Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production.

The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action, high potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism. Previously, we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum. Four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization. Therefore, we used a metabolic engineering approach to increase heterologous production of brasilicardin in A. japonicum. Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate (IPP) and to channel the precursor into the direction of diterpenoid biosynthesis. Both approaches contributed to an elevated heterologous production of the brasilicardin backbone, which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1.

C: Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( xS, EAF, G P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints xS, DCF and G P. In all thiolases, OAH2 is formed by the main chain NH groups of two catalytic loops. In the well-studied CNH-thiolases, OAH1 is formed by a water (of the Wat-Asn(NEAF) dyad) and NE2 (of the GHP-histidine). In the two described liganded Lm-thiolase structures, it is seen that in this CHH-thiolase, OAH1 is formed by NE2 of His338 (HDCF) and His388 (GHP). Analysis of the OAH1 hydrogen bond networks suggests that the GHP-histidine is doubly protonated and positively charged in these complexes, whereas the HDCF histidine is neutral and singly protonated.

Functional characterization of thiolase-encoding genes from Xanthophyllomyces dendrorhous and their effects on carotenoid synthesis.

BACKGROUND: The basidiomycetous yeast Xanthophyllomyces dendrorhous has been described as a potential biofactory for terpenoid-derived compounds due to its ability to synthesize astaxanthin. Functional knowledge of the genes involved in terpenoid synthesis would create opportunities to enhance carotenoid production. A thiolase enzyme catalyzes the first step in terpenoid synthesis. RESULTS: Two potential thiolase-encoding genes were found in the yeast genome; bioinformatically, one was identified as an acetyl-CoA C-acetyltransferase (ERG10), and the other was identified as a 3-ketoacyl Co-A thiolase (POT1). Heterologous complementation assays in Saccharomyces cerevisiae showed that the ERG10 gene from X. dendrorhous could complement the lack of the endogenous ERG10 gene in S. cerevisiae, thereby allowing cellular growth and sterol synthesis. X. dendrorhous heterozygous mutants for each gene were created, and a homozygous POT1 mutant was also obtained. This mutant exhibited changes in pigment composition and higher ERG10 transcript levels than the wild type strain. CONCLUSIONS: The results support the notion that the ERG10 gene in X. dendrorhous is a functional acetyl-CoA C-acetyltransferase essential for the synthesis of mevalonate in yeast. The POT1 gene would encode a functional 3-ketoacyl Co-A thiolase that is non-essential for cell growth, but its mutation indirectly affects pigment production.

Structural insights into bacterial resistance to cerulenin.

UNLABELLED: Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant protein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichia coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl-ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a different phenotype in B. subtilis. The structural, biochemical and physiological data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin. DATABASE: Crystallographic data (including atomic coordinates and structure factors) have been deposited in the Protein Data Bank under accession codes 4LS5, 4LS6, 4LS7 and 4LS8.

Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches.

Acetoacetyl-CoA thiolase (EC 2.3.1.9), commonly named thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA and CoA. This enzyme acts in anabolic processes as the first step in the biosynthesis of isoprenoids and polyhydroxybutyrate in eukaryotes and bacteria, respectively. We have recently reported the evolutionary and functional equivalence of these enzymes, suggesting that thiolase II could be the rate limiting enzyme in these pathways and presented evidence indicating that this enzyme modulates the availability of reducing equivalents during abiotic stress adaptation in bacteria and plants. However, these results are not sufficient to clarify why thiolase II was evolutionary selected as a critical enzyme in the production of antioxidant compounds. Regarding this intriguing topic, we propose that thiolase II could sense changes in the acetyl-CoA/CoA ratio induced by the inhibition of the tricarboxylic acid cycle under abiotic stress. Thus, the high level of evolutionary and functional constraint of thiolase II may be due to the connection of this enzyme with an ancient and conserved metabolic route.