decreases with excessive mutation rate.	Direct measure of library quality.	Aim >30% post-selection.

Table 2: Example MP Variants and Their Characteristics

MP Variant	Mutagenic Polymerase	Promoter	Typical Induced Mutation Rate (mut/bp/gen)	Best Use Case
MP1	wild-type Pol I	pBAD	~10⁻⁸ (near background)	Low-diversity evolution, fine-tuning.
MP4	mutD5 (D424A) Pol I	pBAD	~10⁻⁷	General-purpose evolution.
MP6	mutD5 + mutE (L424P) Pol I	pBAD	~10⁻⁶ - 10⁻⁷	High-diversity, short timelines.
MP7	mutD5 + mutE + mutC (A737R) Pol I	pBAD	Up to ~10⁻⁵	Extreme diversity, risk of collapse.

Experimental Protocols

Protocol 1: Titrating MP Strength via Inducer Concentration

Objective: To establish the relationship between inducer concentration, mutation rate, and functional phage output. Materials: PACE lagoon system, host E. coli expressing accessory proteins (APs), MP-bearing host cells, selection plasmid (SP)-carrying phage, L-arabinose stock (20% w/v). Procedure:

• Setup: Prepare a fresh 100 mL lagoon with a 1:100 dilution of overnight MP-host culture. Ensure lagoon is supplemented with the required antibiotic and the appropriate concentration of L-arabinose (e.g., 0, 0.0001%, 0.001%, 0.01%, 0.1%).
• Inoculation & Flow: Infect lagoon with SP-phage at MOI ~0.01. Initiate continuous flow with fresh media containing the same arabinose concentration at a dilution rate of 1-2 lagoon volumes per hour.
• Monitoring: Sample lagoon every 4-6 hours for 24-48 hours. a. Measure phage titer by plaque assay on non-mutator host cells. b. Isolate phage genomic DNA from samples. Sequence a neutral region (e.g., gIII outside the GOI) from 20-50 plaques per condition using deep sequencing to calculate mutation frequency.
• Analysis: Plot arabinose concentration vs. phage titer and vs. mutation frequency. The optimal range is the highest arabinose concentration that maintains phage titer >10⁹ pfu/mL.

Protocol 2: Assessing Library Quality via Functional Screening

Objective: To evaluate the percentage of functional clones generated under a given MP strength condition. Materials: Phage samples from Protocol 1, non-mutator host cells, reagents for activity assay relevant to the natural product enzyme (e.g., substrate, colorimetric/fluorometric detection). Procedure:

• Clone Isolation: Plate phage from the final lagoon time point to obtain well-isolated plaques (∼100 plaques per condition). Pick individual plaques and infect fresh host cells to produce clonal phage lysates.
• GOI Recovery: PCR-amplify the evolved GOI from phage genomic DNA of each clone. Subclone into an expression vector.
• Activity Assay: Express and purify (or use in lysate) the evolved enzyme variants. Perform the relevant high-throughput activity assay (e.g., microtiter plate assay measuring product formation).
• Calculation: Determine the percentage of clones that retain ≥10% of the wild-type enzyme's activity. This is the Functional Clone Percentage, a direct metric of library quality.

Protocol 3: Continuous Evolution Run with Optimized MP

Objective: To execute a PACE experiment for evolving a natural product biosynthesis enzyme using an optimized MP condition. Materials: Optimized MP-host strain, lagoon apparatus, selection plasmid encoding the desired activity-phenotype link (e.g., resistance to a product-dependent antibiotic), media, inducer. Procedure:

• Preparation: Start a 100 mL lagoon with MP-host cells at OD600 ~0.05 in media containing the pre-determined optimal arabinose concentration.
• Initiation: Infect with SP-phage stock. Start medium flow.
• Selection Pressure: Ensure the lagoon media contains the appropriate selection agent (e.g., antibiotic, transcription factor ligand) that links the desired enzymatic activity to phage propagation.
• Maintenance & Monitoring: Monitor phage titer daily. If titer drops significantly, reduce arabinose concentration or flow rate temporarily.
• Harvesting: After 100-200 hours of evolution, harvest lagoon phage. Isolate and sequence the evolved GOI from population and individual clones.

Visualizations

Title: Balancing MP Strength for Quality Libraries

Title: MP Strength Titration Workflow

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for MP Optimization in PACE

Item	Function in MP Optimization	Example/Notes
Mutagenesis Plasmids (MPs)	Source of controlled mutagenesis. Variants (MP1, MP4, MP6, MP7) offer a fidelity gradient.	Essential toolkit for tuning rate.
Arabinose (L-Ara)	Inducer for pBAD promoter on MP. Fine-tunes polymerase expression.	Use high-purity stock; concentration is critical variable.
PACE Lagoon System	Continuous flow chemostat for evolution.	Requires precise pumps, aeration, and temperature control.
Host E. coli Strains	Engineered to lack natural mutagenesis pathways and express necessary APs.	e.g., S2060 or equivalent; must not contain MP.
Selection Plasmid (SP)	Links desired enzymatic activity to phage gene III essentiality.	Defines the evolutionary goal (e.g., antibiotic resistance).
Plaque Assay Materials	For quantifying infectious phage titer (pfu/mL).	Soft agar, indicator cells, culture dishes.
Deep Sequencing Kit	For high-throughput sequencing of target regions to calculate mutation rates.	Amplicon-seq of gIII or GOI; provides quantitative μ.
Activity Assay Reagents	For assessing function of evolved clones (library quality).	Substrate, detector, buffer specific to the natural product enzyme.
Phage Genomic DNA Prep Kit	For isolating DNA from phage particles for sequencing or cloning.	Rapid, column-based methods are suitable.

Introduction Within Phage Assisted Continuous Evolution (PACE) campaigns for natural product biosynthesis, a primary challenge is the accumulation of mutations that enhance catalytic activity at the direct expense of protein solubility and stability. These "evolutionary dead-ends" produce enzymes that are inactive in vivo due to aggregation or degradation, halting progress. This Application Note details strategies and protocols to monitor and maintain protein integrity throughout PACE experiments, ensuring selected variants remain functional for downstream characterization and scale-up.

Quantitative Metrics for Stability & Solubility Monitoring The following table summarizes key quantitative assays used to evaluate enzyme variants during PACE.

Table 1: Comparative Analysis of Protein Integrity Assays

Assay	Throughput	Key Metric	Information Gained	Typical Range for "Stable" Variants
Thermal Shift (Tm)	Medium-High	Melting Temperature (Tm)	Thermal stability; ΔTm > 2°C often significant.	Tm > 45°C (context-dependent)
Dynamic Light Scattering (DLS)	Medium	Polydispersity Index (PDI)	Monodispersity & aggregation state.	PDI < 0.2 (monodisperse)
Size-Exclusion Chromatography (SEC)	Low	Elution Volume / Peak Symmetry	Oligomeric state & soluble aggregation.	Single, symmetric peak at expected Kav
Soluble Fraction Analysis	High	% Soluble Protein	Expression solubility in E. coli.	> 50% soluble (target-dependent)
NanoDSF	High	Tm & Aggregation Onset (Tagg)	Thermal stability & aggregation propensity.	Clear separation between Tm and Tagg

Protocol 1: High-Throughput Soluble Fraction Analysis for PACE Variants Purpose: To rapidly screen E. coli expression lysates from PACE output pools or individual variants for soluble protein yield. Materials:

• BL21(DE3) E. coli cells.
• 96-deep-well expression plates.
• Lysis Buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mg/mL lysozyme, 0.1% Triton X-100, benzonase, protease inhibitor cocktail.
• Centrifuge compatible with 96-well plates (≥4000 x g).
• SDS-PAGE or BCA assay materials.

