Zhang Guo-Chang, Kong In Iok, Wei Na, Peng Dairong, Turner Timothy L, Sung Bong Hyun, Sohn Jung-Hoon, Jin Yong-Su
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois.
Biotechnol Bioeng. 2016 Dec;113(12):2587-2596. doi: 10.1002/bit.26021. Epub 2016 Sep 21.
Xylose fermentation by engineered Saccharomyces cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD -linked xylitol dehydrogenase (XDH) suffers from redox imbalance due to cofactor difference between XR and XDH, especially under anaerobic conditions. We have demonstrated that coupling of an NADH-dependent acetate reduction pathway with surplus NADH producing xylose metabolism enabled not only efficient xylose fermentation, but also in situ detoxification of acetate in cellulosic hydrolysate through simultaneous co-utilization of xylose and acetate. In this study, we report the highest ethanol yield from xylose (0.463 g ethanol/g xylose) by engineered yeast with XR and XDH through optimization of the acetate reduction pathway. Specifically, we constructed engineered yeast strains exhibiting various levels of the acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) activities. Engineered strains exhibiting higher activities of AADH and ACS consumed more acetate and produced more ethanol from a mixture of 20 g/L of glucose, 80 g/L of xylose, and 8 g/L of acetate. In addition, we performed environmental and genetic perturbations to further improve the acetate consumption. Glucose-pulse feeding to continuously provide ATPs under anaerobic conditions did not affect acetate consumption. Promoter truncation of GPD1 and gene deletion of GPD2 coding for glycerol-3-phosphate dehydrogenase to produce surplus NADH also did not lead to improved acetate consumption. When a cellulosic hydrolysate was used, the optimized yeast strain (SR8A6S3) produced 18.4% more ethanol and 41.3% less glycerol and xylitol with consumption of 4.1 g/L of acetate than a control strain without the acetate reduction pathway. These results suggest that the major limiting factor for enhanced acetate reduction during the xylose fermentation might be the low activities of AADH and ACS, and that the redox imbalance problem of XR/XDH pathway can be exploited for in situ detoxification of acetic acid in cellulosic hydrolysate and increasing ethanol productivity and yield. Biotechnol. Bioeng. 2016;113: 2587-2596. © 2016 Wiley Periodicals, Inc.
通过表达与NADPH相关的木糖还原酶（XR）和与NAD相关的木糖醇脱氢酶（XDH）的工程化酿酒酵母进行木糖发酵，由于XR和XDH之间的辅因子差异，会出现氧化还原失衡，尤其是在厌氧条件下。我们已经证明，将依赖NADH的乙酸还原途径与产生过量NADH的木糖代谢相偶联，不仅能够实现高效的木糖发酵，还能通过同时利用木糖和乙酸对纤维素水解产物中的乙酸进行原位解毒。在本研究中，我们报道了通过优化乙酸还原途径，工程化酵母利用XR和XDH从木糖中获得的最高乙醇产量（0.463克乙醇/克木糖）。具体而言，我们构建了具有不同水平乙酰化乙醛脱氢酶（AADH）和乙酰辅酶A合成酶（ACS）活性的工程化酵母菌株。表现出较高AADH和ACS活性的工程化菌株从含有20克/升葡萄糖、80克/升木糖和8克/升乙酸的混合物中消耗了更多的乙酸，并产生了更多的乙醇。此外，我们进行了环境和基因扰动以进一步提高乙酸的消耗。在厌氧条件下通过葡萄糖脉冲补料持续提供ATP并不影响乙酸的消耗。对编码甘油-3-磷酸脱氢酶以产生过量NADH的GPD1进行启动子截短和对GPD2进行基因缺失也并未导致乙酸消耗的改善。当使用纤维素水解产物时，与没有乙酸还原途径的对照菌株相比，优化后的酵母菌株（SR8A6S3）消耗了4.1克/升的乙酸，乙醇产量提高了18.4%，甘油和木糖醇产量降低了41.3%。这些结果表明，木糖发酵过程中增强乙酸还原的主要限制因素可能是AADH和ACS的低活性，并且XR/XDH途径的氧化还原失衡问题可用于纤维素水解产物中乙酸的原位解毒以及提高乙醇的生产力和产量。《生物技术与生物工程》2016年；113：2587 - 2596。©2016威利期刊公司
