molecules

Review Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of

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 ), saframycin (tetrahydroisoquinoline alkaloid), (monoterpene ), 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 , such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.

Keywords: alkaloids; biosynthesis; ; 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]. Compound and 1 [7] diversity and its related of metabolites compound being Ditryptophenaline (1) is a dimeric diketopiperazine alkaloid [5] and has a complicated structure biosynthesized.WIN 64821 It(Figure can 2) also [8] originally increase isolated the valency from Aspergillus of a compound, versicolor exhibit affording useful biological the molecule activities, a higher (Figure 2). Dimerization is found relatively frequently among natural products, as it is considered to be affinitysuch for itsas targetan inhibition or higher of substance stability P receptor upon formingfor potential a complex analgesic withand anti-inflammatory its target [6]. Compound activities. 1 [7] an efficient way of enhancing the chemical complexity and diversity of metabolites being biosynthesized. and itsDespite relatedIt can alsotheir compound increase complex the WINvalencychemical 64821 of ast compound,ructures, (Figure their afford2)[ formation8ing] originallythe molecule seemed isolateda higher relatively affinity from simple, forAspergillus its target involving or versicolor exhibit usefuldimerizationhigher biological stability of diketopiupon activities, formingperazine a such comple monomers asx anwith inhibition itsformed target [6].through of Compound substance condensation 1 [7] P receptorand itsof relatedtwo for amino compound potential acids analgesicat the initial stage. Judging from the structure of the diketopiperazine monomer of 1, it appeared that and anti-inflammatoryWIN 64821 (Figure activities. 2) [8] originally Despite isolated their from complex Aspergillus chemicalversicolor exhibit structures, useful biological their formation activities, seemed L-tryptophansuch as an inhibition and L-phenylalanine of substance Pcould receptor be condensedfor potential togeth analgesicer to and form anti-inflammatory the diketopiperazine activities. core relatively simple, involving dimerization of diketopiperazine monomers formed through condensation structureDespite of their the monomer.complex chemicalOnce the stphenylalanine-derivedructures, their formation amide seemed nitrogen relatively of the diketopiperazinesimple, involving core of two aminois Ndimerization-methylated, acids at of a the ringdiketopi initial closureperazine stage. within monomers Judgingthe monomer formed from and thethrough a subsequent structure condensation monomer–monomer of the of diketopiperazine two amino crosslinking acids at monomer of 1, it appearedthroughthe initial oxidation thatstage.L Judging -tryptophanreactions from involving the and struct Ltheure-phenylalanine tryptophanof the diketopiperazine indole could ring bemonomer can condensed lead of to 1 , theit appeared togetherformation that toof forma the diketopiperazinehomodimerL-tryptophan coreof 1 and. structure L-phenylalanine of the could monomer. be condensed Once togeth theer phenylalanine-derived to form the diketopiperazine amide core nitrogen structure of the monomer. Once the phenylalanine-derived amide nitrogen of the diketopiperazine core N of the diketopiperazineis N-methylated, a corering closure is -methylated, within the monomer a ring and closure a subsequent within monomer–monomer the monomer crosslinking and a subsequent monomer–monomerthrough oxidation crosslinking reactions involving through the oxidation tryptophan reactions indole ring involving can lead to the the tryptophan formation of indolea ring can lead tohomodimer the formation of 1. of a homodimer of 1.

Figure 2. Chemical structures of dimeric diketopiperazine alkaloids, (–)-ditryptophenaline 1 [7] and (+)-WIN 64821 [8].

