Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis
Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis
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DOI：
10.1099/mic.0.2007/007856-0
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发表时间：
2007-09-01
期刊：
影响因子：
2.8
通讯作者：
Makino, Keisuke
中科院分区：
文献类型：
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作者：
Watanabe, Seiya;Abu Saleh, Ahmed;Makino, Keisuke
用毕赤酵母木糖还原酶（xylose reductase， XR）和木糖醇脱氢酶（xylitol dehydrogenase， XDH）基因（分别为PsXR和PsXDH）转化的重组酿酒酵母菌株具有木糖转化为乙醇的能力，但木糖醇的排出不利，这可能是由于偏好nadph的XR和依赖NAD（+）的XDH之间的辅酶特异性不同导致细胞间氧化还原失衡。在本研究中，我们重点研究了突变的nadh偏好PsXR在发酵中的作用。与野生型（WT）相比，R276H和K270R/N272D突变体的NADH/NADPH比值（k(cat)/ k -m与NADH）/（k(cat)/ k -m与NADPH）分别提高了52倍和46倍，这是由于R276H突变体的k（cat）与NADPH降低，K270R/N272D突变体的k（cat）与NADPH升高所致。此外，R276H突变导致PsXR显著的热稳定性。与表达PsXR R276H突变体和PsXDH WT的Y-R276H菌株相比，表达PsXR R276H突变体和PsXDH WT的Y-R276H菌株对木糖发酵成乙醇的效果最为显著：与表达PsXR WT和PsXDH WT的Y-WT菌株相比，其乙醇产量增加20%，木糖醇排泄量减少52%。细胞内辅酶浓度的测定表明，维持NADPH/NADP（+）和NADH/NAD（+）的比例对重组酿酒酵母将木糖高效发酵成乙醇至关重要。
A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-pref erring XR and NAD(+)-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k(cat)/K-m with NADH)/(k(cat)/K-m with NADPH)] compared with the wild-type (WT), which was due to decrease of k(cat) with NADPH in the R276H mutant and increase of K-m with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20% increase of ethanol production and 52% decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP(+) and NADH/NAD(+) ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.