Metabolic Regulation Of Corynebacterium Glutamicum To Synthesize N-acetylglucosamine

Glucosamine(GlcN)and its derivative N-acetylglucosamine(GlcNAc)are highly recognized drugs for the treatment of osteoarticular diseases in the world today,which are widely used in the fields of health care,medicine,agriculture,and chemical industry.They are listed as dietary supplements and pharmaceuticals in many countries,with a very broad application prospect.At present,the production methods of GlcNAc are mainly acid hydrolysis of chitin,microbial enzyme conversion and microbial fermentation.Compared with other methods,the microbial fermentation method showed the advantages of environmental protection,mild conditions,abundant raw material sources,and difficulty in sensitization.In this study,the food-safety strain Corynebacterium glutamicum S9114 was used as the starting strain.Through in-depth analysis of the biosynthesis pathway of GlcNAc,metabolic flow distribution between the GlcNAc synthesis pathway module and each metabolic pathway module was balanced by using metabolic engineering and protein engineering methods,and an engineering strain of C.glutamicum capable of efficiently synthesizing GlcNAc was constructed.Moreover,a genome base editing tool and a gene expression regulation tool in the C.glutamicum S9114 strain were constructed to facilitate subsequent genome transformation and gene expression regulation.The main research results are divided into the following four parts:(1)On the basis of sequencing the whole genome of C.glutamicum S9114 and analyzing key functional gene modules,an exogenous GlcN6 P acetylase(Gna1)was introduced to improve the synthetic pathway of GlcNAc in C.glutamicum,and the obtained titer of GlcNAc in shake flask fermentation was 1.7 g/L;Besides,the expression of Gna1-encoding gene GNA1 was enhanced by using synthetic extra-gene repetitive palindrome sequence(REP),and the titer of GlcNAc reached 3.1 g/L;The related coding genes of GlcNAc catabolism pathway enzymes GlcNAc6 P deacetylase(Nag A)and GlcN6 P deaminase(Nag B)were further knocked out,and the titer of GlcNAc was increased to 4.8 g/L;Knocking out the ldh coding gene of lactate dehydrogenase and pta coding gene of acetate dehydrogenase to block the synthesis of byproducts lactic acid and acetic acid,and further improving the titer of GlcNAc to 5.4 g/L;Further optimization of the expression of the GlcN6 P synthetase(Glm S)-encoding gene glm S resulted in an increase in the titer of GlcNAc to 6.9 g/L(in the shake flask)and 17.7 g/L by fedbatch fermentation(in the 50-L fermentor).(2)In order to develop a gene editing and regulating tool that can be used in C.glutamicum S9114,we introduced cytosine deaminase and adenine deaminase into C.glutamicum and obtained a single base editing tool that can realize base conversion of C-T,C-G and A-G in the editing window of 28 bp nucleotide upstream of a target site after a series of optimization,which is beneficial to the construction of mutant libraries on the genome of C.glutamicum.Meanwhile,a method for quickly connecting a plurality of g RNA on an expression vector was developed,and the connection efficiency of a plurality of sg RNA targets on the vector was greatly shortened.In addition,we constructed a single-gene and multiple-gene interference system based on CRISPR-d Cpf1(Clustered Regularly Interspaced Short Palindromic Repeat sequences and CRISPR?associated DNase deactivated Cas protein from Prevotella and Francisella 1),which could inhibit the expression of multiple genes individually or simultaneously.In conclusion,we have further improved the gene editing and regulation tools for C.glutamicum S9114,providing effective tools for genome editing and metabolic engineering in C.glutamicum S9114 cell factory for the production of various biochemical products.(3)Global transcription machinery engineering(gTME)was used to rebalance the central carbon and nitrogen metabolism of C.glutamicum S9114 to promote the production of target metabolite GlcNAc.Seven transcription regulators of carbon metabolism and one transcription regulator of nitrogen metabolism were selected for research.RamA,the global transcription regulator with the most obvious promotion effect on GlcNAc synthesis,was selected from the seven kinds of carbon metabolism regulators,and a mutant library of two domains of RamA was constructed.The forward mutants were screened from the two mutant libraries and combined mutation was carried out,and finally the mutant strain with GlcNAc shake flask titer of 16 g/L was obtained.Furthermore,transcriptome analysis revealed the reasons for the high titer of GlcNAc by mutant strains.The expression of Lld R and Amt R,the global negative transcription regulators of GlcNAc synthesis,was inhibited by CRISPR-d Cpf1 gene regulation system,which promoted the GlcNAc titer to 27.5 g/L(in shake flask)and 62.6 g/L(in 50-L fermentor).(4)As a provider of redox power,cofactors play an important role in regulating metabolic flux.However,every 1 mol of GlcNAc synthesized by recombinant C.glutamicum will produce3 mol of NADH.With the continuous improvement of GlcNAc synthesis ability of cells,it is very likely to lead to imbalance of cofactors in recombinant engineering bacteria.Therefore,this study firstly confirmed that the synthesis of GlcNAc in C.glutamicum would cause NADH accumulation in cells,and changed the cofactor preference of key dehydrogenases in anabolic pathway through rational protein modification.As a result,the intracellular NADH level was reduced by 59.7%,and the intracellular redox state was balanced,and the titer of the target metabolite GlcNAc was increased to 36.9 g/L(in the shake flask),and the titer in the 50-L fermentor was increased to 104.9 g/L.