Wang Lu, Liu Guiming, Zhang Yingzi, Wang Yu, Ding Jiuyuan, Weng Weiqi
Wei Sheng Wu Xue Bao. 2015 Feb 4;55(2):164-75.
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.
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.
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 D-xylose (0.09 mol/L) mixed sugars as fermentative substrate.
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.
通过改造真养产碱杆菌W50-EAB中的磷酸戊糖途径（PPP）和醛还原酶基因h16_A3186，进一步提高木糖发酵效率。
通过PCR从真养产碱杆菌染色体中克隆转酮醇酶（tktA、cbbT2）和转醛醇酶（tal）基因，并插入表达载体pBBR1MCS-3。将所得重组质粒分别转化到W50-EAB中，构建W50-KAB、W50-CAB和W50-TAB。通过框内缺失将醛还原酶基因h16_A3186从834 bp缩短至135 bp，该缺失操作在染色体整合了编码木糖转运蛋白的xylE基因的W50-E菌株中进行，以构建重组菌株W50'-E。然后将来自大肠杆菌的编码木糖异构酶和木酮糖激酶的xylAB基因在W50'-E中表达，构建重组菌株W50'-EAB。将重组质粒pWL1-TAL转化到W50'-EAB中，构建菌株W50'-TAB。研究了工程菌株的发酵特性。
通过酶活性测定证实了真养产碱杆菌W50-EAB中tktA、cbbT2和tal基因的表达。通过PCR分析和酶活性测定证实了h16_A3186基因的缺失。真养产碱杆菌W50-EAB中转酮醇酶活性的增强对细胞生长和D-木糖消耗产生了负面影响。当在0.1 mol/L D-木糖上培养时，重组菌株W50-TAB和W50'-EAB的生长速度比W50-EAB快，最大比生长速率分别为0.039 h⁻¹和0.040 h⁻¹。基于细胞干重，W50-TAB和W50'-EAB的聚羟基丁酸酯（PHB）积累分别达到16.2±1.01%和19.8±1.05%。此外，重组菌株W50'-TAB表现出更好的发酵性能，最大比生长速率为0.042 h⁻¹，PHB含量为27.9±0.47%。同时，当使用葡萄糖（0.01 mol/L）和D-木糖（0.09 mol/L）混合糖作为发酵底物时，重组菌株W50-TAB、W50'-EAB和W50'-TAB表现出更高的生物量和更多的PHB积累。
tal基因的过表达导致D-木糖消耗增加。醛还原酶的缺失减轻了对D-木糖代谢的抑制。两种策略的结合有助于提高工程化真养产碱杆菌菌株对D-木糖的利用效率和更多的PHB积累。
