CRISPRi-Engineered Bacillus subtilis for GlcNAc Co-utilization
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Solution Overview
Problem
Current methods for producing N-acetylglucosamine (GlcNAc) face challenges such as environmental pollution, allergic reactions due to seafood-derived byproducts, and insufficient production capacity in recombinant Bacillus subtilis, particularly in efficiently co-utilizing glucose and xylose for enhanced yield.
Innovation Solution
A genetically engineered bacterium is developed using CRISPRi to regulate the glycolysis, pentose phosphate, and peptidoglycan synthesis pathways by integrating and expressing the dCas9 protein and specific sgRNAs, allowing for efficient co-utilization of glucose and xylose, thereby increasing GlcNAc production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the glycolysis pathway and pentose phosphate pathway are blocked to increase GlcNAc production, then the GlcNAc titer and yield improve, but cell growth is adversely affected
Solution Approach 1:
The patent employs a dynamic regulation system using CRISPRi technology with an inducible promoter (Ptac) to control the expression of dCas9. By adding IPTG at different stages, the system can dynamically adjust the inhibition of glycolysis and pentose phosphate pathways, allowing cell growth during early stages and GlcNAc production during later stages, thus resolving the contradiction between productivity and reliability
Solution Approach 2:
The patent performs preliminary action by first allowing cells to grow and adapt using glucose as the sole carbon source, then subsequently inducing the CRISPRi system to inhibit competing pathways when cells are in the stationary phase. This sequential approach ensures cells are sufficiently developed before pathway inhibition begins, maintaining cell viability while enabling high GlcNAc production
2Productivity
If only the xylose metabolism pathway is strengthened without regulating glucose metabolism, then xylose utilization improves, but glucose carbon resources are wasted and GlcNAc yield is insufficient
Solution Approach 1:
The patent implements dynamic control of glucose metabolism through the inducible CRISPRi system. During the growth phase, glucose metabolism proceeds normally to support cell proliferation. In the production phase, upon IPTG induction, the system dynamically inhibits glycolysis and peptidoglycan synthesis pathways, redirecting glucose carbon flux toward GlcNAc production while xylose continues to be utilized, thereby eliminating carbon waste and maximizing yield
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method significantly increases the titer and yield of GlcNAc to 20.5 g/L, providing a foundation for industrialization while avoiding environmental and allergic issues, and demonstrates efficient carbon source management for cell growth and product synthesis.
Implementation Method 1
Method for producing N-acetylglucosamine by co-utilizing glucose and xylose based on CRISPR interference (CRISPRi)
Data Source
AI summary
The present invention discloses a method for producing N-acetylglucosamine (GlcNAc) by co-utilizing glucose and xylose based on CRISPRi, and belongs to the field of genetic engineering. According to the method, Bacillus subtilis BSGNY-Pveg-glmS-P43-GNA1 is used as an original strain, dCas9 induced by xylose and three sgRNA expression fragments targeting to genes zwf, pfkA and glmM respectively are integrated on the genome, and the strain is fermented in a shake flask, so that the titer of GlcNAc reaches 20.5 g/L, the yield of GlcNAc is 0.612 g/g glucose, at the same time, the efficient co-utilizing of glucose and xylose by the recombinant B.s subtilis is achieved, and the foundation for further metabolic engineering transformation of the B. subtilis to produce GlcNAc and industrialization thereof is laid.