Tetsuo Kushiro

The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′‐hydroxylases: key enzymes in ABA catabolism

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8′‐hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8′‐hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1–CYP707A4, encode ABA 8′‐hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six‐fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.

CYP707A1 and CYP707A2, which encode abscisic acid 8'-hydroxylases, are indispensable for proper control of seed dormancy and germination in Arabidopsis

Endogenous abscisic acid (ABA) levels are regulated by both biosynthesis and catabolism of the hormone. ABA 8′-hydroxylase is considered to be the key catabolic enzyme in many physiological processes. We have previously identified that four members of the Arabidopsis (Arabidopsis thaliana) CYP707A gene family (CYP707A1 to CYP707A4) encode ABA 8′-hydroxylases, and that the cyp707a2 mutants showed an increase in ABA levels in dry and imbibed seeds. In this study, we showed that the cyp707a1 mutant accumulated ABA to higher levels in dry seeds than the cyp707a2 mutant. Expression analysis showed that the CYP707A1 was expressed predominantly during mid-maturation and was down-regulated during late-maturation. Concomitantly, the CYP707A2 transcript levels increased from late-maturation to mature dry seed. Phenotypic analysis of single and double cyp707a mutants indicates that the CYP707A1 is important for reducing ABA levels during mid-maturation. On the other hand, CYP707A2 is responsible for the regulation of ABA levels from late-maturation to germination. Moreover, CYP707A1 and CYP707A3 were also shown to be involved in postgermination growth. Spatial expression analysis suggests that CYP707A1 was expressed predominantly in embryo during mid-maturation, whereas CYP707A2 expression was detected in both embryo and endosperm from late-maturation to germination. Our results demonstrate that each CYP707A gene plays a distinct role during seed development and postgermination growth.

Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng.

Panax ginseng produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane type and oleanane type. Dammarane‐type ginsenosides dominate over oleanane type not only in amount but also in structural varieties. However, their sapogenin structure is restricted to two aglycones, protopanaxadiol and protopanaxatriol. So far, the genes encoding oxidosqualene cyclase (OSC) responsible for formation of dammarane skeleton have not been cloned, although OSC yielding oleanane skeleton (β‐amyrin synthase) has been successfully cloned from this plant. In this study, cDNA cloning of OSC producing dammmarane triterpene was attempted from hairy root cultures of P. ginseng by homology based PCR method. A new OSC gene (named as PNA) obtained was expressed in a lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. LC‐MS and NMR analyses identified the accumulated product in the yeast transformant to be dammarenediol‐II, demonstrating PNA to encode dammarenediol‐II synthase.

Identification of β‐amyrin and sophoradiol 24‐hydroxylase by expressed sequence tag mining and functional expression assay

Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the initial origin of structural diversity of skeletons in their biosynthesis, and subsequent regio‐ and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. The enzymes responsible for this hydroxylation were thought to be cytochrome P450‐dependent monooxygenase, although their cloning has not been reported. To mine these hydroxylases from cytochrome P450 genes, five genes (CYP71D8, CYP82A2, CYP82A3, CYP82A4 and CYP93E1) reported to be elicitor‐inducible genes in Glycine max expressed sequence tags (EST), were amplified by PCR, and screened for their ability to hydroxylate triterpenes (β‐amyrin or sophoradiol) by heterologous expression in the yeast Saccharomyces cerevisiae. Among them, CYP93E1 transformant showed hydroxylating activity on both substrates. The products were identified as olean‐12‐ene‐3β,24‐diol and soyasapogenol B, respectively, by GC‐MS. Co‐expression of CYP93E1 and β‐amyrin synthase in S. cerevisiae yielded olean‐12‐ene‐3β,24‐diol. This is the first identification of triterpene hydroxylase cDNA from any plant species. Successful identification of a β‐amyrin and sophoradiol 24‐hydroxylase from the inducible family of cytochrome P450 genes suggests that other triterpene hydroxylases belong to this family. In addition, substrate specificity with the obtained P450 hydroxylase indicates the two possible biosynthetic routes from triterpene‐monool to triterpene‐triol.

Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

A plant growth retardant, uniconazole, is a potent inhibitor of ABA catabolism in Arabidopsis

Plant growth retardants (PGRs) reduce the shoot growth of plants by inhibiting gibberellin biosynthesis. In this study, we performed detailed analyses of the inhibitory effects of PGRs on Arabidopsis abscisic acid (ABA) 8′-hydroxylase, a major ABA catabolic enzyme, recently identified as CYP707As. In an in vitro assay with CYP707A3 microsomes expressed in insect cells, uniconazole-P inhibited CYP707A3 activity more effectively than paclobutrazol or tetcyclacis, whereas the other PGRs tested did not inhibit it significantly. Uniconazole-P was found to be a strong competitive inhibitor (K i=8.0 nM) of ABA 8′-hydroxylase. Uniconazole-P-treated Arabidopsis plants showed enhanced drought tolerance. In uniconazole-P-treated plants, endogenous ABA levels increased 2-fold as compared with the control, and co-application of GA4 did not suppress the effects, indicating that the effects were not due to gibberellin deficiency. Thus uniconazole-P effectively inhibits ABA catabolism in Arabidopsis plants. We also discuss the structure-activity relationship of the azole-type compounds on ABA 8′-hydroxylase inhibitory activity.

