System Metabolic Engineering Of Corynebacterium Glutamicum For The Production Of L-valine

L-valine is one of three kinds of branched-chain amino acids,it is also an essential amino acid for human body.Due to its variety of physiological functions,it has a broad application prospects in the fields of food,medicine,cosmetics and feed.Corynebacterium glutamicum VWB-1 is a high L-valine producing strain obtained through multiple rounds of mutagenesis screening.It is very difficult to increase its L-valine production by traditional methods such as mutagenesis.This study focuses on the research of C.glutamicum VWB-1,with the employment of systematic metabolic engineering strategies,the production and production efficiency of L-valine is improved through the analysis of transcriptomics and proteomics and the transformation of genetic engineering.The main conclusions are as follows:?1?Analysis of the high L-valine producing mechanism in C.glutamicum VWB-1.During batch fermentation experiments,the L-valine production level of the L-valine producing strain C.glutamicum VWB-1 was 20-fold higher than that of the wild-type strain C.glutamicum ATCC 13869,and the level of the mixed acids were relatively low,such as L-alanine.According to the analysis based on transcriptomics and proteomics,the transcription level of 1055 genes and the translation level of 96 genes between VWB-1 and ATCC 13869 were different.The high L-valine yielding mechanism of C.glutamicum VWB-1 was as follows:?1?The enhancement of the pentose phosphate pathway provides sufficient reducing power for the L-valine synthesis enzymatic reaction with the more production of NADPH;?2?The weakening of the parabranch pathway employing pyruvate as the metabolic substrate reduces the formation of acetic acid and lactic acid,pyruvate was accumulated to provide more substrates for L-valine synthesis;?3?All the genes involved in the biosynthesis of L-valine were transcriptional up-regulated and partial were translational up-regulated,such as ilvBN,ilvC,ilvD,and ilvE,increasing the carbon flow of carbon towards the synthesis of L-valine;?4?The transcriptional upregulation of the gene brnFE encoding the L-valine transporter enhances the L-valine secretion ability of VWB-1;?5?Down-regulation of genes involved in the side-branch pathways such as L-alanine,L-isoleucine and L-leucine were favorable to reduce the accumulation of hetero acids;?6?The regulation of intracellular elongation factors,ribosomal proteins,sigma factors and cell division related genes,etc.,indirectly affect L-valine production by correlating to cellular protein synthesis and biomass,etc.?2?Comparison of AHAS enzyme activities from different sources and anti-feedback inhibition construction.Amino acids sequence alignment of the AHAS between C.glutamicum VWB-1 and C.glutamicum ATCC 13869 showed that there are four mutation sites in the catalytic subunit of AHAS in C.glutamicum VWB-1,and the key site Ala138Val is located in the catalytic structure of the enzyme,domains which might affect its catalytic activity.The E.coli expression vectors for ilvBN and ilvBN1 were constructed,and the amino acids at position20-22 of the ilvN gene encoding regulatory subunits were subjected to point mutations to obtain ilvBN^M and ilvBN1^M.The in vitro enzymatic activity assay showed that the AHAS containing the Ala138Val mutation in its catalytic subunit and the Gly20Asp,Ile21Asp and Ile22Phe in its regulatory subunit mutation sites had higher activity,which could also be completely resistant to the feedback inhibition of the three branched-chain amino acids 10 mM combinations.The overexpression effect of four AHAS on the yield of L-valine was analyzed.It was found that the expression of AHAS carrying the catalytic subunit mutation site Ala138Val and the regulatory subunit mutation sites Gly20Asp,Ile21Asp,and Ile22Phe can significantly increase the level of L-valine production,while having no significant effect on cell growth and glucose consumption.During a 5 L fed-batch fermentation,the recombinant strain VWB-1/pEC-XK99E-ilvBN1^M showed that the yield of L-valine was increased by 26.9%compared to the original strain,reaching to a concentration of 37.9 g·L^-1.?3?The production of L-valine was increased by reducing the production of hetero acids with the modifying the branched pathway.Mutant strains VWB-1?alaT:kan,VWB-1?pyc:kan,VWB-1?poxB:kan and VWB-1?panB:kan were constructed using the gene knockout system based on homologous recombination-site-specific recombination.The levels of L-valine showed that the VWB-1?pyc:kan and VWB-1?poxB:kan and VWB-1?panB:kan had no significant increase effect in the yield of VWB-1 L-valine except for the mutant strain VWB-1?alaT:kan.The sacB-based integrated vector was used to completely knock out alaT to obtain the mutant VWB-2.The 5 L level fermentation of VWB-2 showed that the knock out of alaT had a greater effect on the cell's ability to produce L-alanine which was decreased by 83.9%.The L-valine yield was higher than that of the original strain VWB-1.Transmission electron microscopy experiments showed that the morphological character VWB-2 cells were changed to longer and thinner from short rod ones.The reason may be that the sudden drop in the intracellular L-alanine content affected the normal metabolism of cell morphology maintaining.?4?High L-valine producing level were realized through overexpression of the key genes involved in the L-valine synthesis pathway and secretory system in C.glutamicum.Expression vectors with the different tandem combination of the L-valine pathway involved genes were constructed,and their effects over the production of L-valine were compared.Shake flask fermentation experiments showed that the L-valine yields of the recombinant strains VWB-1/pEC-XK99E-ilvBN1^MC,VWB-1/pEC-XK99E-ilvBN1^MCD and VWB-1/pEC-XK99E-ilvBN1^MCE were all increased.The L-valine production of VWB-1/pEC-XK99E-ilvBN1^MCE was most pronounced,with a final concentration of 43.4 g·L^-1 and 73.1%increase compared to the control strain.The pEC-XK99E-ilvBN1^MCD and pEC-XK99E-ilvBN1^MCE were ligated with lrp1-brnFE containing the mutation site Arg39Trp on Lrp1,respectively.The shake flask level fermentation showed that the L-valine producing ability of the recombinant strains VWB-1/pEC-XK99E-ilvBN1^MCD-lrp1-brnFE and VWB-1/pEC-XK99E-ilvBN1^MCE-lrp1-brnFE were both increased,reaching to 45.7 g·L^-1 and 47.3 g·L^-1,respectively.The recombinant plasmid with the most prominent L-valine promotion ability was overexpressed in VWB-2.During the 5 L fed-batch fermentation experiments,the L-valine production of the strains VWB-2/pEC-XK99E-ilvBN1^MCD-lrp1-brnFE and VWB-2/pEC-XK99E-ilvBN1^MCE-lrp1-brnFE reached to 51.1 g·L^-1 and 54.1 g·L^-1 after 96 h of fermentation,respectively,which were increased by 71.3%and 81.1%compared to the original strain with the glucose and acid conversion rates of 0.292 g·g^-1 glucose and 0.312 g·g^-1 glucose,respectively.In addition,the levels of L-alanine and other hybrid acids in the fermentation broth were significantly reduced,indicating that two L-valine producing strains with excellent acid production performance and low yield of mixed acids were successfully constructed.