重组菌Ralstonia eutropha W50-EAB D-木糖代谢相关的限速靶点

Limiting metabolic steps in the utilization of D-xylose by recombinant Ralstonia eutropha W50-EAB

摘要:

摘要:【目的】通过代谢工程改造真养罗氏菌(Ralstonia eutropha)W50-EAB木糖代谢的相关限速靶点，进一步提高R.eutropha W50-EAB的D-木糖利用效率，为获得高效利用纤维素水解液的菌株奠定基础。【方法】利用PCR技术扩增R.eutropha 转酮酶基因tktA，cbbT2和转醛酶基因tal，将扩增的tktA，cbbT2和tal基因分别构建到表达载体pBBR1MCS-3上，获得重组质粒pWL1-TKT，pWL1-CBBT2，pWL1-TAL。通过电转的方式将质粒分别转化W50-EAB获得重组菌W50-KAB，W50-CAB和W50-TAB。利用基因敲除的方法，获得醛还 原酶基因h16_A3186敲除株W50'-EAB。通过电转的方式将重组质粒pWL1-TAL导入敲除株W50'-EAB获得重组菌株W50'-TAB。通过摇瓶发酵研究重组菌株W50-KAB，W50-CAB，W50-TAB，W50'-EAB 以及W50'-TAB的发酵特性。【结果】酶活分析结果表明，转酮酶和转醛酶基因实现表达。摇瓶发酵结果表明，转酮酶基因过表达菌株W50-KAB和W50-CAB相比于对照菌株W50-EAB/p3，表现出降低的木糖利用能力;而转醛酶基因过表达重组菌株W50-TAB以及敲除菌株W50'-EAB对木糖的利用得到一定的提高。在0.1 mol/L木糖的发酵培养基中，W50-EAB的最大比生长速率为0.035 h－1，PHB干重比为16.2±1.01%;而W50-TAB的最大比生长速率提高到0.039 h－1，PHB干重比达到20.5±0.76%;醛还原酶基因敲除菌株W50'-EAB最大比生长速率提高到0.040 h－1，PHB含量提高到19.8±1.05%。结果显示转醛酶基因的过表达与醛还原酶基因的敲除对木糖利用均表现出一定的优势，将这两种优势组合获得菌株W50'-TAB，摇瓶发酵分析结果为最大比生长速率达到0.042 h－1，PHB积累达到27.9±0.47%，相比于对照菌株提高了72.2%。另外，在含有葡萄糖(0.01 mol /L)和木糖(0.09 mol/L)的混合糖培养下，重组菌株W50-TAB，W50'-EAB和W50'-TAB相比于在纯木糖培养下都表现出更高的生物量和胞内PHB积累量。【结论】磷酸戊糖途径关键 酶转醛酶基因的过表达加速了木糖代谢流，从而可以高效利用木糖积累一定量的PHB。醛还原酶对木糖代谢有阻碍作用，敲除该酶基因后木糖代谢能力有了一定的提高，而两者协同作用可以进一步提高重组菌株的木糖利用效率和PHB积累能力。

Abstract:

Abstract:［Objective］ To further improve the efficiency of xylose fermentation by modifying the pentose phosphate pathway (PPP) and the aldehyde reductase gene h16_A3186 in Ralstonia eutropha W50-EAB．［Methods］The transketolase (tktA，cbbT2) and transaldolase (tal) gene were cloned from R. eutropha chromosome by PCR and inserted into expressing vector pBBR1MCS-3．The resulting recombinant plasmids were transformed into W50-EAB to generate W50-KAB，W50-CAB and W50-TAB，respectively．The aldehyde reductase gene h16_A3186 was shortened from 834 bp to 135 bp by in-frame deletion from strain W50-E in which the xylE gene coding for xylose transporter was chromosomally integrated to construct recombinant strain W50'-E．Then the xylAB gene coding for xylose isomerase and xylulokinase from Escherichia coli were expressed in W50'-E to generate recombinant strain W50'-EAB．Recombinant plasmid pWL1-TAL was transformed into W50'-EAB to construct the strain W50'-TAB．The fermentation characteristics of the engineered strains were investigated． ［Results］The expression of tktA，cbbT2 and tal genes in R.eutropha W50-EAB was confirmed by enzyme assay．The deletion of h16 _A3186 gene was confirmed by PCR analysis and enzyme assay．Amplification of transketolase activity in R.eutropha W50-EAB showed negative effect on cell growth and D-xylose consumption．The recombinant strain W50-TAB and W50'-EAB exhibited a faster growth than W50-EAB with the maximum specific growth rate of 0.039 h-1 and 0.040 h－1，respectively，when cultivated on 0.1 mol/L D-xylose．And the PHB accumulation of W50-TAB and W50'-EAB reached 16.2±1.01% and 19.8±1.05% on the basis of cell dry weight，respectively．Furthermore，recombinant strain W50'-TAB exhibited better fermentation performance with the maximum specific growth rate of 0.042 h－1 and PHB content of 27.9±0.47%，respectively．Meanwhile，the recombinant strains W50-TAB，W50'-EAB and W50'-TAB showed higher biomass and more PHB accumulation when using glucose (0.01 mol/L) and Dxylose (0.09mol/L) mixed sugars as fermentative substrate.［Conclusion］Overexpression of the tal gene resulted in incressed D-xylose consumption．Deficiency of the aldehyde reductase relieved inhibition to D-xylose metabolism．Combination of the two strategies contributed to a higher efficiency of D-xylose utilisation and more PHB accumulation of the engineered R.eutropha strain.