Yohei Katsuyama

Curcuminoid Biosynthesis by Two Type III Polyketide Synthases in the Herb Curcuma longa

Curcuminoids found in the rhizome of turmeric, Curcuma longa, possess various biological activities. Despite much attention regarding the biosynthesis of curcuminoids because of their pharmaceutically important properties and biosynthetically intriguing structures, no enzyme systems have been elucidated. Here we propose a pathway for curcuminoid biosynthesis in the herb C. longa, which includes two novel type III polyketide synthases. One of the type III polyketide synthases, named diketide-CoA synthase (DCS), catalyzed the formation of feruloyldiketide-CoA by condensing feruloyl-CoA and malonyl-CoA. The other, named curcumin synthase (CURS), catalyzed the in vitro formation of curcuminoids from cinnamoyldiketide-N-acetylcysteamine (a mimic of the CoA ester) and feruloyl-CoA. Co-incubation of DCS and CURS in the presence of feruloyl-CoA and malonyl-CoA yielded curcumin at high efficiency, although CURS itself possessed low activity for the synthesis of curcumin from feruloyl-CoA and malonyl-CoA. These findings thus revealed the curcumin biosynthetic route in turmeric, in which DCS synthesizes feruloyldiketide-CoA, and CURS then converts the diketide-CoA esters into a curcuminoid scaffold.

In vitro synthesis of curcuminoids by type III polyketide synthase from Oryza sativa.

Curcuminoids, major components of the spice turmeric, are used as a traditional Asian medicine and a food additive. Curcumin, a representative curcuminoid, has received a great deal of attention because of its anti-inflammatory, anticarcinogenic, and antitumor activities. Here we report a novel type III polyketide synthase named curcuminoid synthase from Oryza sativa, which synthesizes bisdemethoxycurcumin via a unique mechanism from two 4-coumaroyl-CoAs and one malonyl-CoA. The reaction begins with the thioesterification of the thiol moiety of Cys-174 by a starter molecule, 4-coumaroyl-CoA. Decarboxylative condensation of the first extender substrate, malonyl-CoA, onto the thioester of 4-coumarate results in the formation of a diketide-CoA intermediate. Subsequent hydrolysis of the intermediate yields a β-keto acid, which in turn acts as the second extender substrate. The β-keto acid is then joined to the Cys-174-bound 4-coumarate by decarboxylative condensation to form bisdemethoxycurcumin. This reaction violates the traditional head-to-tail model of polyketide assembly; the growing diketide intermediate is hydrolyzed to a β-keto acid that subsequently serves as the second extender to form curcuminoids. Curcuminoid synthase appears to be capable of the synthesis of not only diarylheptanoids but also gingerol analogues, because it synthesized cinnamoyl(hexanoyl)methane, a putative intermediate of gingerol, from cinnamoyl-CoA and 3-oxo-octanoic acid.

Molecular Basis of Elansolid Biosynthesis: Evidence for an Unprecedented Quinone Methide Initiated Intramolecular Diels–Alder Cycloaddition/Macrolactonization

Elansolids A1/A2 (1) and B1–B3 (2–4) and the structurally unusual and highly reactive elansolid A3 (5) are new metabolites from the gliding bacterium Chitinophaga sancti (formerly Flexibacter spec. ; Scheme 1). While elansolid A2 (1*) shows antibiotic activity against Gram-positive bacteria in the range of 0.2 to 64 mgmL 1 and cytotoxicity against L929 mouse fibroblast cells with an IC50 value of 12 mgmL , the atropisomer elansolid A1 (1) is significantly less active. 3] The elansolids feature a bicyclo[4.3.0]nonane core which in the case of elansolids A1/A2 is part of a 19-membered macrolactone. Elansolid B1 is the corresponding seco acid of elansolids A1/A2, while the elansolids B2 and B3 are workup artifacts that result from nucleophilic addition of methanol and NH3, respectively, to

Production of curcuminoids by Escherichia coli carrying an artificial biosynthesis pathway

Curcuminoids, which are produced specifically by plants of the order Zingiberales, have long been used as food additives because of their aromatic, stimulant and colouring properties and as traditional Asian medicines because of their anti-tumour, antioxidant and hepatoprotective activities. Curcuminoids are therefore attractive targets for metabolic engineering. An artificial curcuminoid biosynthetic pathway, including reactions of phenylalanine ammonia-lyase (PAL) from the yeast Rhodotorula rubra, 4-coumarate : CoA ligase (4CL) from Lithospermum erythrorhizon and curcuminoid synthase (CUS) from rice (Oryza sativa), a type III polyketide synthase, was constructed in Escherichia coli for the production of curcuminoids. Cultivation of the recombinant E. coli cells in the presence of tyrosine or phenylalanine, or both, led to production of bisdemethoxycurcumin, dicinnamoylmethane and cinnamoyl-p-coumaroylmethane. Another E. coli system carrying 4CL and CUS genes was also used for high-yield production of curcuminoids from exogenously supplemented phenylpropanoid acids: p-coumaric acid, cinnamic acid and ferulic acid. The yields of curucminoids were up to approximately 100 mg l(-1). Furthermore, this system gave approximately 60 mg curcumin l(-1) from 10 g rice bran pitch, an industrial waste discharged during rice edible oil production, as a source of ferulic acid.

