Biodiesel is an alternative energy source and a substitute for petroleum-based diesel fuel. It is produced from renewable biomass by transesterification of triacylglycerols from plant oils, yielding monoalkyl esters of long-chain fatty acids with short-chain alcohols such as fatty acid methyl esters and fatty acid ethyl esters (FAEEs). Despite numerous environmental benefits, a broader use of biodiesel is hampered by the extensive acreage required for sufficient production of oilseed crops. Therefore, processes are urgently needed to enable biodiesel production from more readily available bulk plant materials like sugars or cellulose. Toward this goal, the authors established biosynthesis of biodiesel-adequate FAEEs, referred to as Microdiesel, in metabolically engineeredEscherichia coli. This was achieved by heterologous expression inE. coliof theZymomonas mobilispyruvate decarboxylase and alcohol dehydrogenase and the unspecific acyltransferase fromAcinetobacter baylyistrain ADP1. By this approach, ethanol formation was combined with subsequent esterification of the ethanol with the acyl moieties of coenzyme A thioesters of fatty acids if the cells were cultivated under aerobic conditions in the presence of glucose and oleic acid. Ethyl oleate was the major constituent of these FAEEs, with minor amounts of ethyl palmitate and ethyl palmitoleate. FAEE concentrations of 1.28 g l⁻¹and a FAEE content of the cells of 26 % of the cellular dry mass were achieved by fed-batch fermentation using renewable carbon sources. This novel approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing engineered micro-organisms, enabling a broader use of biodiesel-like fuels in the future.

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

Triacylglycerols (TAGs) and wax esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates wax esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS) as well as acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA acyltransferase is not related to known acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatismc²155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinantPseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.

Summary: Neutral lipid accumulation is frequently observed in some Gram‐negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation of wax ester‐ and triacylglycerol (TAG)‐bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods. A general model for prokaryotic lipid‐body formation is proposed, clearly differing from the current models for the formation of lipid inclusions in eukaryotes and of poly(hydroxyalkanoic acid) (PHA) inclusions in prokaryotes. Formation of lipid‐bodies starts with the docking of wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase (WS/DGAT) to the cytoplasm membrane. Both, analyses of in vivo and in vitro lipid‐body synthesis, demonstrated the formation of small lipid droplets (SLDs), which remain bound to the membrane‐associated enzyme. SLDs conglomerated subsequently to membrane‐bound lipid‐prebodies which are then released into the cytoplasm. The formation of matured lipid‐bodies in the cytoplasm occurred by means of coalescence of SLDs inside the lipid prebodies, which are surrounded by a half‐unit membrane of phospholipids.

Mycobacterium tuberculosis, the etiological agent of TB, possesses a cholesterol catabolic pathway implicated in pathogenesis. This pathway includes an iron-dependent extradiol dioxygenase, HsaC, that cleaves catechols. Immuno-compromised mice infected with a ΔhsaC mutant of M. tuberculosis H37Rv survived 50% longer than mice infected with the wild-type strain. In guinea pigs, the mutant disseminated more slowly to the spleen, persisted less successfully in the lung, and caused little pathology. These data establish that, while cholesterol metabolism by M. tuberculosis appears to be most important during the chronic stage of infection, it begins much earlier and may contribute to the pathogen's dissemination within the host. Purified HsaC efficiently cleaved the catecholic cholesterol metabolite, DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione; k_cat/K_m = 14.4±0.5 µM⁻¹ s⁻¹), and was inactivated by a halogenated substrate analogue (partition coefficient<50). Remarkably, cholesterol caused loss of viability in the ΔhsaC mutant, consistent with catechol toxicity. Structures of HsaC:DHSA binary complexes at 2.1 Å revealed two catechol-binding modes: bidentate binding to the active site iron, as has been reported in similar enzymes, and, unexpectedly, monodentate binding. The position of the bicyclo-alkanone moiety of DHSA was very similar in the two binding modes, suggesting that this interaction is a determinant in the initial substrate-binding event. These data provide insights into the binding of catechols by extradiol dioxygenases and facilitate inhibitor design. Mycobacterium tuberculosis, the etiological agent of TB, is the most devastating infectious agent of mortality worldwide: it is carried by one-third of all humans and kills nearly two million people annually. Recent work has established that the pathogen metabolizes cholesterol, although the role of this metabolism in pathogenesis remains unclear. In the current study, we demonstrate that HsaC is a key enzyme in the cholesterol catabolic pathway and that it can be inactivated by compounds that resemble its substrate. Using molecular genetic approaches, we demonstrated that the enzyme is essential for the growth of M. tuberculosis on cholesterol and that a lack of this enzyme impairs the survival of the pathogen in each of two animal models. These studies provide definitive evidence that M. tuberculosis metabolizes cholesterol during infection and that this metabolism occurs during the early stages of infection. The oxygen-utilizing enzymes of the cholesterol catabolic pathway, of which HsaC is but one example, are intriguing potential chemotherapeutic targets, as their inhibition can lead to toxic metabolites, including reactive oxygen species. Overall, our study combines a variety of approaches to provide novel insights into a disease of global importance and into the mechanism of an interesting class of enzymes.