【摘要】 【目的】拓宽高产聚-β-羟基丁酸酯(poly-β-hydroxybutyrate,PHB)罗氏真养菌(Ralstonia eutropha)W50的碳源使用范围,使其获得D-木糖代谢能力。【方法】运用PCR技术扩增大肠杆菌(Escherichia coli)K-12W3110来源的D-木糖转运蛋白基因xylE,利用同源重组技术将xylE基因整合到R.eutropha W50的染色体上构建菌株W50-E。运用PCR技术扩增E.coli K-12 W3110来源的D-木糖代谢基因xylAB和R.eutropha H16来源的PHA合酶基因phaC1的启动子片段P pha C1,同表达载体连接后构建重组质粒p1-AB。将重组质粒分别转入菌株R.eutropha W50和W50-E中构建工程菌株W50-AB和W50-EAB。通过摇瓶发酵研究W50-AB和W50-EAB的D-木糖代谢特性。【结果】酶活分析结果表明,xylA和xylB基因在菌株R.eutropha W50中得到表达。摇瓶发酵结果表明,W50-AB在含0.1 mol/L D-木糖的基础发酵培养基中的最大比生长速率为0.025 h-1,在含0.01 mol/L D-木糖的基础发酵培养基中没有生长;W50-EAB在含0.01 mol/L D-木糖的基础发酵培养基中表现出一定生长,在含0.1 mol/L D-木糖的基础发酵培养基中最大比生长速率为0.035 h-1。PHB含量分析结果表明,摇瓶发酵终点时,W50-AB和W50-EAB菌株内的PHB含量分别为细胞干重的15.07±1.01%和15.07±1.64%,其相应的D-木糖-PHB转化率分别为0.0920 g·g-1和0.0838 g·g-1,低于两重组菌株利用葡萄糖发酵的糖-PHB转化率(>0.22 g·g-1)。另外,重组菌株W50-AB和W50-EAB在含葡萄糖(0.01 mol/L)和D-木糖(0.09 mol/L)的混合糖培养基中的发酵结果表明,两重组菌株均表现出更高的生长速率和D-木糖消耗速率以及胞内PHB积累量。【结论】来源于E.coli K-12W3110菌株的xylAB基因的表达使R.eutropha W50获得了一定的D-木糖代谢能力,通过D-木糖转运蛋白基因xylE的表达能提高菌株的D-木糖代谢能力,同时重组菌株利用D-木糖能积累一定量PHB。更多还原

【Abstract】 [Objective]This study aimed to broaden the substrate spectrum of Ralstonia eutropha W50 to use D-xylose, which can produce poly-β-hydroxybutyrates(PHB) at a high level.[Methods]The D-xylose transporter gene xylE from Escherichia coli K-12 W3110 was cloned by PCR technique and integrated into the R.eutropha W50 chromosome.The recombinant strain W50-E was obtained.The D-xylose catabolic genes xylAB from E.coli K-12 W3110 and the promotor of PHA synthase gene phaC1 from R.eutropha H16 were cloned into pBBR1MCS to construct a recombinant plasmid.The plasmid was transformed into R.eutropha W50 and W50-E to generate the recombinant strains W50-AB and W50-EAB respectively.The characteristics of D-xylose utilization by W50-AB and W50-EAB were investigated.[Results] The expression of xylA and xylB genes in R.eutropha W50 was confirmed by enzyme assay.The recombinant strain W50-AB could grow on 0.1 mol/ L D-xylose with the maximum specific growth rate of 0.025 h-1,but no growth and D-xylose consumption were observed when cultivated on 0.01 mol/ L D-xylose.The recombinant strain W50-EAB exhibited a faster growth than W50-AB on 0.1 mol/ L D-xylose,with the maximum specific growth rate of 0.035 h-1.Furthermore,it exhibited a slow but defined growth and D-xylose consumption on 0.01 mol/ L D-xylose.The PHB content assay showed that both recombinant strains accumulated a small amount of PHB,with a proportion of 15.07 ± 1.01% and 15.07 ± 1.64% on the basis of dry cell weight respectively,by using D-xylose(0.1 mol/ L) as substrate.And their final D-xylose- PHB conversion rates were 0.0920 g·g-1 and 0.0838 g·g-1 respectively,which were much lower than their glucose-PHB conversion rates(> 0.22 g·g-1).However,the recombinant strains W50-AB and W50-EAB exhibited better fermentation performance and more PHB accumulation when using glucose(0.01 mol/ L) and D-xylose(0.09 mol/ L) mixed sugars as fermentative substrate.[Conclusion]The recombinant strain W50-AB can metabolize D-xylose by the expression of xylAB genes,and the further expression of xylE gene is able to improve its D-xylose consumption rate.Meanwhile,the two recombinant strains can accumulate a small amount of PHB by using D-xylose as the sole carbon source.更多还原