Figure 2. ChemicalFigure 2. Chemical structures structures of dimeric of dimeric diketopiperazine diketopiperazine alkaloids, alkaloids, (–)-ditryptophenaline (–)-ditryptophenaline 1 [7] and 1 [7] and (+)-WIN 64821 [8]. (+)-WIN 64821 [8]. Molecules 2016, 21, 1078 3 of 19

MoreMolecules recently, 2016, 21, 10781 was found to be produced by another fungus, Aspergillus flavus [9]. To3 elucidate of 19 the biosynthetic pathway of 1, experiments of deficiency of the biosynthetic gene were performed to identify ditryptophenalineMore recently, 1 was biosynthetic found to be produced gene cluster. by another Based fungus, on the Aspergillus homology flavus of amino [9]. To acid elucidate sequences deducedtheMolecules biosynthetic from 2016 the, 21A. ,pathway 1078 flavus genomeof 1, experiments sequence of deficiency [10] to known of the enzymes,biosynthetic three gene genes,were performeddtpA3 of, 19dtpB to and identify ditryptophenaline biosynthetic gene cluster. Based on the homology of amino acid sequences dtpC, clusteredMore together recently, 1 on was the foundA. flavus to be producedgenome by were another predicted fungus, Aspergillus to code for flavus a nonribosomal [9]. To elucidatepeptide deduced from the A. flavus genome sequence [10] to known enzymes, three genes, dtpA, dtpB and dtpC, synthetasethe biosynthetic (NRPS), an pathwayN-methyltransferase of 1, experiments and of deficiency a cytochrome of the biosynthetic P450 (P450), gene respectively were performed (Figure to 3)[ 9]. clustered together on the A. flavus genome were predicted to code for a nonribosomal peptide synthetase Knockoutidentify of any ditryptophenaline one of the three biosynthetic genes ingene the clusteA. flavusr. Based∆ onpyrG the /homology∆ku70 strain of amino A1421 acid sequences [11] resulted in (NRPS), an N-methyltransferase and a (P450), respectively (Figure 3) [9]. Knockout the lossdeduced of production from the A. of flavus1, indicating genome sequence that these [10] to three known genes enzymes, were three involved genes, dtpA in the, dtpB biosynthesis and dtpC, of 1. of clusteredany one togeof thether three on the genes A. flavus in thegenome A. flavus were ∆predictedpyrG/∆ku70 to code strain for a A1421 nonribosomal [11] resulted peptide in synthetase the loss of Based on the in vitro assays of DtpB using a 2 isolated from the dtpB knockout mutant, DtpB production(NRPS), an of N 1-methyltransferase, indicating that these and three a cytochrome genes were P450 involved (P450), respectively in the biosynthesis (Figure 3) of [9]. 1. Based Knockout on the was shownin vitroof any toassays one catalyze of of the DtpB anthree Nusing -methylationgenes a substratein the A.of flavus2 isolated2 to ∆ biosynthesizepyrG from/∆ku70 the straindtpB the knockout A1421 intermediate [11] mutant, resulted3 .DtpB Interestingly,in the was loss shown of 3 was directlyto productioncatalyze converted an of N to 1-methylation, 1indicatingby DtpC that (P450),of these2 to whichthreebiosynthesize genes was were prepared the involved intermediate as in a the microsomal biosynthesis3. Interestingly, fraction of 1. Based3 was of Saccharomyces on directly the cerevisiaeconvertedin vitroexpressing assaysto 1 by of DtpCdtpC DtpB (P450), recombinantly.using awhich substrate was 2prepared Theisolated analysis fromas a microsomal the of dtpB the knockoutin fraction vitro mutant,experiments of Saccharomyces DtpB was suggested shown cerevisiae that DtpCexpressing catalyzesto catalyze dtpC the an recombinantly.C2–N10N-methylation ring closureofThe 2 analysisto biosynthesize within of the the in monomerthe vitro intermediate experiments and the3. suggestedInterestingly, dimerization that 3 DtpCwas of thedirectly catalyzes tetracyclic monomers,theconverted C2–N10 both ringto of 1 by closure which DtpC within(P450), likely whichthe proceed monomer was throughprepared and th as ae adimerization radical microsomal pathway. fraction of the tetracyclic of In Saccharomyces the proposed monomers, cerevisiae mechanism both of whichexpressing likely dtpC proceed recombinantly. through Thea radical analysis pathway. of the in Invitro the experiments proposed mechanism suggested that (Figure DtpC 4),catalyzes a radical (Figure4), a radical formed at N10 through hydrogen atom abstraction by the heme iron of P450 can formedthe C2–N10 at N10 ring through closure hydrogen within the atom monomer abstraction and th bye dimerizationthe heme iron of ofthe P450 tetracyclic can migrate monomers, to C3 both with a migrate to C3 with a concomitant C2–N10 ring closure and undergo a radical-mediated coupling with concomitantof which likely C2–N10 proceed ring throughclosure anda radical undergo pathway. a radical-mediated In the proposed coupling mechanism with (Figure another 4), aC3 radical radical- anotherbearingformed C3 radical-bearing monomer at N10 through to form monomerhydrogen the bridging atom to form bondabstraction thein the bridging by dimer the heme like bond 1iron. in of the P450 dimer can migrate like 1 .to C3 with a concomitant C2–N10 ring closure and undergo a radical-mediated coupling with another C3 radical- bearing monomer to form the bridging bond in the dimer like 1.

