Screening Genes Regulating The Expression Of Glycerol Dehydratase From Klebiella Pneumoniae 2e And Their Corresponding Regulations Mechanism

Crude glycerol is a by-product of biodiesel production.With the large-scale development of biodiesel industry,the output of its low value by-product crude glycerol has also increased rapidly.Transforming crude glycerol into 1,3-propanediol(1,3-PDO)which is widely used in chemical industry is a very promising green and efficient utilization way of crude glycerol.Klebsiella has attracted much attention because of its excellent production performance of 1,3-PDO.Glycerol dehydratase(GDHt)is the key rate limiting enzyme for microbial synthesis of 1,3-propanediol.This enzyme has an important impact on the synthesis rate and yield of 1,3-PDO.The upstream regulatory factors of glycerol dehydratase have not been reported.In this study,a crude glycerol tolerant strain Klebsiella pneumoniae 2e was selected as the research object.The transcription factors upstream of glycerol dehydratase were screened by DNA pull-down method,and the screening results were bioinformatics analyzed to select the transcription factors,The electrophoretic mobility shift assay(EMSA)method was used to validate the selected transcription factors.After the transcription factor was knocked out and overexpressed,the growth rate of the recombinant strain,the expression of glycerol dehydratase gene and enzyme activity were determined.The main results obtained were as follows:1.Four proteins were screened and identified as possible transcription factors of K.pneumoniae 2e glycerol dehydratase115 differential proteins were screened by DNA pull-down with glycerol dehydratase promoter as probe.Four candidate transcription factors with high reliability were selected through bioinformatics analysis:RpoE(KP2e_GM001383)、RpoS(KP2e_GM001184)、CRP(KP2e_GM000532)and H-NS(KP2e_GM002164).2.Construction of four selected K.pneumoniae 2e glycerol dehydratase transcription factor coding gene deletion and overexpression strainsThe four transcription factor coding gene deletion strains KP.2e_ΔrpoE、KP.2e_ΔrpoS、KP.2e_ΔCRP、KP.2e_ΔH-NS was constructed by λRed homologous recombination;The cloned transcription factor gene fragment was connected to pUC19K plasmid to construct the overexpression vector,and transferred into K.pneumoniae 2e by electric shock transformation.KP.2e_rpoE-OE、KR2e_rpoS-OE、KP.2e_CRP-OE、KP.2e H-NS-OE was successfully constructed.3.The regulation mechanism of the selected transcription factors on the expression of glycerol dehydratase by K.pneumoniae 2e during pure glycerol medium fermentation was exploredThe transcription level and enzyme activity of glycerol dehydratase in pure glycerol culture of transcription factors selected from K.pneumoniae 2e wild-type and recombinant strains were detected by fluorescence quantitative PCR and enzyme activity assay.The results showed that at the transcriptional level,the dhaB expression of glycerol dehydratase gene in CRP and H-NS knockout strains was significantly lower than that in the wild type,the lowest was 84.9%and 84.4%lower than that in the wild type,while the dhaB gene expression of rpoE,rpoS,CRP and H-NS overexpression strains was 1.67,2.05,3.71 and 3.54 times higher than that in the wild type.The positive and negative results showed that the selected transcription factors had a positive regulatory effect on the expression of glycerol dehydratase at the transcriptional level.Compared with the wild-type glycerol dehydratase(GDHt),the enzyme activities of rpoE,rpoS,CRP and H-NS knockout strains decreased by 12.9%,14.4%,15.9%and 15.6%respectively after 16 hours of culture.The enzyme activity of rpoS overexpressing strains increased significantly,up to 20.59%higher than the wild type.The enzyme activities of rpoE,CRP and H-NS overexpressing strains also increased slightly.The above results showed that the selected transcription factors had a positive regulatory effect on glycerol dehydratase at the transcription level and enzyme activity level.4.The regulation mechanism of selected transcription factors on the expression and of glycerol dehydratase by K.pneumoniae 2e in glycerol medium containing different concentrations of crude glycerol were researchedUnder the conditions of different concentrations of NaCl and methanol,the expression of glycerol dehydratase of the recombinant strain was similar to pure glycerol.NaCl or methanol in a certain concentration range had little effect on the regulation of the selected transcription factors.Oleic acid and linoleic acid had a intense inhibitory effect on the expression of glycerol dehydratase in K.pneumoniae 2e.Although the knockout strain had a more sensible inhibitory effect on the expression of glycerol dehydratase under oleic acid and linoleic acid culture compared with the wild type,the expression of glycerol dehydratase in the overexpressed strain could still maintain a higher level compared with the wild type.These results suggested that the four selected transcription factors play an important positive regulatory role in maintaining the expression of glycerol dehydratase of K.pneumoniae 2e in crude glycerol environment.5.Binding analysis of selected transcription factors with glycerol dehydratase gene promoterAccording to literature reports and bioinformatics prediction methods,the 180bp sequence before the sense chain on the glycerol dehydratase promoter was selected for biotin labeling,and the recombinant candidate transcription factors obtained from prokaryotic expression were verified by electrophoretic mobility shift assay(EMSA).The experimental results showed that the candidate transcription factors RpoE,RpoS and CRP could not directly bind to the glycerol dehydratase promoter sequence,These three candidate transcription factors may regulate the gene dhaB encoding glycerol dehydratase indirectly,and the selected transcription factor H-NS can combine with the dhaB promoter in a concentration dependent manner,indicating that H-NS can directly regulate the expression of dhaB.