Systems Metabolic Engineering Of Bacillus Subtilis For N-acetylglucosamine Production

N-acetyl-D-glucosamine（GlcNAc）and its derivatives,such as glucosamine,have been widely used in food,health food products and pharmaceuticals,especially in osteoarthritis treatment and enhancing immunity effect.GlcNAc,as a kind of pharmaceutical excipient,owing to its good solubility,high safety and low side effects,is widely used for promoting drug absorption in the diagnosis of tumors.In this dissertation,Bacillus subtilis strain BSGN6,which was constructed previously,was used as the starting strain.This aim could be realized by combination of systems metabolic engineering and synthetic biology.In this way,some chanllenges in metabolic engineering can be improved at least three aspects:balancing the carbon flux between N-acetylglucosamine synthesis pathway and other competing metabolic pathway,enhancing intermediates biosynthesis in pathways and enhancing the dephosphorylation of GlcNAc-6-phosphate.And reducing carbon flux loss to produce GlcNAc.The main results were described as follows:1.The expression of glmS（encoding glucosamine-6-phosphate synthase）was strictly feedback inhibited by glmS riboswitch.And the limited step of GlcNAc synthesis was the glucosamine-6-phosphate（GlcN6P）synthesis catalyzed by glucosamine-6-phosphate synthase（GlmS）.Using different strategies to delete the feedback inhibition of glmS ribozyme.And the deleting the glmS ribozyme with inserting a trp terminator-P₄₃ sequence behind the glmS ribozyme 5’cleavage fragment works well.As a result,it can release the feedback inhibition of glmS ribozyme and enhance the expression of GlmS.As a result, the GlcNAc was increased from 9.2 g/L to 12.2 g/L with the yield increased from 0.106 to 0.167 g/g glucose.The overexpression of glmS ribozyme 5’cleavage fragment can promote the cell growth.Searching the potential target of glmS ribozyme 5’cleavage fragment and westernblot analysis demonstrated that there is no interaction between glmS ribozyme 5’cleavage fragment and ypqE mRNA.2.GlcNAc synthesis pathway compete the carbon flux with competitive glycolysis,peptidoglycan synthesis and pentose phosphate pathways.Therefore,we chose pfkA and glmM as targets for downregulating the competitive glycolysis and peptidoglycan synthesis pathways using the glmS ribozyme.To increase the flux toward the GlcNAc synthesis pathway,the glmS ribozyme mutant M9（cleavage site AG→CC）was embedded into the 5’end of pgi mRNA to increase the expression of pgi.This strategy increased the GlcNAc titer from 12.2 to 16.3 g/L,and the yield of GlcNAc reached 0.394 g/g glucose and eliminated acetoin in engineered strain SFMI-G.We used both glmS ribozyme and RBS sequence to control the expression of pfkA and compared their effects on GlcNAc synthesis.But all the strains having RBS substitution produced considerably less GlcNAc compared to that of strain SFMI-G.The pgi expression was fine-tuned by different elements promoter,glmS ribozyme mutant,RBS sequence to promote metabolic balance.The GlcNAc titer was increased to 18.45 g/L with the yield decreased to 0.283 g/g glucose,while the mutant N3531-G shows better cell growth（15.2 g/L）and produced more acetoin （18.2 g/L）than that of SFMI-G.3.The expression of a key enzyme,GlcN6P N-acetyltransferase（GNA1）was enhanced by engineering the promoter and ribosome binding site.This strategy shows that the GNA1 activity increase by 30%,and the GlcNAc titier reached 17.8 g/L.Next,we investigated that glutamate and urea can promote nitrogen assimilation for GlcNAc synthesis.The glutamate synthesis was improved by deleting the two endogenous glutamate dehydrogenase genes（rocG and gudB）and integrating one exogenous glutamate dehydrogenase gene（gdh）.The synergetic engineering of central carbon and nitrogen metabolisms increased the GlcNAc titer from 14.0 to 22.2 g/L in the shaker flask and produced 1.2 g/L acetoin,with the yield of 0.271 g/g glucose.4.Enhancing glucose non-phosphorylation transportation and the expression of key gene GNA1,reducing the metabolic flux of pentose phosphate pathway,make the overaccumulation of GlcNAc6P.As a result,the phosphoglucose stress response was activated and decreased the GlcNAc titer.The results demonstrated that GlcNAc6P dephosphorylation is the limitation step of GlcNAc synthesis.Using the Kegg database alignment,phosphatase YwpJ was screened and identified.The high concentration of phosphosugar was decreased by overexpression of YwpJ.As a results,the DCW and GlcNAc titer reached 9.99 g/L and 21.1 g/L,respectively,with a yield of 0.412 g/g glucose.Transcriptome analysis guided deleting up-regulated genes murQ（encoding a N-acetylmuramic acid 6-phosphate etherase）and nagBB（encoding a glucosamine-6-phosphate deaminase 2）enabled further improvement in GlcNAc production in FMIP34（26.1 g/L）with a yield of 0.483 g/g glucose.Finally,the titer of GlcNAc reached 87.5 g/L in a 30 L fermentor with no acetoin produced and added glutamate and urea three times,and the yiled reached 0.435 g/g glucose.