Si-Hyung Park

Characterization of alkyl thiosulfinate in Allium hookeri root using HPLC-ESI-MS

Allicin produced by alliinase system of Allium hookeri was evaluated via high performance liquid chromatography (HPLC). Allicin contents of A. hookeri were 56.6±3.5 μg per g of fresh root and 12.7±3.2 μg per g of fresh stem. These values were relatively low as compared with garlic. HPLC-electrospray ionization-mass spectrometry analyses showed A. hookeri root extract contained ten alkyl thiosulfinates, and the chemical structures were characterized by MS/MS analyses.

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.

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.

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.

Delineating citrinin biosynthesis: Ctn-ORF3 dioxygenase-mediated multi-step methyl oxidation precedes a reduction-mediated pyran ring cyclization

Citrinin (3) is a polyketide-derived mycotoxin, that is, produced by Monascus, Penicillium, and Aspergillus spp. and is a common contaminant in a number of agricultural products. ctPKS, a non-reducing type iterative polyketide synthase with a C-terminal reductive domain, is proposed to generate the polyketide backbone of 3. The targeted gene inactivation of ctn-orf1 or ctn-orf3 gene resulted in the accumulation of a benzaldehyde derivative 6, and the ectopic expression of ctPKS/ctnB in yeast produced 6, demonstrating that ctPKS generates 6 with the support of CtnB and suggesting that Ctn-ORF1/Ctn-ORF3 converts 6 into 3. The Δctn-orf1 mutant also produced a novel benzdialdehyde derivative 10. When either 6 or 10 was fed into a ΔctPKS mutant, 3 was readily detected, which confirms that both 6 and 10 are involved in the biosynthesis of 3. A bioconversion experiment of 6 in the ectopic expression system demonstrated that ctn-orf3 expression, but not ctn-orf1 expression, efficiently consumed 6. The resulting metabolite(s) of 6 could not be identified, however. A recombinant Ctn-ORF3 enzyme was demonstrated to convert 6 into 10 and a hypothetical carboxylic derivative 8, which substantiates that Ctn-ORF3 oxidizes the exocyclic methyl moiety of 6. Ctn-ORF1 is thus proposed to reduce 8 and the subsequent non-enzymatic reactions to complete the biosynthesis of 3. The present study delineates the biosynthetic route of 3, proposing the biochemical mechanism, that is, involved in producing the natural dihydropyranoquinone structure.

A Single Module Type I Polyketide Synthase Directs De Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

Background: Galbonolide (GAL) was proposed to be synthesized by a modular type I polyketide synthase (PKS) with GalA-E serving a supporting role. Results: GalA-C constitute a sole type I PKS that is involved in GAL biosynthesis in S. galbus. Conclusion: GalA-C catalyze the de novo formation of GAL macrolactone. Significance: GalA-C constitute a novel iterative PKS that incorporates methylmalonate units with highly programmed β-keto group modifications. Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-13C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed β-keto group modifications.

Transformation of menthane monoterpenes by Mentha piperita cell culture

Abstract(+)-Isopiperitenone (100 mg l−1) was converted into (4S,6R)-6-hydroxy- and (4S,8R)-8,9-epoxyisopiperitenone, aside from the already known (+)-7-hydroxyisopiperitenone, by suspension cell culture of Mentha piperita. As (–)-isopiperitenone was hydroxylated similarly, this implies that the hydroxylating enzyme(s) have a broad substrate stereospecificity in regards to the stereochemistry at C4. (–)-(4R)-Carvone was reduced by the Mentha cells both at carbonyl and C1-C6 double bond to give (1R,2S,4R)-neodihydrocarveol and (1R,2R,4R)-dihydrocarveol with the former being the major product. (+)-(4S)-Carvone had a similar reduction pattern, producing (1S,2R,4S)-neodihydrocarveol and (1S,4S)-dihydrocarvone. Formation of these compounds indicates that the peppermint cell culture cannot only hydroxylate the allylic position but also reduce the α,β-unsaturated carbonyl system.

Inactivation of the oxidase gene mppG results in the selective loss of orange azaphilone pigments in Monascus purpureus

Monascus species are filamentous ascomycetes fungi and produce azaphilone (Az) pigment that is a well-known food colorant. Az is a class of fungal polyketides that bears a highly oxygenated pyranoquinone bicyclic core and is produced by a nonreducing fungal polyketide synthase with a reductive release domain (NR-fPKS-R). MpPKS5 encodes an NR-fPKS-R for Monascus Az (MAz) and is clustered with four oxidoreductase genes including mppG; mpp designates Monascus pigment production. MAz pigments are classified as yellow and orange MAz, and their structures differ in two hydride reductions with yellow MAz as the reduced type. The biosynthesis of yellow MAz (monascin, Y-1 and ankaflavin, Y-2) is completed by a reductive pathway involving a reductase gene mppE. This reductive pathway is diverged from a common MAz pathway involving two other reductase genes of mppA and mppC. This suggests that the biosynthesis of orange MAz (rubropunctatin, O-1 and monascorubrin, O-2) is completed by an oxidative branch pathway and the cognate oxidative role of mppG is genetically characterized in the present study. A targeted gene inactivation mutant of ΔmppG displayed a severe impairment in the production of orange MAz with no significant alteration in the level of yellow MAz. The feeding experiment with Y-1 in ΔMpPKS5 indicated that Y-1 could not be converted into O-1, which excludes the possibility that mppG mediates the conversion of yellow into orange MAz. This study supports the existence of divergent pathways in MAz biosynthesis and creates a recombinant strain for the selective production of yellow MAz.

Immunological activities of cationic methylan derivatives

Methylan polysaccharide was aminated to add dialkylaminoethyl and free amino groups at hydroxyl sites in the methylan backbone, and these derivatives were quaternized to produce pH-independent cationic polyelectrolytes. The immunological activities of the resulting methylan derivatives were investigated. Diethylaminoethyl (DEAE)-methylan derivatives inhibited the classical pathway of the complement system in a dose-dependent way. Quaternized DEAE-methylan exhibited the highest anticomplementary activities among the all derivatives. Anticomplementary activities increased significantly as the cationic charge of the methylan derivatives increased via aminoderivatization followed by quaternization, indicating that there is an electrostatic interaction between the methylan derivatives and the negatively charged functional residues on the cell.