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Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics

机译：生物质发酵细菌抵抗木质素酚的进化。

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Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, long-term growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors.IMPORTANCE Fermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacterium Clostridium phytofermentans resists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors.
机译：提高植物发酵细菌对木质纤维素抑制剂的抗性对于理解微生物适应性和开发用于整合生物加工的候选菌株非常有用。在这里，我们研究和提高了Clostridium phytofermentans（也称为Lachnoclostridium phytofermentans）的抑制剂抗性，这是一种发酵木质纤维素生物质的厌氧菌模型。我们调查了这种细菌对一系列生物量抑制剂的抗性，然后通过在厌氧GM3自动机中进行自动化，长期的生长选择，进化出了在浓度越来越高的木质素酚，阿魏酸中生长的菌株。最终，菌株抵抗多种抑制剂，并在阿魏酸的溶解度极限下强劲生长，同时保留了发酵纤维素的能力。我们分析了在阿魏酸胁迫期间发生的全基因组转录模式以及在阿魏酸生长选择过程中出现的基因组变异，揭示了细胞如何通过基因剂量和调节，膜脂肪酸结构和表面层的变化来适应抑制剂。总的来说，这项研究展示了一个针对厌氧菌体内定向进化的自动化框架，并洞察了细菌在暴露于化学抑制剂后存活的遗传机制。重要信息植物生物量的发酵是多种生态系统中碳循环的关键部分。此外，工业生物质发酵可以提供化石燃料的可再生替代品。植物主要由木质纤维素，多糖和多酚木质素的基质组成。因此，当微生物降解木质纤维素以获取糖时，它们还会释放酚类和酸性抑制剂。在这里，我们研究了使用木质素酚醛酸，阿魏酸的植物发酵细菌梭菌Clostridium phytofermentans如何抵抗植物抑制剂。我们通过测量基因表达的变化来检查细胞如何应对突然的阿魏酸胁迫。我们通过在逐渐提高的阿魏酸浓度下进行自动化，长期培养来进化出越来越具有抗药性的菌株，并对它们的基因组进行测序，以鉴定与获得性阿魏酸抗性相关的突变。我们的研究开发出了一种能抑制纤维素发酵的抗抑制剂细菌，并为抗化学抑制剂的基因组进化提供了见识。

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