Figure 3. Aspergillus flavus ditryptophenaline biosynthetic gene cluster and predicted functions of the Figure 3. Aspergillus flavus ditryptophenaline biosynthetic gene cluster and predicted functions of the genesFigure in the 3. Aspergillus cluster. flavus ditryptophenaline biosynthetic gene cluster and predicted functions of the genes in the cluster. genes in the cluster.

Figure 4. Proposed mechanism of DtpC-catalyzed dimerization of intermediates 3 and 4 to form FigureFigure 4. Proposed 4. Proposed mechanism mechanism of of DtpC-catalyzed DtpC-catalyzed dimerization dimerization of ofintermediates intermediates 3 and3 and4 to 4formto form (−)-ditryptophenaline 1, (−)-dibrevianamide F 5 and (−)-tryprophenaline 6 via a radical route [9]. DtpA, (−)-ditryptophenaline(−)-ditryptophenaline1 ,(1, −(−)-dibrevianamide)-dibrevianamide F 5 Fand5 and(−)-tryprophenaline (−)-tryprophenaline 6 via a radical6 via route a radical [9]. DtpA, route [9]. ditryptophenalineditryptophenaline nonribosomal nonribosomal peptidepeptide synthetase (NRPS);(NRPS); DtpB,DtpB, NN-methyltransferase;-methyltransferase; DtpC, DtpC, DtpA, ditryptophenalinecytochrome P450; FtmA, nonribosomal fumitremorgin peptide NRPS. synthetase (NRPS); DtpB, N-methyltransferase; DtpC, cytochrome P450; FtmA, fumitremorgin NRPS.

