Significance: We have studied the functionality of an incomplete acetyl-CoA “Wood–Ljungdahl” pathway in a strictly organohalide-respiring bacterium, Dehalococcoides mccartyi . We found that in addition to its ability to incorporate exogenous formate, this pathway cleaves acetyl-CoA to generate methyl-tetrahydrofolate for methionine biosynthesis, serving as a unique substitute of the missing methylene-tetrahydrofolate reductase function. We also found that accumulation of carbon monoxide (CO), an obligate by-product from acetyl-CoA cleavage, inhibits D. mccartyi axenic cultures, but can be ameliorated by the presence of a CO-oxidizing organism, resulting in an unusual syntrophic association. The understanding of the products and biosynthetic functions of this incomplete Wood–Ljungdahl pathway improves our knowledge of alternate central metabolic strategies used by environmental microorganisms.

Lignocellulosic biomass is a promising source of renewable biofuels. However, pretreatment of lignocellulosic biomass generates fermentation inhibitors that adversely affect the growth of industrial microorganisms such as Saccharomyces cerevisiae and prevent economic production of lignocellulosic biofuels. A critical challenge on developing S. cerevisiae with improved inhibitor resistance lies in incomplete understanding of molecular basis for inhibitor stress response and limited information on effective genetic targets for increasing yeast resistance to mixed fermentation inhibitors. In this study, we applied comparative transcriptomic analysis to determine the molecular basis for acetic acid and/or furfural resistance in S. cerevisiae. We recently developed a yeast strain YC1 with superior resistance to acetic acid, furfural, and their mixture through inverse metabolic engineering. In this study, we first determined transcriptional changes through RNA sequencing in YC1 versus the wild-type strain S-C1 under three different inhibitor conditions, including acetic acid alone, furfural alone, and mixture of acetic acid and furfural. The genes associated with stress responses of S. cerevisiae to single and mixed inhibitors were revealed. Specifically, we identified 184 consensus genes that were differentially regulated in response to the distinct inhibitor resistance between YC1 and S-C1. Bioinformatic analysis next revealed key transcription factors (TFs) that regulate these consensus genes. The top TFs identified, Sfp1p and Ace2p, were experimentally tested as overexpression targets for strain optimization. Overexpression of the SFP1 gene improved specific ethanol productivity by nearly four times, while overexpression of the ACE2 gene enhanced the rate by three times in the presence of acetic acid and furfural. Overexpression of SFP1 gene in the resistant strain YC1 further resulted in 42 % increase in ethanol productivity in the presence of acetic acid and furfural, suggesting the effect of Sfp1p in optimizing the yeast strain for improved tolerance to mixed fermentation inhibitor. Transcriptional regulation underlying yeast resistance to acetic acid and furfural was determined. Two transcription factors, Sfp1p and Ace2p, were uncovered for the first time for their functions in improving yeast resistance to mixed fermentation inhibitors. The study demonstrated an omics-guided metabolic engineering framework, which could be developed as a promising strategy to improve complex microbial phenotypes.

The unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 (Cyanothece 51142) is able to grow aerobically under nitrogen-fixing conditions with alternating light–dark cycles or continuous illumination. This study investigated the effects of carbon and nitrogen sources on Cyanothece 51142 metabolism via ¹³C-assisted metabolite analysis and biochemical measurements. Under continuous light (50 μmol photons m⁻² s⁻¹) and nitrogen-fixing conditions, we found that glycerol addition promoted aerobic biomass growth (by twofold) and nitrogenase-dependent hydrogen production [up to 25 μmol H₂ (mg chlorophyll) ⁻¹ h⁻¹], but strongly reduced phototrophic CO₂ utilization. Under nitrogen-sufficient conditions, Cyanothece 51142 was able to metabolize glycerol photoheterotrophically, and the activity of light-dependent reactions (e.g. oxygen evolution) was not significantly reduced. In contrast, Synechocystis sp. PCC 6803 showed apparent mixotrophic metabolism under similar growth conditions. Isotopomer analysis also detected that Cyanothece 51142 was able to fix CO₂ via anaplerotic pathways, and to take up glucose and pyruvate for mixotrophic biomass synthesis.