Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, C.P. 62209, Cuernavaca, Morelos, Mexico.
Departamento de Ingeniería Celular Y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, C.P. 62210, Cuernavaca, Morelos, Mexico.
Appl Microbiol Biotechnol. 2022 Jan;106(1):383-399. doi: 10.1007/s00253-021-11730-z. Epub 2021 Dec 16.
Saccharomyces cerevisiae scarcely grows on minimal media with acetic acid, acidic pH, and high temperatures. In this study, the adaptive laboratory evolution (ALE), whole-genome analysis, and reverse engineering approaches were used to generate strains tolerant to these conditions. The thermotolerant strain TTY23 and its parental S288C were evolved through 1 year, in increasing concentrations of acetic acid up to 12 g/L, keeping the pH ≤ 4. Of the 18 isolated strains, 9 from each ancestor, we selected the thermo-acid tolerant TAT12, derived from TTY23, and the acid tolerant AT22, derived from S288C. Both grew in minimal media with 12 g/L of acetic acid, pH 4, and 30 °C, and produced ethanol up to 29.25 ± 6 mmol/g/h-neither of the ancestors thrived in these conditions. Furthermore, only the TAT12 grew on 2 g/L of acetic acid, pH 3, and 37 °C, and accumulated 16.5 ± 0.5 mmol/g/h of ethanol. Whole-genome sequencing and transcriptomic analysis of this strain showed changes in the genetic sequence and transcription of key genes involved in the RAS-cAMP-PKA signaling pathway (RAS2, GPA2, and IRA2), the heat shock transcription factor (HSF1), and the positive regulator of replication initiation (SUM1), among others. By reverse engineering, the relevance of the combined mutations in the genes RAS2, HSF1, and SUM1 to the tolerance for acetic acid, low pH, and high temperature was confirmed. Alone, the RAS2 mutation yielded acid tolerance and HSF1 nutation thermotolerance. Increasing the thermo-acidic niche and acetic acid tolerance of S. cerevisiae can contribute to improve economic ethanol production. KEY POINTS: • Thermo-acid tolerant (TAT) yeast strains were generated by adaptive laboratory evolution. • The strain TAT12 thrived on non-native, thermo-acidic harmful conditions. • Mutations in RAS2, HSF1, and SUM1 genes rendered yeast thermo and acid tolerant.
酿酒酵母在含有乙酸、酸性 pH 值和高温的最低培养基上几乎无法生长。在这项研究中，采用适应实验室进化（ALE）、全基因组分析和反向工程方法来产生耐受这些条件的菌株。耐热菌株 TTY23 和其亲本 S288C 经过 1 年的进化，在不断增加的乙酸浓度下达到 12 g/L，同时保持 pH 值≤4。从每个祖先中分离出 18 株菌株，我们选择了耐热耐酸的 TAT12（来自 TTY23）和耐酸的 AT22（来自 S288C）。这两种菌株都可以在含有 12 g/L 乙酸、pH 值为 4 和 30°C 的最低培养基中生长，并产生高达 29.25±6 mmol/g/h 的乙醇，而其祖先都无法在这些条件下生长。此外，只有 TAT12 可以在 2 g/L 乙酸、pH 值为 3 和 37°C 下生长，并积累 16.5±0.5 mmol/g/h 的乙醇。对该菌株的全基因组测序和转录组分析表明，参与 RAS-cAMP-PKA 信号通路（RAS2、GPA2 和 IRA2）、热休克转录因子（HSF1）和复制起始正调控因子（SUM1）等关键基因的遗传序列和转录发生了变化。通过反向工程，证实了基因 RAS2、HSF1 和 SUM1 的组合突变对乙酸、低 pH 值和高温的耐受性的相关性。单独的 RAS2 突变产生了耐酸，而 HSF1 突变产生了耐热性。提高酿酒酵母的耐热酸性生态位和乙酸耐受性可以有助于提高经济乙醇生产。关键点：• 通过适应实验室进化生成了耐热耐酸（TAT）酵母菌株。• 菌株 TAT12 在非天然的、耐热酸性有害条件下茁壮成长。• RAS2、HSF1 和 SUM1 基因的突变使酵母具有耐热性和耐酸性。
