Breeding Of Klebsiella For High-Yield1,3-Propanediol And Study On Transcriptional Regulation Of Key Enzyme Genes

As a three carbon platform compound,1,3-propanediol has broad prospect of application.1,3-propanediol-producing strains with high yield were bred by ultraviolet mutagenesis, andthe transcription level of key enzyme genes were studied by semi-quantitative RT-PCRtechnology, which aimed to explore the high yield1,3-propanediol mechanism of mutant andprovided theoretical basis for modifying Klebsiella.Klebsiella B0038(named K) which produced the highest yield of1,3-propanediol in tenKlebsiella strains was used for original strain. After twice ultraviolet mutagenesis, a mutantnamed A-39-3was obtain, and the yield of1,3-propanediol was23.51g/L. Compared to thewild strain, the1,3-propanediol concentrations increased by58%, and the by-products of2,3-butanediol and ethanol concentrations decreased by27%and19%respectively. A-39-3was subcultured ten generations, and it was found that A-39-3produced1,3-propanediolsteady and has good genetic stability.The tracking analysis of key enzyme activities during fermentation was carried out. Itwas found that the activity of glycerol dehydratase increased obviously compared with wildstrain, the activity of glycerol dehydrogenase, aldcohol dehydrogenase and2,3-butanedioldehydrogenase decreased a little, the activity of1,3-propanediol oxidoreductase was lessnoticeable than wild strain. Glycerol dehydratase gene, glycerol dehydrogenase gene and1,3-propanediol oxidoreductase gene were studied on transcription level by thesemi-quantitative RT-PCR technology in these two strains. It showed that the relative mRNAtranscription level of glycerol dehydratase gene, glycerol dehydrogenase gene and1,3-propanediol oxidoreductase gene in A-39-3was2.58,0.60and1.17times respectively asmuch as the level in wild strain.Transcriptional regulation of glycerol dehydratase gene under different fermentationconditions was studied. It showed that the relative mRNA transcription level of glyceroldehydratase gene was1.58when glycerol was the only carbon source. When glucose was theonly carbon source, glycerol dehydratase gene could transcribe, but the relative mRNAtranscription level was just0.30. Then the double substrates fermentation was studied. Whenthe concentrations of glucose was less than15g/L, the relative mRNA transcription level ofglycerol dehydratase gene showed no obvious difference. When the concentrations of glucosewas more than15g/L, the relative mRNA transcription level of glycerol dehydratase genedecreased obviously. It provided theoretical foundation for improving1,3-propanediolproduction rate by adding glucose as auxiliary substrate. When different concentrations of1,3-propanediol was added to fermentation culture medium, the relative mRNA transcriptionlevel of glycerol dehydratase gene was very low. It showed that1,3-propanediol which wasadded exogenously inhibited the transcription leve of glycerol dehydratase gene.