Xu Jian-Ren, Zhao Xin-Qing, Liu Chen-Guang, Bai Feng-Wu
School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China.
State Key Laboratory of Microbial Metabolism, School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Protein Pept Lett. 2018;25(2):202-207. doi: 10.2174/0929866525666180122142609.
The major carbohydrate components of lignocellulosic biomass are cellulose and hemicelluloses. Saccharomyces cerevisiae cannot efficiently utilize xylose derived upon the hydrolysis of hemicelluloses. Although engineering the yeast with xylose metabolic pathway has been intensively studied, challenges are still ahead for developing robust strains for lignocellulosic bioethanol production.
The main objective of this study was to reveal the role of the MIG1 mutant isolated from the self-flocculating S. cerevisiae SPSC01 in xylose utilization, glucose repression and ethanol fermentation by S. cerevisiae.
The MIG1 mutant was amplified from S. cerevisiae SPSC01 by PCR and MIG1- overexpression-cassette was transformed into S. cerevisiae S288c and xylose-metabolizing strain YB-2625-T through homologous recombination. Yeast growth was measured by colony assay on plates with or without xylose supplementation. Then xylose utilization and ethanol production were further evaluated through flask fermentation when mixed sugars of glucose and xylose at 3:1 and 2:1, respectively, were supplied. Fermentation products were detected by HPLC, and activities of xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) were also measured. The transcription of genes regulated by the expression of the MIG1 mutant was analyzed by RTqPCR. Evolutionary relationship of various MIG1s was developed by gene sequencing and sequence alignment.
No difference was observed for S288c growing with xylose when it was engineered with the overexpression or deletion of its native MIG1, but its growth was enhanced when overexpressing the MIG1 mutant from SPSC01. The submerged culture of YB-2625-T MIG1-SPSC engineered with xylose-metabolic pathway and the MIG1 mutant indicated that xylitol accumulation was decreased, and consequently, more biomass was accumulated. Furthermore, improved activities of the key enzymes such as XR, XDH and XK were detected in YB-2625-T MIG1-SPSC. Evolutionary analysis of MIG1s amplified from S. cerevisiae strains commonly used for ethanol production revealed a close relationship of SPSC01 and YB-2625.
Our results demonstrated the effect of the overexpression of the MIG1 mutant from SPSC01 on xylose utilization of S. cerevisiae. This study could be an alternative strategy for engineering S. cerevisiae with improved xylose utilization.
木质纤维素生物质的主要碳水化合物成分是纤维素和半纤维素。酿酒酵母不能有效利用半纤维素水解产生的木糖。尽管对构建具有木糖代谢途径的酵母进行了深入研究，但开发用于木质纤维素生物乙醇生产的优良菌株仍面临挑战。
本研究的主要目的是揭示从自絮凝酿酒酵母SPSC01中分离得到的MIG1突变体在酿酒酵母木糖利用、葡萄糖阻遏和乙醇发酵中的作用。
通过PCR从酿酒酵母SPSC01中扩增MIG1突变体，并通过同源重组将MIG1过表达盒转化到酿酒酵母S288c和木糖代谢菌株YB-2625-T中。通过在添加或不添加木糖的平板上进行菌落测定来测量酵母生长。然后，当分别以3:1和2:1的比例供应葡萄糖和木糖的混合糖时，通过摇瓶发酵进一步评估木糖利用和乙醇生产情况。通过高效液相色谱法检测发酵产物，并测量木糖还原酶（XR）、木糖醇脱氢酶（XDH）和木酮糖激酶（XK）的活性。通过逆转录定量聚合酶链反应分析受MIG1突变体表达调控的基因转录情况。通过基因测序和序列比对建立各种MIG1的进化关系。
当对酿酒酵母S288c进行其天然MIG1的过表达或缺失改造时，观察到其在木糖上生长无差异，但当过表达来自SPSC01的MIG1突变体时其生长得到增强。对构建了木糖代谢途径和MIG1突变体的YB-2625-T MIG1-SPSC进行深层培养表明，木糖醇积累减少，因此积累了更多生物量。此外，在YB-2625-T MIG1-SPSC中检测到关键酶如XR、XDH和XK的活性提高。对从常用于乙醇生产的酿酒酵母菌株中扩增得到的MIG1进行进化分析，发现SPSC01和YB-2625关系密切。
我们的结果证明了过表达来自SPSC01的MIG1突变体对酿酒酵母木糖利用的影响。本研究可能是一种用于构建木糖利用能力提高的酿酒酵母的替代策略。
