Exogenous butyrate inhibits butyrogenic metabolism and alters virulence phenotypes in Clostridioides difficile.

The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short-chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile-inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells and is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and (S)-3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of a butyrogenic pathway(s) in C. difficile coincides with alterations in toxin release and sporulation. Together, this work highlights butyrate as a marker of a C. difficile-inhospitable environment to which C. difficile responds by releasing its diarrheagenic toxins and producing environmentally resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of how C. difficile growth is inhibited by butyrate and how butyrate alters C. difficile virulence in the face of a highly competitive and dynamic gut environment.IMPORTANCEThe gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood, which hinders the development of novel therapeutic interventions for C. difficile infection (CDI). We investigated how C. difficile responds to butyrate, an end-product of gut microbiome community metabolism which inhibits C. difficile growth. We show that exogenously produced butyrate is internalized into C. difficile, which inhibits C. difficile growth by interfering with its own butyrate production. This growth inhibition coincides with increased toxin release from C. difficile cells and the production of environmentally resistant spores necessary for transmission between hosts. Future work to disentangle the molecular mechanisms underlying these growth and virulence phenotypes will likely lead to new strategies to restrict C. difficile growth in the gut and minimize its pathogenesis during CDI.

C-di-AMP accumulation disrupts glutathione metabolism and inhibits virulence program expression in Listeria monocytogenes.

C-di-AMP is an essential second messenger in many bacteria but its levels must be regulated. Unregulated c-di-AMP accumulation attenuates the virulence of many bacterial pathogens, including those that do not require c-di-AMP for growth. However, the mechanisms by which c-di-AMP regulates bacterial pathogenesis remain poorly understood. In Listeria monocytogenes , a mutant lacking both c-di-AMP phosphodiesterases, denoted as the ΔPDE mutant, accumulates a high c-di-AMP level and is significantly attenuated in the mouse model of systemic infection. All key L. monocytogenes virulence genes are transcriptionally upregulated by the master transcription factor PrfA, which is activated by reduced glutathione (GSH) during infection. Our transcriptomic analysis revealed that the ΔPDE mutant is significantly impaired for the expression of virulence genes within the PrfA core regulon. Subsequent quantitative gene expression analyses validated this phenotype both at the basal level and upon PrfA activation by GSH. A constitutively active PrfA * variant, PrfA G145S, which mimics the GSH-bound conformation, restores virulence gene expression in ΔPDE but only partially rescues virulence defect. Through GSH quantification and uptake assays, we found that the ΔPDE strain is significantly depleted for GSH, and that c-di-AMP inhibits GSH uptake. Constitutive expression of gshF (encoding a GSH synthetase) does not restore GSH levels in the ΔPDE strain, suggesting that c-di-AMP inhibits GSH synthesis activity or promotes GSH catabolism. Taken together, our data reveals GSH metabolism as another pathway that is regulated by c-di-AMP. C-di-AMP accumulation depletes cytoplasmic GSH levels within L. monocytogenes that leads to impaired virulence program expression. IMPORTANCE: C-di-AMP regulates both bacterial pathogenesis and interactions with the host. Although c-di-AMP is essential in many bacteria, its accumulation also attenuates the virulence of many bacterial pathogens. Therefore, disrupting c-di-AMP homeostasis is a promising antibacterial treatment strategy, and has inspired several studies that screened for chemical inhibitors of c-di-AMP phosphodiesterases. However, the mechanisms by which c-di-AMP accumulation diminishes bacterial pathogenesis are poorly understood. Such understanding will reveal the molecular function of c-di-AMP, and inform therapeutic development strategies. Here, we identify GSH metabolism as a pathway regulated by c-di-AMP that is pertinent to L. monocytogenes replication in the host. Given the role of GSH as a virulence signal, nutrient, and antioxidant, GSH depletion impairs virulence program expression and likely diminishes host adaptation.

Lipid membrane remodeling and metabolic response during isobutanol and ethanol exposure in Zymomonas mobilis.

