Regulated redirection of central carbon flux enhances anaerobic production of bioproducts in Zymomonas mobilis.

The microbial production of chemicals and fuels from plant biomass offers a sustainable alternative to fossilized carbon but requires high rates and yields of bioproduct synthesis. Z. mobilis is a promising chassis microbe due to its high glycolytic rate in anaerobic conditions that are favorable for large-scale production. However, diverting flux from its robust ethanol fermentation pathway to nonnative pathways remains a major engineering hurdle. To enable controlled, high-yield synthesis of bioproducts, we implemented a central-carbon metabolism control-valve strategy using regulated, ectopic expression of pyruvate decarboxylase (Pdc) and deletion of chromosomal pdc. Metabolomic and genetic analyses revealed that glycolytic intermediates and NADH accumulate when Pdc is depleted and that Pdc is essential for anaerobic growth of Z. mobilis. Aerobically, all flux can be redirected to a 2,3-butanediol pathway for which respiration maintains redox balance. Anaerobically, flux can be redirected to redox-balanced lactate or isobutanol pathways with ≥65% overall yield from glucose. This strategy provides a promising path for future metabolic engineering of Z. mobilis.

OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate.

Efficient microbial production of the next-generation biofuel isobutanol (IBA) is limited by metabolic bottlenecks. Overcoming these bottlenecks will be aided by knowing the optimal ratio of enzymes for efficient flux through the IBA biosynthetic pathway. OptSSeq (Optimization by Selection and Sequencing) accomplishes this goal by tracking growth rate-linked selection of optimal expression elements from a combinatorial library. The 5-step pathway to IBA consists of Acetolactate synthase (AlsS), Keto-acid reductoisomerase (KARI), Di-hydroxy acid dehydratase (DHAD), Ketoisovalerate decarboxylase (Kivd) and Alcohol dehydrogenase (Adh). Using OptSSeq, we identified gene expression elements leading to optimal enzyme levels that enabled theoretically maximal productivities per cell biomass in Escherichia coli. We identified KARI as the rate-limiting step, requiring the highest levels of enzymes expression, followed by AlsS and AdhA. DHAD and Kivd required relatively lower levels of expression for optimal IBA production. OptSSeq also enabled the identification of an Adh enzyme variant capable of an improved rate of IBA production. Using models that predict impacts of enzyme synthesis costs on cellular growth rates, we found that optimum levels of pathway enzymes led to maximal IBA production, and that additional limitations lie in the E. coli metabolic network. Our optimized constructs enabled the production of ~3 g IBA per hour per gram dry cell weight and was achieved with 20 % of the total cell protein devoted to IBA-pathway enzymes in the molar ratio 2.5:6.7:2:1:5.2 (AlsS:IlvC:IlvD:Kivd:AdhA). These enzyme levels and ratios optimal for IBA production in E. coli provide a useful starting point for optimizing production of IBA in diverse microbes and fermentation conditions.

Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis.

Zymomonas mobilis is an ethanologenic bacterium currently being developed for production of advanced biofuels. Recent studies have shown that Z. mobilis can fix dinitrogen gas (N2) as a sole nitrogen source. During N2 fixation, Z. mobilis exhibits increased biomass-specific rates of ethanol production. In order to better understand the physiology of Z. mobilis during N2 fixation and during changes in ammonium (NH4+) availability, we performed liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomics and shotgun proteomics under three regimes of nitrogen availability: continuous N2 fixation, gradual NH4+ depletion, and acute NH4+ addition to N2-fixing cells. We report dynamic changes in abundance of proteins and metabolites related to nitrogen fixation, motility, ammonium assimilation, amino acid biosynthesis, nucleotide biosynthesis, isoprenoid biosynthesis, and Entner-Doudoroff (ED) glycolysis, providing insight into the regulatory mechanisms that control these processes in Z. mobilis. Our analysis identified potential physiological mechanisms that may contribute to increased specific ethanol production during N2 fixation, including decreased activity of biosynthetic pathways, increased protein abundance of alcohol dehydrogenase (ADHI), and increased thermodynamic favorability of the ED pathway. Of particular relevance to advanced biofuel production, we found that intermediates in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis were depleted during N2 fixation, coinciding with decreased protein abundance of deoxyxylulose 5-phosphate synthase (DXS), the first enzyme in the pathway. This implies that DXS protein abundance serves as a native control point in regulating MEP pathway activity in Z. mobilis. The results of this study will inform metabolic engineering to further develop Z. mobilis as a platform organism for biofuel production. IMPORTANCE Biofuels and bioproducts have the potential to serve as environmentally sustainable replacements for petroleum-derived fuels and commodity molecules. Advanced fuels such as higher alcohols and isoprenoids are more suitable gasoline replacements than bioethanol. Developing microbial systems to generate advanced biofuels requires metabolic engineering to reroute carbon away from ethanol and other native products and toward desired pathways, such as the MEP pathway for isoprenoid biosynthesis. However, rational engineering of microbial metabolism relies on understanding metabolic control points, in terms of both enzyme activity and thermodynamic favorability. In Z. mobilis, the factors that control glycolytic rates, ethanol production, and isoprenoid production are still not fully understood. In this study, we performed metabolomic, proteomic, and thermodynamic analysis of Z. mobilis during N2 fixation. This analysis identified key changes in metabolite levels, enzyme abundance, and glycolytic thermodynamic favorability that occurred during changes in NH4+ availability, helping to inform future efforts in metabolic engineering.

