Hyung-Jin Kwon

Genetic localization and in vivo characterization of a Monascus azaphilone pigment biosynthetic gene cluster

Monascus spp. produce several well-known polyketides such as monacolin K, citrinin, and azaphilone pigments. In this study, the azaphilone pigment biosynthetic gene cluster was identified through T-DNA random mutagenesis in Monascus purpureus. The albino mutant W13 bears a T-DNA insertion upstream of a transcriptional regulator gene (mppR1). The transcription of mppR1 and the nearby polyketide synthase gene (MpPKS5) was significantly repressed in the W13 mutant. Targeted inactivation of MpPKS5 also gave rise to an albino mutant, confirming that mppR1 and MpPKS5 belong to an azaphilone pigment biosynthetic gene cluster. This M. purpureus sequence was used to identify the whole biosynthetic gene cluster in the Monascus pilosus genome. MpPKS5 contains SAT/KS/AT/PT/ACP/MT/R domains, and this domain organization is preserved in other azaphilone polyketide synthases. This biosynthetic gene cluster also encodes fatty acid synthase (FAS), which is predicted to assist the synthesis of 3-oxooactanoyl-CoA and 3-oxodecanoyl-CoA. These 3-oxoacyl compounds are proposed to be incorporated into the azaphilone backbone to complete the pigment biosynthesis. A monooxygenase gene (an azaH and tropB homolog) that is located far downstream of the FAS gene is proposed to be involved in pyrone ring formation. A homology search on other fungal genome sequences suggests that this azaphilone pigment gene cluster also exists in the Penicillium marneffei and Talaromyces stipitatus genomes.

Effects of extracellular ATP on the physiology of Streptomyces coelicolor A3(2)

Because ATP is an extracellular effector in animal and plant systems and derivatives of ATP, such as S-adenosylmethionine and cAMP, can control antibiotic production and morphological differentiation in Streptomyces, we hypothesized that extracellular ATP (exATP) can also affect physiologies of Streptomyces. We found that the addition of 10 microM exATP to Streptomyces coelicolor A3(2) cultures resulted in enhanced actinorhodin and undecylprodigiosin production and morphological differentiation on solid medium. However, these phenotypes were reduced by the addition of a 10-fold higher concentration of exATP (100 microM). Intracellular ATP concentrations were also modulated in response to changes in exATP. ATP analogs, added at a 100-fold lower concentration, affected Streptomyces similarly to that seen for 10 microM exATP. The enhanced promoter activity of actII-orf4 indicated that 10 microM exATP affect the transcriptional level for actinorhodin production. Results from this study suggest that exATP is an effector for the physiology of S. coelicolor and careful manipulation of exATP may significantly enhance the high-yield production of antibiotics by S. coelicolor.

Gene inactivation study of gntE reveals its role in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis.

A gene inactivation study was performed on gntE, a member of the gentamicin biosynthetic gene cluster in Micromonospora echinospora. Computer-aided homology analysis predicts a methyltransferase-related cobalamin-binding domain and a radical S-adenosylmethionine domain in GntE. It is also found that there is no gntE homolog within other aminoglycoside biosynthetic gene clusters. Inactivation of gntE was achieved in both M. echinospora ATCC 15835 and a gentamicin high-producer GMC106. High-performance liquid chromatographic analysis, coupled with mass spectrometry, revealed that gntE mutants accumulated gentamicin A2 and its derivative with a methyl group installed on the glucoamine moiety. This result substantiated that GntE participates in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis, though the catalytic nature of this unusual oxidoreductase/methyltransferase candidate is not resolved. The present gene inactivation study also demonstrates that targeted genetic engineering can be applied to produce specific gentamicin structures and potentially new gentamicin derivatives in M. echinospora.

Monascus azaphilone pigment biosynthesis employs a dedicated fatty acid synthase for short chain fatty acyl moieties

The biosynthetic gene cluster of Monascus azaphilone pigments (MAzPs) encodes a canonical fatty acid synthase, MpFAS2. It is thus proposed that MpFAS2 plays a role in MAzP biosynthesis by supplying short chain (C8 and C10) fatty acyl moieties. Targeted gene inactivation of MpfasB2 in Monascus purpureus generated an F9 mutant, which developed white hyphae that discharged a yellow color on potato dextrose agar. High-performance liquid chromatography analysis demonstrated that F9 was incapable of producing MAzP and accumulated a wide array of chromophoric compounds instead. The main compound found in F9 was monascusone A, a hydrogenated azaphilone lacking a fatty acyl moiety. Gas chromatography analysis of the fatty acid methyl esters indicated that there was no significant difference in the cellular fatty acyl (C16 and C18) contents between WT and F9. The present study demonstrates that the dedicated fatty acid synthase is required to decorate the azaphilone polyketides in MAzP biosynthesis.

