Engineering Central Pathways For Industrial-level D-(-)-acetoin Biosynthesis In Corynebacterium Glutamicum

In this study,cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies to realize a high production of D-（-）-acetoin.First,by knocking out ppc,succinate production was reduced by 98.6%and the production of acetoin increased from 11.36 g/L to 11.95 g/L.As for the pyc knocked out strain,it accumulated 11.75 g/L acetoin and almost the same amount of succinate as CGR6,indicating that the replenishing pathway of succinate is mainly from phosphoenol pyruvate rather than pyruvate under aerobic condition.A double deletion of ppc and pyc is generally lethal for C.glutamicum due to a lack of oxaloacetate.We introduced the mutant ICD^G407Sthat recovered the growth,but the growth rate was also reduced in constrast to CGR6 and the production of acetoin was only 11.37 g/L with a lower productivity,indicating that carbon fluxes from acetoin synthesis rather than its competing pathways were redirected to regain biomass synthesis.Then,we down-regulated gltA in CGS6 yielding CGS7,acetoin production increased form 11.36 g/L to 13.36 g/L.In addition,the same gene modification was used to construct strain CGS8 in ppc-knockout strain CGS1,acetoin production increased to 14.56 g/L,indicating that down-regulating gltA had a significant effect on acetoin production and deleting ppc and down-regulating gltA had a significant synergistic effect on acetoin production.Finally,two more copies of alsSD were inserted into the chromosome of the CGS8.The activities of acetolactate synthas（ALS）and acetolactate decarboxylase（ALDC）increased by 2.33-fold and 1.47-fold in the middle log phase respectively.After introduction of p EC-XK99E-alsSD-Δlac Iq in CGS10,the optimal engineered strain CGS11 accumulated 15.70 g/L acetoin in shake flasks and accumulated 102.48g/L acetoin at a rate of 1.86 g/（L·h）with a yield of 0.419 g/g glucose during the fed-batch fermentation process which is the highest titer of highly enantiomerically enriched D-（-）-acetoin together with a competitive product yield and productivity that opens the possibility for industrial application.