Studies On The Evolution Of Acetohydroxyacid Synthase And Its Key Role On The Biosynthesis Of L-valine

Acetohydroxyacid synthase（AHAS）is a member of thiamin diphosphate-dependent（ThDP）enzymes.It catalyzes the first reaction in the bisosynthetic pathway of the branched-chain amino acids.It either condenses two pyruvate to yield acetolactate,which leads to produce L-valine,or condenses pyruvate with 2-ketobutyrate to yield acetohydroxybutyrate,which leads to the bisosynthesis of L-isoleucine.L-valine and L-isoleucine are both essential amino acids,which have broad application prospects in food,medicine and other fields.In enterobacteria,acetohydroxyacid synthase have three isozymes,each isozyme could efficiently produce L-isoleucine or L-valine.In Corynebacterium glutamicum,there is only one AHAS.Untill now,how and why AHAS could produce L-valine efficiently is still unknown.In this study,based on the sequences,structure and phylogenetic analysis,a plausible evolutionary model was proposed.Meanwhile,the differences of IlvBNs on producing L-valine were investigated,including IlvBN-I from C.glutamicum IWJ001 and IlvBN-V from C.glutamicum VWB-1.Several amino acids of AHAS were perpared for site-specific mutation.Then the effects of these AHAS mutations on producing L-valine were investigated in C.glutamicum.And then metabolic engineering in both strains were used to increase the production of L-valine.The main results are as follows:（1）Through bioinformatics analysis,the evolutionary model of AHAS was proposed.AHASs mainly participate in the biosynthesis of branched chain amino acids,and catabolic acetolactate synthase（CALS）paly role in catabolic pathway.CALS and catabolism related genes form gene clusters,thus from therories of evolution,CALS appeared later than AHAS.Based on sequences,phylogenetic analysis,and the distribution of related genes in the genomes,AHAS and CALS were not from a common ancestor.They were likely to be enzymes with the same function.Among AHASs,the single-copy AHAS occured more frequently in bacteria than isozymes,thus the single-copy AHAS was more likely to be the ancestor.It is similar to the isozyme AHAS III.Among isozymes,it was inferred that isozyme AHAS III appeared the earliest,while isozyme AHAS I appeared the latest.The reason for the emergence of isozyme AHAS II and AHAS I was the biological differentiation ofγ-proteobacteria strains.In these strains,gene rearrangement occurred.Initally,genes in branched chain amino acid synthesis pathway separated from each other in the genome of bacteria,gradually,they formed gene clusters to catalyze the synthesis of amino acids more efficiently.（2）By site-specific mutation,we found that the 138th amino acid of C.glutamicum AHAS affected L-valine biosynthesis.Sequences alignment of IlvBN-I(IlvBN^138A404V)from IWJ001and IlvBN-V(IlvBN^138V404A)from VWB-1 showed that,the sequences of their regulatory subunit were identical,while the 138th and 404th amino acids of the catalytic subunit are different.When overexpressed of IlvBN^138V404V in ATCC14067,the production of L-valine was9.06 g·L^-1,which was 50.0%higher than overexpression of IlvBN^138A404V;When overexpressed of IlvBN^138A404A in ATCC14067,the production of L-valine was 5.17 g·L^-1,which was 42.5%lower than overexpression of IlvBN^138V404A.When the 138th amino acid of IlvBN was valine,it could increase the production of l-valine,while the 404th amino acid not change the production of L-valine.Through enzyme activity assay,it found that when the 138th amino acid changing from alanine to valine,the optimal pH of AHAS was reduced,and it is proper to low pH.When overexpressing ilvBN in IWJ001 and strains constructed by knocking out genes form IWJ001,the production of L-valine all increased.（3）The effects of highly conserved amino acids in isozyme AHAS I,II,III were analyzed by protein structure modeling and molecular biology methods.AHAS I,II and III have highly conserved amino acids,separately.Studies on IlvBN site-specific mutations have shown that,these conserved amino acids could effect the catalytic activity of the enzyme.When expressed IlvBN^H78F,IlvBN^A79I and IlvBN^M499L,the production of L-valine reduced.When amino acids in AHAS III were partially conserved,but the corresponding sites were highly conserved in AHAS I and AHAS II,these amino acid were likely related to the specific synthesis of branched chain amino acids.The 47th amino acid of CgIlvBN was conserved isoleucine residue（I）in AHAS I,but was conserved tyrosine residue（Y）in AHAS II.When expressed IlvBN^I47Y mutant,the production of L-valine reduced 61.3%,while the production of L-isoleucine increased by12%.It means the 47th amino acid effects the preference of strains for the synthesis of L-valine or L-isoleucine.Meanwhile it showed that this isoleucine residue of 47th amino acid was important for producing L-valine.（4）YTW-104（ATCC14067△ilvA△aceE△alr△leuA）constructed in our lab previously was metabolic engineered into an L-valine production strain by overexpressing the mutant ilvBN^A138V and other L-valine synthesis pathway genes.When added a small amount of sodium pyruvate at the beginning of fermentation,and supplied potassium acetate in batches,YTW-104 could produce L-valine 9.32 g·L^-1.When co-expressing gene ilvBN^A138V,ilvC and ilvE,the recombinant strain YTW-104/pJYW-4-ilvBN^A138VC-ilvE could synthesize the highest L-valine after 84 h.The production of L-vaine was 25.93 g·L^-1,and the glucose conversion rate was0.489 g·g^-1.