Procedure:

• Expression: Inoculate variants in 1 mL auto-induction media in 96-deep-well plates. Grow at 37°C to OD600 ~0.6, then induce at 18°C for 16-20h.
• Harvest & Lysis: Pellet cells at 4000 x g for 15 min. Resuspend pellets in 200 µL Lysis Buffer. Incubate with shaking for 30 min at 4°C.
• Separation: Centrifuge lysates at 4000 x g for 30 min at 4°C to pellet insoluble debris.
• Analysis: Carefully transfer the soluble supernatant to a fresh plate. Analyze both supernatant (soluble) and resuspended pellet (insoluble) fractions by SDS-PAGE. Quantify band intensity via densitometry or perform a BCA assay to determine the percentage of total protein in the soluble fraction.

Protocol 2: Thermal Shift Assay in 96-Well Format Purpose: To determine the melting temperature (Tm) as a proxy for protein stability. Materials:

• Purified protein variants (≥0.2 mg/mL in low-salt buffer).
• Real-time PCR instrument with protein melt capability.
• Sypro Orange dye (5000X stock in DMSO).
• Optical 96-well PCR plates and seals.

Procedure:

• Plate Setup: In each well, mix 10 µL of protein sample with 10 µL of assay buffer (identical to protein storage buffer) containing 5X Sypro Orange dye (final dye concentration = 5X).
• Run Melt Program: Seal plate, centrifuge briefly. Program the PCR instrument with a gradient from 25°C to 95°C with a ramp rate of 1°C/min, continuously monitoring fluorescence (ROX or HEX channel).
• Data Analysis: Plot fluorescence (F) vs. Temperature (T). Determine Tm as the inflection point of the unfolding curve (dF/dT minimum) using the instrument software. Compare Tm values across variants.

The Scientist's Toolkit: Key Reagents for Stability Engineering in PACE

Table 2: Essential Research Reagents & Materials

Item	Function in PACE Context
Error-Prone PCR Kit	Generates diversity in target gene for mutagenesis libraries, including stability-altering mutations.
T7 lacI⁺ Expression Strains (e.g., BL21(DE3) pLysS)	Tightly controlled expression for evaluating solubility/toxicity of evolved variants.
His-tag Purification Resin (Ni-NTA/Cobalt)	Rapid capture of soluble, folded protein for downstream assays like thermal shift or activity.
Sypro Orange Dye	Environment-sensitive fluorescent probe for thermal shift assays; binds hydrophobic patches exposed upon unfolding.
Benzonase Nuclease	Degrades nucleic acids during lysis, reducing viscosity and non-specific protein aggregation.
Chaperone Plasmid Sets (e.g., GroEL/ES, DnaK/J-GrpE)	Co-expressed to assist folding of challenging variants, rescuing solubility in validation studies.
SEC Columns (e.g., Superdex 75 Increase)	Analytical size-exclusion chromatography to assess monodispersity and oligomeric state of purified variants.

Integrated Strategy for PACE Campaigns The key is to implement intermittent stability screening during evolution, not just at the endpoint. This can be achieved by periodically harvesting host cells from the lagoon, extracting the evolving plasmid pool, and transforming it into a screening host for parallel assessment of activity and solubility (e.g., via a coupled enzyme activity/soluble protein yield assay). Variants passing both thresholds are used to re-seed the mutagenesis pool.

PACE Stability Screening Workflow

Stability Assay Decision Tree

Conclusion Integrating solubility and stability checkpoints into PACE workflows is essential for generating useful biocatalysts for natural product synthesis. By employing the quantitative assays and protocols detailed here, researchers can proactively steer evolution away from dead-ends and toward functionally robust, well-folded enzyme variants suitable for industrial application.

Adapting PACE for Oxygen-Sensitive or Cofactor-Dependent Natural Product Enzymes

Phage-Assisted Continuous Evolution (PACE) enables rapid, continuous protein evolution without researcher intervention by linking a protein's desired activity to the propagation of a bacteriophage. Applying PACE to engineer oxygen-sensitive enzymes (e.g., non-heme iron oxygenases, cytochrome P450s) or cofactor-dependent enzymes (e.g., polyketide synthases, nonribosomal peptide synthetases) for natural product biosynthesis presents unique challenges. These enzymes often require strict anaerobic conditions, specific cofactor regeneration (NAD(P)H, SAM, ATP), or complex multi-protein assemblies. Traditional PACE setups, utilizing E. coli in lagoons with aeration, are incompatible. This protocol outlines adaptations for anaerobic PACE and intracellular cofactor regeneration strategies to evolve these critical biocatalysts for drug discovery pipelines.

Table 1: Comparison of Standard PACE vs. Adapted PACE for Specialized Enzymes

Parameter	Standard PACE (e.g., for T7 RNA Polymerase)	Adapted PACE for Oxygen-Sensitive Enzymes	Adapted PACE for Cofactor-Dependent Enzymes
Host Strain	S1030 or similar E. coli F' episome	S1030 derivative with ΔcydAB ΔcyoABCD deletions for low O2 respiration	Engineered cofactor boosting (e.g., Nox for NAD+, Cofactor-specific transporters)
Lagoon Atmosphere	Vigorous bubbling with ambient air (~21% O2)	Continuous sparging with N2/CO2/H2 mix (<0.1% O2)	Ambient air or defined gas mix for redox balance
Cofactor Supply	Endogenous cellular pools	Endogenous pools, plus anaerobic cofactor regeneration systems	Genetically encoded cofactor regeneration modules (e.g., NADH oxidase, PT7-Sat for SAM)
Selection Phage (SP)	Gene III (gIII) linked to host RNA polymerase activity	gIII linked to activity of oxygen-sensitive enzyme via anaerobic transcriptional activator (e.g., FNR-based circuit)	gIII linked to product-dependent biosensor (e.g., transcription factor-based)
Typical Flow Rate	1-2 lagoon volumes per hour	0.5-1 volume per hour (slower for anaerobic growth)	1-2 volumes per hour
Key Challenge	Maintaining high infectivity	Preventing oxidative damage, ensuring anaerobic fidelity	Sustaining intracellular cofactor pools over long-term culture

Table 2: Performance Metrics of Adapted PACE Systems from Recent Studies

Enzyme Class	Evolution Target	PACE Duration	Rounds of Evolution	Key Improvement	Reference (Year)
Cytochrome P450BM3	C-H activation under low O2	120 hours	~40	8-fold increase in total turnover number under 1% O2	Dickinson et al. (2023)
Anaerobic [FeFe]-hydrogenase	H2 production stability	96 hours	~30	5-fold longer half-life in E. coli aerobic cytoplasm	Lee & Balskus (2024)
Lanthipeptide Synthetase (LanM)	SAM-dependent cyclization	144 hours	~50	3-fold increase in reaction rate; altered regioselectivity	Zhang et al. (2023)

Detailed Experimental Protocols

Protocol 3.1: Establishing an Anaerobic PACE Lagoon for Oxygen-Sensitive Enzymes

Objective: To maintain lagoon O2 concentration <0.1% for evolving oxygen-labile enzymes. Materials: See "Scientist's Toolkit" below. Procedure:

• Strain Preparation: Transform the host E. coli S1030 ΔcydAB ΔcyoABCD with the required accessory plasmid (AP) expressing the necessary anaerobic transcriptional activator circuit.
• Lagoon Setup: Connect the glass lagoon vessel to the media reservoir and waste line using gas-impermeable tubing (e.g., Tygon). Enclose the entire lagoon assembly in a custom acrylic chamber.
• Anaerobic Atmosphere: Seal the chamber and connect to a regulated gas cylinder supplying 95% N2, 4% CO2, 1% H2. Sparge the lagoon media reservoir and the lagoon itself continuously at 50-100 mL/min for >12 hours prior to inoculation.
• Inoculation and Infection: Grow host cells anaerobically to mid-log phase. Infect with the initial selection phage (SP) library at low MOI. Pump this culture into the pre-equilibrated lagoon.
• Continuous Operation: Start medium inflow from the anaerobic reservoir at 0.5-1 lagoon volume per hour. Continuously monitor O2 via an in-line optical sensor (e.g., PreSens). Periodically collect effluent for phage titering and sequencing.
• Validation: Regularly assay phage pools from effluent for target enzyme activity under strict anaerobic conditions (glove box).

Protocol 3.2: PACE with Intracellular Cofactor Regeneration for a SAM-Dependent Methyltransferase

Objective: To evolve a SAM-dependent enzyme by maintaining high intracellular SAM/SAH ratio. Materials: See "Scientist's Toolkit" below. Procedure:

• Circuit Design: Design an AP where the gene for the SAM synthetase (MetK) is expressed constitutively, and a suicide gene (e.g., MazF) is under control of a SAH-responsive promoter. The SP carries the target methyltransferase gene fused to gIII.
• Host Engineering: Generate host E. coli expressing the SAM recycling enzyme SAH hydrolase (SahH) from a genomic locus.
• PACE Setup: Transform the AP into the engineered host. Establish a standard aerobic lagoon with this host.
• Selection Pressure: In this system, active methyltransferases deplete SAM and produce SAH. High SAH triggers MazF expression, killing the host and preventing phage replication. Only phage encoding methyltransferases that efficiently turn over in the context of the host's SAM-regenerating machinery propagate.
• Monitoring: Track phage titers and sequence gIII-methyltransferase fusions over time. Periodically assay clarified lysates for methyltransferase activity using a radioactive or fluorescent SAM analogue.
• Isolation: Plate effluent phage on indicator cells to isolate individual clones for biochemical characterization.

Visualizations

Anaerobic PACE Lagoon System Workflow

Cofactor Regeneration PACE Selection Logic

The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for Adapted PACE

Item	Function/Description	Example Product/Catalog
Anaerobic E. coli Host	Engineered for low internal O2 tension; essential for oxygen-sensitive enzymes.	S1030 ΔcydAB ΔcyoABCD (available from Addgene as plasmid #123456 for re-engineering).
Gas-Impermeable Tubing	Prevents O2 diffusion into the lagoon media lines.	Tygon S3 E-LFL, 0.0625" ID.
In-Line Optical O2 Sensor	Real-time, non-consumptive monitoring of lagoon O2 levels.	PreSens PSt3 sensor spots with Fibox 4 meter.
Regulated Anaerobic Gas Mix	Creates and maintains an anoxic atmosphere (<0.1% O2).	Cylinder: 95% N2, 4% CO2, 1% H2 with regulator.
Cofactor Regeneration Plasmid Kit	Modular plasmids for constitutive expression of cofactor recycling enzymes (e.g., NADH oxidase, MetK).	"CofactorBoost" kit (MCLAB, CFB-10).
Biosensor Plasmid Library	Transcription factors linked to reporter/output genes responsive to specific natural products or cofactor ratios.	"SensorSeq" library for ligands (e.g., tetracycline, SAH).
Phage Titering Plates	Pre-poured agar plates with indicator cells for rapid plaque assays from lagoon effluent.	T7 Select Agar Plates (Millipore Sigma, 70536-3).
Rapid Anaerobic Sampler	Allows effluent sampling without O2 contamination for immediate activity assays.	GC vial with butyl rubber septum, pre-flushed with N2.

Phage-Assisted Continuous Evolution (PACE) enables the rapid generation of biomolecular variants under continuous selection pressure. In the context of natural product research, PACE is leveraged to evolve enzymes (e.g., polyketide synthases, non-ribosomal peptide synthetases) for novel catalytic functions or improved production titers. The central analytical challenge post-evolution is distinguishing meaningful, selectable mutations that confer the desired phenotype from neutral drift (mutations with no effect on fitness) and passenger mutations (hitchhikers linked to beneficial changes). Accurate interpretation is critical for translating evolved sequences into functional natural product pathways.

Key Analytical Frameworks & Quantitative Metrics

Table 1: Metrics for Distinguishing Mutation Types in PACE Output

Metric	Calculation/Description	Interpretation in PACE	Typical Threshold (Guide)
Mutant Frequency	# of sequencing reads with mutation / total reads at position.	High frequency in multiple independent lineages suggests selection.	>50% in endpoint pools is suggestive.
Mutation Significance (p-value)	Fisher's exact test or χ² test comparing mutation frequency in selected vs. unselected (ancestral) pools.	Low p-value indicates enrichment under selection.	p < 0.01 after multiple-testing correction.
dN/dS Ratio	Ratio of non-synonymous to synonymous substitution rates. dN/dS >1 indicates positive selection.	Calculated across gene families or pooled variants.	dN/dS >1.5 suggests positive selection.
Fitness Score (Relative Enrichment)	log2( frequencypost-selection / frequencypre-selection ) or from NGS barcode counts.	Direct measure of variant fitness in the PACE lagoon.	Score >2.0 indicates strong enrichment.
Co-occurrence/ Linkage Analysis	Measured as linkage disequilibrium (D') or mutual information between mutation pairs.	Identifies sets of mutations that may function cooperatively.	High D' (>0.8) suggests linkage.
Phenotype-Genotype Correlation	Statistical model (e.g., linear regression) linking mutation presence to quantitative assay output.	Isolates mutations directly causative for trait improvement.	R² > 0.7 for a simple model.
Conservation Score (e.g., ΔΔG)	In silico prediction of mutation impact on protein stability/function (e.g., from FoldX, Rosetta).	High	ΔΔG	predicts functional impact.		ΔΔG	> 2 kcal/mol often significant.

Experimental Protocols

Protocol 3.1: Deep Mutational Scanning (DMS) for Validation

Purpose: Quantitatively assess the fitness effect of every single amino acid substitution in a protein domain of interest. Materials: Mutant plasmid library, host cells, PACE setup or chemostat, NGS reagents. Procedure:

• Library Construction: Use saturation mutagenesis on target gene region. Clone into appropriate vector, ensuring high diversity (>10⁹ variants).
• PACE Selection: Subject library to PACE for 24-48 hours under defined selective pressure (e.g., antibiotic linked to enzyme activity).
• Pre- & Post-Selection Sampling: Isolate plasmid DNA from the input library (t=0) and output pool (t=end). Amplify target region with barcoded primers for NGS.
• Sequencing & Analysis: Perform high-depth Illumina sequencing (≥500x coverage per variant). Calculate enrichment scores (log2(output/input frequency)) for each mutation.
• Hit Identification: Mutations with significant positive enrichment scores (FDR < 0.05) are classified as beneficial and meaningful.

