Tamakawa Hideyuki, Tomita Yasuyuki, Yokoyama Aki, Konoeda Yuki, Ikushima Shigehito, Yoshida Satoshi
Central Laboratories for Key Technologies, KIRIN Company, Ltd, Yokohama, Kanagawa 236-0004, Japan.
Biosci Biotechnol Biochem. 2013;77(7):1441-8. doi: 10.1271/bbb.130093. Epub 2013 Jul 7.
We have reported that a recombinant Candida utilis strain expressing a Candida shehatae xylose reductase K275R/N277D, a C. shehatae xylitol dehydrogenase, and xylulokinase from Pichia stipitis produced ethanol from xylose, but its productivity was low. In the present study, metabolomic (CE-TOF MS) and transcriptomic (microarray) analyses were performed to characterize xylose metabolism by engineered C. utilis and to identify key genetic changes contributing to efficient xylose utilization. The metabolomic analysis revealed that the xylose-fermenting strain accumulated more pentose phosphate pathway intermediates, more NADH, and more glycolytic intermediates upstream of glyceraldehyde 3-phosphate than the wild-type. Transcriptomic analysis of the strain grown on xylose indicated a significant increase in expression of the genes encoding tricarboxylic acid cycle enzymes, respiratory enzymes, and enzymes involved in ethanol oxidation. To decrease the NADH/NAD(+) ratio and increase the ethanol yield of the fermentation of xylose, ADH1 encoding NADH-dependent alcohol dehydrogenase was overexpressed. The resulting strain exhibited a 17% increase in ethanol production and a 22% decrease in xylitol accumulation relative to control.
我们曾报道过，一种重组产朊假丝酵母菌株表达了树干毕赤酵母木糖还原酶K275R/N277D、树干毕赤酵母木糖醇脱氢酶以及来自树干毕赤酵母的木酮糖激酶，该菌株能够利用木糖生产乙醇，但其生产效率较低。在本研究中，进行了代谢组学（CE-TOF MS）和转录组学（微阵列）分析，以表征工程化产朊假丝酵母的木糖代谢，并确定有助于高效利用木糖的关键基因变化。代谢组学分析表明，与野生型相比，木糖发酵菌株积累了更多的磷酸戊糖途径中间产物、更多的NADH以及更多位于3-磷酸甘油醛上游的糖酵解中间产物。对在木糖上生长的该菌株进行转录组学分析表明，编码三羧酸循环酶、呼吸酶以及参与乙醇氧化的酶的基因表达显著增加。为了降低NADH/NAD(+)比率并提高木糖发酵的乙醇产量，对编码NADH依赖性乙醇脱氢酶的ADH1进行了过表达。相对于对照，所得菌株的乙醇产量提高了17%，木糖醇积累量降低了22%。