Transcriptomic, Protein-DNA Interaction, and Metabolomic Studies of VosA, VelB, and WetA in Aspergillus nidulans Asexual Spores.

In filamentous fungi, asexual development involves cellular differentiation and metabolic remodeling leading to the formation of intact asexual spores. The development of asexual spores (conidia) in Aspergillus is precisely coordinated by multiple transcription factors (TFs), including VosA, VelB, and WetA. Notably, these three TFs are essential for the structural and metabolic integrity, i.e., proper maturation, of conidia in the model fungus Aspergillus nidulans To gain mechanistic insight into the complex regulatory and interdependent roles of these TFs in asexual sporogenesis, we carried out multi-omics studies on the transcriptome, protein-DNA interactions, and primary and secondary metabolism employing A. nidulans conidia. RNA sequencing and chromatin immunoprecipitation sequencing analyses have revealed that the three TFs directly or indirectly regulate the expression of genes associated with heterotrimeric G-protein signal transduction, mitogen-activated protein (MAP) kinases, spore wall formation and structural integrity, asexual development, and primary/secondary metabolism. In addition, metabolomics analyses of wild-type and individual mutant conidia indicate that these three TFs regulate a diverse array of primary metabolites, including those in the tricarboxylic acid (TCA) cycle, certain amino acids, and trehalose, and secondary metabolites such as sterigmatocystin, emericellamide, austinol, and dehydroaustinol. In summary, WetA, VosA, and VelB play interdependent, overlapping, and distinct roles in governing morphological development and primary/secondary metabolic remodeling in Aspergillus conidia, leading to the production of vital conidia suitable for fungal proliferation and dissemination.IMPORTANCE Filamentous fungi produce a vast number of asexual spores that act as efficient propagules. Due to their infectious and/or allergenic nature, fungal spores affect our daily life. Aspergillus species produce asexual spores called conidia; their formation involves morphological development and metabolic changes, and the associated regulatory systems are coordinated by multiple transcription factors (TFs). To understand the underlying global regulatory programs and cellular outcomes associated with conidium formation, genomic and metabolomic analyses were performed in the model fungus Aspergillus nidulans Our results show that the fungus-specific WetA/VosA/VelB TFs govern the coordination of morphological and chemical developments during sporogenesis. The results of this study provide insights into the interdependent, overlapping, or distinct genetic regulatory networks necessary to produce intact asexual spores. The findings are relevant for other Aspergillus species such as the major human pathogen Aspergillus fumigatus and the aflatoxin producer Aspergillus flavus.

Metabolic flux analysis and fluxomics-driven determination of reaction free energy using multiple isotopes.

Metabolite concentrations, fluxes, and free energies constitute the basis for understanding and controlling metabolism. Mass spectrometry and stable isotopes are integral tools in quantifying these metabolic features. For absolute metabolite concentration and flux measurement, 13C internal standards and tracers have been the gold standard. In contrast, no established methods exist for comprehensive thermodynamic quantitation under physiological environments. Recently, using high-resolution mass spectrometry and multi-isotope tracing, flux quantitation has been increasingly adopted in broader metabolism. The improved flux quantitation led to determination of Gibbs free energy of reaction (ΔG) in central carbon metabolism using a relationship between reaction reversibility and thermodynamic driving force. Here we highlight recent advances in multi-isotope tracing for metabolic flux and free energy analysis.

Systems-Level Analysis of Oxygen Exposure in Zymomonas mobilis: Implications for Isoprenoid Production.

