The biosynthetic gene cluster for the enediyne antitumor antibiotic maduropeptin (MDP) from Actinomadura madurae ATCC 39144 was cloned and sequenced. Cloning of the mdp gene cluster was confirmed by heterologous complementation of enediyne polyketide synthase (PKS) mutants from the C-1027 producer Streptomyces globisporus and the neocarzinostatin producer Streptomyces carzinostaticus using the MDP enediyne PKS and associated genes. Furthermore, MDP was produced, and its apo-protein isolated and N-terminal sequenced; the encoding gene, mdpA, was found to reside within the cluster. The biosynthesis of MDP is highlighted by two iterative type I PKSs -the enediyne PKS and a 6-methylsalicylic acid PKS; generation of (S)-3-(2-chloro-3-hydroxy-4-methoxyphenyl)-3-hydroxypropionic acid derived from L-α-tyrosine; a unique type of enediyne apo-protein; and a convergent biosynthetic approach to the final MDP chromophore. The results demonstrate a platform for engineering new enediynes by combinatorial biosynthesis and establish a unified paradigm for the biosynthesis of enediyne polyketides.

Mycobacterium tuberculosis produces a large number of structurally diverse lipids generated from the carboxylation products of acetyl-CoA and propionyl-CoA. A biotin-dependent acyl-CoA carboxylase was purified from M. tuberculosis H37Rv by avidin affinity chromatography, and the three major protein components were determined by N-terminal sequencing to be the 63-kDa ␣3-subunit (AccA3, Rv3285), the 59-kDa ␤5-subunit (AccD5, Rv3280), and the 56-kDa ␤4-subunit (AccD4, Rv3799). A minor protein of about 24 kDa that co-purified with the above subunits was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to be the product of Rv3281 that is located immediately downstream of the open reading frame encoding the ␤5-subunit. This protein displays identity over a short stretch of amino acids with the recently discovered ⑀-subunits of Streptomyces coelicolor, suggesting that it might be an ⑀-subunit of the mycobacterial acyl-CoA carboxylase. To test this hypothesis, the carboxylase subunits were expressed in Escherichia coli and purified. Acyl-CoA carboxylase activity was successfully reconstituted for the first time from purified subunits of the acyl-CoA carboxylase of M. tuberculosis. The reconstituted ␣3-␤5 showed higher activity with propionyl-CoA than with acetyl-CoA, and the addition of the ⑀-subunit stimulated the carboxylation by 3.2-and 6.3-fold, respectively. The ␣3-␤4 showed very low activity with the above substrates but carboxylated long chain acyl-CoA. This ⑀-subunit contains five sets of tandem repeats at the N terminus that are required for maximal enhancement of carboxylase activity. The Rv3281 open reading frame is co-transcribed with Rv3280 in the mycobacterial cell, and the level of ⑀-protein was highest during the log phase and decreased during the stationary phase.Biotin-dependent carboxylases are involved in the synthesis of malonyl-CoA and methylmalonyl-CoA. Most biotin-dependent enzymes contain three functional components: the biotin carboxylase (BC), 2 the biotin carboxyl carrier protein (BCCP), and the carboxyltransferase (CT) (1). The acyl-CoA carboxylation reaction occurs in two steps in two separate subsites of the enzyme. The first partial reaction involves the fixation of CO 2 on biotin and requires the cooperation of BC and BCCP components; the biotin group is moved to interact with the BC component, resulting in the formation of carboxyl biotin. This carboxyl biotin then swings out to the carboxyltransferase component, resulting in the formation of the carboxylated product (2).Acyl-CoA carboxylases from Mycobacterium tuberculosis and Mycobacterium bovis BCG have been purified previously and shown to have both propionyl-CoA carboxylase (PCC) and acetyl-CoA carboxylase (ACC) activities (3). Mycobacterium smegmatis PCC was reported to be composed of two subunits with the biotin being associated with the heavier subunit (4, 5), as found also in M. tuberculosis (3). M. tuberculosis has three accA genes annotated as an ␣-subunit that contains the BC and BCC...

Zorbamycin (1, ZBM) is a glycopeptide antitumor antibiotic first reported in 1971. The partial structures of 1 were speculated on the basis of its acid hydrolysis products, but the structure of the intact molecule has never been established. The low titer of 1 from the wild-type strain, combined with its acid-instability, has so far hampered its isolation. By random mutagenesis of Streptomyces flavoviridis ATCC21892, a wild-type producer of 1, with UV irradiation, two high-producing strains of 1, S. flavoviridis SB9000 and SB9001, were isolated. Under the optimized fermentation conditions, these two strains produced about 10 mg/L of 1, which was about 10-fold higher than the wild-type ATCC21892 strain, as estimated by HPLC analysis. Finally, 1 was isolated as both a 1-Cu complex and Cu-free molecule, and the intact structure of 1 was established on the basis of a combination of mass spectrometry and 1H and 13C NMR spectroscopic analyses.

The biosynthetic gene cluster for the glycopeptide-derived antitumor antibiotic zorbamycin (ZBM) was cloned by screening a cosmid library of Streptomyces flavoviridis ATCC 21892. Sequence analysis revealed 40 ORFs belonging to the ZBM biosynthetic gene cluster. However, only 23 and 22 ORFs showed striking similarities to the biosynthetic gene clusters for the bleomycins (BLMs) and tallysomycins (TLMs), respectively; the remaining ORFs do not show significant homology to ORFs from the related BLM and TLM clusters. The ZBM gene cluster consists of 16 nonribosomal peptide synthetase (NRPS) genes encoding eight complete NRPS modules, three incomplete didomain NRPS modules, and eight freestanding single NRPS domains or associated enzymes, a polyketide synthase (PKS) gene encoding one PKS module, six sugar biosynthesis genes, as well as genes encoding other biosynthesis and resistance proteins. A genetic system using Escherichia coli-Streptomyces flavoviridis intergeneric conjugation was developed to enable ZBM gene cluster boundary determinations and biosynthetic pathway manipulations.

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we reveal the genome sequence of S. tora at the chromosome level with 526 Mb (96%) assembled into 13 chromosomes. Comparison among related plant species shows that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora. Combining genomics, transcriptomics, metabolomics, and biochemistry, we identify a CHS-L gene contributing to the biosynthesis of anthraquinones. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants.