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Microbial tolerance to toxic compounds formed during biomass pretreatment is a significant challenge to produce bio-based products from lignocellulose cost effectively. Rational engineering can be problematic due to insufficient prerequisite knowledge of tolerance mechanisms. Therefore, adaptive laboratory evolution was applied to obtain 20 tolerant lineages of Bacillus subtilis strains able to utilize Distiller's Dried Grains with Solubles-derived (DDGS) hydrolysate. Evolved strains showed both improved growth performance and retained heterologous enzyme production using 100% hydrolysate-based medium, whereas growth of the starting strains was essentially absent. Whole-genome resequencing revealed that evolved isolates acquired mutations in the global regulator codY in 15 of the 19 sequenced isolates. Furthermore, mutations in genes related to oxidative stress (katA, perR) and flagella function appeared in both tolerance and control evolution experiments without toxic compounds. Overall, tolerance adaptive laboratory evolution yielded strains able to utilize DDGS-hydrolysate to produce enzymes and hence proved to be a valuable tool for the valorization of lignocellulose.

Abstract Background Lactobacillus reuteri is a heterofermentative Lactic Acid Bacterium (LAB) that is commonly used for food fermentations and probiotic purposes. Due to its robust properties, it is also increasingly considered for use as a cell factory. It produces several industrially important compounds such as 1,3-propanediol and reuterin natively, but for cell factory purposes, developing improved strategies for engineering and fermentation optimization is crucial. Genome-scale metabolic models can be highly beneficial in guiding rational metabolic engineering. Reconstructing a reliable and a quantitatively accurate metabolic model requires extensive manual curation and incorporation of experimental data. Results A genome-scale metabolic model of L. reuteri JCM 1112T was reconstructed and the resulting model, Lreuteri_530, was validated and tested with experimental data. Several knowledge gaps in the metabolism were identified and resolved during this process, including presence/absence of glycolytic genes. Flux distribution between the two glycolytic pathways, the phosphoketolase and Embden–Meyerhof–Parnas pathways, varies considerably between LAB species and strains. As these pathways result in different energy yields, it is important to include strain-specific utilization of these pathways in the model. We determined experimentally that the Embden–Meyerhof–Parnas pathway carried at most 7% of the total glycolytic flux. Predicted growth rates from Lreuteri_530 were in good agreement with experimentally determined values. To further validate the prediction accuracy of Lreuteri_530, the predicted effects of glycerol addition and adhE gene knock-out, which results in impaired ethanol production, were compared to in vivo data. Examination of both growth rates and uptake- and secretion rates of the main metabolites in central metabolism demonstrated that the model was able to accurately predict the experimentally observed effects. Lastly, the potential of L. reuteri as a cell factory was investigated, resulting in a number of general metabolic engineering strategies. Conclusion We have constructed a manually curated genome-scale metabolic model of L. reuteri JCM 1112T that has been experimentally parameterized and validated and can accurately predict metabolic behavior of this important platform cell factory.

Fermentation of gases provides a promising opportunity for the production of biochemicals from renewable resources, which has resulted in a growing interest in acetogenic bacteria. Thermophilic organisms provide potential advantages for the fermentation of, e.g., syngas into for example volatile compounds, and the thermophiles Moorella thermoacetica and Moorella thermoautotrophica have become model organisms of acetogenic metabolism. The justification for the recognition of the closely related species M. thermoautotrophica has, however, recently been disputed. In order to expand knowledge on the genus, we have here genome sequenced a total of 12 different M. thermoacetica and M. thermoautotrophica strains. From the sequencing results, it became clear that M. thermoautotrophica DSM 1974T consists of at least two different strains. Two different strains were isolated in Lyngby and Ulm from a DSM 1974T culture obtained from the DSMZ (Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Brunswick, Germany). Phylogenetic analysis revealed a close relationship between all the sequenced genomes, suggesting that the two strains detected in the type strain of the species M. thermoautotrophica could not be distinguished at the species level from M. thermoacetica. Despite genetic similarities, differences in genomic features were observed between the strains. Differences in compounds that can serve as carbon and energy sources for selected strains were also identified. On the contrary, strain DSM 21394, currently still named M. thermoacetica, obviously represents a new Moorella species. In addition, based on genome analysis and comparison M. glycerini NMP, M. stamsii DSM 26217T, and M. perchloratireducens An10 cannot be distinguished at the species level. Thus, this comprehensive analysis provides a significantly increased knowledge of the genetic diversity of Moorella strains.

A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.