Automated selection was used to evolve an Escherichia coli strain unable to synthesize thymine nucleotides into a chemically modified organism whose DNA genome is composed of adenine, cytosine, guanine, and an artificial base, the thymine analogue 5‐chlorouracil. Evolving cells were initially observed as irregular filaments and progressively recovered the appearance of short rods typical of wild‐type E. coli (see picture).
Durch automatisierte Selektion wurde ein E.‐coli‐Stamm, der keine Thyminnucleotide synthetisieren kann, in einen chemisch modifizierten Organismus umgewandelt, dessen DNA‐Genom aus Adenin, Cytosin, Guanin und dem künstlichen Thyminanalogon 5‐Chloruracil besteht. Sich entwickelnde Zellen wurden zu Beginn als irreguläre Filamente beobachtet und nahmen dann immer stärker wieder die Gestalt kurzer Stäbe an, wie sie typisch für Wildtyp‐E. coli ist (siehe Bild).
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, longterm 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.KEYWORDS clostridia, evolution, genomics F ermentation of lignocellulosic biomass by bacteria like Clostridium phytofermentans is central to the function of soil, aquatic, and intestinal microbiomes. In addition, industrial fermentation of lignocellulosic biomass into fuels and chemicals could contribute significantly to global energy needs without impacting food production (1). Plant biomass is primarily composed of a macromolecular network of polysaccharides linked with lignin, a polymer of phenylpropanoid subunits with aromatic rings of various degrees of methoxylation (2). Thus, when microorganisms hydrolyze lignocel-
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