Delineating Monascus azaphilone pigment biosynthesis: oxidoreductive modifications determine the ring cyclization pattern in azaphilone biosynthesis†

The product profiles of mppF, mppA, and mppC mutants substantiate that MppA-mediated ω-2 ketoreduction is a prerequisite for the synthesis of the pyranoquinone bicyclic core of the Monascus azaphilone pigment and that MppC activity determines the regioselectivity of the spontaneous Knoevenagel condensation.

Characterization of 2-Octenoyl-CoA Carboxylase/Reductase Utilizing pteB from Streptomyce avermitilis

The filipin biosynthetic gene cluster of Streptomyces avermitilis contains pteB, a homolog of crotonyl-CoA carboxylase/reductase. PteB was predicted to be 2-octenoyl-CoA carboxylase/reductase, supplying hexylmalonyl-CoA to filipin biosynthesis. Recombinant PteB displayed selective reductase activity toward 2-octenoyl-CoA while generating a broad range of alkylmalonyl-CoAs in the presence of bicarbonate.

A reductase gene mppE controls yellow component production in azaphilone polyketide pathway of Monascus

ObjectivesTo characterize a biosynthetic gene that is selectively involved in the biosynthesis of yellow or orange components in the azaphilone polyketide pathway of Monascus.ResultsA reductive modification is predicted to control the relative levels of reduced (yellow) and oxidized (orange and red) components in the pathway of azaphilone pigment biosynthesis in Monascus. Targeted inactivation of a reductase gene mppE enhanced orange and red pigment production whereas overexpression of the gene promoted yellow pigment production. The effect of mppE overexpression was dependent on culture methods, and augmented yellow pigmentation was evident in a submerged culture employing a chemically defined medium.ConclusionsMppE controls the biosynthesis of the yellow pigments, ankaflavin and monascin, as a reductive enzyme in the azaphilone polyketide pathway.

Catalytic Domain of AfsKav Modulates Both Secondary Metabolism and Morphologic Differentiation in Streptomyces avermitilis ATCC 31272

Genetic characterization of afsK-av (SAV3816) in Streptomyces avermitilis ATCC 31272 was performed to evaluate the role(s) of this eukaryotic-type serine–threonine protein kinase (STPK) in the regulation of morphologic differentiation and secondary metabolism. The afsK-av::neo mutant (SJW4001) was defective in sporulation, melanogenesis, and avermectin production. These phenotypic defects were complemented by introduction of either the intact afsK-av or the 900-nt catalytic domain region. The catalytic domain restored sporulation and melanogenesis to SJW4001 whereas it partially recovered avermectin production. This study reveals that AfsKav is a pleiotropic regulator and demonstrates in vivo that the C-region of AfsKav is not essential for its regulatory role in S. avermitilis differentiations.

The methoxymalonyl-acyl carrier protein biosynthesis locus and the nearby gene with the β-ketoacyl synthase domain are involved in the biosynthesis of galbonolides in Streptomyces galbus, but these loci are separate from the modular polyketide synthase gene cluster.

Galbonolides A and B are antifungal compounds, which are produced by Streptomyces galbus. A multimodular polyketide synthase (PKS) was predicted to catalyze their biosynthesis, and a methoxymalonyl-acyl carrier protein (methoxymalonyl-ACP) was expected to be involved in the biosynthesis of galbonolide A. Cloning of a methoxymalonyl-ACP biosynthesis locus (galGHIJK) and the flanking regions has revealed that the locus is colocalized with beta-ketoacyl synthase (KAS)-related genes (orf3, 4, and 5), but separated from any multimodular PKS gene cluster in S. galbus. A galI-disruption mutant (SK-galI-5) is unable to produce galbonolide A, but can synthesize galbonolide B, indicating that galGHIJK is involved in the biosynthesis of galbonolide A. A disruption mutant of orf4 is severely impaired in the production of both galbonolides A and B. These results indicate that galGHIJK and the KAS genes are involved in the biosynthesis of galbonolides, although they are not colocalized with a multimodular PKS gene cluster. We further propose that a single galbonolide PKS generates two discrete structures, galbonolides A and B, by alternatively incorporating methoxymalonate and methylmalonate, respectively.

Gene Inactivation Study on gntK, a Putative C-methyltransferase Gene in Gentamicin Biosynthesis from Micromonospora echinospora

GntK harbors methyltransferase-related cobalamin-binding domain and radical S-adenosylmethionine domain. The gntK-inactivation mutant of Micromonospora echinospora accumulated higher levels of gentamicin Cla and lower levels of gentamicin C1 and C2 isomers compared to the wild-type strain. Based on these results, we propose that GntK is involved in C-methylation on C-6′ in gentamicin X2 but is dispensable in gentamicin biosynthesis.