Matsushika Akinori, Watanabe Seiya, Kodaki Tsutomu, Makino Keisuke, Inoue Hiroyuki, Murakami Katsuji, Takimura Osamu, Sawayama Shigeki
Biomass Technology Research Center, National Institute of Advanced Industrial Science and Technology, 2-2-2 Hirosuehiro, Kure, Hiroshima, 737-0197, Japan.
Appl Microbiol Biotechnol. 2008 Nov;81(2):243-55. doi: 10.1007/s00253-008-1649-1. Epub 2008 Aug 27.
A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD(+)-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP(+). In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP(+)-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP(+)-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.
用来自树干毕赤酵母的木糖还原酶（XR）和木糖醇脱氢酶（XDH）基因转化的重组酿酒酵母菌株能够将木糖转化为乙醇，但同时会不利地分泌木糖醇，这可能是由于偏好NADPH的XR和依赖NAD⁺的XDH之间的辅因子失衡所致。为了减少木糖醇的形成，我们已经构建了几种辅酶特异性逆转至NADP⁺的XDH突变体。在本研究中，我们构建了一组具有木糖发酵能力的重组酿酒酵母菌株，包括蛋白质工程化的表达依赖NADP⁺的XDH的菌株。通过使用一种名为MA-N5的菌株发现了对木糖到乙醇发酵最积极的影响，该菌株是通过将依赖NADP⁺的XDH基因与XR和内源性木酮糖激酶基因进行染色体整合构建而成。与表达野生型XDH的参考菌株相比，MA-N5菌株在发酵木糖以及含有葡萄糖和木糖的混合糖时，乙醇产量增加，木糖醇排泄减少。此外，MA-N5菌株在木屑的非硫酸水解物中产生乙醇的产率高达0.49 g乙醇/g总消耗糖。结果表明，这种氧化还原工程菌株能够有效地发酵木质纤维素水解物中存在的葡萄糖和木糖。