BACKGROUND: Recent engineering efforts have targeted the ethanologenic bacterium Zymomonas mobilis for isobutanol production. However, significant hurdles remain due this organism's vulnerability to isobutanol toxicity, adversely affecting its growth and productivity. The limited understanding of the physiological impacts of isobutanol on Z. mobilis constrains our ability to overcome these production barriers. RESULTS: We utilized a systems-level approach comprising LC-MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis. Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis' CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure. Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology-including cell swelling and formation of intracellular protein aggregates -when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis' primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates. CONCLUSIONS: This study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis. These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars.

ACAD10 and ACAD11 enable mammalian 4-hydroxy acid lipid catabolism.

Fatty acid ß-oxidation (FAO) is a central catabolic pathway with broad implications for organismal health. However, various fatty acids are largely incompatible with standard FAO machinery until they are modified by other enzymes. Included among these are the 4-hydroxy acids (4-HAs)-fatty acids hydroxylated at the 4 (γ) position-which can be provided from dietary intake, lipid peroxidation, and certain drugs of abuse. Here, we reveal that two atypical and poorly characterized acyl-CoA dehydrogenases (ACADs), ACAD10 and ACAD11, drive 4-HA catabolism in mice. Unlike other ACADs, ACAD10 and ACAD11 feature kinase domains N-terminal to their ACAD domains that phosphorylate the 4-OH position as a requisite step in the conversion of 4-hydroxyacyl-CoAs into 2-enoyl-CoAs-conventional FAO intermediates. Our ACAD11 cryo-EM structure and molecular modeling reveal a unique binding pocket capable of accommodating this phosphorylated intermediate. We further show that ACAD10 is mitochondrial and necessary for catabolizing shorter-chain 4-HAs, whereas ACAD11 is peroxisomal and enables longer-chain 4-HA catabolism. Mice lacking ACAD11 accumulate 4-HAs in their plasma while comparable 3- and 5-hydroxy acids remain unchanged. Collectively, this work defines ACAD10 and ACAD11 as the primary gatekeepers of mammalian 4-HA catabolism and sets the stage for broader investigations into the ramifications of aberrant 4-HA metabolism in human health and disease.

Relative Activities of the ß-ketoacyl-CoA and Acyl-CoA Reductases Influence Product Profile and Flux in a Reversed ß-Oxidation Pathway.

The ß-Oxidation pathway, normally involved in the catabolism of fatty acids, can be functionally made to act as a fermentative, iterative, elongation pathway when driven by the activity of a trans-enoyl-CoA reductase. The terminal acyl-CoA reduction to alcohol can occur on substrates with varied chain lengths, leading to a broad distribution of fermentation products in vivo. Tight control of the average chain length and product profile is desirable as chain length greatly influences molecular properties and commercial value. Lacking a termination enzyme with a narrow chain length preference, we sought alternative factors that could influence the product profile and pathway flux in the iterative pathway. In this study, we reconstituted the reversed ß-oxidation (R-ßox) pathway in vitro with a purified tri-functional complex (FadBA) responsible for the thiolase, enoyl-CoA hydratase and hydroxyacyl-CoA dehydrogenase activities, a trans-enoyl-CoA reductase (TER), and an acyl-CoA reductase (ACR). Using this system, we determined the rate limiting step of the elongation cycle and demonstrated that by controlling the ratio of these three enzymes and the ratio of NADH and NADPH, we can influence the average chain length of the alcohol product profile.

Deuterated water as a substrate-agnostic isotope tracer for investigating reversibility and thermodynamics of reactions in central carbon metabolism.

Stable isotope tracers are a powerful tool for the quantitative analysis of microbial metabolism, enabling pathway elucidation, metabolic flux quantification, and assessment of reaction and pathway thermodynamics. 13C and 2H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of 2H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to 13C-tracers. Furthermore, isotope tracer studies in industrially relevant bacteria that metabolize complex substrates such as cellulose, hemicellulose, or lignocellulosic biomass, are challenging given the difficulty in obtaining these as isotopically labeled substrates. In this study, we examine the potential of deuterated water (2H2O) as an affordable, substrate-neutral isotope tracer for studying central carbon metabolism. We apply 2H2O labeling to investigate the reversibility of glycolytic reactions across three industrially relevant bacterial species -C. thermocellum, Z. mobilis, and E. coli-harboring distinct glycolytic pathways with unique thermodynamics. We demonstrate that 2H2O labeling recapitulates previous reversibility and thermodynamic findings obtained with established 13C and 2H labeled nutrient substrates. Furthermore, we exemplify the utility of this 2H2O labeling approach by applying it to high-substrate C. thermocellum fermentations -a setting in which the use of conventional tracers is impractical-thereby identifying the glycolytic enzyme phosphofructokinase as a major bottleneck during high-substrate fermentations and unveiling critical insights that will steer future engineering efforts to enhance ethanol production in this cellulolytic organism. This study demonstrates the utility of deuterated water as a substrate-agnostic isotope tracer for examining flux and reversibility of central carbon metabolic reactions, which yields biological insights comparable to those obtained using costly 2H-labeled nutrient substrates.

