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December 2014 Chem. Pharm. Bull. 62(12) 1153–1165 (2014) 1153 Review Effective Use of Heterologous Hosts for Characterization of Biosynthetic Enzymes Allows Production of Natural Products and Promotes New Natural Product Discovery Kenji Watanabe Department of Pharmaceutical Sciences, University of Shizuoka; Shizuoka 422–8526, Japan. Received June 23, 2014 In the past few years, there has been impressive progress in elucidating the mechanism of biosynthesis of various natural products accomplished through the use of genetic, molecular biological and biochemical techniques. Here, we present a comprehensive overview of the current results from our studies on fungal natural product biosynthetic enzymes, including nonribosomal peptide synthetase and polyketide synthase– nonribosomal peptide synthetase hybrid synthetase, as well as auxiliary enzymes, such as methyltransferases and oxygenases. Specifically, biosynthesis of the following compounds is described in detail: (i) Sch210972, potentially involving a Diels–Alder reaction that may be catalyzed by CghA, a functionally unknown protein identified by targeted gene disruption in the wild type fungus; (ii) chaetoglobosin A, formed via multi-step oxidations catalyzed by three redox enzymes, one flavin-containing monooxygenase and two cytochrome P450 oxygenases as characterized by in vivo biotransformation of relevant intermediates in our engineered Saccharomyces cerevisiae; (iii) ()-ditryptophenaline, formed by a cytochrome P450, revealing the dimeriza- tion mechanism for the biosynthesis of diketopiperazine alkaloids; (iv) pseurotins, whose variations in the C- and O-methylations and the degree of oxidation are introduced combinatorially by multiple redox enzymes; and (v) spirotryprostatins, whose spiro-carbon moiety is formed by a flavin-containing monooxygenase or a cytochrome P450 as determined by heterologous de novo production of the biosynthetic intermediates and final products in Aspergillus niger. We close our discussion by summarizing some of the key techniques that have facilitated the discovery of new natural products, production of their analogs and identification of bio- synthetic mechanisms in our study. Key words natural product; biosynthesis; polyketide synthase; nonribosomal peptide synthetase; de novo production; reaction mechanism 1. Introduction practical to obtain those enzymes in their active form in large To date, numerous secondary metabolites from various quantities from the producing organisms to be used as cata- natural sources, such as bacteria, fungi, and plants, have been lysts, if we can isolate the genes that encode such enzymes isolated and characterized. Many of those metabolites are from the original host and express them in a more convenient important and valuable, because they often exhibit a broad strain or heterologous host, desired natural products can be spectrum of biological activities. Their remarkable properties synthesized more readily. To achieve this, the development can be attributed to their structural complexity and diversity, of expression systems for heterologous production of complex and enzymes are responsible for the formation and diversifica- natural products has been pursued with success in various tion of those compounds. Each biosynthetic enzyme catalyzes organisms, including Escherichia coli,1–4) yeast (Saccharomy- a reaction that constructs a certain chemical structure, and ces cerevisiae)5) and fungi.6) On the other hand, isolation of collection of such transformations along a biosynthetic path- new secondary metabolites from natural sources has become way results in the formation of a natural product. Chemists more difficult in recent times because numerous secondary who are interested in the study of biosynthesis are attracted metabolites have already been isolated from Streptomyces and to carbon–carbon bond-forming enzymes having the ability to other microorganisms. Moreover, culturability of the organ- produce the carbon skeleton of a molecule and auxiliary en- isms significantly restricts the available pool of microbes for zymes catalyzing various modifications to the skeleton, such natural product isolation. Therefore, if drug discovery contin- as C- and O-methylations and oxidations. While it is often im- ues to rely on such traditional methods, the isolation of new natural products may encounter significant difficulties. More The author declares no conflict of interest. recent technology has explored the culturing of anaerobic mi- e-mail: [email protected] © 2014 The Pharmaceutical Society of Japan 1154 Vol. 62, No. 12 croorganisms for production of metabolites that are expected synthesis of mRNA from a target gene cluster. Once mRNA to be new products. However, the methodology is not new—it can be