Gao Jiaoqi, Gao Ning, Zhai Xiaoxin, Zhou Yongjin J
Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China.
CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, PR China.
iScience. 2021 Feb 9;24(3):102168. doi: 10.1016/j.isci.2021.102168. eCollection 2021 Mar 19.
Methanol biotransformation can expand biorefinery substrate spectrum other than biomass by using methylotrophic microbes. () , a representative methylotrophic yeast, attracts much attention due to its thermotolerance, but the low homologous recombination (HR) efficiency hinders its precise genetic manipulation during cell factory construction. Here, recombination machinery engineering (rME) is explored for enhancing HR activity together with establishing an efficient CRISPR-Cas9 system in . Overexpression of HR-related proteins and down-regulation of non-homologous end joining (NHEJ) increased HR rates from 20%-30% to 60%-70%. With these recombination perturbation mutants, a competition between HR and NHEJ is observed. This HR up-regulated system has been applied for homologous integration of large fragments and assembly of multiple fragments, which enables the production of fatty alcohols in . These findings will simplify genetic engineering in non-conventional yeasts and facilitate the adoption of as an attractive cell factory for industrial application.
甲醇生物转化可以通过利用甲基营养型微生物来扩大生物炼制底物谱，而不仅仅局限于生物质。（某一种）作为代表性的甲基营养型酵母，因其耐热性而备受关注，但低同源重组（HR）效率阻碍了其在细胞工厂构建过程中的精确基因操作。在此，探索了重组机制工程（rME）以增强HR活性，并在（该酵母中）建立了高效的CRISPR-Cas9系统。HR相关蛋白的过表达和非同源末端连接（NHEJ）的下调使HR率从20%-30%提高到60%-70%。在这些重组扰动突变体中，观察到了HR和NHEJ之间的竞争。这种HR上调系统已应用于大片段的同源整合和多个片段的组装，从而能够在（该酵母中）生产脂肪醇。这些发现将简化非传统酵母中的基因工程，并促进（该酵母）成为具有吸引力的工业应用细胞工厂。
