Gene Modification To Reduce The2,3-butanediol Synthesis On1,3-propandediol Production In Klebsiella Pneumoniae

1,3-propanediol (1,3-PDO) is recognized as the most prospective three carboncompounds in this century and increasing attention has been paid by many researchers toKlebsiella pneumoniae because of its high1,3-PDO productivity. During the process of1,3-PDO fermentation by K.pneumoniae, many byproducts are formed, such as2,3-butanediol(2,3-BD), acetic acid, lactic acid, succinic acid and ethanol, among which2,3-BD is the majorone. It not only consumes large amounts of carbon source and NADH to diminish synthesis of1,3-PDO, but also serves as an obstacle in downstream processes because of the difficulty toseparate it from1,3-PDO. This study is an attempt to reduce the main byproduct2,3-butanediol by adjusting the metabolic pathway of K. pneumoniae based on the genetictools to provide reference for strain reforming of1,3-propanediol industrialization.Based on the analysis of K. pneumoniae metabolism, the author tries to structure thegene knockout fragments of budA and budC gene, coding two key enzymes of2,3-BDsynthetic pathways. The budA and budC gene on genome are knocked out using Redrecombination technology and identification of PCR indicates that the two target genes areremoved in the two mutants. Enzyme activity results showthat there is40%2,3-BDdehydrogenase (BDH) activity in K. pneumoniae ZG38(budC) and no α-acetolactatedecarboxylase activity in K. pneumoniae ZG38(budA). Two mutants K. pneumoniaeZG38(budC) and K. pneumoniae ZG38(budA) are thus obtained.Fermentation experiments show that growth of the two mutants are inhibited at differentlevel. The budA deficient strain didn’t produce1,3-PDO and2,3-BD, but the final titer oflactic acid, succinic acid, ethanol and acetic acid increased remarkably compared with theparent strain. The budC deficient strain fermentation results show that1,3-PDO concentrationincreased to110%and2,3-butanediol concentration dropped to71%of the parent strain. It ispresumed from the further analysis of budC deficient strain fermentation that K. pneumoniaemay possess the2,3-BD cycle as a replenishment pathway.In order to solve the problem of excessive accumulation of organic acid in K.pneumoniae ZG38(budC) fermentation, pH control strategy is utilized to optimize K.pneumoniae ZG38(budC) fermentation. The pH-start optimization results show that1,3-PDO production was highest at pH9. The final titers of1,3-PDO and2,3-BD were22.1g/L and3.5g/L respectively, which accounted for109%and74%of those of parent strain. Bycontrast, the fermentation results under constant initial pH show that1,3-PDO production washighest at pH7. The final titers of1,3-PDO and2,3-BD were24.2g/L and1.1g/Lrespectively, accounting for115%and50%of those of parent strain. Obviously, constant pHis much better than pH-start strategy. The author also carried out Fed-batch fermentations in5L stirring bioreactor by feeding glycerol under constant initial pH. The maximum amount of1,3-PDO and2,3-BD of K. pneumoniae ZG38(budC) were66.9g/L and6.2g/L, accountingfor110%and70%of those of the wild strain. This research provides the basis for reforminglow-byproduct K. pneumoniae.