obtained, the corresponding cDNA can be prepared in still relies on traditional processes that involve culturing of a streamlined fashion even for a polyketide synthase (PKS) or the organisms and screening, isolating and characterizing the a nonribosomal peptide synthetase (NRPS) gene, which can compounds.7,8) Thus, the development of new approaches for exceed 20 kilobases (kb) in length.16) Availability of cDNA al- isolating novel compounds from various microorganisms has lows transfer of the gene into a convenient host, such as yeast, also intensified in recent years. to achieve heterologous production of a biosynthetic enzyme During the past decade, many secondary metabolite gene for detailed analysis of the reaction mechanism. If sufficient clusters, including polyketide biosynthetic genes, have been genes from the cluster can be transferred to yeast, it becomes discovered through fungal genome sequencing. While typi- possible to biosynthesize the otherwise inaccessible new com- cally 30 to 40 gene clusters are identified in a single Asper- pounds heterologously. gillus genome, much fewer polyketide, peptide and terpene In this review, we will focus on heterologous production products can be isolated from a fungal culture grown under of natural products in yeast and fungi systems that have been typical growth conditions.9) Based on transcriptome analysis developed in our laboratory. Using our own work as examples, of secondary metabolism gene expression, the transcription we will illustrate how those systems have allowed us to gain level of some gene clusters responsible for secondary me- insight into natural product biosynthetic mechanisms. We will tabolite biosynthesis has been observed at a very low level, also touch on how the system can be exploited for engineering existing essentially as silent gene clusters, under conventional and identifying natural product biosynthetic pathways to ad- culture conditions.10) Consequently, natural products that those dress the obstacles described above in our efforts toward the gene clusters can biosynthesize are not attainable using con- production of potentially valuable analogs and discovery of ventional methods. To circumvent these situations and mine new natural products. for new natural products, techniques to artificially activate those silent biosynthetic gene clusters in fungal chromosomes 2. Sch210972 Biosynthesis Involvement with Diels– have been devised. Currently, there are two approaches avail- Alder Reaction Catalyzing Enzyme Responsible for the able for achieving gene cluster activation using molecular bio- Formation of Decalin Core Structure logical techniques. One is to decrease or increase epigenetic Discovering novel enzymes along with deciphering their regulation. For secondary metabolites, decreasing or increas- mechanism of enzymatic reaction is an arduous undertaking ing epigenetic regulation can increase transcription from a requiring collaboration among biochemistry, natural product biosynthetic gene cluster, where the increase of transcription chemistry, molecular biology, and structural biology. However, is achieved at the chromatin level by reducing or inducing such enzymes can shed light on how biology accomplishes modifications of histones.11) The alternate approach is to over- complex chemical synthesis, providing potential hints for in- express or knock out a transcriptional regulator (TR) that is novation in our drug discovery efforts. In this chapter, we will associated with a silent biosynthetic gene cluster. When a TR discuss approaches sought to obtain proof for the existence is located away from its corresponding gene cluster, such a of a Diels–Alder reaction-catalyzing enzyme in nature. It has TR often represses transcription from the gene cluster and been proposed that many natural product biosyntheses involve suppresses the production of its associated natural products.12) the participation of a Diels–Alder reaction.17–20) However, On the other hand, overexpression of a TR located within a only five examples of biotransformation and their reaction gene cluster frequently enhances the biosynthesis of its re- mechanisms have been examined in detail using purified spective secondary metabolites. Lack of expression of this natural enzymes that catalyze a pericyclic reaction. Those type of regulator is typically the cause of the low expression five enzymes, SpnF in spinosyn biosynthesis,21) solanapyrone of biosynthetic genes that results in poor production of the synthase (Sol5),22) LovB from lovastatin biosynthesis,23,24) corresponding natural product.13,14) In our recent study,15) we macrophomate synthase (MPS),22,25) and riboflavin synthase showed that overexpression of a TR encoded within a silent, (RibC)26,27) were reported previously

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