Integrated Services for Enzyme Directed Evolution and Library Construction

CD Biosynsis offers specialized services for Enzyme Directed Evolution and Library Construction, which are crucial for successfully optimizing enzyme properties. Our core capability lies in utilizing advanced molecular biology techniques to build high-coverage, high-efficiency mutant libraries tailored to client needs. These libraries are generated through strategies like random mutagenesis (e.g., DNA Shuffling) or site-directed mutagenesis (e.g., Saturation Mutagenesis) to maximize genetic diversity. By integrating various ultra-high-throughput display technologies (such as Phage Display, Yeast Display, and mRNA Display), we ensure the efficient screening of rare clones with desired characteristics (e.g., enhanced activity, stability, and specificity) from millions or even billions of variants. Our integrated platform significantly accelerates the discovery cycle for industrial biocatalysts and functional enzymes.

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The Synergy of Library Diversity and Screening Throughput

The success of directed evolution depends on two factors: library diversity (i.e., the number of variants) and screening efficiency (i.e., speed and accuracy). We ensure the library covers the target amino acid sequence space by offering various strategies, including Error-Prone PCR, DNA Shuffling, and NNS/NNK Saturation Mutagenesis. Subsequently, we connect these high-diversity libraries to our range of display technology platforms, such as 10^9 capacity Phage Display or 10^13 capacity mRNA Display, to establish a physical "genotype-phenotype" linkage. This linkage, coupled with automated sorting methods like FACS, allows screening tasks, which were once time-consuming, to be completed rapidly, quickly identifying rare enzyme variants with subtle performance improvements.

Core Library Construction and Ultra-High-Throughput Display Technologies

High-Diversity Library Construction Strategies Affinity-Based Display Screening Activity-Based Display Screening

Creating Genetic Diversity

Random and Site-Specific Mutagenesis Strategies

Enzyme Library Generation Services for Directed Evolution

Offers various library construction methods, including random mutagenesis (e.g., Error-Prone PCR) and site-directed mutagenesis (e.g., Saturation Mutagenesis), ensuring high coverage and high transformation efficiency.

DNA Shuffling

Integrates multiple beneficial mutations into a single enzyme molecule through gene recombination, suitable for rapid functional evolution of homologous genes.

Saturation Mutagenesis (NNS/NNK)

Performs NNS or NNK mutagenesis at critical residue sites to precisely explore all amino acid variants at the target position, avoiding the generation of unnecessary stop codons.

Ultra-High Throughput and Affinity Enrichment

Utilizing Phage and Cell-Free Systems

Phage Display for Enzyme Engineering Service

Displays enzyme variants on the surface of bacteriophage and screens/enriches based on affinity binding to a target molecule (substrate or inhibitor). Library capacity up to 10^9.

mRNA Display for Enzyme Engineering Service

Cell-free in vitro display system, linking the enzyme variant to its mRNA template, achieving the highest library capacity (up to 10^13), suitable for de novo design.

Ribosome Display

Another cell-free system that links the enzyme to its mRNA via the ribosomal complex, an effective tool for screening high stability and affinity variants.

High Precision and Quantitative Activity Sorting

Utilizing Yeast and Cell Surface Systems

Yeast Surface Display for Enzyme Engineering Service

Yeast cell surface display system, capable of eukaryotic folding and modification, combined with FACS for 10^7 scale quantitative activity sorting.

Cell Surface Display for Enzyme Engineering Service

Enzymes anchored on the surface of bacterial (e.g., E coli) or other host cells, combined with FACS, suitable for screening under specific physiological or industrial conditions.

FACS-Based Screening

Utilizing high-precision Fluorescence-Activated Cell Sorting to rapidly and accurately sort high-activity cells from millions based on the fluorescent signal generated by enzyme activity.

Directed Evolution and Library Construction Service Flow

From diversity creation to active variant selection.

Target Design and Strategy Selection

High-Diversity Library Construction

Library Display and Enrichment

Variant Validation and Delivery

Performance Goals Defined: Specify required enzyme property improvements (e.g., activity, stability).

