Institute of Cell Biology, NAS of Ukraine, Drahomanov St, 14/16, 79005, Lviv, Ukraine.
University of Rzeszow, Zelwerowicza 4, 35-601, Rzeszow, Poland.
Microb Cell Fact. 2022 Aug 13;21(1):162. doi: 10.1186/s12934-022-01889-z.
Fuel ethanol from lignocellulose could be important source of renewable energy. However, to make the process feasible, more efficient microbial fermentation of pentose sugars, mainly xylose, should be achieved. The native xylose-fermenting thermotolerant yeast Ogataea polymorpha is a promising organism for further development. Efficacy of xylose alcoholic fermentation by O. polymorpha was significantly improved by metabolic engineering. Still, genes involved in regulation of xylose fermentation are insufficiently studied.
We isolated an insertional mutant of O. polymorpha with impaired ethanol production from xylose. The insertion occurred in the gene HXS1 that encodes hexose transporter-like sensor, a close homolog of Saccharomyces cerevisiae sensors Snf3 and Rgt2. The role of this gene in xylose utilization and fermentation was not previously elucidated. We additionally analyzed O. polymorpha strains with the deletion and overexpression of the corresponding gene. Strains with deletion of the HXS1 gene had slower rate of glucose and xylose consumption and produced 4 times less ethanol than the wild-type strain, whereas overexpression of HXS1 led to 10% increase of ethanol production from glucose and more than 2 times increase of ethanol production from xylose. We also constructed strains of O. polymorpha with overexpression of the gene AZF1 homologous to S. cerevisiae AZF1 gene which encodes transcription activator involved in carbohydrate sensing. Such transformants produced 10% more ethanol in glucose medium and 2.4 times more ethanol in xylose medium. Besides, we deleted the AZF1 gene in O. polymorpha. Ethanol accumulation in xylose and glucose media in such deletion strains dropped 1.5 and 1.8 times respectively. Overexpression of the HXS1 and AZF1 genes was also obtained in the advanced ethanol producer from xylose. The corresponding strains were characterized by 20-40% elevated ethanol accumulation in xylose medium. To understand underlying mechanisms of the observed phenotypes, specific enzymatic activities were evaluated in the isolated recombinant strains.
This paper shows the important role of hexose sensor Hxs1 and transcription factor Azf1 in xylose and glucose alcoholic fermentation in the native xylose-fermenting yeast O. polymorpha and suggests potential importance of the corresponding genes for construction of the advanced ethanol producers from the major sugars of lignocellulose.
从木质纤维素中提取燃料乙醇可能是可再生能源的重要来源。然而，为了使这一过程可行，需要提高微生物对戊糖（主要是木糖）的发酵效率。产朊假丝酵母（Ogataea polymorpha）是一种耐热的天然木糖发酵微生物，具有很大的发展潜力。通过代谢工程，可显著提高产朊假丝酵母的木糖酒精发酵效率。然而，参与木糖发酵调控的基因还没有得到充分研究。
我们从一株以木糖为碳源的产朊假丝酵母突变株中分离到一个插入失活突变体，该突变体的插入发生在 HXS1 基因中，该基因编码己糖转运蛋白样传感器，是酿酒酵母传感器 Snf3 和 Rgt2 的密切同源物。该基因在木糖利用和发酵中的作用以前没有被阐明。我们还分析了 HXS1 基因缺失和过表达的产朊假丝酵母菌株。HXS1 基因缺失菌株对葡萄糖和木糖的消耗速率较慢，比野生型菌株产生的乙醇少 4 倍，而 HXS1 过表达导致葡萄糖乙醇产量增加 10%，木糖乙醇产量增加 2 倍以上。我们还构建了产朊假丝酵母的过表达菌株，该菌株过表达与酿酒酵母 AZF1 基因同源的 AZF1 基因，该基因编码参与碳水化合物感知的转录激活因子。这种转化体在葡萄糖培养基中产生的乙醇增加了 10%，在木糖培养基中产生的乙醇增加了 2.4 倍。此外，我们还在产朊假丝酵母中敲除了 AZF1 基因。在这种缺失菌株中，木糖和葡萄糖培养基中乙醇的积累分别下降了 1.5 倍和 1.8 倍。在从木糖生产乙醇的先进生产菌中，也实现了 HXS1 和 AZF1 基因的过表达。相应的菌株在木糖培养基中乙醇积累提高了 20-40%。为了了解观察到的表型的潜在机制，我们在分离的重组菌株中评估了特定的酶活性。
本文表明，己糖传感器 Hxs1 和转录因子 Azf1 在产朊假丝酵母的木糖和葡萄糖酒精发酵中起着重要作用，并提示这些基因在构建木质纤维素主要糖基的先进乙醇生产菌方面具有潜在的重要性。
