Influence Of Genes Expression Of 2,3-butanediol Synthesis On 1,3-propandediol Fermentation In Klebsiella Pneumoniae

As a valuable chemical,1,3-propandediol (1,3-PD) is the essential monomer of polytrimethylene terephthalate (PTT), a novel textile fiber. As the main byproduct of 1,3-propandiol synthesis in Klebsiella pneumoniae,2,3-butanediol(2,3-BD) not only suppresses production of 1,3-PD but also multiply difficulties for the purification. Therefore, The research of 2,3-BD in synthesis mechanism and regulation for 1,3-PD production in K. pneumoniae has considerable significance for 1,3-PD fermentation in K. pneumoniae.The influences of different carbon sources to the carbon fluxes were studied during biosynthesis of 1,3-PD. The results showed that the biomass of K. pneumoniae was highest using glucose as the sole carbon source, and 1,3-PD was not detected.1,3-PD yield of K. pneumoniae using glycerol as sole carbon source was achieved only 9 g·L-1. Although there was still by-products using mixed carbon source (35 g·L-1 glycerol and 5 g·L-1 glucose), the biomass and the yield of 1,3-PD were increased by 66.7% and 150% respectively. The results suggested that the mixed carbon sources strategy is the most efficient carbon source recipe.BudR(transcription activator) was overexpressed to explore its regulation on 2,3-BD synthesis and 1,3-PD fermentation in K. pneumoniae. The results indicated that the biomass and the activity of BudC were increased by 22.6% and 68.78% respectively. This led to the production of 1,3-PD decreased by 12.6% while 2,3-BD increased 66.7%. Interrelated analyses concluded that overerpression of budR increased 2,3-butanediol carbon flow but caused a sharp decline in 1,3-propandiol production. All of these proved that expression of budR either directly linkage to BudC, controlled the synthesis of 2,3-BD and regulate the generation of 1,3-PD.For further analysing regulation of 2,3-BD synthesis, coding region and noncoding region of bud operon were transcriptional silenced. We constructed K. pneumoniae/pETRH-budA, K. pneumoniae/pETRH-budB, K. pneumoniae/pETRH-budC, K. pneumoniae/pETRH-budR, K. pneumoniae/pETRH-budAF and K. pneumoniae/pETRH-budRF. Recombinant strains were analyzed from levels of transcription, enzyme and fermetation. Transcriptional of supressed gene in K. pneumoniae/pETRH-budA, K. pneumoniae/pETRH-budB and K. pneumoniae/pETRH-budC were decreased by 61%,63% and 76%, corresponding enzyme activity were reduced by 60%-70%. But other genes were upregulated, different from the above recombinant strains. All transcription levels of genes on bud operon and corresponding enzyme activity in K. pneumoniae/pETRH-budR were remarkable downregulated.2,3-BD production in K. pneumoniae/pETRH-budA, K. pneumoniae/pETRH-budB and K. pneumoniae/pETRH-budR were decreased by about 30%. Transcription of budA, budB and budC in K. pneumoniae/pETRH-budAF were significantly downregulated while budR was upregulated by 1 fold. The transcription of bud genes and corresponding enzyme activity in K. pneumoniae/pETRH-budRF were similar like K. pneumoniae/pETRH-budR but less significantly.