罗氏真养菌W50的L-阿拉伯糖代谢途径工程改造

Engineering of an L-arabinose metabolic pathway in Ralstonia eutropha W50

【目的】在产聚-β-羟基丁酸酯(Poly-β-hydroxybutyrate,PHB)的罗氏真养菌(Ralstonia eutropha)H16突变株W50中建立完整的阿拉伯糖代谢途径,引入高亲和力阿拉伯糖转运蛋白,获得能利用L-阿拉伯糖的重组菌株,为获得能高效利用纤维质降解物并积累PHB的工程菌株奠定基础。【方法】利用PCR技术扩增R.eutropha H16的PHB合酶启动子P pha C1、大肠杆菌(Escherichia coli)W3110的阿拉伯糖代谢酶基因araBAD和高亲和力阿拉伯糖转运蛋白基因araFGH。将P pha C1、araBAD与表达载体pBBR1MCS连接,构建带有阿拉伯糖代谢酶基因的表达载体,转化R.eutropha W50得到重组菌株W50-1。利用双质粒和染色体重组两种方法将araFGH导入W50-1菌,分别得到重组菌株W50-2和W50-3。通过摇瓶发酵研究重组菌株W50-1、W50-2和W50-3的发酵特性。【结果】酶活分析结果表明,阿拉伯糖代谢酶基因实现了表达。重组菌株W50-1、W50-2和W50-3均能利用L-阿拉伯糖,并且表达了转运蛋白基因的重组菌利用L-阿拉伯糖的能力提高。摇瓶发酵结果表明,W50-1可以在含0.1 mol/L阿拉伯糖的发酵培养基中生长,但不能利用低浓度(0.01 mol/L)阿拉伯糖。W50-2、W50-3菌株能够利用低浓度阿拉伯糖生长,并且在含0.1 mol/L阿拉伯糖的培养基中,W50-3的生物量是W50-1的2.5倍,合成的PHB占菌体干重的38.6%。【结论】在R.eutropha W50中表达阿拉伯糖代谢酶基因及转运蛋白基因,可以使其高效利用L-阿拉伯糖生长并积累一定水平的PHB。

[Objective] To broaden the substrate spectrum including L-arabinose,Ralstonia eutropha W50,a mutant strain with high yield of poly-β-hydroxybutyrate （PHB）,was metabolically engineered by expressing the genes encoding L-arabinose catabolic enzymes and high-affinity L-arabinose transporter from Escherichia coli.[Methods]The promoter fragment of PHB synthase gene phaC1 （Ppha C1） from R.eutropha H16 and the araBAD genes from E.coli W3110 were cloned by PCR and inserted into expression vector pBBR1MCS. The resulting recombinant plasmid was transformed into W50 to generate W50-1.The araFGH gene from E.coli W3110 was introduced into W50-1 by plasmid system or homologous recombination,yielding W50-2 and W50-3 respectively. The fermentation characteristics of the three engineered strains were investigated.[Results] The flask fermentation experiments of the engineered strains show that W50-1 carrying the arabinose catabolic genes under the control of Ppha C1 could grow in the fermentation medium containing 0.1 mol/L arabinose as the sole carbon source,but could not utilize low concentration arabinose （0.01 mol/L）. However,W50-2 and W50-3 containing the gene of high-affinity arabinose transporter were able to utilize low concentration arabinose. In the fermentation medium containing 0.1 mol/L arabinose,the biomass of W50-3 was 2.5 fold higher than that of W50-1,and the PHB accumulation amount of W50-3 accounted for 38.6% of the cell dry weight.[Conclusion] R.eutropha W50 was able to metabolize L-arabinose by the expression of araBAD genes,and the simultaneous expression of araFGH genes could further improve its ability of L-arabinose utilization. By using L-arabinose as the sole carbon source,the recombinant strain W50-3 can accumulate a noticeable level of PHB.