Genome Engineering of Corynebacterium glutamicum

Genome Engineering of Corynebacterium glutamicum

1. Research Background

Corynebacterium glutamicum is a universally recognized core strain in the industrial production of amino acids and classified as a Generally Recognized as Safe (GRAS) strain. Its exceptional capacity for amino acid synthesis, well-characterized genetic background and excellent industrial production traits have established its central role in the fields of food additives, feed nutrition and biomedicine. Precise and efficient gene editing technology serves as a pivotal tool for optimizing its production performance and constructing high-efficiency cell factories.

Against this backdrop, GeneRulor has developed genomic modification strategies for Corynebacterium glutamicum based on the CRISPR/Cas9 system and a variety of novel CRISPR systems. We provide customized services including target gene knockout, large-fragment deletion, point mutation and gene integration (overexpression) in different strains, with the delivery of positive mutant strains. We are committed to offering professional and reliable customized gene editing solutions for Corynebacterium glutamicum to scientific research and industrial clients worldwide.

2. Strain Characteristics and Biological Background

(1) Gram-staining property: Corynebacterium glutamicum is a Gram-positive bacterium with a high-GC-content genome.

(2) Physical characteristics: It possesses a unique cell wall structure comprising arabinogalactan polymers and an outer mycolic acid layer, which forms an additional permeability barrier and endows the strain with certain hydrophobicity and environmental tolerance.

(3) Industrial significance: As a model strain for amino acid production, it is an ideal model for investigating amino acid biosynthesis, metabolic regulation and the optimization of industrial fermentation. Discovered in 1957, it has been widely used for the large-scale industrial production of amino acids such as glutamic acid and lysine across the globe.

(4) Genetic transformation: Exogenous DNA delivery can be achieved via electroporation or conjugation, yet the transformation efficiency requires optimization due to its distinctive cell wall structure. A variety of inducible promoter systems have been developed for regulated gene expression.

Figure 1 Growth of Corynebacterium glutamicum on culture medium plates

3. Reported Editing Strategies in Literatures

Targeting the unique cell wall barrier of Corynebacterium glutamicum and the demands of industrial production, the mainstream editing strategies adopted in current literatures and scientific research practices are as follows:

3.1 CRISPR/Cas9 System (Classic Protocol):

(1) Principle: The Cas9 protein mediates site-specific cleavage of the target genomic DNA under the guidance of sgRNA to generate double-strand breaks (DSBs), and precise editing is achieved through homology-directed repair (HDR).

(2) Advantages: Enables efficient gene knockout, large-fragment deletion and gene integration.

3.2 CRISPR-Cpf1 (Cas12a) System (Emerging Protocol) :

(1) Principle: The Cpf1 protein recognizes T-rich PAM sequences, and its intrinsic ability to process crRNA simplifies the editing operation.

(2) Advantages: Exhibits high editing efficiency in Corynebacterium glutamicum and is applicable for multiplex gene editing and transcriptional regulation.

3.3 Base Editing System:

(1) Principle: A catalytically inactive Cas protein is fused with a deaminase to achieve precise single-base conversion without inducing double-strand breaks.

(2) Advantages: Eliminates the need for donor DNA templates, features high editing efficiency and low byproduct formation, making it suitable for functional studies of essential genes and fine regulation of metabolic flux.

4. Core Application Fields

(1) Industrial amino acid production: Significantly improve the yields of amino acids such as L-glutamic acid, L-lysine and L-serine through metabolic pathway optimization and byproduct pathway blocking.

(2) Terpenoid synthesis: Realize high lycopene production by knocking out the competitive pathway genes crtEb and crtR using the CRISPR/MAD7 system combined with key gene screening.

(3) Functional protein expression: Achieve the efficient synthesis of soybean hemoglobin, clover hemoglobin, bovine myoglobin and other proteins by optimizing heme supply.

(4) Chassis cell streamlining: Perform large-fragment deletion to remove unnecessary metabolic burden genes and construct industrial strains with higher stability.

(5) Functional genomics: Construct a knockout library covering 98.1% of the genome using base editors to screen genes associated with drug resistance or environmental tolerance.

5. Project Process and Verification

We provide one-stop services from design to delivery to ensure the accuracy of editing results:

(1) Protocol design and vector construction: Design knockout vectors for target loci.

(2) Bacterial transformation and screening: Introduce the editing system via natural transformation or electroporation technology.

(3) Multiple verification: Confirm positive mutations through PCR identification and Sanger sequencing.

Figure 2 Schematic diagram of the project process

6. Introduction to Gene Editing Projects

6.1 Core Services

(1) Gene knockout/inactivation: Precisely delete protease genes or metabolic byproduct genes to optimize chassis performance.

(2) Gene knock-in/overexpression: Integrate exogenous expression cassettes at specific loci or enhance the expression of endogenous rate-limiting enzymes.

(3) Point mutation/modification: Perform site-directed mutation on key enzymes to improve their thermal stability or catalytic activity.

(4) Multiplex gene editing: Conduct consecutive editing of multiple genes to construct strains with modified complex metabolic networks.

6.2 Technical Advantages

(1) High success rate: Extensive experience in editing industrial strains and laboratory model strains.

(2) Customized design: Devise the optimal editing strategy according to production objectives (e.g., enzyme activity enhancement, growth improvement).

(3) Full-process verification: Provide a complete closed-loop report from protocol design to final genotype confirmation.

7. Case Introduction

We have successfully provided services for numerous top universities, research institutions and biotechnology companies at home and abroad, with a partial example presented as follows:

Case: Construction of mutant strains of Corynebacterium glutamicum ATCC13032

Project content: Successful integration of exogenous gene fragments

Figure 3 Verification of transposon strains by single colony picking of Corynebacterium glutamicum

(Upper panel: Verification of resistance gene insertion; Lower panel: Verification of plasmid existence)

8. References

[1] Wu M, et al. Homing endonucleaseI-SceI-mediated Corynebacteriumglutamicum ATCC 13032 genome engineering. ApplMicrobiol Biotechnol. 2020Apr;104(8):3597-3609.

[2] Cho JS, et al. CRISPR/Cas9-coupledrecombineering formetabolic engineering of Corynebacterium glutamicum. MetabEng. 2017Jul;42:157-167.

[3] Zhao N, et al. Multiplex gene editing andlarge DNA fragmentdeletion by the CRISPR/Cpf1-RecE/T system in Corynebacteriumglutamicum. J IndMicrobiol Biotechnol. 2020 Aug;47(8):599-608.

[4] Jiang Y, et al. CRISPR-Cpf1 assistedgenome editing ofCorynebacterium glutamicum. Nat Commun. 2017 May 4;8:15179.

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