cytochrome P450; FtmA, fumitremorgin NRPS. Molecules 2016, 21, 1078 4 of 19

To examine the substrate tolerance of DtpC and to biosynthesize new dimeric products using DtpC in this study, brevianamide F (4) was provided to this as a substrate [9]. When adding Molecules 2016, 21, 1078 4 of 19 only 4, compound 5 was produced. Furthermore, adding both the natural substrate 2 and the substrate analog 4Toprovided examine compounds the substrate6 toleranceas well as of5 .DtpC Using an NMRd to biosynthesize and X-ray crystallographic new dimeric products data on using5 and 6, the chemicalDtpC in this structures study, brevianamide of these compounds, F (4) was includingprovided to their this absoluteenzyme as configurations, a substrate [9]. wereWhen determined. adding Theonly homodimeric 4, compound 5 was produced.5 was named Furthermore, (−)-dibrevianamide adding both the F, andnatural the substrate heterodimeric 2 and the product substrate6 was namedanalog (− )-tryprophenaline4 provided compounds as new 6 as alkaloidswell as 5. Using (Figure NMR5). As and in X-ray1, the crystallographic stereochemistry data of on chiral 5 and carbon 6, atomsthe in chemical5 and 6 structureswas determined of these compounds, to be all in the includS configurationing their absolute by theconfigur Flackations, parameter were determined. value obtained fromThe the homodimeric crystallographic product data. 5 was Thus, named the biosynthetic (−)-dibrevianamide mechanism F, and for the the heterodimeric formation ofproduct5 and 6 wasis likely the samenamed as (− that)-tryprophenaline described above as new for alkaloids the formation (Figure 5). of 1As(Figure in 1, the4). stereochemistry Furthermore, of as chiral an example carbon of atoms in 5 and 6 was determined to be all in the S configuration by the Flack parameter value obtained engineered biosynthesis of non-native dimeric products, the heterologous host Aspergillus niger A1179 from the crystallographic data. Thus, the biosynthetic mechanism for the formation of 5 and 6 is likely modified [12] with the introduction of ftmA (fumitremorgin NRPS [13]) genes was provided with the the same as that described above for the formation of 1 (Figure 4). Furthermore, as an example of dtpCengineeredgene [14]. biosynthesis This engineered of non-native strain was dimeric able products, to isolate the additional heterologous dimeric host compounds Aspergillus niger7 and 8 (FigureA11796). Thesemodified two [12] compounds with the introduction were analogs of ftmA of the (fumitremorgin homodimeric NRPS product [13]) 5genespreviously was provided produced (Figurewith4 ).the Elucidation dtpC gene [14]. of the This chemical engineered structures strain was7 andable to8 showedisolate additional7 to be a dimeric new compound compounds and 7 and8 to be naseseazine8 (Figure B6). isolated These two recently compounds from awere marine-dwelling analogs of the streptomycetehomodimeric product bacterium 5 previously [15]. Dimerization produced in 7 and(Figure8 did 4). not Elucidation form the of C3–C3’ the chemical linkage structures found 7 in and1, 58 showedand 6. In7 to7 ,be dimerization a new compound occurred and 8 betweento be C3 andnaseseazine N1’, whereas B isolated positions recently C3 from and C6a marine-dwe were involvedlling instreptomycete the dimerization bacterium that formed[15]. Dimerization8. Those dimeric in compounds7 and 8 did are not speculated form the alsoC3–C3’ as productslinkage found of a radical-mediated in 1, 5 and 6. In 7, oxidativedimerization dimerization occurred between of tryptophan C3 derivatives,and N1’, whereas where the positions linkages C3 areand formedC6 were atinvolved various in differentthe dimerization positions. that DtpCformed is 8 thought. Those dimeric to extract the hydrogencompounds from are speculated the