Study On Metabolic Regulation And Transcription Elements In Klebsiella Pneumoniae And Escherichia Coli

3-Hydroxypropionic acid(3-HP) is an achiral organic acid with active chemical properties. It can be easily converted into a series of important compounds. At present, the commercial production of 3-HP still relies on chemical synthesis method. Because of its high cost, low conversion rate, and problems with downstream separation and purification, efficient 3-HP production methods are highly desirable. With the development of synthetic biology, althrough microbial production of 3-HP comes to light, it is still challenging. To address it, more strategies are required, including metabolic engineering, transcription regulation, etc. In this study, the metabolic regulation and transcription elements in Klebsiella pneumoniae and Escherichia coli were investigated. The experiment protocols and results mainly include the following three aspects:1. We sought to enhanced the promoter activity by replenishment of sigma factor rpoE in Klebsiella pneumoniae. Towards this goal, sigma factors were examined for their affinity with pk, the native promoter of glycerol dehydratase. We expected that increasing the expression of 3-HP synthetic genes, could lead to enhancement of 3-HP production. A total of three factors including rpoE, rpoS and dksA were studied. The Pkana promoter and gene gfp were applied to screen the most appropriate factor. The results showed that the sigma factor rpoE is most appropriate for gfp expression. To further validate the role of rpoE in enhancing gene expression, the gfp gene on the vector was replaced by aldehyde dehydrogenase gene (ald4), leading to recombinant strain K. pneumoniae (pET-pk-ald4-pkana-rpoE). Enzyme activity assay and SDS-PAGE analysis showed that the ald4 was correctly expressed and its activity was elavated. Shake flask fermentation indicated that the yield of 3-HP was increased by 0.6 fold compared to the control K. pneumoniae(pET-pk-ald4). In bioreactor cultivation, the conversion rates glycerol to 3-HP and 1,3-PD were 0.105 mol/mol and 0.34 mol/mol, respectively. And the yield of 3-HP and 1,3-PD reached significant levels. Clearly, rpoE expression elevated the activity of pk promoter.2. To investigate the mixed culture of E. coli and K. pneumoniae, we constructed two recombinant strains:E.coli (pET-cw) and K. pneumoniae (pET-28a). The recombinant strain E. coli (pET-c/w) harbored the chloramphenicol and kanamycin resistance, while K. pneumoniae (pET-28a) carried the kanamycin resistance gene. To examine the relative colonies and survival rates of E. coli and K. pneumoniae in mixed culture, the above two recombinant strains were co-cultured or separately. We found that K. pneumoniae strongly inhibited the growth of E. coli, and K. pneumoniae was the predominant species in co-culture system.3. Improving the production of 3-hydroxypropionic acid depends on regulation of by-products metabolism in K. pneumoniae, expecially the attenuation of by-products. In this experiment, a total of eight genes associated with glycerol metabolism were knocked out separately, including pmd (KPN 01632), poxB(KPN 00904),frdB(KPN 04552),fumC(KPN 01517), dhaT(KPN 03491),ilvH(KPN 00083), adhP(KPN 01853) and pflB(KPN 00931), generating eight mutants. In view of the levels of metabolites in mutants, the adhP and pflB genes were subjected to double deletion. Next, the recombinant plasmid was transformed into double-knockout mutant, leading to recombinant strain K. pneumoniae ?adhP?pflB(tac-puuC). This strain produced 66.91 g/L 3-HP and 3.85 g/L 1,3-PD. More over, this strain produced only 19.40 g/L lactic acid, which is a third of the control strain. Collectively, we believe that K. pneumoniae ?adhP ?pflB(tac-puuC) is a promising industrial strain for production of 3-HP.