The genetics of aerotolerant growth in an alphaproteobacterium with a naturally reduced genome.

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living alphaproteobacterium Zymomonas mobilis has a naturally reduced genome containing fewer than 2,000 protein-coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model alphaproteobacterium. Here, we use genome-scale CRISPRi-seq (clustered regularly interspaced short palindromic repeats interference sequencing) to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as a reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rhodobacter nitrogen fixation (Rnf) respiratory complex are required for the anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts. IMPORTANCE The inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the alphaproteobacterium Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts.

Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence.

Listeria monocytogenes is a remarkably well-adapted facultative intracellular pathogen that can thrive in a wide range of ecological niches. L. monocytogenes maximizes its ability to generate energy from diverse carbon sources using a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions. Cellular respiration maintains redox homeostasis by regenerating NAD+ while also generating a proton motive force. The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respirations. We previously demonstrated that intermediates in the MK biosynthesis pathway, notably 1,4-dihydroxy-2-naphthoate (DHNA), are required for the survival and virulence of L. monocytogenes independent of their role in respiration. Furthermore, we found that restoration of NAD+/NADH ratio through expression of water-forming NADH oxidase could rescue phenotypes associated with DHNA deficiency. Here, we extend these findings to demonstrate that endogenous production or direct supplementation of DHNA restored both the cellular redox homeostasis and metabolic output of fermentation in L. monocytogenes. Furthermore, exogenous supplementation of DHNA rescues the in vitro growth and ex vivo virulence of L. monocytogenes DHNA-deficient mutants. Finally, we demonstrate that exogenous DHNA restores redox balance in L. monocytogenes specifically through the recently annotated NADH dehydrogenase Ndh2, independent of its role in the extracellular electron transport pathway. These data suggest that the production of DHNA may represent an additional layer of metabolic adaptability by L. monocytogenes to drive energy metabolism in the absence of respiration-favorable conditions.

Efficiency of Lactiplantibacillus plantarum JT-PN39 and Paenibacillus motobuensis JT-A29 for Fermented Coffee Applications and Fermented Coffee Characteristics.

To develop a process for low-cost and ecologically friendly coffee fermentation, civet gut bacteria were isolated and screened to be used for fermentation. Among 223 isolates from civet feces, two bacteria exhibited strong protease, amylase, lipase, pectinase, and cellulase activities. By analyzing 16S rDNA phylogeny, those bacteria were identified to be Lactiplantibacillus plantarum JT-PN39 (LP) and Paenibacillus motobuensis JT-A29 (PM), where their potency (pure or mixed bacterial culture) for fermenting 5 L of arabica parchment coffee in 48-72 h was further determined. To characterize the role of bacteria in coffee fermentation, growth and pH were also determined. For mixed starter culture conditions, the growth of PM was not detected after 36 h of fermentation due to the low acid conditions generated by LP. Coffee quality was evaluated using a cupping test, and LP-fermented coffee expressed a higher cupping score, with a main fruity and sour flavor, and a dominant caramel-honey-like aroma. Antioxidant and anti-foodborne pathogenic bacteria activity, including total phenolic compounds of PM and LP fermented coffee extracts, was significantly higher than those of ordinary coffee. In addition, LP-fermented coffee expressed the highest antibacterial and antioxidant activities among the fermented coffee. The toxicity test was examined in the murine macrophage RAW 264.7 cell, and all fermented coffee revealed 80-90% cell variability, which means that the fermentation process does not generate any toxicity. In addition, qualifications of non-volatile and volatile compounds in fermented coffee were examined by LC-MS and GC-MS to discriminate the bacterial role during the process by PCA plot. The flavors of fermented coffee, including volatile and non-volatile compounds, were totally different between the non-fermented and fermented conditions. Moreover, the PCA plot showed slightly different flavors among fermentations with different starter cultures. For both the cupping test and biological activities, this study suggests that LP has potential for health benefits in coffee fermentation.

