Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
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molecules Review Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids Shinji Kishimoto, Michio Sato, Yuta Tsunematsu and Kenji Watanabe * Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan; [email protected] (S.K.); [email protected] (M.S.); [email protected] (Y.T.) * Correspondence: [email protected]; Tel.: +81-54-264-5662; Fax: +81-54-264-5666 Academic Editor: Michael Wink Received: 20 July 2016; Accepted: 15 August 2016; Published: 18 August 2016 Abstract: Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin (tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details. Keywords: alkaloids; biosynthesis; enzymes; engineered biosynthesis; aromatic amino acids 1. Introduction Alkaloids were originally defined as organic compounds of plant origin that possess strong bioactivities and exhibit basicity that is attributed to the presence of nitrogen [1]. However, harmful nitrogen-containing compounds, such as tetrodotoxin and saxitoxin (Figure1), that are isolated from animal or microorganism sources are frequently categorized as alkaloids recently. Similarly, compounds that do not show basicity due to conversion of an amine into an amide in the molecule as seen in colchicine (Figure1) are also generally identified as alkaloid-type compounds. Even synthetic compounds, such as procaine (Figure1), that have similar chemical structures or biological properties to plant alkaloids can be referred to as alkaloids. As such, it has become commonplace nowadays to define alkaloids as nitrogen-containing organic compounds that can produce pronounced physiological responses, with notable exceptions being amino acids, nucleic acids and certain natural nitrogen-containing compounds, such as tetracycline (polyketide) and kanamycin (aminoglycoside). The alkaloid class of natural products has been very important for humanity as a valuable source of pharmaceutical products, because they show a variety of biological activities at a very low dosage. Therefore, alkaloids have been frequently employed as lead compounds for drug discovery. On the other hand, during the past decade, development of new DNA sequencing Molecules 2016, 21, 1078; doi:10.3390/molecules21081078 www.mdpi.com/journal/molecules Molecules 2016, 21, 1078 2 of 19 technologies, in particular next-generation sequencing, has enabled us to easily explore “the master plan of alkaloidMolecules 2016 production”;, 21, 1078 that is, the genes responsible for the biosynthesis of alkaloids.2 By of 19 studying the master plan, we started to gain deep insight into “the implementation of the master plan”, has enabled us to easily explore “the master plan of alkaloid production”; that is, the genes responsible meaningfor the the biosynthesis mechanism of ofalkaloids. the biosynthesis By studying the of mast alkaloids.er plan, we Furthermore, started to gain deep we areinsight using into “the the gained knowledgeimplementation to look for ofnew the master alkaloids plan”, and meaning at the th samee mechanism time modify of the thebiosynthesis “master of plan” alkaloids. to engineer productionFurthermore, of modified we are naturalusing the gained products. knowledge In this to look article, for new we alkaloids focus onand five at the successful same time modify examples of the “master plan” to engineer production of modified natural products. In this article, we focus on five reconstitutionMolecules of 2016 alkaloid, 21, 1078 biosynthetic pathways in heterologous microbial hosts. Those2 five of 19 pathways are responsiblesuccessful for examples the formation of reconstitution of ditryptophenaline of alkaloid biosynthetic (dimeric pathways diketopiperazine in heterologous alkaloid),microbial hosts. saframycin Thosehas enabledfive pathways us to easily are exploreresponsible “the formaster the planformation of alkaloid of ditryptophenaline production”; that is,(dimeric the genes diketopiperazine responsible (tetrahydroisoquinolinealkaloid),for the biosynthesis saframycin alkaloid), of (tetrahydroisoquinoline alkaloids. strictosidineBy studying the alkalo mast (monoterpeneid),er plan, strictosidine we started indole (monoterpene to gain alkaloid), deep insight indole ergotamine into alkaloid), “the (ergot alkaloid)ergotamine andimplementation opiates (ergot (benzylisoquinoline alkaloid)of the master and opiatesplan”, andmeaning(benzylisoquinoline morphinan the mechanism alkaloid). and morphinanof the Discussion biosynthesis alkaloid). of of theDiscussion alkaloids. use of of microbes as catalyststheFurthermore, use for of themicrobes bioconversion we are as usingcatalysts the gainedfor of the advanced knowledge bioconversio alkaloidto lnook of foradvanced new intermediates alkaloids alkaloid and intermediates at can the same be found time can modify be elsewhere found [2]. Similarly,elsewhere readersthe “master [2]. are plan” Similarly, referred to engineer toreaders other production are review referred of articlesmodified to otherfor natural review the recentproducts. articles efforts In forthis the towardarticle, recent we engineering focusefforts on toward five plants for successful examples of reconstitution of alkaloid biosynthetic pathways in heterologous microbial hosts. improvedengineering production plants of for alkaloids improved and production their analogs of alka [3loids,4]. and We willtheir alsoanalogs discuss [3,4]. We the will recent also progress discuss in the theThose recent five progress pathways in theare responsibledevelopment for ofthe meth formationodology of ditryptophenalinefor generating va (dimericrious compounds diketopiperazine through development of methodology for generating various compounds through heterologous reconstitution heterologousalkaloid), saframycin reconstitution (tetrahydroisoquinoline and engineering of alkalo biosynthesisid), strictosidine pathways, (monoterpene especially indole those alkaloid),involved in ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). Discussion of and engineeringthe bioproduction of biosynthesis of opioids.pathways, especially those involved in the bioproduction of opioids. the use of microbes as catalysts for the bioconversion of advanced alkaloid intermediates can be found elsewhere [2]. Similarly, readers are referred to other review articles for the recent efforts toward engineering plants for improved production of alkaloids and their analogs [3,4]. We will also discuss the recent progress in the development of methodology for generating various compounds through heterologous reconstitution and engineering of biosynthesis pathways, especially those involved in the bioproduction of opioids. Figure 1. Chemical structures of alkaloids in a broad sense: natural alkaloids of non-plant origin, Figure 1. Chemical structures of alkaloids in a broad sense: natural alkaloids of non-plant origin, tetrodotoxin and saxitoxin; a natural but non-basic alkaloid, colchicine; and a synthetic alkaloid, procaine. tetrodotoxin and saxitoxin; a natural but non-basic alkaloid, colchicine; and a synthetic alkaloid, procaine. 2. Ditryptophenaline 2. Ditryptophenaline Ditryptophenaline (1) is a dimeric diketopiperazine alkaloid [5] and has a complicated structure Figure 1. Chemical structures of alkaloids in a broad sense: natural alkaloids of non-plant origin, (Figure 2). Dimerization is found relatively frequently among natural products, as it is considered to be Ditryptophenalinetetrodotoxin and (1 saxitoxin;) is a dimeric a natural but diketopiperazine non-basic alkaloid, colchicine; alkaloid and [a5 sy]nthetic and hasalkalo aid, complicated procaine. structure an efficient way of enhancing the chemical complexity and diversity of metabolites being biosynthesized. (Figure2). Dimerization is found relatively frequently among natural products, as it is considered It can2. Ditryptophenaline also increase the valency of a compound, affording the molecule a higher affinity for its target or to be anhigher efficient stability way upon of forming enhancing a comple thex with chemical its target complexity [6].