Near-equilibrium glycolysis supports metabolic homeostasis and energy yield.

Glycolysis plays a central role in producing ATP and biomass. Its control principles, however, remain incompletely understood. Here, we develop a method that combines 2H and 13C tracers to determine glycolytic thermodynamics. Using this method, we show that, in conditions and organisms with relatively slow fluxes, multiple steps in glycolysis are near to equilibrium, reflecting spare enzyme capacity. In Escherichia coli, nitrogen or phosphorus upshift rapidly increases the thermodynamic driving force, deploying the spare enzyme capacity to increase flux. Similarly, respiration inhibition in mammalian cells rapidly increases both glycolytic flux and the thermodynamic driving force. The thermodynamic shift allows flux to increase with only small metabolite concentration changes. Finally, we find that the cellulose-degrading anaerobe Clostridium cellulolyticum exhibits slow, near-equilibrium glycolysis due to the use of pyrophosphate rather than ATP for fructose-bisphosphate production, resulting in enhanced per-glucose ATP yield. Thus, near-equilibrium steps of glycolysis promote both rapid flux adaptation and energy efficiency.

The role of neuropeptide F in a transition to parental care.

The genetics of complex social behaviour can be dissected by examining the genetic influences of component pathways, which can be predicted based on expected evolutionary precursors. Here, we examine how gene expression in a pathway that influences the motivation to eat is altered during parental care that involves direct feeding of larvae. We examine the expression of neuropeptide F, and its receptor, in the burying beetle Nicrophorus vespilloides, which feeds pre-digested carrion to its begging larvae. We found that the npf receptor was greatly reduced during active care. Our research provides evidence that feeding behaviour was a likely target during the evolution of parental care in N. vespilloides Moreover, dissecting complex behaviours into ethologically distinct sub-behaviours is a productive way to begin to target the genetic mechanisms involved in the evolution of complex behaviours.

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.

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.

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.

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.

Genetic conservation of Cytauxzoon felis antigens and mRNA expression in the schizont life-stage.

Cytauxzoonosis is a highly fatal disease of domestic cats caused by the apicomplexan protozoan Cytauxzoon felis, which is most closely related to Theileria spp. The growing prevalence, high morbidity and mortality, and treatment cost of cytauxzoonosis emphasize the need for vaccine development. Traditional approaches for vaccine development, however, have been hindered by the inability to culture C. felis in vitro. Recent availability of the annotated C. felis genome combined with genome-based vaccine design and protein microarray immunoscreening allowed for high-throughput identification of C. felis antigens that could serve as vaccine candidates. This study assessed the suitability of three of these vaccine candidates (cf30, cf63, cf58) in addition to a previously reported vaccine candidate (cf76) based on two criteria: genetic conservation among diverse C. felis geographic isolates and expression in tissues containing the C. felis schizont life stage, which has been previously associated with the development of a protective immune response. A comparison of seventeen C. felis isolates across seven states demonstrated high sequence identity (99-100%) for cf30, cf63, and cf58, similar to the degree of conservation previously reported for cf76. RNAscope® in situ hybridization using acutely infected feline splenic tissue revealed robust levels of all transcripts in the schizont life stage of the parasite. These data support the suitability of these three antigens for further investigation as vaccine candidates against cytauxzoonosis.