ABSTRACT
Tryprostatin A and B are indole alkaloid-based fungal products that inhibit mammalian cell cycle at the G2/M phase. They are biosynthetic intermediates of fumitremorgins produced by a complex pathway involving a nonribosomal peptide synthetase (FtmA), a prenyltransferase (FtmB), a cytochrome P450 hydroxylase (FtmC), an O-methyltransferase (FtmD), and several additional enzymes. A partial fumitremorgin biosynthetic gene cluster (ftmABCD) from Aspergillus sp. was reconstituted in Escherichia coli BL21(DE3) cells, with or without the co-expression of an Sfp-type phosphopantetheinyltransferase gene (Cv_sfp) from Chromobacterium violaceum No. 968. Several recombinant E. coli strains produced tryprostatin B up to 106 mg/l or tryprostatin A up to 76 mg/l in the fermentation broth under aerobic condition, providing an effective way to prepare those pharmaceutically important natural products biologically.
ABSTRACT
The biosynthetic gene cluster of FK228, an FDA-approved anticancer natural product, was identified and sequenced previously. The genetic organization of this gene cluster has now been delineated through systematic gene deletion and transcriptional analysis. As a result, the gene cluster is redefined to contain 12 genes: depA through depJ, depM, and a newly identified pathway regulatory gene, depR.
Subject(s)
Chromobacterium/genetics , Depsipeptides/biosynthesis , Genes, Bacterial , Antibiotics, Antineoplastic/biosynthesis , Base Sequence , Chromatography, Liquid , Gene Deletion , Gene Expression Regulation, Bacterial , Mass Spectrometry , Multigene Family , Operon , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Functional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide in Chromobacterium violaceum strain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway in Escherichia coli cells, two essential genes, fabD1 and fabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineered E. coli strains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of the trans complementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.
Subject(s)
Chromobacterium/genetics , Chromobacterium/metabolism , Depsipeptides/biosynthesis , Escherichia coli/genetics , Fatty Acid Synthases/metabolism , Polyketide Synthases/metabolism , Acyl-Carrier Protein S-Malonyltransferase/genetics , Acyl-Carrier Protein S-Malonyltransferase/metabolism , Anaerobiosis , Antibiotics, Antineoplastic/biosynthesis , Bacterial Proteins/metabolism , Biosynthetic Pathways , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Fatty Acid Synthase, Type II/genetics , Fermentation , Gene Expression , Genetic Engineering , Molecular Sequence Data , Multigene Family , Peptide Biosynthesis, Nucleic Acid-Independent , Polymerase Chain Reaction , Sequence Deletion , Transferases (Other Substituted Phosphate Groups)/metabolismABSTRACT
Disulfide bonds are rare in bacterial natural products, and the mechanism of disulfide bond formation in those products is unknown. Here we characterize a gene and its product critical for a disulfide bond formation in FK228 anticancer depsipeptide in Chromobacterium violaceum. Deletion of depH drastically reduced FK228 production, whereas complementation of the depH-deletion mutant with a copy of depH on a medium copy-number plasmid not only fully restored the FK228 production but also significantly increased the FK228 yield. Purified 6xHis-tagged DepH fusion protein in native form is a homodimer of 71.0 kDa, with each monomer containing one molecule of FAD. DepH efficiently converts an immediate FK228 precursor to FK228 in the presence of NADP(+). We conclude that DepH is an FAD-dependent pyridine nucleotide-disulfide oxidoreductase, specifically and efficiently catalyzing a disulfide bond formation in FK228.