diketopiperazine also as products core of a amideradical-mediated nitrogen N10oxidative of 2 dimerizationto yield a radical. of tryptophan Then, the derivatives, where the linkages are formed at various different positions. DtpC is thought to extract the radical can migrate to C3 through the C2–N10 ring closure, and the subsequent coupling can proceed hydrogen from the diketopiperazine core amide nitrogen N10 of 2 to yield a radical. Then, the radical to form the C3–C3’ linkage, giving the dimeric product 1. However, when DtpC was provided with can migrate to C3 through the C2–N10 ring closure, and the subsequent coupling can proceed to form the unnaturalthe C3–C3’ substrate linkage, giving4, which the lacks dimeric the product bulky phenyl 1. However, side chain when group, DtpC thewas positioning provided with of 4 thewithin the activeunnatural site substrate of DtpC 4 may, which be lacks shifted the from bulky where phenyl2 sidewould chain be group, bound. the This positioning may cause of 4 anwithin occasional the extractionactive site of the of hydrogenDtpC may frombe shifted the indole from nitrogenwhere 2 would to yield be a bound. radical This at N1, may which cause can an start occasional migrating fromextraction the indole of the nitrogen hydrogen to C6from and the backindole to nitrogen N1 (Figure to yield6, right a radical column). at N1, which Coupling can start of the migrating radical at C3 infrom one the monomer indole nitrogen formed to viaC6 and the N10back extractionto N1 (Figure pathway 6, right column). (labeled Coupling as C3 in Figureof the radical6, left column)at C3 within the one radical monomer at N1formed in the via other the N10 monomer extraction formed pathway via (labeled the N1 as extraction C3 in Figure pathway 6, left column) (labeled with as N1 in Figurethe radical6, right at N1 column) in the other can monomer produce formed the dimeric via the N1 compound extraction pathway7. Similarly, (labeled coupling as N1 in ofFigure the 6, same C3 radicalright column) in one can monomer produce withthe dimeric the C6 compound radical in 7. the Similarly, other monomercoupling of (labeled the same asC3 C6 radicalN1 in in Figure one 6, N1 rightmonomer column) with can producethe C6 radical8. The in titer the ofother these monomer compounds (labeled was as significantly C6 in Figure lower 6, right than column) that of the can produce 8. The titer of these compounds was significantly lower than that of the native product native product 1 (approximately 350 µg/L of culture for 7 and 8 as compared to 2.3 mg/L of culture 1 (approximately 350 µg/L of culture for 7 and 8 as compared to 2.3 mg/L of culture for 1) [9,14], for 1)[9,14], presumably because of the low frequency of the N1 hydrogen extraction happening inside presumably because of the low frequency of the N1 hydrogen extraction happening inside the active the of DtpC. There are other known natural products having similar dimerization patterns. site of DtpC. There are other known natural products having similar dimerization patterns. For Forexample, (–)-asperazine (–)-asperazine A [16] A [and16] (+)-pestalazine and (+)-pestalazine B [17,18] B have [17,18 the] havesame C3–N1 the same linkage C3–N1 as 7, linkage whereas as 7, whereas(+)-asperazine (+)-asperazine [19] and [19 (+)-pestalazine] and (+)-pestalazine A [17,20] A ha [17ve, 20a ]C3–C7 have alinkage C3–C7 that linkage was not that observed was not in observed our in ourstudy study (Figure (Figure 6, boxed).6, boxed). Presumably, Presumably, the biosynthes the biosynthesisis of those dimeric of those diketopiperazine dimeric diketopiperazine compounds compoundsis also catalyzed is also catalyzedby P450 enzymes by P450 with enzymes relaxed with substrate relaxed specificity, substrate much specificity, like DtpC. much like DtpC.

