Antunes Miguel, Mota Marta N, Sá-Correia Isabel
iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.
Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.
Biotechnol Biofuels Bioprod. 2024 May 28;17(1):71. doi: 10.1186/s13068-024-02518-0.
The red oleaginous yeast Rhodotorula toruloides is a promising cell factory to produce microbial oils and carotenoids from lignocellulosic hydrolysates (LCH). A multi-stress tolerant strain towards four major inhibitory compounds present in LCH and methanol, was derived in our laboratory from strain IST536 (PYCC 5615) through adaptive laboratory evolution (ALE) under methanol and high glycerol selective pressure.
Comparative genomic analysis suggested the reduction of the original strain ploidy from triploid to diploid, the occurrence of 21,489 mutations, and 242 genes displaying copy number variants in the evolved strain. Transcriptomic analysis identified 634 genes with altered transcript levels (465 up, 178 down) in the multi-stress tolerant strain. Genes associated with cell surface biogenesis, integrity, and remodelling and involved in stress-responsive pathways exhibit the most substantial alterations at the genome and transcriptome levels. Guided by the suggested stress responses, the multi-stress tolerance phenotype was extended to osmotic, salt, ethanol, oxidative, genotoxic, and medium-chain fatty acid-induced stresses.
The comprehensive analysis of this evolved strain provided the opportunity to get mechanistic insights into the acquisition of multi-stress tolerance and a list of promising genes, pathways, and regulatory networks, as targets for synthetic biology approaches applied to promising cell factories, toward more robust and superior industrial strains. This study lays the foundations for understanding the mechanisms underlying tolerance to multiple stresses in R. toruloides, underscoring the potential of ALE for enhancing the robustness of industrial yeast strains.
红色油脂酵母粘红酵母是一种有潜力的细胞工厂，可从木质纤维素水解物（LCH）中生产微生物油脂和类胡萝卜素。我们实验室通过在甲醇和高甘油选择压力下的适应性实验室进化（ALE），从菌株IST536（PYCC 5615）衍生出了对LCH和甲醇中存在的四种主要抑制化合物具有多重胁迫耐受性的菌株。
比较基因组分析表明，进化菌株的原始菌株倍性从三倍体降低到二倍体，发生了21489个突变，并且有242个基因显示出拷贝数变异。转录组分析确定了多重胁迫耐受菌株中634个转录水平发生改变的基因（465个上调，178个下调）。与细胞表面生物合成、完整性和重塑相关且参与应激反应途径的基因在基因组和转录组水平上表现出最显著的变化。在建议的应激反应指导下，多重胁迫耐受表型扩展到渗透、盐、乙醇、氧化、遗传毒性和中链脂肪酸诱导的胁迫。
对该进化菌株的综合分析为深入了解多重胁迫耐受性的获得机制提供了机会，并提供了一系列有前景的基因、途径和调控网络清单，作为应用于有潜力的细胞工厂的合成生物学方法的靶点，以培育更健壮和更优良的工业菌株。本研究为理解粘红酵母对多种胁迫的耐受机制奠定了基础，强调了ALE在增强工业酵母菌株鲁棒性方面的潜力。