A novel multifunctional triterpene synthase from Arabidopsis thaliana

The Arabidopsis thaliana genome sequencing project has identified several triterpene synthase homologues. One cDNA of these clones, YUP8H12R.43, was obtained and functionally expressed in yeast. At least nine triterpenes have been identified as its products based on NMR and LCMS analysis. The products include extensively migrated triterpenes, multiflorenol and bauerenol. Several chimeric clones were constructed between YUP8H12R.43 and A. thaliana lupeol synthase LUP1, to reveal that a C-terminal half and a part of N-terminus is important for such product multiplicity. The presence of such multifunctional triterpene synthase in plants is noteworthy from both a mechanistic and a physiological point of view.

Functional genomics approach to the study of triterpene biosynthesis

The Arabidopsis thaliana genome-sequencing project has identified the presence of 13 oxidosqualene cyclase homologs in this plant. In addition to the already identified clones, namely, CAS1 cycloartenol synthase, LUP1 lupeol synthase, and YUP8H12R.43 multifunctional triterpene synthase, two new cDNAs of the putative oxidosqualene cyclase genes, F1019.4 and T30F21.16, were obtained by polymerase chain reaction (PCR) and functionally expressed in yeast. Liquid chromatography/mass spectrometry (LC/MS) analysis led to the identification of some of their reaction products. Interestingly, except for CAS1 for sterol biosynthesis of primary metabolism, so-far-obtained all triterpene synthases of this plant are multifunctional, producing more than one cyclization product. A feeding experiment of 13C-labeled acetate with LUP1 lupeol synthase transformant demonstrated the stereospecific water addition to lupenyl cation intermediate, yielding 3β,20 dihydroxylupane, which accounts for the multiproduct nature of this synthase.

Biosynthesis of Steroidal Antibiotic Fusidanes: Functional Analysis of Oxidosqualene Cyclase and Subsequent Tailoring Enzymes from Aspergillus fumigatus

Three putative oxidosqualene cyclase (OSC) genes exist in the genome of the fungus Aspergillus fumigatus that produces a steroidal antibiotic, helvolic acid. One of these genes, Afu4g14770, designated AfuOSC3, is clustered with genes of cytochrome P450 monooxygenases (P450s), a short-chain dehydrogenase/reductase (SDR), and acyltransferases, which presumably function in triterpene tailoring steps, suggesting that this gene cluster codes for helvolic acid biosynthesis. AfuOSC3 was PCR amplified from A. fumigatus IFO8866 genomic DNA and expressed in yeast. The yeast transformant accumulated protosta-17(20)Z,24-dien-3beta-ol, an established precursor for helvolic acid. Its structural isomer, (20R)-protosta-13(17),24-dien-3beta-ol, was also isolated from the transformed yeast. To further identify the function of triterpene tailoring enzymes, four P450 genes (CYP5081A1-D1) and a SDR gene (AfuSDR1) in the cluster were each coexpressed with AfuOSC3 in yeast. As a result, coexpression of AfuSDR1 gave a 3-keto derivative of protostadienol. On the other hand, coexpression with CYP5081A1 gave protosta-17(20)Z,24-diene-3beta,29-diol and protosta-17(20)Z,24-dien-3beta-ol-29-oic acid. These metabolites are in well accord with the oxidative modification involved in helvolic acid biosynthesis. AfuSDR1 and CYP5081A1 presumably function together to catalyze demethylation of C-29 methyl group. These results provided a firm ground for identification of the present gene cluster to be involved in helvolic acid biosynthesis.

Linking genotype and phenotype of Saccharomyces cerevisiae strains reveals metabolic engineering targets and leads to triterpene hyper-producers

Background Metabolic engineering is an attractive approach in order to improve the microbial production of drugs. Triterpenes is a chemically diverse class of compounds and many among them are of interest from a human health perspective. A systematic experimental or computational survey of all feasible gene modifications to determine the genotype yielding the optimal triterpene production phenotype is a laborious and time-consuming process. Methodology/Principal Findings Based on the recent genome-wide sequencing of Saccharomyces cerevisiae CEN.PK 113-7D and its phenotypic differences with the S288C strain, we implemented a strategy for the construction of a β-amyrin production platform. The genes Erg8, Erg9 and HFA1 contained non-silent SNPs that were computationally analyzed to evaluate the changes that cause in the respective protein structures. Subsequently, Erg8, Erg9 and HFA1 were correlated with the increased levels of ergosterol and fatty acids in CEN.PK 113-7D and single, double, and triple gene over-expression strains were constructed. Conclusions The six out of seven gene over-expression constructs had a considerable impact on both ergosterol and β-amyrin production. In the case of β-amyrin formation the triple over-expression construct exhibited a nearly 500% increase over the control strain making our metabolic engineering strategy the most successful design of triterpene microbial producers.