Mutagenesis Strategy Selection: Choose random or site-directed saturation mutagenesis based on target and known structural information.

Gene Synthesis and Mutagenesis: Generate the parent gene and perform the selected mutagenesis method.

Cloning and Transformation: Clone the mutant library into the display vector and transform the host cells for maximum library size.

Assay Customization: Develop an HTS-compatible assay (e.g., fluorescence, FACS-based) for the target property.

Display and Selection: Utilize Phage, Yeast, mRNA, or Cell Surface Display for high-throughput selection and enrichment.

Re-Screening and Characterization: Confirm the top variants and perform detailed kinetic and stability analysis.
Iteration: Implement beneficial mutations into the next round of library construction if required.
Delivery: Final optimized gene sequence, glycerol stocks, and a comprehensive characterization report.

Key Advantages of Our Integrated Platform

Maximum Diversity Coverage

Construction of libraries with up to 10^13 variants, ensuring the exploration of vast sequence space.

High-Precision Sorting

Utilizing FACS and specific display systems for quantitative, single-cell selection based on enzyme activity.

Genotype-Phenotype Linkage

Display technologies ensure the enzyme activity is physically linked to its encoding DNA for efficient recovery.

Customized Evolution

Tailored strategies combining specific mutagenesis methods and the most suitable display platform for each project.

Client Testimonials on Directed Evolution Services

"Their expertise in saturation mutagenesis delivered a high-quality library that yielded an enzyme with 15^\circC enhanced thermostability after only two rounds of Yeast Display screening."

Dr. Samuel Liu, R&D Director

"The integrated DNA Shuffling and Phage Display service allowed us to quickly combine beneficial mutations from related enzyme families, significantly speeding up our lead optimization."

Ms. Janet Chen, Lead Bioengineer

"For our challenging de novo design project, the 10^13 capacity of mRNA Display was the only viable path to identify the rare functional clones in a vast sequence space."

Dr. Kenji Tanaka, Principal Scientist

FAQs about Enzyme Directed Evolution and Library Construction

Still have questions?

What is the key advantage of an integrated directed evolution service?

The integrated service ensures seamless transition and optimization between library generation (where diversity is created) and screening (where performance is measured), minimizing technical compatibility issues and accelerating the overall campaign timeline.

How do you choose between random mutagenesis (like Error-Prone PCR) and targeted mutagenesis (like Saturation Mutagenesis)?

Random mutagenesis is used when little is known about the enzyme structure or function to explore global sequence space. Targeted mutagenesis is used when specific active sites or loops are known to be critical for the desired property (e.g., activity or specificity).

Can I use this service for novel or uncharacterized enzymes?

Yes. Our experts can start with rational design and random mutagenesis to create initial variants, followed by HTS to quickly identify beneficial starting points for subsequent targeted evolution.

What is the maximum throughput for screening services?

While microplate systems screen up to 10^5 variants per day, display technologies such as FACS-sorted Yeast Display and mRNA Display enable the functional screening and recovery of up to 10^8 to 10^13 variants per cycle.

How much does Metabolic Engineering services cost?

The cost of Metabolic Engineering services depends on the project scope, complexity of the target compound, the host organism chosen, and the required yield optimization. We provide customized quotes after a detailed discussion of your specific research objectives.

Do your engineered strains meet regulatory standards?

We adhere to high quality control standards in all strain construction and optimization processes. While we do not handle final regulatory approval, our detailed documentation and compliance with best laboratory practices ensure your engineered strains are prepared for necessary regulatory filings (e.g., GRAS, FDA).

What to look for when selecting the best gene editing service?

We provide various gene editing services such as CRISPR-sgRNA library generation, stable transformation cell line generation, gene knockout cell line generation, and gene point mutation cell line generation. Users are free to select the type of service that suits their research.

Does gene editing allow customisability?

Yes, we offer very customised gene editing solutions such as AAV vector capsid directed evolution, mRNA vector gene delivery, library creation, promoter evolution and screening, etc.

What is the process for keeping data private and confidential?

We adhere to the data privacy policy completely, and all customer data and experimental data are kept confidential.