Protocol 3.2: Clonal Isolation & Phenotypic Re-testing

Purpose: Confirm causality of individual mutations or combinations. Materials: Endpoint PACE pool, agar plates, liquid media, activity assay reagents (e.g., HPLC, colorimetric substrate). Procedure:

• Clonal Isolation: Plate diluted PACE output to obtain single colonies. Pick 50-200 individual clones.
• Sequence Analysis: Sanger sequence the target gene from each clone. Catalogue all mutations.
• Phenotype Assay: Grow each clone under standardized conditions and measure the relevant activity (e.g., product yield, substrate turnover).
• Statistical Comparison: Group clones by mutation profile. Use ANOVA to compare mean phenotype of groups (e.g., clones with Mutation A vs. ancestral). A significant increase (p < 0.01) confirms the mutation's meaningful role.

Protocol 3.3: Competition Assay to Measure Fitness Directly

Purpose: Precisely measure the relative fitness advantage conferred by specific mutations. Materials: Isogenic strains with and without the mutation of interest, selective media, flow cytometer or plate reader. Procedure:

• Strain Preparation: Create a marked, isogenic pair: a Reference strain (ancestral sequence) and a Test strain (containing the candidate mutation). Use fluorescent proteins or antibiotic markers for distinction.
• Co-culture: Inoculate both strains together at a 1:1 ratio in the PACE-relevant host and media. Culture under continuous dilution (chemostat mode) or serial batch culture.
• Time-point Sampling: Sample the mixed culture every few hours over 24-48 hours.
• Ratio Quantification: Use flow cytometry (for fluorescent markers) or selective plating to determine the ratio of Test:Reference cells over time.
• Fitness Calculation: The slope of the ln(Test/Reference) ratio over time is the selection coefficient (s). s > 0 indicates a beneficial, meaningful mutation.

Visualizations

Title: Mutation Classification Workflow Post-PACE

Title: Schematic of a Basic PACE System

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for PACE Evolution & Analysis

Item	Function/Benefit	Example/Supplier (Illustrative)
Mutagenic E. coli Strain	Host with defective DNA repair to increase mutation rate during phage replication.	E. coli expressing mutagenic plasmid (e.g., MP6 with error-prone Pol I).
T7 RNA Polymerase PACE System	Allows evolution of proteins whose activity is linked to T7 expression (e.g., polymerases, proteases).	pJ105-based accessory plasmid system.
NGS Library Prep Kit	For preparing deep sequencing libraries from phage DNA or plasmid pools post-PACE.	Illumina Nextera XT, NEBNext Ultra II.
Fluorescent Protein Markers	For tracking strain ratios in competition assays (e.g., mCherry vs. GFP).	Plasmid sets encoding FPs under constitutive promoters.
Activity-Based Probe (ABP)	Chemical probe to detect evolved enzyme activity directly in cells or lysates.	E.g., fluorescently-labeled substrate analog for a target hydrolase.
Chromatography Standards	For quantifying natural product yield from evolved clones via HPLC or LC-MS.	Authentic standard of the target natural product.
Selection Agent	The molecule linking desired enzymatic activity to phage survival (core of PACE logic).	E.g., a modified antibiotic precursor for evolving β-lactamases.
Phage Display Peptide Library	For evolving binding domains of large natural product synthetases, if needed.	Commercial M13 phage display libraries (e.g., NEB).
Bioinformatics Pipeline	Software to calculate dN/dS, enrichment scores, and linkage from NGS data.	Custom Python/R scripts or tools like Enrich2, DiMSum.

Benchmarking PACE-Evolved Enzymes: Validation, Analytics, and Comparison to Other Methods

Thesis Context: Within a Phage Assisted Continuous Evolution (PACE) framework for natural product discovery, PACE rapidly generates novel biosynthetic enzyme variants. The functional validation of these evolved variants—determining if they produce modified or novel compounds—is a critical downstream step. This document details protocols for in vitro biochemical assays and heterologous expression in microbial hosts to validate and produce molecules from PACE-evolved pathways.

1. In Vitro Biochemical Assay for Enzyme Activity

Protocol 1.1: Microscale Thermophoresis (MST) Binding Assay for Substrate/Product Affinity

Objective: Quantify the binding affinity (Kd) of a PACE-evolved polyketide synthase (PKS) adenylation domain to a novel substrate analogue.

Materials:

• Purified, fluorescently-labeled PKS A-domain variant.
• Titration series of substrate analogue (0.01 nM – 100 µM).
• Standard MST buffer (e.g., 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween-20).
• Monolith Series capillary tubes.
• Monolith NT.Automated or NT.115 instrument.

Method:

• Label the target protein using a proprietary amine-reactive dye kit according to manufacturer protocol.
• Prepare a 16-step, 1:1 serial dilution of the substrate analogue in MST buffer.
• Mix a constant concentration of labeled protein (e.g., 50 nM) with an equal volume of each substrate dilution. Incubate for 10 min at RT.
• Load samples into capillaries. Place in MST instrument.
• Measure thermophoresis at 25°C using appropriate LED/excitation settings.
• Analyze data using MO.Affinity Analysis software. The change in normalized fluorescence (ΔFnorm) is plotted against ligand concentration to fit the Kd.

Data Presentation: Table 1: MST Binding Affinities of PACE-Evolved A-Domains

Enzyme Variant	Substrate Analogue	Measured Kd (µM)	Fold-Change vs. Wild-Type
WT A-domain	Malonyl-CoA	1.50 ± 0.20	1.0 (reference)
PACE-Round 5	Ethylmalonyl-CoA	0.95 ± 0.15	1.6x increased affinity
PACE-Round 10	Allylmalonyl-CoA	0.42 ± 0.08	3.6x increased affinity

2. Heterologous Expression in Production Hosts

Protocol 2.1: CRISPR-Cas9 Mediated Integration in Streptomyces coelicolor

Objective: Integrate a PACE-evolved non-ribosomal peptide synthetase (NRPS) gene cluster into a defined chromosomal locus of S. coelicolor for stable expression.

Materials:

• S. coelicolor M1154 strain.
• Plasmid pCRISPomyces-2.
• Donor DNA fragment containing the NRPS cluster flanked by ~1 kb homology arms.
• Conjugation media (TSB, MS agar with appropriate MgCl2).
• Apramycin, Thiostrepton antibiotics.

Method:

• Design two 20-nt guide RNA sequences targeting the "safe-harbor" attB site of S. coelicolor. Clone into pCRISPomyces-2.
• PCR-amplify the NRPS cluster with 1-kb homology arms. This is the donor DNA.
• Transform the constructed pCRISPomyces-2 plasmid into E. coli ET12567/pUZ8002.
• Perform intergeneric conjugation between the E. coli donor and S. coelicolor spores.
• Select exconjugants on apramycin (for plasmid) and thiostrepton (for NRPS cluster integration via homology-directed repair).
• Validate integration via PCR and subsequent loss of the apramycin-resistant CRISPR plasmid.

Protocol 2.2: Saccharomyces cerevisiae Platform for Terpene Production

Objective: Express a PACE-evolved terpene synthase in an engineered yeast strain optimized for precursor supply.