Zymomonas mobilis is an aerotolerant anaerobe and prolific ethanologen with attractive characteristics for industrial bioproduct generation. However, there is currently insufficient knowledge of the impact that environmental factors have on flux through industrially relevant biosynthetic pathways. Here, we examined the effect of oxygen exposure on metabolism and gene expression in Z. mobilis by combining targeted metabolomics, mRNA sequencing, and shotgun proteomics. We found that exposure to oxygen profoundly influenced metabolism, inducing both transient metabolic bottlenecks and long-term metabolic remodeling. In particular, oxygen induced a severe but temporary metabolic bottleneck in the methyl erythritol 4-phosphate pathway for isoprenoid biosynthesis caused by oxidative damage to the iron-sulfur cofactors of the final two enzymes in the pathway. This bottleneck was resolved with minimal changes in expression of isoprenoid biosynthetic enzymes. Instead, it was associated with pronounced upregulation of enzymes related to iron-sulfur cluster maintenance and biogenesis (i.e., flavodoxin reductase and the suf operon). We also detected major changes in glucose utilization in the presence of oxygen. Specifically, we observed increased gluconate production following exposure to oxygen, accounting for 18% of glucose uptake. Our results suggest that under aerobic conditions, electrons derived from the oxidation of glucose to gluconate are diverted to the electron transport chain, where they can minimize oxidative damage by reducing reactive oxygen species such as H2O2. This model is supported by the simultaneous upregulation of three membrane-bound dehydrogenases, cytochrome c peroxidase, and a cytochrome bd oxidase following exposure to oxygen. IMPORTANCE Microbially generated biofuels and bioproducts have the potential to provide a more environmentally sustainable alternative to fossil-fuel-derived products. In particular, isoprenoids, a diverse class of natural products, are chemically suitable for use as high-grade transport fuels and other commodity molecules. However, metabolic engineering for increased production of isoprenoids and other bioproducts is limited by an incomplete understanding of factors that control flux through biosynthetic pathways. Here, we examined the native regulation of the isoprenoid biosynthetic pathway in the biofuel producer Zymomonas mobilis. We leveraged oxygen exposure as a means to perturb carbon flux, allowing us to observe the formation and resolution of a metabolic bottleneck in the pathway. Our multi-omics analysis of this perturbation enabled us to identify key auxiliary enzymes whose expression correlates with increased production of isoprenoid precursors, which we propose as potential targets for future metabolic engineering.

Cytochrome P450 Monooxygenase-Mediated Metabolic Utilization of Benzo[a]Pyrene by Aspergillus Species.

Soil-dwelling fungal species possess the versatile metabolic capability to degrade complex organic compounds that are toxic to humans, yet the mechanisms they employ remain largely unknown. Benzo[a]pyrene (BaP) is a pervasive carcinogenic contaminant, posing a significant concern for human health. Here, we report that several Aspergillus species are capable of degrading BaP. Exposing Aspergillus nidulans cells to BaP results in transcriptomic and metabolic changes associated with cellular growth and energy generation, implying that the fungus utilizes BaP as a growth substrate. Importantly, we identify and characterize the conserved bapA gene encoding a cytochrome P450 monooxygenase that is necessary for the metabolic utilization of BaP in Aspergillus We further demonstrate that the fungal NF-κB-type velvet regulators VeA and VelB are required for proper expression of bapA in response to nutrient limitation and BaP degradation in A. nidulans Our study illuminates fundamental knowledge of fungal BaP metabolism and provides novel insights into enhancing bioremediation potential.IMPORTANCE We are increasingly exposed to environmental pollutants, including the carcinogen benzo[a]pyrene (BaP), which has prompted extensive research into human metabolism of toxicants. However, little is known about metabolic mechanisms employed by fungi that are able to use some toxic pollutants as the substrates for growth, leaving innocuous by-products. This study systemically demonstrates that a common soil-dwelling fungus is able to use benzo[a]pyrene as food, which results in expression and metabolic changes associated with growth and energy generation. Importantly, this study reveals key components of the metabolic utilization of BaP, notably a cytochrome P450 monooxygenase and the fungal NF-κB-type transcriptional regulators. Our study advances fundamental knowledge of fungal BaP metabolism and provides novel insight into designing and implementing enhanced bioremediation strategies.

Recent applications of metabolomics to advance microbial biofuel production.

Biofuel production from plant biomass is a promising source of renewable energy [1]. However, efficient biofuel production involves the complex task of engineering high-performance microorganisms, which requires detailed knowledge of metabolic function and regulation. This review highlights the potential of mass-spectrometry-based metabolomic analysis to guide rational engineering of biofuel-producing microbes. We discuss recent studies that apply knowledge gained from metabolomic analyses to increase the productivity of engineered pathways, characterize the metabolism of emerging biofuel producers, generate novel bioproducts, enable utilization of lignocellulosic feedstock, and improve the stress tolerance of biofuel producers.