Cloning and elucidation of the FR901464 gene cluster revealing a complex acyltransferase-less polyketide synthase using glycerate as starter units.

FR901464, an antitumor natural product, represents a new class of potent anticancer small molecules targeting spliceosome and inhibiting both splicing and nuclear retention of pre-mRNA. Herein we describe the biosynthetic gene cluster of FR901464, identified by degenerate primer PCR amplification of a gene encoding the 3-hydroxy-3-methylglutaryl-CoA synthase (HCS) postulated to be involved in the biosynthesis of a ß-branched polyketide from Pseudomonas sp. No. 2663. This cluster consists of twenty open reading frames (ORFs) and was localized to 93-kb DNA segment, and its involvement in FR901464 biosynthesis was confirmed by gene inactivation and complementation. FR901464 is biosynthesized by a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS), HCS, and acyltransferases (AT)-less system. The PKS/NRPS modules feature unusual domain organization including multiple domain redundancy, inactivation, and tandem. Biochemical characterization of a glyceryl transferase and an acyl carrier protein (ACP) in the start module revealed that it incorporates D-1,3-bisphosphoglycerate, which is dephosphorylated and transferred to ACP as the starter unit. Furthermore, an oxidative Baeyer-Villiger reaction followed by chain release was postulated to form a pyran moiety. On the basis of in silico analysis and genetic and biochemical evidances, a biosynthetic pathway for FR901464 was proposed, which sets the stage to further investigate the complex PKS biochemically and engineer the biosynthetic machinery for the production of novel analogues.

Genetic polymorphism of catechol-O-methyltransferase modulates the association of green tea consumption and lung cancer.

Tea polyphenols are strong antioxidants, which can be rapidly O-methylated by catechol-O-methyltransferase (COMT). Thus, it is possible that the genetic polymorphism of COMT can modulate the association of green tea consumption and lung cancer. Here, we designed a case-control study to evaluate the combined effect of green tea consumption and COMT genotypes on the risk of lung cancer. A total of 237 lung cancer patients and 474 healthy controls were recruited. Questionnaires were administered to obtain demographic data, smoking status, green tea consumption, fruits and vegetables intake, exposure to cooking fumes, and family history of lung cancer. Genotypes for COMT were identified by PCR. Smoking, green tea consumption, exposure to cooking fumes, and family history of lung cancer were associated with the development of lung cancer. When green tea drinkers carrying COMT HL/LL genotypes were selected as the reference group, drinkers carrying the COMT HH genotype had a higher risk for the development of lung cancer (odds ratio: 1.97, 95% confidence interval: 0.99-3.91). Among the current and ever smokers, the elevated risk for lung cancer was more apparent in green tea drinkers carrying the COMT HH genotype compared with green tea drinkers carrying COMT HL/LL genotypes (odds ratio: 5.84, 95% confidence interval: 1.75-19.45). Green tea drinkers with greater activity of the COMT genotype, whereby polyphenols are effectively excluded, will gain fewer protective benefits against lung cancer development.

Unconventional origin and hybrid system for construction of pyrrolopyrrole moiety in kosinostatin biosynthesis.

Kosinostatin (KST), an antitumor antibiotic, features a pyrrolopyrrole moiety spirally jointed to a five-membered ring of an anthraquinone framework glycosylated with a γ-branched octose. By a combination of in silico analysis, genetic characterization, biochemical assay, and precursor feeding experiments, a biosynthetic pathway for KST was proposed, which revealed (1) the pyrrolopyrrole moiety originates from nicotinic acid and ribose, (2) the bicyclic amidine is constructed by a process similar to the tryptophan biosynthetic pathway, and (3) a discrete adenylation enzyme and a peptidyl carrier protein (PCP) are responsible for producing a PCP-tethered building block parallel to type II polyketide synthase (PKS) rather than for the PKS priming step by providing the starter unit. These findings provide an opportunity to further explore the inexplicable enzymatic logic that governs the formation of pyrrolopyrrole moiety and the spirocyclic skeleton.