Bioproduction Of Xylonate And 3,4-dihydroxybutyrate From Xylose

Human society is suffering from increasingly crises of resource and environment,and the utilization of renewable biomass to produce platform compounds has attracted widespread attention.Xylose is the second most abundant monosaccharide in lignocellulose.Xylonate and 3,4-dihydroxybutyric acid(3,4-DHBA)are important platform compounds and have versatile application in the chemical industry and the pharmaceutical industry.At present,the production of xylonate and 3,4-DHBA by chemical processes have many disadvantages,while xylonate and 3,4-DHBA production by biologic methods have advantages such as low cost,high yield,mild reaction conditions and are environmentally friendly.These advantages of biologic methods are in accord with the concept of green sustainable development and favored by researchers.Corn cob is the secondary agricultural residue that can be easily hydrolyzed into hydrolysate.Besides xylose,glucose and arabinose are also utilizable sugars in corn cob hydrolysate.The second section of this thesis established a co-utilization system through which recombinant Escherichia coli can use glucose and arabinose supporting its growth and convert xylose in corn cob hydrolysate into xylonate.Firstly,biosynthetic pathway of xylonate was overexpressed and two xylonate dehydratases were knocked out in E.coli W3110 to enhance xylonate production from xylose.Then,the genes responsible for acetate,ethanol,and lactate synthesis were knocked out to decrease these byproducts production during the growth of the recombinant strain.Three successive strategies including using lactose as the inducer,eliminating carbon catabolite repression,and inactivating the lactose degradation were employed to enhance the xylonate production.Finally,108.2 g/L xylonate was produced with a yield of 1.09 g of xylonate/g of xylose using xylose as the substrate and glucose as the carbon source through fed-batch fermentation.When corn cob hydrolysate was used as the substrate,91.2 g/L xylonate was produced with a specific productivity of 1.52 g/[L·h].The research not only established a new production method of xylonate,but also provided a successful example of co-utilization of different sugars in corncob hydrolysate.Related strategies can be used to produce other important chemicals based on corncob hydrolysate.Traditional chemical approaches for 3,4-DHBA production involving hazardous materials and harsh reaction conditions.The reported biotechnological routes for 3,4-DHBA production rely on microbial mono-cultures and have issues of cytoplasm acidification and high redox,and thus often suffer from low productivity and yield.The third section of this thesis established a co-culture system to produce 3,4-DHBA from xylose using Gluconobacter oxydans and E.coli.Firstly,G.oxydans 621H was confirmed to have the ability to oxidize xylose and 3,4-dihydroxybutanal(3,4-DHB)into xylonate and 3,4-DHBA,respectively.Secondly,xylonate dehydratase andα-keto iso valerate decarboxylase were overexpressed and the competing pathways in the host strain were knocked out for 3,4-DHB biosynthesis from xylonate in E.coli.Thirdly,the conditions for the co-culture of G.oxydans 621H and engineered E.coli were optimized to enhance 3,4-DHBA production from xylose.Finally,the co-culture system produced 3.26 g/L 3,4-DHBA from 7.0 g/L xylose with a high yield of 0.47 g/g,achieving the highest titer and yield of 3,4-DHBA reported so far.Besides the biotechnological production of 3,4-DHBA production,the co-culture of G.oxydans 621H with recombinant E.coli would be a useful engineering strategy in the production of other important chemicals.The production of 3,4-DHBA using modular co-culture engineering avoided the problems of cytoplasm acidification and high redox and increased the titer and yield of 3,4-DHBA.In vitro enzymatic cascade is also an alternative method for 3,4-DHBA production.The fourth section of this thesis established an in vitro enzymatic cascade composed of aldol oxidase,dihydroxy acid dehydratase,α-keto acid decarboxylase and catalase for producing 3,4-DHBA from xylose and achieved 4.55 g/L 3,4-DHBA accumulation with a yield of 0.66 g/g.In addition,this system did not require the addition of expensive redox cofactors.In summary,this thesis has developed three methods for the biological production of xylonate and 3,4-DHBA based on xylose.Among them,the recombinant E.coli constructed by metabolic engineering techniques can produce 91.2 g/L xylonate from the corn cob hydrolysate.The G.oxydans-E.coli co-culture system can produce 3.26 g/L 3,4-DHBA.The in vitro enzymatic cascade can produce 4.55 g/L 3,4-DHBA.Both of the two methods for 3,4-DHBA production in this thesis achieved higher titer of 3,4-DHBA that all of the reports so far.Relevant researches in this thesis not only provide a feasible method for recycling corncob hydrolysate,but also laid theoretical basis for producing important chemicals using non-phosphorylation pathway of xylose.