School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, China.
Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province, Zhejiang University of Science and Technology, Hangzhou, 310023, China.
Appl Microbiol Biotechnol. 2024 Mar 15;108(1):263. doi: 10.1007/s00253-024-13103-8.
Elucidation of the thermotolerance mechanism of erythritol-producing Yarrowia lipolytica is of great significance to breed robust industrial strains and reduce cost. This study aimed to breed thermotolerant Y. lipolytica and investigate the mechanism underlying the thermotolerant phenotype. Yarrowia lipolytica HT34, Yarrowia lipolytica HT36, and Yarrowia lipolytica HT385 that were capable of growing at 34 °C, 36 °C, and 38.5 °C, respectively, were obtained within 150 days (352 generations) by adaptive laboratory evolution (ALE) integrated with Co-γ radiation and ultraviolet ray radiation. Comparative genomics analysis showed that genes involved in signal transduction, transcription, and translation regulation were mutated during adaptive evolution. Further, we demonstrated that thermal stress increased the expression of genes related to DNA replication and repair, ceramide and steroid synthesis, and the degradation of branched amino acid (BCAA) and free fatty acid (FFA), while inhibiting the expression of genes involved in glycolysis and the citrate cycle. Erythritol production in thermotolerant strains was remarkably inhibited, which might result from the differential expression of genes involved in erythritol metabolism. Exogenous addition of BCAA and soybean oil promoted the growth of HT385, highlighting the importance of BCAA and FFA in thermal stress response. Additionally, overexpression of 11 out of the 18 upregulated genes individually enabled Yarrowia lipolytica CA20 to grow at 34 °C, of which genes A000121, A003183, and A005690 had a better effect. Collectively, this study provides novel insights into the adaptation mechanism of Y. lipolytica to thermal stress, which will be conducive to the construction of thermotolerant erythritol-producing strains. KEY POINTS: • ALE combined with mutagenesis is efficient for breeding thermotolerant Y. lipolytica • Genes encoding global regulators are mutated during thermal adaptive evolution • Ceramide and BCAA are critical molecules for cells to tolerate thermal stress.
阐明赤藓糖醇产生酵母解脂假丝酵母的耐热机制对于培育强壮的工业菌株和降低成本具有重要意义。本研究旨在培育耐热解脂假丝酵母，并研究耐热表型的机制。通过适应性实验室进化（ALE）与 Co-γ 辐射和紫外线辐射相结合，在 150 天（352 代）内获得了分别能够在 34°C、36°C 和 38.5°C 下生长的解脂假丝酵母 HT34、解脂假丝酵母 HT36 和解脂假丝酵母 HT385。比较基因组学分析表明，在适应性进化过程中，信号转导、转录和翻译调节相关的基因发生了突变。此外，我们证明了热应激增加了与 DNA 复制和修复、神经酰胺和类固醇合成以及支链氨基酸（BCAA）和游离脂肪酸（FFA）降解相关的基因的表达，同时抑制了糖酵解和柠檬酸循环相关基因的表达。耐热菌株中赤藓糖醇的产量显著受到抑制，这可能是由于赤藓糖醇代谢相关基因的差异表达所致。外源性添加 BCAA 和大豆油促进了 HT385 的生长，突出了 BCAA 和 FFA 在热应激反应中的重要性。此外，18 个上调基因中的 11 个单独过表达使解脂假丝酵母 CA20 能够在 34°C 下生长，其中基因 A000121、A003183 和 A005690 的效果更好。总的来说，本研究为解脂假丝酵母适应热应激的机制提供了新的见解，这将有助于构建耐热赤藓糖醇生产菌株。
ALE 与诱变相结合是培育耐热解脂假丝酵母的有效方法。
热适应性进化过程中，编码全局调控因子的基因发生突变。
神经酰胺和 BCAA 是细胞耐受热应激的关键分子。
