Zhang Ming-Ming, Xiong Liang, Tang Ya-Jie, Mehmood Muhammad Aamer, Zhao Zongbao Kent, Bai Feng-Wu, Zhao Xin-Qing
1State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240 China.
3School of Life Science and Biotechnology, Dalian University of Technology, Dalian, 116024 China.
Biotechnol Biofuels. 2019 May 10;12:116. doi: 10.1186/s13068-019-1456-1. eCollection 2019.
Yeast strains that are tolerant to multiple environmental stresses are highly desired for various industrial applications. Despite great efforts in identifying key genes involved in stress tolerance of budding yeast , the effects of de novo purine biosynthesis genes on yeast stress tolerance are still not well explored. Our previous studies showed that zinc sulfate addition improved yeast acetic acid tolerance, and key genes involved in yeast stress tolerance were further investigated in this study.
Three genes involved in de novo purine biosynthesis, namely, , , and , showed significantly increased transcription levels by zinc sulfate supplementation under acetic acid stress, and overexpression of these genes in BY4741 enhanced cell growth under various stress conditions. Meanwhile, ethanol productivity was also improved by overexpression of the three genes under stress conditions, among which the highest improvement attained 158.39% by overexpression in the presence of inhibitor mixtures derived from lignocellulosic biomass. Elevated levels of adenine-nucleotide pool "AXP" ([ATP] + [ADP] + [AMP]) and ATP content were observed by overexpression of , both under control condition and under acetic acid stress, and is consistent with the better growth of the recombinant yeast strain. The global intracellular amino acid profiles were also changed by overexpression of the genes. Among the changed amino acids, significant increase of the stress protectant γ-aminobutyric acid (GABA) was revealed by overexpression of the genes under acetic acid stress, suggesting that overexpression of the genes exerts control on both purine biosynthesis and amino acid biosynthesis to protect yeast cells against the stress.
We proved that the de novo purine biosynthesis genes are useful targets for metabolic engineering of yeast stress tolerance. The engineered strains developed in this study with improved tolerance against multiple inhibitors can be employed for efficient lignocellulosic biorefinery to produce biofuels and biochemicals.
对于各种工业应用而言，非常需要能够耐受多种环境压力的酵母菌株。尽管在鉴定参与出芽酵母应激耐受性的关键基因方面付出了巨大努力，但从头嘌呤生物合成基因对酵母应激耐受性的影响仍未得到充分研究。我们之前的研究表明，添加硫酸锌可提高酵母对乙酸的耐受性，本研究进一步探究了参与酵母应激耐受性的关键基因。
在乙酸胁迫下，通过添加硫酸锌，参与从头嘌呤生物合成的三个基因，即[基因名称1]、[基因名称2]和[基因名称3]，转录水平显著增加，在BY4741中过表达这些基因可增强细胞在各种胁迫条件下的生长。同时，在胁迫条件下过表达这三个[基因名称]基因也提高了乙醇生产率，其中在存在木质纤维素生物质衍生的抑制剂混合物的情况下，[基因名称3]过表达提高幅度最大，达到158.39%。在对照条件和乙酸胁迫下，过表达[基因名称1]均观察到腺嘌呤核苷酸库“AXP”（[ATP]+[ADP]+[AMP]）水平和ATP含量升高，这与重组酵母菌株更好的生长情况一致。过表达[基因名称]基因也改变了细胞内整体氨基酸谱。在变化的氨基酸中，在乙酸胁迫下过表达[基因名称]基因可显著增加应激保护剂γ-氨基丁酸（GABA），这表明过表达[基因名称]基因对嘌呤生物合成和氨基酸生物合成均有调控作用，以保护酵母细胞免受胁迫。
我们证明了从头嘌呤生物合成基因是酵母应激耐受性代谢工程的有用靶点。本研究中开发的对多种抑制剂耐受性提高的工程菌株可用于高效木质纤维素生物炼制以生产生物燃料和生化产品。
