Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction
Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction
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DOI：
10.1007/s00253-009-2053-1
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发表时间：
2009-09-01
影响因子：
5
通讯作者：
Gorwa-Grauslund, Marie-F.
中科院分区：
文献类型：
--
作者：
Almeida, Joao R. M.;Bertilsson, Magnus;Gorwa-Grauslund, Marie-F.
能够利用木糖的工业酿酒酵母菌株已经通过分别编码来自树干毕赤酵母（Pichia stipitis）的NADPH偏好性木糖还原酶（XR）和NAD（+）依赖性木糖醇脱氢酶（XDH）的XYL 1和XYL 2基因的过表达而构建。然而，XR和XDH使用不同的辅因子导致NAD（+）缺乏，随后是木糖醇排泄和降低的产物产率。糠醛5-羟甲基糠醛（HMF）和糠醛抑制酵母代谢，延长滞后期，并降低乙醇产率。最近，编码糠醛还原酶的基因被鉴定出来，它们的过表达被证明可以改善S。在含有HMF的培养基和木质纤维素水解产物中，酿酒酵母的生长和发酵速率。在本研究中，我们构建了一个木糖消耗的S。酿酒酵母菌株使用来自树干毕赤酵母的XR/XDH途径。然后，编码NADH和NADPH依赖性HMF还原酶的基因，分别为ADH 1-S110 P-Y295 C和ADH 6，在此背景下单独过表达。在厌氧条件下，在不存在或存在HMF的情况下，在分批发酵中评价了这些菌株的性能，这些菌株用于HMF还原的辅因子使用不同。在厌氧连续培养中，获得了同时消耗木糖和还原HMF的碳通量。我们的研究结果表明，用于HMF还原的辅因子主要影响乙醇以外的产物的形成，并且依赖于NADH的HMF还原比依赖于NADPH的HMF还原更影响产物的形成。特别地，NADH依赖性HMF还原有助于碳保护，使得以木糖醇和甘油形成为代价产生生物质。
Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD(+)-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD(+) deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.