Regulation Of Key Metabolic Processes To Biosynthesize O-acetyl-L-homoserine In Corynebacterium Glutamicum

O-Acetyl-L-homoserine?OAH?is an intermediate metabolite,which is widely used in food,agriculture and other fields.In particular,OAH can be used as a precursor to directly combine with methanethiol for L-methionine production through enzymatic conversion.This method has been paid more and more attention in the production of L-methionine,thus the microbial fermentation of OAH has also attracted more and more attention.Corynebacterium glutamicum,a safe and popular industrial microorganism that can use cheap feedstock to biosynthesize many amino acids and other useful compounds,has rarely been reported for production of OAH.In this study,C.glutamicum ATCC 13032 was used as the starting strain,and a high-level L-homoserine producing strain was constructed as a platform strain through metabolic engineering and synthetic biology.Then,the synthetic biology tools of C.glutamicum was further improved by PUTR?promoter-5'-UTR?screening and CRISPR-Cas9system optimization.Based on this,the key factors of OAH accumulation were explored through enhancement of metabolic pathway and substrate addition.Finally,CRISPRi was used to identify the key nodes of OAH accumulation and regulate the expression of key genes to achieve the efficient accumulation of OAH.Major results achieved in this study are highlighted below:?1?Efficient production of L-homoserine by redistribution of metabolic flux.Firstly,we created an initial L-homoserine-producing strain with the L-homoserine titer of 1.5 g/L by overexpressing genes involved in amino acid-biosynthesis pathways and deletion of those involved in amino acid degradation,regulation,and import.Importantly,we identified the brn FE operon encoding the two-component export system as responsible for L-homoserine export.Following its overexpression,we ultimately generated an efficient L-homoserine-producing strain by additional overexpression of lys C^T311I,asd from C.glutamicum ATCC 13032,and thr A^S345F from E.coli K12-MG1655 after screening high-efficiency enzymes,resulting in the L-homoserine titer of 8.8 g/L.?2?Obtaining a series of native gradient promoter-5'-UTR sequences.A total of 90 PUTR sequences with different Transcripts Per Million?TPM?were selected based on RNA-Seq data.Then the PUTR fragments were inserted into the backbone plasmid,and the PUTR library was constructed by inserting the PUTR fragments.These 90 PUTRs exhibited a span of expression levels,and 16 strong PUTRs and a strongest PUTR(P_NCgl1676UTR)were selected based on the strong P_sodUTR in stationary phase.Nine common typical chemicals related to the biosynthesis pathways of O-acetyl-L-homoserine and L-methionine were added to the culture medium to explore their potential effects on these PUTRs.?3?Optimizing and expanding the CRISPR-Cas9 in C.glutamicum ATCC 13032.According to previous studies,CRISPR-Cas9 was established in C.glutamicum.By analyzing the sg RNA expression cassette of CRISPR-Cas9 system,it was found that redundant base had negative effect on editing efficiency.To expand the application of the system,we used other promoters(P₄₅,P_trc,and P_H36)to replace the inducible promoter P_{gly A} for the expression of sg RNA and improved the efficiency of sg RNA expression plasmid curing combined with the expression of fluorescent protein.?4?Promotion of OAH accumulation based on precursors supply.The strain was able to accumulate OAH with the titer of 0.97 g/L by expressing different L-homoserine acetyltransferases.Through the introduction of the different biosynthesis pathways of acetyl-Co A and the addition of substrates,it was found that the biosynthesis of acetyl-Co A was not the rate limiting step of OAH accumulation,but the metabolic flux of L-aspartate downstream pathway was the key factor.Finally,the strain was able to accumulate OAH with the titer of 5.2 g/L by addition of acetate and overexpression of L-homoserine dehydrogenase and L-homoserine acetyltransferase.?5?Identification of limiting factors for OAH accumulation by CRISPRi system.Based on CRISPR-Cas9 system with the chromosome-borne expression of cas9 gene,a more suitable CRISPRi system was constructed.The CRISPRi was used to inhibit the expression of key genes in glucose transport,glycolysis,pentose phosphate pathway and TCA cycle.It was found that the inhibition of glucose transport and glycolysis had adverse effects on OAH,while the inhibition of glt A gene expression level had a positive effect on OAH accumulation.Finally,the accumulation of OAH was increased with the titer of 7.8 g/L by regulating the glt A gene expression level.