Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics

Abstract

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-

ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans

“…Growth curves were measured in medium containing 5 g liter Ϫ1 glucose (G5767; Sigma), galactose (G0750; Sigma), galacturonic acid (73960; Sigma), cellobiose (C7252; Sigma), galactan (P-GALLU; Megazyme), or homogalacturonan (P8471; Sigma). Log-phase cultures were resuspended in an equal volume of medium without sugars and inoculated at 1:10 volume into 100-well microtiter plates (9502550; Bioscreen) in an anaerobic chamber (2% H 2 , 98% N 2 ), and plates were sealed by press-fitting adhesive pads (1018104; Qiagen) (33). Growth (optical density at 600 nm [OD 600 ]) was measured using a Thermo Scientific Bioscreen C at 37°C with 30 s of shaking before readings each 15 min.…”

Colonic aberrant crypt formation accompanies an increase of opportunistic pathogenic bacteria in C57BL/6 mice fed a high-fat diet

“…One of the health effects attributed to the production of SCFA is to inhibit pathogenic microorganisms and to maintain the gut barrier function [38]. Although the abundance of certain SCFA-producing bacteria (e.g., Barnesiella,) [38][39][40][41] was lower in the HFD and HFD-AOM groups, of the fecal SCFA measured, only acetic acid was lower in the HFD-AOM group compared with the AIN and AIN-AOM groups (Table 3; Fig. 8).…”

“…6, Tables 3 and 4). At genus level, three major (high abundance) bacterial taxa (Bacteroides, Barnesiella, Lachnoclostridium), which are responsible for SCFA production [38][39][40][41], were at least 1-fold higher in the AIN group when compared with the HFD group (Table 3). We also found that the abundance of Anaeroplasma (an opportunistic pathogen) and Values are means ± S.D., n=8/group.…”

Synthetic Metabolic Pathway for the Production of 1-Alkenes from Lignin-derived Molecules

“…LDMs are known to be toxic to microorganisms (Cerisy et al, 2017; Ibraheem and Ndimba, 2013; Mills et al, 2009), which also hinders the use of LDMs as substrates. In order to develop a tolerant strain, adaptive laboratory evolution (ALE) can be employed.…”

“…The approach can lead to the strains with beneficial changes allowing them to adapt to the stressful condition. ALE has been successfully applied on various microorganisms to improve their tolerance against inhibitory compounds (Almario et al, 2013; Atsumi et al, 2010; Cerisy et al, 2017).…”