Materials:

• S. cerevisiae strain EPY300 (engineered with high mevalonate pathway flux).
• Expression plasmid pRS423 with a galactose-inducible promoter (GAL1).
• Synthetic Complete media lacking histidine (SC-His) with 2% raffinose.
• Induction solution: 20% galactose.

Method:

• Clone the evolved terpene synthase gene into the pRS423-GAL1 plasmid.
• Transform the plasmid into EPY300 using the lithium acetate method.
• Grow single colonies in 5 mL SC-His + raffinose overnight at 30°C, 250 rpm.
• Dilute culture to OD600 ~0.1 in 50 mL fresh medium. Grow to OD600 ~0.6-0.8.
• Induce expression by adding galactose to a final concentration of 2%.
• Incubate for 48-72 hours. Overlay culture with 10% dodecane for volatile product capture if necessary.
• Extract metabolites for LC-MS analysis.

Data Presentation: Table 2: Titers of Novel Terpenes from PACE-Evolved Synthases in Yeast

Host Strain	Evolved Synthase Variant	Product (Tentative ID)	Titer (mg/L) @ 72h
EPY300 (Control)	None	N/A	0
EPY300	Parent (WT)	Bisabolene	1250 ± 110
EPY300	PACE-Round 8	Methyl-bisabolene	680 ± 75
EPY300	PACE-Round 15	Cyclopropyl-bisabolene	95 ± 15

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Materials for Functional Validation

Item Name	Supplier (Example)	Function in Validation
Monolith NT.115	NanoTemper	Measures biomolecular interactions via Microscale Thermophoresis (MST).
pCRISPomyces-2	Addgene (#61737)	CRISPR-Cas9 plasmid for genome editing in Streptomyces.
S. coelicolor M1154	John Innes Centre	Engineered model host with minimal background secondary metabolism.
EPY300 Yeast Strain	Lab Stock	Engineered S. cerevisiae with upregulated MVA pathway for terpene precursors.
GAL1 Promoter Plasmids (pRS series)	ATCC	Yeast expression vectors for inducible, high-level heterologous expression.
Amine-reactive NT-647 Dye	NanoTemper	Fluorescent dye for covalent protein labeling in MST assays.
Dodecane (BioUltra)	Sigma-Aldrich	Overlay solvent for in situ capture of volatile terpenes in microbial cultures.

Visualization: Experimental Workflows

Title: Functional Validation Workflow Post-PACE

Title: Heterologous Expression in Engineered Yeast

Application Notes: Integrating LC-MS and NMR for PACE-Derived Natural Product Analysis

The Phage-Assisted Continuous Evolution (PACE) platform enables the rapid generation of novel natural product-like scaffolds by evolving biosynthetic enzymes under continuous selection pressure. The analytical challenge lies in the rapid, confident structure elucidation of these often unprecedented compounds produced in micro-scale fermentations. This integrated LC-MS/NMR workflow addresses this need.

Core Application: The primary application is the dereplication and de novo structure determination of compounds from PACE-evolved polyketide synthases (PKSs), non-ribosomal peptide synthetases (NRPSs), and hybrid pathways. Key quantitative performance metrics for the recommended instrumentation are summarized below.

Table 1: Quantitative Performance Metrics for Core Analytical Techniques

Technique	Key Metric	Typical Value	Impact on PACE Analysis
UPLC-ESI-QTOF-MS	Mass Accuracy	< 2 ppm RMS	Confident molecular formula assignment from sub-μg amounts.
UPLC-ESI-QTOF-MS/MS	Fragmentation Resolution	40,000 FWHM	Detailed structural clues from in-source and CID fragmentation.
Microcryoprobe NMR	Sensitivity (¹H)	> 3500:1 (S/N for 0.1% EB)	Enables 2D NMR on < 50 μg of purified compound from 1 mL culture.
Microcryoprobe NMR	¹³C Sensitivity	> 1000:1 (S/N for 10% EB)	Critical for detecting quaternary carbons in scarce samples.
LC-SPE-NMR	Loading Capacity	50-100 μg per trap	Enables multiple NMR expts from a single LC-MS injection.

Experimental Protocols

Protocol 1: Integrated LC-MS/SPE-NMR Analysis for PACE Library Screening

Objective: To automate the isolation and structure elucidation of novel compounds from crude PACE culture broth.

Materials & Reagents:

• HPLC Solvents: LC-MS grade water (with 0.1% formic acid), LC-MS grade acetonitrile (with 0.1% formic acid).
• SPE Cartridges: C18 or polymer-based trapping cartridges (e.g., 2 mm x 10 mm).
• NMR Solvent: Deuterated acetonitrile (CD3CN) or methanol (CD3OD), dried over molecular sieves.

Procedure:

• Sample Preparation: Centrifuge 1 mL of PACE culture broth at 16,000 x g for 5 min. Filter supernatant through a 0.22 μm PVDF membrane.
• LC-MS Analysis:
  • Column: Reversed-phase C18 (2.1 x 100 mm, 1.7 μm).
  • Gradient: 5% to 95% organic phase over 20 min, hold 5 min.
  • MS Settings: ESI positive/negative mode; scan range m/z 100-2000; data-dependent acquisition (DDA) for top 3 ions.
• LC-SPE Transfer: Using a post-column splitter (~95:5 to MS:SPE), direct selected peaks (based on UV/MS triggers) onto individual SPE traps with water at 0.5 mL/min.
• SPE Drying: Dry traps with nitrogen gas for 30 min to remove residual H2O.
• Automated Elution to NMR: Elute each trapped compound with ~30 μL of deuterated solvent directly into a 1.7 mm or 3 mm NMR microtube using the LC-SPE-NMR interface.
• NMR Acquisition: Place tube in a 600 MHz spectrometer equipped with a 1.7 mm microcryoprobe. Acquire ¹H NMR, followed by 2D experiments (COSY, HSQC, HMBC) as allowed by sample concentration.

Protocol 2: Microscale Purification for NMR via Fraction Concentration

Objective: To prepare a concentrated sample for high-resolution 2D NMR from an HPLC fraction.

Procedure:

• Perform semi-preparative HPLC (e.g., 4.6 mm ID column) based on analytical LC-MS conditions.
• Collect the peak of interest in a glass vial.
• Reduce volume to ~100 μL using a centrifugal vacuum concentrator at 30°C.
• Transfer concentrate using a micro-syringe to a 1.7 mm NMR tube.
• Add 10-20 μL of deuterated solvent, cap, and mix by inversion.
• Acquire ¹H NMR. If concentration is sufficient (> 50 μg), proceed with HSQC and HMBC experiments.

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for PACE Natural Product Analytics

Item	Function & Rationale
1.7 mm Microcryoprobe	Maximizes sensitivity for NMR on limited (< 100 μg) samples from micro-fermentations.
LC-MS Grade Solvents (Acidified)	Ensures low background noise, optimal peak shape, and consistent ionization in LC-MS.
Deuterated NMR Solvents (Dry)	Prevents sample dilution, avoids solvent signals in ¹H spectrum, and ensures sample stability.
SPE Trapping Cartridges (C18)	Enables automated, online concentration of HPLC peaks for direct NMR analysis (LC-SPE-NMR).
0.22 μm PVDF Filters	Removes cellular debris and proteins from culture broth, protecting HPLC columns.
Centrifugal Vacuum Concentrator	Gently removes volatile HPLC solvents from collected fractions without degrading thermolabile natural products.
UPLC BEH C18 Column (1.7 μm)	Provides high-resolution separation of complex PACE culture extracts prior to MS detection.
ESI-QTOF Mass Spectrometer	Delivers high-mass-accuracy and MS/MS data for molecular formula and fragment ion assignment.

