The Construction And Application Of Xylulose Cleavage Pathway In Escherichia Coli

Xylose is the second simplest sugar in nature,with the advantages of large reserves,environmental protection and renewable.However,compared with glucose,there are fewer microorganisms that can naturally use xylose,which greatly restricts the research on the conversion of xylose into high value-added products by microorganisms.In this paper,the natural xylose metabolism pathway of Escherichia coli was modified,and the xylulose cleavage pathway of lactic acid bacteria was introduced to improve the utilization efficiency of E.coli to xylose,and poly-3-hydroxybutyrate(PHB)synthesis.The thesis first used Red recombinant gene knockout method to knock out the rpe gene encoding ribulose-5-phosphate isomerase in the E.coli xylose metabolism pathway,blocking the original xylose metabolism pathway in E.coli,rpe Mutants cannot grow with xylose.Next,two pentose phosphate ketolase genes ptk and xfp from lactic acid bacteria capable of normal function in E.coli were screened,forcing the E.coli with rpe gene deletion to use xylulose cleavage pathway to metabolize xylose for growth.Recombinant bacteria that knocked out rpe and expressed the ptk and xfp genes,respectively,were able to completely consume 10 g/L xylose within 96 h,and produced 2.43 g/L and 3.12 g/L of acetic acid,respectively.The accumulation of large amounts of acetic acid hinders the efficient use of xylose by recombinant bacteria and inhibits the growth of E.coli.In this paper,the branched metabolic pathways related to acetate production in E.coli were modified,and the genes encoding pyruvate oxidase poxB and acetate kinase ackA were knocked out.The mutants lacking the poxB and ackA genes accumulated only a small amount of acetic acid during fermentation,and the yield was only 0.07 g/L.Finally,this paper expressed the PHB synthetic operon phaCAB in the above-mentioned recombinant bacteria to realize the production of PHB by using the xylulose cleavage pathway with xylose as the carbon source.PHB synthesis is a NADPH-dependent process,and the NADPH produced by recombinant bacteria in xylose metabolism may not be sufficient to support effective PHB production.The paper attempts to express NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapC,NAD kinase gene yfjB and transhydrogenase genepnt,etc.,to regulate the supply of coenzymes in cells.Recombinant bacteria expressing gapC,yfjB and pnt genes successfully accumulated PHB intracellularly with yields of 0.84 g/L,0.09 g/L and 0.55 g/L,respectively.The highest yield was 10 times that of the control strain PHB.