Vasylkivska Maryna, Branska Barbora, Sedlar Karel, Jureckova Katerina, Provaznik Ivo, Patakova Petra
Department of Biotechnology, University of Chemistry and Technology, Prague, Prague, Czechia.
Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, Czechia.
Front Bioeng Biotechnol. 2020 Nov 5;8:598392. doi: 10.3389/fbioe.2020.598392. eCollection 2020.
-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.
丁醇是一种有价值的溶剂和潜在的燃料添加剂，可通过丙酮-丁醇-乙醇（ABE）发酵生产。ABE发酵的主要缺点之一是丁醇对生产细胞的高毒性，导致细胞膜破坏、培养活力低，因此丁醇的生产浓度也低。本研究的目的是通过随机化学诱变获得具有更高丁醇耐受性的NRRL B-598突变菌株，描述它们与野生型菌株相比的表型变化，并揭示基因组中的变化，这些变化解释了耐受性的提高或其他表型变化。通过三种不同的方法获得了九个具有稳定改良特性的突变菌株，其中两个菌株使用了已知的外排泵底物溴化乙锭（EB）进行选择或作为诱变剂。这是该方法首次用于开发产溶剂梭菌的耐丁醇突变体，对于产溶剂梭菌，人们普遍缺乏对丁醇外排或外排机制及其调控的了解。突变菌株的丁醇耐受性提高了36%至127%不等，以EB作为诱变剂的菌株取得了最大的改善。此外，在所有突变体中都观察到对其他外排泵底物（EB和乙醇）的耐受性增加，并且在一些菌株中观察到更高的抗生素耐受性。对突变菌株的完整基因组进行了测序，结果表明，丁醇耐受性的提高可归因于编码典型应激反应（趋化性、自溶或细胞膜结构变化）的基因突变，也可归因于编码外排泵调节因子的X276_07980和X276_24400基因的突变。后一观察结果证实了外排在该菌株丁醇应激反应中的重要性，并为合理的菌株工程提供了新的靶点。
