罗氏真养菌W50的D-木糖代谢途径工程改造

Engineering of a D-xylose metabolic pathway in Ralstonia eutropha W50

摘要:

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-12 W3110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Ppha 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-12 W3110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。

Abstract:

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．