Exogenous butyrate inhibits butyrogenic metabolism and alters expression of virulence genes in Clostridioides difficile.

The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile-inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells, is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and (S)-3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of butyrogenic pathway(s) in C. difficile coincides with alterations in toxin production and sporulation. Together, this work highlights butyrate as a signal of a C. difficile inhospitable environment to which C. difficile responds by producing its diarrheagenic toxins and producing environmentally-resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of how C. difficile growth is inhibited by butyrate and how butyrate serves as a signal to alter C. difficile virulence in the face of a highly competitive and dynamic gut environment.

Listeria monocytogenes GlmR Is an Accessory Uridyltransferase Essential for Cytosolic Survival and Virulence.

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions against these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell-wall stress, cytosolic survival, inflammasome avoidance, and, ultimately, virulence in vivo. In this study, a genetic suppressor screen revealed that blocking utilization of UDP N-acetylglucosamine (UDP-GlcNAc) by a nonessential wall teichoic acid decoration pathway restored resistance to lysozyme and partially restored virulence of ΔglmR mutants. In parallel, metabolomic analysis revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggests that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell-wall stress responses and virulence in vivo. Taken together, these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes. IMPORTANCE Bacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK), to survive in the host cytosol. GlmR is important for resistance to some cell-wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell-wall metabolite, UDP-GlcNAc, and suppressors that increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc, and this enzymatic activity is conserved in both Bacillus subtilis and Staphylococcus aureus. These results highlight the importance of accessory cell wall metabolism enzymes in responding to cell-wall stress in a variety of Gram-positive bacteria.

Systematic Analysis of Metabolic Bottlenecks in the Methylerythritol 4-Phosphate (MEP) Pathway of Zymomonas mobilis.

Zymomonas mobilis is an industrially relevant aerotolerant anaerobic bacterium that can convert up to 96% of consumed glucose to ethanol. This highly catabolic metabolism could be leveraged to produce isoprenoid-based bioproducts via the methylerythritol 4-phosphate (MEP) pathway, but we currently have limited knowledge concerning the metabolic constraints of this pathway in Z. mobilis. Here, we performed an initial investigation of the metabolic bottlenecks within the MEP pathway of Z. mobilis using enzyme overexpression strains and quantitative metabolomics. Our analysis revealed that 1-deoxy-d-xylulose 5-phosphate synthase (DXS) represents the first enzymatic bottleneck in the Z. mobilis MEP pathway. DXS overexpression triggered large increases in the intracellular levels of the first five MEP pathway intermediates, of which the buildup in 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) was the most substantial. The combined overexpression of DXS, 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBDP) synthase (IspG), and HMBDP reductase (IspH) mitigated the bottleneck at MEcDP and mobilized carbon to downstream MEP pathway intermediates, indicating that IspG and IspH activity become the primary pathway constraints during DXS overexpression. Finally, we overexpressed DXS with other native MEP enzymes and a heterologous isoprene synthase and showed that isoprene can be used as a carbon sink in the Z. mobilis MEP pathway. By revealing key bottlenecks within the MEP pathway of Z. mobilis, this study will aid future engineering efforts aimed at developing this bacterium for industrial isoprenoid production. IMPORTANCE Engineered microorganisms have the potential to convert renewable substrates into biofuels and valuable bioproducts, which offers an environmentally sustainable alternative to fossil-fuel-derived products. Isoprenoids are a diverse class of biologically derived compounds that have commercial applications as various commodity chemicals, including biofuels and biofuel precursor molecules. Thus, isoprenoids represent a desirable target for large-scale microbial generation. However, our ability to engineer microbes for the industrial production of isoprenoid-derived bioproducts is limited by an incomplete understanding of the bottlenecks in the biosynthetic pathway responsible for isoprenoid precursor generation. In this study, we combined genetic engineering with quantitative analyses of metabolism to examine the capabilities and constraints of the isoprenoid biosynthetic pathway in the industrially relevant microbe Zymomonas mobilis. Our integrated and systematic approach identified multiple enzymes whose overexpression in Z. mobilis results in an increased production of isoprenoid precursor molecules and mitigation of metabolic bottlenecks.

