No abstract

Maytansinoids are potent antitumor agents found in plants and microorganisms. To elucidate their biosynthesis at the biochemical and genetic level and to set the stage for their structure modification through genetic engineering, we have cloned two gene clusters required for the biosynthesis of the maytansinoid, ansamitocin, from a cosmid library of Actinosynnema pretiosum ssp. auranticum ATCC 31565. This is a rare case in which the genes involved in the formation of a secondary metabolite are dispersed in separate regions in an Actinomycete. A set of genes, asm22-24, asm43-45, and asm47, was identified for the biosynthesis of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA). Remarkably, there are two AHBA synthase gene homologues, which may have different functions in AHBA formation. Four type I polyketide synthase genes, asmA-D, followed by the downloading asm9, together encode eight homologous sets of enzyme activities (modules), each catalyzing a specific round of chain initiation, elongation, or termination steps, which assemble the ansamitocin polyketide backbone. Another set of genes, asm13-17, encodes the formation of an unusual ''methoxymalonate'' polyketide chain extension unit that, notably, seems to be synthesized on a dedicated acyl carrier protein rather than as a CoA thioester. Additional ORFs are involved in postsynthetic modifications of the initial polyketide synthase product, which include methylations, an epoxidation, an aromatic chlorination, and the introduction of acyl and carbamoyl groups. Tentative functions of several asm genes were confirmed by inactivation and heterologous expression.

The authors note that Fig. 2 and its corresponding legend appeared incorrectly. The corrected figure and its corrected legend appear below.As a result of this correction, the authors note that on page 1288, right column, first full paragraph, lines 6-7, "the first substrate" should instead appear as "the second substrate"; that in the subsequent paragraph, line 2, "the A3 domain" should instead appear as "the A4 domain"; that in the same paragraph, lines 5-6, "A3 (for Val/Ile) and A4 (Val)" should instead appear as "A5 (for Val) and A6 (Ala)"; that on page 1290, right column, first full paragraph, line 10, "the A3 domain" should instead appear as "the A4 domain"; and that in the same paragraph, line 15, "A2 domain" should instead appear as "A3 domain."Also as a result of this correction, Fig. S1 appeared incorrectly. Please see separate SI Correction. These errors do not affect the conclusions of the article.

Covering: 1957 to 2011. 3-Amino-5-hydroxy benzoic acid (3,5-AHBA) is a precursor for a large group of natural products, including the family of naphthalenic and benzenic ansamycins, the unique saliniketals, and the family of mitomycins. This review covers the biosynthesis of AHBA-derived natural products from a molecular genetics, chemical, and biochemical perspectives, and 174 references are cited.

The complete gene cluster for biosynthesis of a polyene complex, FR-008, spans 137.2 kb of the genome of Streptomyces sp. FR-008 consisting of six genes for a modular PKS and 15 additional genes. The extensive similarity to the partially characterized candicidin gene cluster in Streptomyces griseus IMRU3570, especially for genes involved in mycosamine biosynthesis, prompted us to compare the compounds produced by Streptomyces sp. FR-008 and Streptomyces griseus IMRU3570, and we found that FR-008 and candicidin complex are identical. A model for biosynthesis of a set of four structurally related FR-008/candicidin compounds was proposed. Deletion of the putative regulatory genes abolished antibiotic production, while disruption of putative glycosyltransferase and GDP-ketosugar aminotransferase functionalities led to the productions of a set of nonmycosaminated aglycones and a novel polyene complex with attachment of altered sugar moiety, respectively.

A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics.

The unusual "glycolate" extender unit at C-9/C-10 of ansamitocin is not derived from 2-hydroxymalonyl-CoA or 2-methoxymalonyl-CoA, as demonstrated by feeding experiments with the corresponding 1-13C-labeled N-acetylcysteamine thioesters but is formed from an acyl carrier protein (ACP)-bound substrate, possibly 2-methoxymalonyl-ACP, elaborated by enzymes encoded by a subcluster of five genes, asm12-17, from the ansamitocin bisosynthetic gene cluster.

A 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.