PDF(Pubmed)
Abstract：
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 60Co-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.
摘要：
阐明赤藓糖醇生产解脂耶氏酵母的耐热性机理对于培育健壮的工业菌株和降低成本具有重要意义。本研究旨在繁殖耐热Y.Lipolytica，并研究耐热表型的潜在机制。能够在34°C下生长的YarrowialipolyticaHT34，YarrowialipolyticaHT36和YarrowialipolyticaHT385，36°C,和38.5°C，分别,通过与60Co-γ辐射和紫外线辐射结合的适应性实验室进化（ALE）在150天内（352代）获得。比较基因组学分析表明,参与信号转导的基因,转录,翻译调控在适应性进化过程中发生了突变。Further,我们证明了热应激增加了与DNA复制和修复相关的基因的表达，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）的降解，同时抑制参与糖酵解和柠檬酸循环的基因的表达。耐热菌株中赤藓糖醇的产生受到明显抑制，这可能是赤藓糖醇代谢相关基因表达差异的结果。外源添加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个的过表达使YarrowialipolyticaCA20能够在34°C下生长，其中基因A000121、A003183和A005690效果较好。总的来说，这项研究提供了新的见解Y.lipolytica适应热应激的机制，这将有利于耐热赤藓糖醇生产菌株的构建。关键要点：•ALE与诱变相结合可有效繁殖耐热性Y。脂解作用•编码全局调节因子的基因在热适应性进化过程中发生突变•神经酰胺和BCAA是细胞耐受热应激的关键分子。