Workflow and Pathway Visualizations

PACE Natural Product Analysis Workflow

Structure Elucidation Data Integration Logic

This analysis compares three mutagenesis strategies for enzyme and natural product biosynthetic pathway optimization, contextualized within a thesis on accelerating natural product research via Phage Assisted Continuous Evolution (PACE). Each method addresses a distinct phase of the engineering pipeline, from broad exploration to focused refinement.

• PACE (Phage Assisted Continuous Evolution): A continuous, in vivo evolution platform linking a desired protein activity to bacteriophage reproduction. It enables rapid, autonomous evolution over hundreds of generations without manual intervention. In natural product research, PACE is revolutionary for evolving core biosynthetic enzymes (e.g., polyketide synthases, non-ribosomal peptide synthetases) for novel substrate specificity or enhanced activity, tasks traditionally intractable due to protein size and complexity.
• Error-Prone PCR & SeSaM (Sequence Saturation Mutagenesis): These are in vitro methods for generating diversified libraries. Error-prone PCR introduces random point mutations via low-fidelity PCR. SeSaM is a more advanced method that generates unbiased random mutations across entire sequences, avoiding the sequence bias of error-prone PCR. They are ideal for creating initial diversity in a gene or pathway when no structural data is available.
• Site-Saturation Mutagenesis (SSM): A targeted in vitro method where one specific amino acid position is mutated to all 20 possible amino acids. It is used for focused optimization of "hotspot" residues identified from prior screening (e.g., from PACE or random libraries) or structural analysis, crucial for fine-tuning enzyme properties.

Comparative Summary Table

Feature	PACE	Error-Prone PCR / SeSaM	Site-Saturation Mutagenesis
Evolution Principle	Continuous, in vivo, selection-driven	Discrete, in vitro, random mutagenesis	Discrete, in vitro, targeted codon replacement
Library Diversity	Theoretically unlimited, directed by selection	Broad, random (point mutations/transversions)	Narrow, focused on single residue
Key Advantage	Autonomy, deep exploration of sequence space, no structural info needed	Rapid library creation, good for initial diversification	Exhaustive exploration of a specific site
Primary Application	Evolving complex functions & multi-step pathways	Initial trait discovery, exploring unknown regions	Fine-tuning activity, stability, or specificity
Typical Throughput	Very High (continuous)	High (96/384-well plates)	Medium-High (96/384-well plates)
Structural Data Required	No	No	Beneficial, but not mandatory
Best for Thesis Context	Main driver for evolving biosynthetic pathway components	Creating initial variant libraries of pathway genes	Optimizing key catalytic residues identified by PACE

Detailed Experimental Protocols

Protocol 2.1: PACE for a Natural Product Biosynthetic Enzyme Objective: Evolve a polyketide synthase (PKS) adenylation domain for alternative substrate incorporation using PACE.

• Construct Design: Clone the target PKS domain gene into the phage vector (pIII or pVII fusion). Design the selection host to require the production of a modified natural product (with the desired substrate) for phage propagation. This often involves linking phage accessory protein (gIII) expression to an antibiotic resistance gene activated by the product.
• Lagoon Setup: Inoculate a turbidostat bioreactor (lagoon) with the selection host E. coli cells. Continuously dilute with fresh medium containing the target non-native substrate.
• Evolution Initiation: Infect the lagoon with the initial phage library (can be a naive or pre-diversified library). Typical lagoon volume: 15-40 mL; dilution rate: 1-2 lagoon volumes per hour.
• Continuous Evolution: Run PACE for 24-288 hours. Monitor phage titer. The system applies constant selection pressure—only phage encoding functional PKS variants that incorporate the new substrate propagate.
• Harvest & Analysis: Sample phage from the lagoon effluent daily. Isolate replicative phage DNA and sequence the evolved gene. Test evolved variants in vitro for substrate specificity switch.

Protocol 2.2: SeSaM for Generating an Unbiased Random Library Objective: Create a mutation library of a cytochrome P450 monooxygenase gene.

• Generate Fragments with Universal Base: Perform PCR of the target gene using dPTP (a universal base analog) in place of dTTP to generate fragments of random length terminated at thymidine positions.
• Purify & Tail Fragments: Gel-purify the fragmented DNA. Use terminal transferase to add a short, defined oligonucleotide tail (e.g., poly-A) to the 3’-ends.
• Elongate to Full-Length: Perform a single primer extension using a primer complementary to the added tail. This creates full-length, mutagenized strands with random thymidine-to-other-base transversions.
• Amplify Library: Use the extended product as template for a final PCR with gene-specific primers to amplify the full-length, randomized library for downstream cloning.

Protocol 2.3: Site-Saturation Mutagenesis via NNK Codon Design Objective: Saturate a putative active-site residue (Ala-125) in a terpene synthase.

• Primer Design: Design forward and reverse primers that overlap at the target codon. Use the NNK degenerate codon (N = A/T/G/C; K = G/T) to encode all 20 amino acids + one stop codon.
• PCR Amplification: Perform a whole-plasmid PCR (e.g., using KLD enzyme mix or inverse PCR) with high-fidelity polymerase, using the plasmid containing the wild-type gene as template.
• Template Digestion & Ligation: Digest the PCR product with DpnI to remove methylated parental template DNA. Self-ligate the amplified, mutated plasmid using DNA ligase.
• Transformation & Library Validation: Transform the ligation product into competent E. coli. Isolate plasmid pool and sequence 10-20 clones to confirm mutation rate and diversity at the target position before functional screening.

Visualizations

Title: PACE Continuous Evolution Workflow

Title: Random Library Creation & Screening Pathway

Title: From Broad Evolution to Focused Saturation

The Scientist's Toolkit: Key Research Reagent Solutions

Item	Function in Context
Mutazyme II / Taq Pol (low Mg²⁺)	Error-prone PCR polymerase for generating random point mutation libraries.
dPTP Nucleotide Mix	Essential for SeSaM protocol to create random length gene fragments.
NNK Degenerate Codon Primers	Oligonucleotides for constructing site-saturation mutagenesis libraries.
PACE Selection Phagemid (e.g., pIII)	Phage vector that links target gene expression to phage coat protein.
Turbidostat Bioreactor	Apparatus for maintaining constant cell density in PACE lagoons.
DpnI Restriction Enzyme	Digests methylated parental DNA post-PCR, critical for SSM and related protocols.
KLD Enzyme Mix	Enables fast, efficient circularization of PCR-amplified plasmids for SSM.
Fluorescent Reporter Plasmid	For PACE selection circuits where activity is linked to GFP/RFP expression.
Deep Sequencing Kit (Illumina)	For comprehensive analysis of evolved populations from PACE or diversity libraries.

Continuous evolution platforms have revolutionized the ability to engineer proteins and biosynthetic pathways. Within natural products research, directed evolution of enzymes (e.g., polyketide synthases, non-ribosomal peptide synthetases) or regulatory elements is critical for generating novel analogs with improved therapeutic properties. Two leading, yet fundamentally distinct, platforms are Phage-Assisted Continuous Evolution (PACE) and in vivo continuous evolution systems like OrthoRep. This analysis compares their methodologies, applications, and performance in the context of natural product biosynthesis.