Evaluation of 1,2-diacyl-3-acetyl triacylglycerol production in Yarrowia lipolytica.

Plants produce many high-value oleochemical molecules. While oil-crop agriculture is performed at industrial scales, suitable land is not available to meet global oleochemical demand. Worse, establishing new oil-crop farms often comes with the environmental cost of tropical deforestation. The field of metabolic engineering offers tools to transplant oleochemical metabolism into tractable hosts while simultaneously providing access to molecules produced by non-agricultural plants. Here, we evaluate strategies for rewiring metabolism in the oleaginous yeast Yarrowia lipolytica to synthesize a foreign lipid, 3-acetyl-1,2-diacyl-sn-glycerol (acTAG). Oils made up of acTAG have a reduced viscosity and melting point relative to traditional triacylglycerol oils making them attractive as low-grade diesels, lubricants, and emulsifiers. This manuscript describes a metabolic engineering study that established acTAG production at g/L scale, exploration of the impact of lipid bodies on acTAG titer, and a techno-economic analysis that establishes the performance benchmarks required for microbial acTAG production to be economically feasible.

Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence.

Listeria monocytogenes is a remarkably well-adapted facultative intracellular pathogen that can thrive in a wide range of ecological niches. L. monocytogenes maximizes its ability to generate energy from diverse carbon sources using a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions. Cellular respiration maintains redox homeostasis by regenerating NAD + while also generating a proton motive force (PMF). The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respiration. We previously demonstrated that intermediates in the MK biosynthesis pathway, notably 1,4-dihydroxy-2-naphthoate (DHNA), are required for the survival and virulence of L. monocytogenes independent of their role in respiration. Furthermore, we found that restoration of NAD + /NADH ratio through expression of water-forming NADH oxidase (NOX) could rescue phenotypes associated with DHNA deficiency. Here we extend these findings to demonstrate that endogenous production or direct supplementation of DHNA restored both the cellular redox homeostasis and metabolic output of fermentation in L. monocytogenes . Further, exogenous supplementation of DHNA rescues the in vitro growth and ex vivo virulence of L. monocytogenes DHNA-deficient mutants. Finally, we demonstrate that exogenous DHNA restores redox balance in L. monocytogenes specifically through the recently annotated NADH dehydrogenase Ndh2, independent of the extracellular electron transport (EET) pathway. These data suggest that the production of DHNA may represent an additional layer of metabolic adaptability by L. monocytogenes to drive energy metabolism in the absence of respiration-favorable conditions.

Metabolic Promiscuity of an Orphan Small Alarmone Hydrolase Facilitates Bacterial Environmental Adaptation.