Identification and characterization of multiple curcumin synthases from the herb Curcuma longa

Curcuminoids are pharmaceutically important compounds isolated from the herb Curcuma longa. Two additional type III polyketide synthases, named CURS2 and CURS3, that are capable of curcuminoid synthesis were identified and characterized. In vitro analysis revealed that CURS2 preferred feruloyl‐CoA as a starter substrate and CURS3 preferred both feruloyl‐CoA and p‐coumaroyl‐CoA. These results suggested that CURS2 synthesizes curcumin or demethoxycurcumin and CURS3 synthesizes curcumin, bisdemethoxycurcumin and demethoxycurcumin. The availability of the substrates and the expression levels of the three different enzymes capable of curcuminoid synthesis with different substrate specificities might influence the composition of curcuminoids in the turmeric and in different cultivars.

Regio- and stereospecific hydroxylation of various steroids at the 16α position of the D ring by the Streptomyces griseus cytochrome P450 CYP154C3.

ABSTRACT Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio- and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, Δ4-androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16α-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio- and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (Kd ) values of the binding between CYP154C3 and these steroids were less than 8 μM as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of Δ4-androstene-3,17-dione were determined (Km , 31.9 ± 9.1 μM; k cat, 181 ± 4.5 s−1). We concluded that CYP154C3 is a steroid D-ring 16α-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16α-specific hydroxylation activity.

Precursor-directed biosynthesis of curcumin analogs in Escherichia coli.

Curcuminoids, natural products in the rhizome of turmeric, show various biological activities, including antioxidant and antitumor activities. For this reason, curcuminoids have been focused on as potential pharmaceuticals. Exogenous supplementation with various carboxylate precursors in genetically engineered Escherichia coli cells carrying an artificially assembled pathway for curcuminoid biosynthesis led to the production of 17 unnatural curcuminoids.

Type III Polyketide Synthases in Microorganisms

Type III polyketide synthases (PKSs) are simple homodimers of ketosynthases which catalyze the condensation of one to several molecules of extender substrate onto a starter substrate through iterative decarboxylative Claisen condensation reactions. Type III PKSs have been found in bacteria and fungi, as well as plants. Microbial type III PKSs, which are involved in the biosynthesis of some lipidic compounds and various secondary metabolites, have several interesting characteristics that are not shared by plant type III PKSs. Further, many compounds produced by microbial type III PKSs have significant biological functions and/or important pharmaceutical activities. Thus, studies on this class of enzymes will expand our knowledge of the biosynthetic machineries that generate natural products and generate new findings about microbial physiology. The recent development of next-generation DNA sequencing has allowed for an increase in the number of microbial genomes sequenced and the discovery of many microbial type III PKS genes. Here, we describe basic methods to study microbial type III PKSs whose genes are easy to clone.

Alkylresorcylic acid synthesis by type III polyketide synthases from rice Oryza sativa

Alkylresorcinols, produced by various plants, bacteria, and fungi, are bioactive compounds possessing beneficial activities for human health, such as anti-cancer activity. In rice, they accumulate in seedlings, contributing to protection against fungi. Alkylresorcylic acids, which are carboxylated forms of alkylresorcinols, are unstable compounds and decarboxylate readily to yield alkylresorcinols. Genome mining of the rice Oryza sativa identified two type III polyketide synthases, named ARAS1 (alkylresorcylic acid synthase) and ARAS2, that catalyze the formation of alkylresorcylic acids. Both enzymes condensed fatty acyl-CoAs with three C(2) units from malonyl-CoA and cyclized the resulting tetraketide intermediates via intramolecular C-2 to C-7 aldol condensation. The alkylresorcylic acids thus produced were released from the enzyme and decarboxylated non-enzymatically to yield alkylresorcinols. This is the first report on a plant type III polyketide synthase that produces tetraketide alkylresorcylic acids as major products.

A nitrous acid biosynthetic pathway for diazo group formation in bacteria

Although some diazo compounds have bioactivities of medicinal interest, little is known about diazo group formation in nature. Here we describe an unprecedented nitrous acid biosynthetic pathway responsible for the formation of a diazo group in the biosynthesis of the ortho-diazoquinone secondary metabolite cremeomycin in Streptomyces cremeus. This finding provides important insights into the biosynthetic pathways not only for diazo compounds but also for other naturally occurring compounds containing nitrogen-nitrogen bonds.