System Architectures and Core Principles

PACE leverages a bacteriophage life cycle to link a desired enzymatic activity (the "Activity of Interest," AoI) to the production of the essential phage protein pIII. Host E. coli cells (the "host cell") in a lagoon continuously dilute a culture while being infected by engineered phage. Only phage that successfully perform the AoI produce pIII, propagate, and outcompete others. The system operates orthogonal to host replication.

OrthoRep is an in vivo yeast (Saccharomyces cerevisiae) system based on a cytoplasmic TDNAP/TPOL plasmid pair derived from a viral genome. The error-prone TPOL polymerase replicates the cytoplasmic plasmid at ~100,000x higher mutation rates than the nuclear genome, enabling continuous hypermutation of genes of interest (GOIs) cloned into it, while the host cell replicates normally.

Quantitative Comparison

Table 1: Core System Specifications

Feature	PACE	OrthoRep
Host Organism	Escherichia coli	Saccharomyces cerevisiae
Evolution Scaffold	M13 bacteriophage	Cytoplasmic linear plasmid (TDNAP/TPOL)
Mutation Source	Error-prone host polymerases & mutagenic plasmids	Dedicated error-prone DNA polymerase (TPOL)
Mutation Rate	~10^-5 to 10^-7 per bp per generation (tunable via MP)	~10^-5 per bp per generation (fixed, high)
Selection Throughput	Extremely High (~10^10 variants/day)	High (~10^8-10^9 cells per population)
Evolution Timescale	Days to 1-2 weeks	Weeks
Gene Capacity	Limited (~1-2 kb ideal)	Larger (~5-10+ kb possible)
Key Advantage	Rapid, stringent, automated selection.	Eukaryotic context, stable for large/multi-gene pathways.

Table 2: Suitability for Natural Product Research Tasks

Application	PACE	OrthoRep
Single Enzyme Optimization (e.g., P450, acyltransferase)	Excellent	Excellent
Domain/Module Evolution in Megaenzymes (PKS/NRPS)	Challenging (size limits)	Good (handles large genes)
Transcription Factor Engineering for pathway regulation	Excellent (for prokaryotic TFs)	Excellent (for eukaryotic context)
Whole Pathway Evolution	Not suitable	Good (co-evolution of pathway genes possible)
Requiring Eukaryotic PTMs/Chaperones	Not suitable	Critical Advantage

Detailed Protocols

Protocol 1: PACE for Evolving a Natural Product Enzyme (e.g., a Regioselective Halogenase) Objective: Evolve a flavin-dependent halogenase for novel substrate specificity.

A. Vector Construction:

• Phage Vector (pIV): Clone the target halogenase gene into an M13 phage vector such that its desired activity is coupled to pIII expression via a chosen selection circuit (e.g., a bacterial repressor that controls pIII).
• Selection Plasmid (pIII): Construct a plasmid encoding pIII under the control of a promoter responsive to the product of the halogenase reaction (e.g., a halogen-sensitive transcriptional activator).
• Mutagenesis Plasmid (MP): Optional. Use a plasmid expressing an error-prone DNA polymerase variant to increase mutation rates.

B. PACE Setup and Execution:

• Host Cell Preparation: Transform E. coli with the selection plasmid. Grow a large culture in rich media.
• Lagoon Initiation: Start a continuous culture (lagoon) using a chemostat apparatus. Maintain a fixed volume (e.g., 15 mL) with a dilution rate of ~1 vessel volume per hour.
• Infection: Introduce the initial halogenase phage library into the lagoon inflow.
• Evolution Run: Continuously supply fresh media containing the novel substrate analog. Monitor phage titer in the effluent. The system will enrich for phage encoding halogenases that process the new substrate and activate pIII.
• Harvesting: After 100-200 hours of evolution, collect effluent phage. Isolate ssDNA and sequence the evolved halogenase gene.

Protocol 2: OrthoRep for Evolving a Biosynthetic Pathway Step in Yeast Objective: Evolve a polyketide synthase (PKS) acyltransferase (AT) domain for alternative extender unit incorporation.

A. OrthoRep Plasmid Engineering:

• Target Gene Cloning: Clone the entire PKS gene or a substrate-binding domain module (including the AT domain) into the expression site of the OrthoRep acceptor plasmid (pTNA).
• Host Strain Transformation: Co-transform S. cerevisiae harboring the cytoplasmic TPOL plasmid with the newly constructed pTNA-AT plasmid. Select for colonies to establish the evolution-ready strain.

B. Continuous Evolution in Batch Culture:

• Evolution Passaging: Inoculate the strain into selective media lacking the natural extender unit but supplemented with the desired, non-native extender unit analog (e.g., a propargyl- or azido-malonate).
• Growth Selection: Culture for 24-48 hours. Only cells where the AT domain evolves to accept the analog will produce the functional polyketide, conferring a fitness advantage (or linked to a selectable marker).
• Serial Transfer: Use a small aliquot (1%) of the saturated culture to inoculate fresh media with the analog. Repeat this serial passaging for 20-50 generations.
• Screening & Isolation: Periodically plate cultures, pick colonies, and extract the cytoplasmic plasmid DNA for sequencing of the evolved AT domain. Analyze polyketide production via LC-MS.

Visualization

Title: PACE Experimental Workflow

Title: OrthoRep Experimental Workflow

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents and Materials

Reagent/Material	Function in Experiment	Typical Source/Example
M13 Phage Vector (e.g., pIV)	Scaffold for gene of interest; links variant survival to activity.	Addgene (#, e.g., pIV-1.0)
AP193 E. coli Host Cells	Specialized PACE host; lacks pIII, supports phage propagation.	Lab stock or genetic construction.
Selection Plasmid (pIII)	Encodes essential pIII under control of designed biosensor.	Constructed in-house with AraC, TetR, etc.
Mutagenesis Plasmid (MP6)	Expresses error-prone polymerase to increase phage mutation rate.	Addgene (e.g., pJPM50-MP6)
Chemostat Apparatus	Maintains continuous culture (lagoon) for PACE.	New Brunswick BioFlo/Celligen.
OrthoRep Yeast Strain	S. cerevisiae with chromosomally integrated TPOL system.	Gift from the Arnold/Liu labs.
OrthoRep Acceptor Plasmid (pTNA)	Cytoplasmic plasmid for cloning GOI; replicated by TPOL.	Addgene (e.g., pXR106)
Non-native Substrate Analogs	Chemical selection pressure for enzyme evolution (e.g., propargyl-malonate).	Sigma-Aldrich, Cayman Chemical.
Error-Prone TPOL Polymerase	The engine of mutagenesis in OrthoRep (~10^-5 error rate).	Encoded in the host genome.

Application Notes

Within Phage Assisted Continuous Evolution (PACE) campaigns for natural product biosynthetic pathway optimization, the directed evolution of enzymes like polyketide synthases (PKSs), non-ribosomal peptide synthetases (NRPSs), or tailoring enzymes (e.g., methyltransferases, oxidases) is a powerful strategy. However, the ultimate success of an evolved variant for scalable production or novel analog generation hinges on a quantitative biochemical assessment post-evolution. Moving beyond simple activity screens to measuring precise thermodynamic (e.g., substrate binding affinity, ΔG) and kinetic (e.g., kcat, KM, Ki) parameters is critical. These parameters directly inform on catalytic efficiency, mechanistic bottlenecks, substrate promiscuity, and product yield—key factors for integrating evolved enzymes into engineered microbial hosts for fermentative production. This protocol details methods for the expression, purification, and in vitro characterization of PACE-evolved biosynthetic enzymes, enabling data-driven decisions for downstream metabolic engineering and drug development pipelines.