Small alarmone hydrolases (SAHs) are alarmone metabolizing enzymes found in both metazoans and bacteria. In metazoans, the SAH homolog Mesh1 is reported to function in cofactor metabolism by hydrolyzing NADPH to NADH. In bacteria, SAHs are often identified in genomes with toxic alarmone synthetases for self-resistance. Here, we characterized a bacterial orphan SAH, i.e., without a toxic alarmone synthetase, in the phytopathogen Xanthomonas campestris pv. campestris (XccSAH) and found that it metabolizes both cellular alarmones and cofactors. In vitro, XccSAH displays abilities to hydrolyze multiple nucleotides, including pppGpp, ppGpp, pGpp, pppApp, and NADPH. In vivo, X. campestris pv. campestris cells lacking sah accumulated higher levels of cellular (pp)pGpp and NADPH compared to wild-type cells upon amino acid starvation. In addition, X. campestris pv. campestris mutants lacking sah were more sensitive to killing by Pseudomonas during interbacterial competition. Interestingly, loss of sah also resulted in reduced growth in amino acid-replete medium, a condition that did not induce (pp)pGpp or pppApp accumulation. Further metabolomic characterization revealed strong depletion of NADH levels in the X. campestris pv. campestris mutant lacking sah, suggesting that NADPH/NADH regulation is an evolutionarily conserved function of both bacterial and metazoan SAHs and Mesh1. Overall, our work demonstrates a regulatory role of bacterial SAHs as tuners of stress responses and metabolism, beyond functioning as antitoxins. IMPORTANCE Small alarmone hydrolases (SAHs) comprise a widespread family of alarmone metabolizing enzymes. In metazoans, SAHs have been reported to control multiple aspects of physiology and stress resistance through alarmone and NADPH metabolisms, but their physiological functions in bacteria is mostly uncharacterized except for a few reports as antitoxins. Here, we identified an SAH functioning independently of toxins in the phytopathogen Xanthomonas campestris pv. campestris. We found that XccSAH hydrolyzed multiple alarmones and NADPH in vitro, and X. campestris pv. campestris mutants lacking sah displayed increased alarmone levels during starvation, loss of interspecies competitive fitness, growth defects, and strong reduction in NADH. Our findings reveal the importance of NADPH hydrolysis by a bacterial SAH. Our work is also the first report of significant physiological roles of bacterial SAHs beyond functioning as antitoxins and suggests that SAHs have far broader physiological roles and share similar functions across domains of life.

Increasing the Thermodynamic Driving Force of the Phosphofructokinase Reaction in Clostridium thermocellum.

Glycolysis is an ancient, widespread, and highly conserved metabolic pathway that converts glucose into pyruvate. In the canonical pathway, the phosphofructokinase (PFK) reaction plays an important role in controlling flux through the pathway. Clostridium thermocellum has an atypical glycolysis and uses pyrophosphate (PPi) instead of ATP as the phosphate donor for the PFK reaction. The reduced thermodynamic driving force of the PPi-PFK reaction shifts the entire pathway closer to thermodynamic equilibrium, which has been predicted to limit product titers. Here, we replace the PPi-PFK reaction with an ATP-PFK reaction. We demonstrate that the local changes are consistent with thermodynamic predictions: the ratio of fructose 1,6-bisphosphate to fructose-6-phosphate increases, and the reverse flux through the reaction (determined by 13C labeling) decreases. The final titer and distribution of fermentation products, however, do not change, demonstrating that the thermodynamic constraints of the PPi-PFK reaction are not the sole factor limiting product titer. IMPORTANCE The ability to control the distribution of thermodynamic driving force throughout a metabolic pathway is likely to be an important tool for metabolic engineering. The phosphofructokinase reaction is a key enzyme in Embden-Mayerhof-Parnas glycolysis and therefore improving the thermodynamic driving force of this reaction in C. thermocellum is believed to enable higher product titers. Here, we demonstrate switching from pyrophosphate to ATP does in fact increases the thermodynamic driving force of the phosphofructokinase reaction in vivo. This study also identifies and overcomes a physiological hurdle toward expressing an ATP-dependent phosphofructokinase in an organism that utilizes an atypical glycolytic pathway. As such, the method described here to enable expression of ATP-dependent phosphofructokinase in an organism with an atypical glycolytic pathway will be informative toward engineering the glycolytic pathways of other industrial organism candidates with atypical glycolytic pathways.

Diadenosine tetraphosphate regulates biosynthesis of GTP in Bacillus subtilis.

Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the underlying molecular mechanisms are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5'-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics, which suggests that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis, which shows increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type strains. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone.

Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway.

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketol-acid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.

Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model.

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13C-metabolic flux analysis (13C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13C labeling data were used with 13C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.

Novel computational and experimental approaches for investigating the thermodynamics of metabolic networks.

Thermodynamic analysis of metabolic networks has emerged as a useful new tool for pathway design and metabolic engineering. Understanding the relationship between the thermodynamic driving force of biochemical reactions and metabolic flux has generated new insights regarding the design principles of microbial carbon metabolism. This review summarizes the various lessons that can be obtained from the thermodynamic analysis of metabolic pathways, illustrates concepts of computational thermodynamic tools, and highlights recent applications of thermodynamic analysis to pathway design in industrially relevant microbes.