FigureFigure 5. 5.Chemical Chemical structures structures ofof thethe newlynewly formed compounds, compounds, (–)-dibrevianamide (–)-dibrevianamide F 5 Fand5 and (–)-tryprophenaline(–)-tryprophenaline6 [ 96]. [9].

Molecules 2016, 21, 1078 5 of 19 Molecules 2016, 21, 1078 5 of 19

Figure 6. 6.ProposedProposed mechanism mechanism of DtpC-catalyzed of DtpC-catalyzed formation of dimerization formation ofproducts dimerization (−)-dibrevianamide products (F− 5)-dibrevianamide [9], a new compound F 5 [ 97], [14] a new and compoundnaseseazine7 B[14 8 ][15]. and Radical-mediated naseseazine B 8 [formation15]. Radical-mediated of 7 and 8 is formationthought to be of initiated7 and 8 byis thoughtDtpC forming to be a initiated radical at by N1 DtpC instead forming of N10 aas radical proposed at N1for the instead formation of N10 of as5. The proposed radical forspecies the formationare labeled ofby5 the. The position radical of speciesthe radical. are To labeled differentiate by the between position radical of the radical.species Toderived differentiate from hydrogen between extraction radical species at N10 derived (left column from hydrogen) vs. N1 (right extraction column at), N10 those (left formed column via) the vs. N1 (hydrogenright column extraction), those are formed given via a label the N1 with hydrogen a superscript extraction N1. areChemical given astructures label with of a superscriptrelated known N1. Chemicalnatural products structures are ofshown related in knownthe box. natural The monomer-bridging products are shown bonds in the are box. shown The in monomer-bridging red. bonds are shown in red. 3. Saframycin 3. SaframycinSaframycin A (9) isolated from Streptomyces lavendulae is a representative member of the tetrahydroisoquinolineSaframycin A (9) alkaloids isolated from(FigureStreptomyces 7) [21]. This lavendulaeclass of naturalis a products representative has been member isolated of from the tetrahydroisoquinolinevarious organisms, including alkaloids soil (Figurebacteria7 )[and21 mari]. Thisne invertebrates, class of natural such products as sponges has and been ascidians isolated from[22]. These various compounds organisms, exhibit including various soil bacteriaantibiotic and activities marine but invertebrates, also possess such a potent as sponges antitumor and ascidiansactivity, which [22]. These is thought compounds to be effected exhibit variousthrough antibiotic covalent activities modification but also of DNA possess by a iminium potent antitumor ions via activity,carbinolamine which moiety is thought and to its be equivalents effected through [23]. Am covalentong the modification saframycin analogs, of DNA ecteinascidin by iminium ions 743 via(10 carbinolaminein Figure 7) [24] moiety showed and a remarkably its equivalents potent [23 antitumor]. Among activity the saframycin against not analogs, only common ecteinascidin tumor 743cell (lines10 in but Figure also7 resistant)[ 24] showed cell lines a remarkably [25]. Recently, potent 10 was antitumor approved activity as an againstanticancer not drug only in common the European tumor cellUnion lines for butpatients also resistantwith soft celltissue lines sarcoma [25]. Recently,[26]. Because10 was of the approved limited supply as an anticancer from natural drug sources, in the European10 is currently Union produced for patients by a semisynthetic with soft tissue process sarcoma [27]. However, [26]. Because this synthe of thesis limited requires supply more fromthan natural25 chemical sources, transformation10 is currently steps, produced making it by a suboptimal a semisynthetic method process for a [commercial27]. However, production this synthesis of 10. requiresEngineered more biosynthesis than 25 chemical for generating transformation this important steps, making alkaloid it a as suboptimal an alternative method strategy for a commercialis strongly productiondesired, especially of 10. Engineeredfrom the pers biosynthesispective of sustainability for generating and this envi importantronmental alkaloid burden as of an the alternative synthetic strategymanufacturing is strongly process. desired, To especiallyput this bioproduction from the perspective approach of sustainabilityinto practice, anddetailed environmental understanding burden of the biosynthetic mechanism of this class of natural products is essential. Therefore, there has been a considerable study on the subject involving gene isolation [28–30], substrate feeding experiments