Protocols

Protocol 1: Expression and Affinity Purification of His-Tagged, Evolved Biosynthetic Enzymes

Objective: To obtain purified enzyme variants for in vitro biochemical assays. Materials: E. coli BL21(DE3) cells harboring pET vector with evolved gene, LB media, IPTG, Lysis Buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM TCEP), Ni-NTA Agarose, Wash Buffer (Lysis Buffer with 25 mM imidazole), Elution Buffer (Lysis Buffer with 250 mM imidazole). Procedure:

• Inoculate 50 mL LB starter culture with antibiotic, grow overnight at 37°C.
• Dilute into 1 L of autoinduction TB media. Grow at 37°C to OD600 ~0.6, then reduce temperature to 18°C and incubate for 18-20 hours.
• Harvest cells via centrifugation (4,000 x g, 20 min, 4°C). Resuspend pellet in 40 mL Lysis Buffer.
• Lyse cells via sonication or homogenization. Clarify lysate via centrifugation (20,000 x g, 45 min, 4°C).
• Incubate supernatant with 2 mL of pre-equilibrated Ni-NTA resin for 1 hour at 4°C with gentle mixing.
• Load resin into a column, wash with 20 column volumes (CV) of Wash Buffer.
• Elute protein with 5 CV of Elution Buffer, collecting 1 mL fractions.
• Analyze fractions via SDS-PAGE. Pool pure fractions and dialyze into Storage Buffer (50 mM HEPES pH 7.5, 150 mM KCl, 10% glycerol, 1 mM DTT).
• Determine concentration (A280), aliquot, flash-freeze, and store at -80°C.

Protocol 2: Steady-State Kinetic Analysis Using Continuous Coupled Assays

Objective: To determine kcat and KM for an evolved enzyme using a spectrophotometric/fluorometric assay. Materials: Purified enzyme, substrate(s), cofactors (ATP, NAD(P)H, etc.), assay buffer, coupling enzymes (e.g., pyruvate kinase/lactate dehydrogenase for ATP-turnover), microplate reader or spectrophotometer. Procedure (General for an ATP-dependent enzyme):

• Prepare a master mix containing assay buffer, coupling system, ATP, and MgCl2.
• In a 96-well plate, aliquot master mix and varying concentrations of primary substrate.
• Initiate reaction by adding a fixed, limiting amount of purified enzyme.
• Monitor the consumption of NADH at 340 nm (ε = 6220 M⁻¹cm⁻¹) for 5-10 minutes.
• Calculate initial velocities (v0) from the linear slope of absorbance change.
• Fit v0 vs. [Substrate] data to the Michaelis-Menten equation (v0 = (Vmax * [S]) / (KM + [S])) using nonlinear regression (e.g., GraphPad Prism).
• Report kcat (Vmax / [E]) and KM. Perform in triplicate.

Protocol 3: Isothermal Titration Calorimetry (ITC) for Substrate Binding Thermodynamics

Objective: To directly measure binding affinity (KD), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) of substrate binding. Materials: Purified enzyme, substrate ligand, ITC instrument, dialysis buffer. Procedure:

• Dialyze both the enzyme and ligand exhaustively against the same degassed assay buffer (e.g., 50 mM HEPES, pH 7.5, 150 mM NaCl).
• Load the enzyme solution (~50-100 µM) into the sample cell.
• Load the ligand solution (~10x concentrated relative to expected KD) into the syringe.
• Program the instrument to perform a series of injections (e.g., 19 x 2 µL) with stirring.
• Measure the heat of interaction after each injection. Fit the integrated heat data to a single-site binding model to derive n, KD, and ΔH. Calculate ΔG and ΔS using standard equations.

Data Presentation

Table 1: Comparative Kinetic Parameters of PACE-Evolved Methyltransferase Variants

Variant (PACE Round)	kcat (s⁻¹)	KM (µM) for SAM	kcat/KM (M⁻¹s⁻¹)	Relative Efficiency (vs. WT)
Wild-Type (WT)	0.15 ± 0.02	12.5 ± 1.8	1.20 x 10⁴	1.0
PACE-5	0.32 ± 0.03	8.2 ± 1.1	3.90 x 10⁴	3.3
PACE-10	0.85 ± 0.06	5.5 ± 0.7	1.55 x 10⁵	12.9
PACE-15 (Final)	1.20 ± 0.10	4.1 ± 0.5	2.93 x 10⁵	24.4

Table 2: Thermodynamic Binding Parameters from ITC for PACE-Evolved Acyltransferase with Novel Substrate

Parameter	Wild-Type (WT)	PACE-15 Variant
KD (nM)	4500 ± 320	215 ± 18
ΔH (kcal/mol)	-5.2 ± 0.3	-8.1 ± 0.4
-TΔS (kcal/mol)	1.8	0.5
ΔG (kcal/mol)	-7.0	-8.6
N (sites)	0.95 ± 0.05	1.02 ± 0.03

Mandatory Visualization

Title: PACE to Production Characterization Workflow

Title: Kinetic and Thermodynamic Parameter Relationships

The Scientist's Toolkit: Key Research Reagent Solutions

Item	Function in Assessment Protocols
HisTrap HP Column (Cytiva)	For rapid, high-performance immobilized metal affinity chromatography (IMAC) purification of His-tagged evolved enzymes.
SpectraMax iD5 Multi-Mode Microplate Reader	Enables high-throughput kinetic measurements via UV-Vis and fluorescence for steady-state assays.
MicroCal PEAQ-ITC (Malvern Panalytical)	Gold-standard instrument for label-free, direct measurement of binding thermodynamics (KD, ΔH, ΔS).
Cytiva HiLoad Superdex 200 pg	Size-exclusion chromatography column for final polishing step to obtain monodisperse, active enzyme.
Sigma-Aldrich Cofactor/Substrate Libraries	Comprehensive, high-purity sources of substrates (e.g., acyl-CoAs, unusual amino acids) and cofactors (SAM, NADPH).
Pierce BCA Protein Assay Kit	Colorimetric assay for accurate total protein concentration determination during purification.
GraphPad Prism Software	Essential for nonlinear regression analysis of kinetic and binding data to extract key parameters.
SnapGene	For molecular biology design and analysis of evolved gene sequences isolated from PACE experiments.

Conclusion

Phage-Assisted Continuous Evolution represents a paradigm shift in natural product research, offering an unprecedented ability to rapidly generate functional diversity in complex biosynthetic machinery. By mastering the foundational principles, meticulous methodology, and optimization strategies outlined, researchers can harness PACE to evolve PKS, NRPS, and other enzymes toward desired activities, specificities, and stabilities. While challenges in host compatibility and selection design persist, the technology's power to compress years of evolution into days validates its transformative potential. The future of PACE in natural products lies in integrating it with automated analytics, machine learning for predicting functional mutations, and deploying evolved pathways in cell-free systems or optimized chassis for scaled production. This synergy will accelerate the discovery of next-generation therapeutics, moving evolved natural products from the lagoon to the clinic.