| Lignocellulose is an abundant renewable resource with broad sources in nature.Xylose is one of the main components in lignocellulose hydrolysate.It is of great significance to produce high-value compounds with xylose as substrate.1,2,4-Butanetriol and 3,4-dihydroxybutyrate are important platform compounds and have important applications in military,pharmaceutical,chemical,and other fields.At present,there are many disadvantages in the production of 1,2,4butanetriol and 3,4-dihydroxybutyrate by chemical processes,such as low yield,high cost,environmental pollution,and so on.The biological methods for 1,2,4-butanetriol and 3,4dihydroxy butyrate production have the advantages of mild reaction conditions and environmental friendliness.In this thesis,Escherichia coli was metabolic engineered to produce 1,2,4-butanetriol and 3,4-dihydroxybutyrate from xylose.The recombinant strain E.coli 4KI,which can produce 1,2,4-butanetriol with xylonate as substrate,was previously constructed in our laboratory.In the second chapter of this thesis,E.coli 4KI was used as the starting strain,and xylose dehydrogenase XylB and xylonolactonase XylC from Caulobacter crescentus were overexpressed in E.coli 4KI to produce 1,2,4butanetriol from xylose.Then,ptsG was knocked out to block the PTS system and eliminate carbon catabolite repression.The transport rate and metabolic rate of xylose were enhanced,and the production of 1,2,4-butanetriol was increased to 9.79 g/L.In addition,after knocking in of xylBC in the genome of E.coli by Red recombination technology,the production of 1,2,4butanetriol was increased to 23.55 g/L through fed-batch fermentation,and the productivity reached 0.49 g/[L·h].Finally,pgi,the coding gene of glucose-6-phosphate isomerase,was knocked out to enhance the efficiency of glucose metabolism via the pentose phosphate pathway to provide NADPH required for 1,2,4-butanetriol synthesis.In a 5 L fermentor,the production of 1,2,4-butanetriol reached 36.63 g/L and the productivity was 1.14 g/[L·h].Using corn cob hydrolysate as the substrate,the concentration of 1,2,4-butanetriol was 43.4 g/L and the productivity of 1,2,4-butanetriol was 1.09 g/[L·h].The concentration and productivity of 1,2,4-butanetriol were the highest reported until now.In the third chapter of this thesis,after knocking in of xylBC in E.coli 4KI genome,knocking out the coding gene of alcohol dehydrogenase YqhD and overexpressing aldehyde dehydrogenase Gox0499 from Gluconobacter oxydans by plasmid pACYCDuet,we constructed the recombinant strain to produce 3,4-dihydroxybutyrate with xylose as substrate.Then,the transport rate and metabolic rate of xylose were enhanced by knocking out ptsG to eliminate carbon catabolite repression,and the production of 3,4-dihydroxybutyrate was also increased.In addition,after overexpressing Gox0499 by plasmid pETPtac with Ptac promoter,the production of 3,4-dihydroxybutyrate was increased to 11.62 g/L.Finally,Gox0499 was knocked into the genome of E.coli.As a result,the production of 3,4-dihydroxybutyrate in 5 L fermentor reached 16.63 g/L and the productivity was 0.35 g/[L·h].Using corn cob hydrolysate as the substrate,the concentration of 3,4-dihydroxybutyrate was 15.7 g/L and the productivity of 3,4-dihydroxybutyrate was 0.39 g/[L·h].The concentration and productivity of 3,4dihydroxybutyrate were the highest reported until now.In summary,the metabolic engineered E.coli constructed in this work can efficiently produce 1,2,4-butanetriol and 3,4-dihydroxybutyrate with xylose as substrate.Relevant researches in this thesis not only provide a feasible method for the efficient utilization of xylose but also laid theoretical guidance for producing high-value compounds using nonphosphorylation metabolic pathway of xy lose. |