Molecules 2016, 21, 1078 6 of 19 of the synthetic manufacturing process. To put this bioproduction approach into practice, detailed Moleculesunderstanding 2016, 21, 1078 of the biosynthetic mechanism of this class of natural products is essential. Therefore,6 of 19 there has been a considerable study on the subject involving gene isolation [28–30], substrate feeding [31,32],experiments biochemical [31,32 ],characterizations biochemical characterizations [30,33] and bioinformatic [30,33] and analyses bioinformatic [30]. In this analyses review, [30 the]. current In this understandingreview, the current of the understanding mechanism of of biosynthesis the mechanism of 9 ofis biosynthesisdescribed. of 9 is described.

Figure 7. 7. ChemicalChemical structures structures of of tetrahydroisoquinoline tetrahydroisoquinoline alkaloids, alkaloids, saframycin saframycin A A 99 [21][21] and and ecteinascidin ecteinascidin 743 10 [24].[24].

Investigation of the biosynthesis of 9 and related compounds has been ongoing for quite some Investigation of the biosynthesis of 9 and related compounds has been ongoing for quite some time after the initial discovery of 9 in 1977 [21]. By 1985, it was determined that the main framework of time after the initial discovery of 9 in 1977 [21]. By 1985, it was determined that the main framework of 9 was comprised of one molecule each of L-alanine and glycine and two molecules of L-tyrosine [31]. 9 was comprised of one molecule each of L-alanine and glycine and two molecules of L-tyrosine [31]. The alanyl-glycyl terminal moiety was found to be modifiable by feeding of particular amino acids, The alanyl-glycyl terminal moiety was found to be modifiable by feeding of particular amino acids, such as 2-amino-n-butylic acid [32]. The initial discovery of a saframycin biosynthetic gene cluster such as 2-amino-n-butylic acid [32]. The initial discovery of a saframycin biosynthetic gene cluster was made in Myxococcus xanthus, where two NRPS-encoding genes and an O-methyltransferase gene was made in Myxococcus xanthus, where two NRPS-encoding genes and an O-methyltransferase gene responsible for the formation of the core structure were identified [28]. The findings from this study responsible for the formation of the core structure were identified [28]. The findings from this study confirmed that the main framework of 9 was biosynthesized by NRPS. The subsequent study on the confirmed that the main framework of 9 was biosynthesized by NRPS. The subsequent study on the gene cluster for the biosynthesis of safracin, a compound closely related to 9, revealed additional gene cluster for the biosynthesis of safracin, a compound closely related to 9, revealed additional genes proposed to be involved in the modification of building block amino acids and post-NRPS genes proposed to be involved in the modification of building block amino acids and post-NRPS intermediates [29]. The most recent study provided the most complete information on the saframycin intermediates [29]. The most recent study provided the most complete information on the saframycin biosynthetic gene cluster with a total of 30 genes that are proposed to be involved in the biosynthetic biosynthetic gene cluster with a total of 30 genes that are proposed to be involved in the biosynthetic process [30]. process [30]. Based on the bioinformatically predicted functions of the genes that were identified as described Based on the bioinformatically predicted functions of the genes that were identified as described above, various biosynthetic schemes were hypothesized [29,31,34]. However, no actual experimental above, various biosynthetic schemes were hypothesized [29,31,34]. However, no actual experimental data were available to verify the proposed mechanisms. Thus, to establish the mechanism of how the data were available to verify the proposed mechanisms. Thus, to establish the mechanism of how the complex core structure of 9 was biosynthesized, in vitro assays on the recombinantly prepared SfmA complex core structure of 9 was biosynthesized, in vitro assays on the recombinantly prepared SfmA and SfmB were carried out [30]. As predicted from the adenylation domain amino acid sequence [30], and SfmB were carried out [30]. As predicted from the adenylation domain amino acid sequence [30], two NRPSs, SfmA and SfmB, were shown to specifically activate L-alanine and glycine, respectively, two NRPSs, SfmA and SfmB, were shown to specifically activate L-alanine and glycine, respectively, indicating that those two enzymes were responsible for the formation of the non-tyrosine dipeptide indicating that those two enzymes were responsible for the formation of the non-tyrosine dipeptide segment of 9 (Figure 8). However, amino acid sequence analysis of SfmA also revealed the presence of segment of 9 (Figure8). However, amino acid sequence analysis of SfmA also revealed the presence of an N-terminal acyl-CoA (AL)-acyl carrier protein (ACP) didomain [30,35], which is commonly an N-terminal acyl-CoA ligase (AL)-acyl carrier protein (ACP) didomain [30,35], which is commonly found in NRPSs that form lipopeptides having a long fatty acyl chain extension at the N-terminus of found in NRPSs that form lipopeptides having a long fatty acyl chain extension at the N-terminus of the the core peptide [36,37]. This finding prompted analysis for the requirement of an N-terminal acyl core peptide [36,37]. This finding prompted analysis for the requirement of an N-terminal acyl chain chain in the L-alanyl-glycyl intermediate. In vitro assays with synthetically prepared L-alanyl-glycyl- in the L-alanyl-glycyl intermediate. In vitro assays with synthetically prepared L-alanyl-glycyl-S-CoA S-CoA substrates having an N-acyl chain of various length and SfmC that was recombinantly prepared substrates having an N-acyl chain of various length and SfmC that was recombinantly prepared in Escherichia coli clearly indicated that the N-myristoylated dipeptide was the actual substrate of SfmC in Escherichia coli clearly indicated that the N-myristoylated dipeptide was the actual substrate of [35]. No reaction took place when substrates lacking a long acyl extension were provided to SfmC. SfmC [35]. No reaction took place when substrates lacking a long acyl extension were provided to Because the final product 9 does not carry any acyl chain, the requirement of SfmC for a cryptic acyl SfmC. Because the final product 9 does not carry any acyl chain, the requirement of SfmC for a cryptic chain in its substrate is a completely unexpected finding. acyl chain in its substrate is a completely unexpected finding. Once the actual substrate for SfmC was identified, the mechanism of the formation of the Once the actual substrate for SfmC was identified, the mechanism of the formation of the pentacyclic tetrahydroisoquinoline core was investigated. Further detailed examinations, which pentacyclic tetrahydroisoquinoline core was investigated. Further detailed examinations, which involved the use of additional synthetic substrates and SfmC mutants [35], demonstrated that SfmC was solely responsible for the formation of the pentacyclic scaffold from two molecules of a L-tyrosine derivative, 3-hydroxy-5-methyl-O-methyltyrosine (Figure 8) [30]. Based on the results from those experiments, the reaction mechanism was suggested with the Re (reduction) domain of SfmC catalyzing three reduction reactions and the C (condensation) domain catalyzing two cycles of the

