Synthetic Biology Research Of Corynebacterium Glutamicum On Biorefinery Synthesis Of Biopolymer And Precursors

The biopolymers have the potential to work as an alternative of petroleum-based plastics,therefore contributes to solving the environmental pollution and greenhouse effect,but the utilization of food crop starch used for biopolymers production increases the production cost and leads to food shortage.The lignocellulose biomass is an ideal feedstock for commercialization of biopolymers owing to its abundance and availability.This thesis aims to produce biopolymer(Poly 3-hydroxybutyrate,PHB)and precursors(glutamic acid and lysine)from lignocellulose.However,some technical bottlenecks remain to be solved such as:(ⅰ)the inhibitors generated in the lignocellulose pretreatment process suppressing the growth and fermentation of microorganisms;(ⅱ)the xylose and other fermentable sugars produced in the pretreatment difficult to preserve and utilize;(ⅲ)the non-inhibitory compounds in lignocellulose adverse to production processes;(ⅳ)the strains to be optimized globally to accommodate for the complex lignocellulose environment.To address these obstacles,the Corynebacterium glutamicum which shows strong tolerance to the inhibitors used as the starting strain and further engineered with a synthetic biology approach to make use of the dry biorefinery hydrolysate of lignocellulose biomass as carbon source.In the end,the high concentration of PHB,glutamic acid,and lysine were produced from lignocellulose by recombinant C.glutamicum.In the first part,the glutamic acid-producing strain C.glutamicum S9114 was used as the starting strain,and the stepwise metabolic engineering was conducted:(ⅰ)the stable xyloseutilizing strain C.glutamicum GJ01 was obtained by promoter engineering and genome integration;(ⅱ)the glutamic acid transport protein MscCG was modified to activate the glutamic acid secretion channel upon high concentration of lignocellulose-originated biotin;(ⅲ)the α-oxoglutarate dehydrogenase activity was decreased to redirect the α-oxoglutarate flux to glutamic acid synthesis;(ⅳ)the pentose transporter was overexpressed to enhance xylose uptake rate.The final strain C.glutamicum GJ04 produced 61.7 g/L of glutamic acid at 1.29 g/L/h from the wheat straw hydrolysate with no need of induction.In the second part,the lysine-producing strain C.glutamicum B253 was used as starting strain,and the xylose-utilizing strain C.glutamicum LJ01 was obtained by promoter engineering and genome integration.Moreover,systematic metabolic engineering was conducted to increase lysine production,including amplifying lysine biosynthetic flux,increasing precursor supply,increasing NADPH regeneration,and enhancing xylose utilization rate.The concentration of NADPH decreased by 46.6%when the C.glutamicum LJ01 was cultured in the xylose medium compared to the glucose medium,and the NADPH content increased by 61.3%when the PntAB gene was overexpressed.The final strain C.glutamicum LJ01-Peftu-PntAB produced 31.3 g/L of lysine,which was approximately 25%greater than that of the control strain.In the third part,the xylose-utilizing strain C.glutamicum GJ01 obtained in the first part was used as the starting strain and the PHB synthesis pathway was constructed and optimized:(ⅰ)the stable PHB-producing strain was obtained by successively integrating the PHB synthetic genes(phaA,phaB,and phaC)into the genome of C.glutamicum GJ01;(ⅱ)the cell growth and division of C.glutamicum were adjusted to change cell morphology and increase cell volume;(ⅲ)the stability of PHA synthase was increased by fixing the PHA synthase in the cell membrane;(ⅳ)The C/N ratio of PHB fermentation was optimized to further increase PHB production.The final obtained strain produced 15.1 g/L of PHB with 32.0%g PHB/g DCW from the synthetic medium,which is the highest PHB production by C.glutamicum.Moreover,the 16.2 g/L of PHB with 39.0%g PHB/g DCW was obtained from the wheat straw hydrolysate,which is the highest PHB production from the practical lignocellulose.In the fourth part,the Gluconobacter oxydans DSM2003 with lignocellulose full spectrum of sugars utilization capacity was used as the starting strain,which was alternately transferred in inhibitors containing hydrolysate and inhibitors free hydrolysate for 420 days to intensify sugars simultaneous utilization.The conversion rate of all non-glucose sugars was significantly improved by several folds and the total fermentation time was reduced to 36 h from 72 h.The significant up-regulation of mGDH gene in the adapted G.oxydans strain(more than 40-fold greater than the parental)was considered as the decisive factor for the improvement of strain performance.This evolution adaptation strategy also could be used to accelerate robust sugar utilization for other fermented strains in lignocellulose biorefinery.In conclusion,the dry biorefinery process combined with synthetic biology were applied to overcome the technical bottlenecks of biopolymer and monomers production from lignocellulose.High concentration of PHB,glutamic acid,and lysine was achieved from lignocellulose which represents a big step toward industrial biopolymer production.