Tuberculosis (TB) is a formidable infectious disease that remains a major cause of death worldwide today. Escalating application of genomic techniques has expedited the identification of increasing number of mutations associated with drug resistance in Mycobacterium tuberculosis. Unfortunately the prevalence of bacillary resistance becomes alarming in many parts of the world, with the daunting scenarios of multidrug-resistant tuberculosis (MDR-TB), extensively drug-resistant tuberculosis (XDR-TB) and total drug-resistant tuberculosis (TDR-TB), due to number of resistance pathways, alongside some apparently obscure ones. Recent advances in the understanding of the molecular/ genetic basis of drug targets and drug resistance mechanisms have been steadily made. Intriguing findings through whole genome sequencing and other molecular approaches facilitate the further understanding of biology and pathology of M. tuberculosis for the development of new therapeutics to meet the immense challenge of global health.

Buruli ulcer (BU) is an emerging infectious disease that causes disfiguring skin ulcers. The causative agent, Mycobacterium ulcerans, secretes toxin called mycolactone that triggers inflammation and immunopathology. Existing treatments are lengthy and consist of drugs developed for tuberculosis. Here, we report that a pyrazolo[1,5-a]pyridine-3-carboxamide, TB47, is highly bactericidal against M. ulcerans both in vitro and in vivo. In the validated mouse model of BU, TB47 alone reduces M. ulcerans burden in mouse footpads by more than 2.5 log10 CFU compared to the standard BU treatment regimen recommended by the WHO. We show that mutations of ubiquinol-cytochrome C reductase cytochrome subunit B confer resistance to TB47 and the dissimilarity of CydABs from different mycobacteria may account for their differences in susceptibility to TB47. TB47 is highly potent against M. ulcerans and possesses desirable pharmacological attributes and low toxicity that warrant further assessment of this agent for treatment of BU.

Advanced bioproduct synthesis via reductive metabolism requires coordinating carbons, ATP, and reducing agents, which are generated with varying efficiencies depending on metabolic pathways. Substrate mixtures with shortcut access concurrently to multiple pathways may optimally satisfy these biosynthetic requirements. However, native regulation favoring preferential utilization precludes cells from co-metabolizing multiple substrates. Here we explore mixed substrate metabolism and tailor pathway usage to synergistically stimulate carbon reduction. By controlled cofeeding of superior ATP-and NADPH-generators as "dopant" substrates to cells primarily utilizing inferior substrates, we circumvent catabolite repression and drive synergy in two divergent organisms. Glucose doping in Moorella thermoacetica stimulates CO 2 reduction (2.3 g/g cell /hr) into acetate by augmenting ATP synthesis via pyruvate kinase. Gluconate doping in Yarrowia lipolytica accelerates acetate-driven lipogenesis (0.046 g/g cell /hr) by obligatory NADPH synthesis through the pentose cycle. Together, synergistic cofeeding produces CO 2 -derived lipids with 38% energetic efficiency and demonstrates potential to convert CO 2 into advanced bioproducts.

Dehalococcoides strains respire a wide variety of chloro-organic compounds and are important for the bioremediation of toxic, persistent, carcinogenic, and ubiquitous ground water pollutants. In order to better understand metabolism and optimize their application, we have developed a pan-genome-scale metabolic network and constraint-based metabolic model of Dehalococcoides. The pan-genome was constructed from publicly available complete genome sequences of Dehalococcoides sp. strain CBDB1, strain 195, strain BAV1, and strain VS. We found that Dehalococcoides pan-genome consisted of 1118 core genes (shared by all), 457 dispensable genes (shared by some), and 486 unique genes (found in only one genome). The model included 549 metabolic genes that encoded 356 proteins catalyzing 497 gene-associated model reactions. Of these 497 reactions, 477 were associated with core metabolic genes, 18 with dispensable genes, and 2 with unique genes. This study, in addition to analyzing the metabolism of an environmentally important phylogenetic group on a pan-genome scale, provides valuable insights into Dehalococcoides metabolic limitations, low growth yields, and energy conservation. The model also provides a framework to anchor and compare disparate experimental data, as well as to give insights on the physiological impact of “incomplete” pathways, such as the TCA-cycle, CO2 fixation, and cobalamin biosynthesis pathways. The model, referred to as iAI549, highlights the specialized and highly conserved nature of Dehalococcoides metabolism, and suggests that evolution of Dehalococcoides species is driven by the electron acceptor availability.

No abstract

Moorella thermoacetica is a strictly anaerobic, endospore-forming, and metabolically versatile acetogenic bacterium capable of conserving energy by both autotrophic (acetogenesis) and heterotrophic (homoacetogenesis) modes of metabolism. Its metabolic diversity and the ability to efficiently convert a wide range of compounds, including syngas (CO + H2) into acetyl-CoA have made this thermophilic bacterium a promising host for industrial biotechnology applications. However, lack of detailed information on M. thermoacetica's metabolism is a major impediment to its use as a microbial cell factory. In order to overcome this issue, a genome-scale constraint-based metabolic model of Moorella thermoacetica, iAI558, has been developed using its genome sequence and physiological data from published literature. The reconstructed metabolic network of M. thermoacetica comprises 558 metabolic genes, 705 biochemical reactions, and 698 metabolites. Of the total 705 model reactions, 680 are gene-associated while the rest are non-gene associated reactions. The model, in addition to simulating both autotrophic and heterotrophic growth of M. thermoacetica, revealed degeneracy in its TCA-cycle, a common characteristic of anaerobic metabolism. Furthermore, the model helped elucidate the poorly understood energy conservation mechanism of M. thermoacetica during autotrophy. Thus, in addition to generating experimentally testable hypotheses regarding its physiology, such a detailed model will facilitate rapid strain designing and metabolic engineering of M. thermoacetica for industrial applications.

No abstract

Mono-ethylene glycol (MEG) is an important petrochemical with widespread use in numerous consumer products. The current industrial MEG-production process relies on non-renewable fossil fuel-based feedstocks, such as petroleum, natural gas, and naphtha; hence, it is useful to explore alternative routes of MEG-synthesis from gases as they might provide a greener and more sustainable alternative to the current production methods. Technologies of synthetic biology and metabolic engineering of microorganisms can be deployed for the expression of new biochemical pathways for MEG-synthesis from gases, provided that such promising alternative routes are first identified. We used the BNICE.ch algorithm to develop novel and previously unknown biological pathways to MEG from synthesis gas by leveraging the Wood-Ljungdahl pathway of carbon fixation of acetogenic bacteria. We developed a set of useful pathway pruning and analysis criteria to systematically assess thousands of pathways generated by BNICE.ch. Published genome-scale models of Moorella thermoacetica and Clostridium ljungdahlii were used to perform the pathway yield calculations and in-depth analyses of seven (7) newly developed biological MEG-producing pathways from gases, including CO, CO, and H. These analyses helped identify not only better candidate pathways, but also superior chassis organisms that can be used for metabolic engineering of the candidate pathways. The pathway generation, pruning, and detailed analysis procedures described in this study can also be used to develop biochemical pathways for other commodity chemicals from gaseous substrates.