Molecules 2016, 21, 1078 7 of 19 involved the use of additional synthetic substrates and SfmC mutants [35], demonstrated that SfmC was solely responsible for the formation of the pentacyclic scaffold from two molecules of a L-tyrosine derivative, 3-hydroxy-5-methyl-O-methyltyrosine (Figure8)[ 30]. Based on the results from those Moleculesexperiments, 2016, 21 the, 1078 reaction mechanism was suggested with the Re (reduction) domain of SfmC catalyzing7 of 19 three reduction reactions and the C (condensation) domain catalyzing two cycles of the Pictet-Spengler Pictet-Spenglerreaction to form reaction the tetrahydroisoquinoline to form the tetrahydroisoquinoline core of 9 [35]. The core last of 9 cyclization [35]. The last reaction cyclization after thereaction third afterreduction the third step reduction may also bestep catalyzed may also by be SfmC, cataly althoughzed by SfmC, the reaction although could the proceedreaction spontaneously.could proceed Whilespontaneously. further investigations While further into investig the biosynthesisations into of 9theand biosynthesis related compounds of 9 and are required,related compounds elucidation areof this required, complex elucidation reaction mechanism of this complex would provide reaction us withmechanism a novel chemoenzymaticwould provide us approach with a toward novel chemoenzymaticsynthesizing sufficient approach amount toward of 10 syandnthesizing its analogs. sufficient amount of 10 and its analogs.

Figure 8. Proposed overall schemescheme forforthe thepentacyclic pentacyclic core core skeleton skeleton assembly assembly during during the the biosynthesis biosynthesis of ofsaframycin saframycin A A9 [935 [35].]. SfmA, SfmA, SfmB SfmB and and SfmC SfmC are are three three saframycinsaframycin AA nonribosomal peptide synthetases. synthetases. C, condensation domain; domain; A, A, adenylation adenylation domain domain;; T, T, thiolation thiolation domain; domain; Re, Re, reductase reductase domain. domain.

4. Strictosidine Strictosidine Terpene indole alkaloids (TIAs) constitute one of the largest and most diverse families of complex nitrogen-containing metabolites found in plantsplants [38,39]. [38,39]. Many TIAs, TIAs, especially especially monoterpene monoterpene indole alkaloids (MIAs) and their analogs, have medicinal properties: and are approved as anticancer therapeutics, whereas whereas reserpine reserpine is is used used as as an antihypertensive [40]. [40]. Physostigmine is an antidote of anticholinergic anticholinergic toxici toxicityty and and is is used used primarily primarily for for treating treating glaucoma glaucoma [41]. [41]. However, these these structurally structurally complex complex compounds compounds can can be be difficult difficult to tosynthesize synthesize chemically chemically [42,43]. [42,43 To]. supplyTo supply these these compounds compounds through through biosynthetic biosynthetic routes, routes,the biosynthetic the biosynthetic pathway for pathway a target for compound a target mustcompound be elucidated. must be elucidated.Once all of Oncethe genes all of involved the genes in involved the pathway in the are pathway identified, are identified,a host organism a host thatorganism is more that convenient is more convenient to cultivate to cultivatethan a plant than can a plant be used canbe for used heterologous for heterologous production production of the desiredof the desired compound. compound. Furthermore, Furthermore, elucidation elucidation of the ofbiosynthetic the biosynthetic pathway pathway and establishment and establishment of a heterologousof a heterologous system system would would allow allow engineering engineering of the ofpathway the pathway for efficient for efficient analog analog production. production. From theseFrom theseviewpoints, viewpoints, the most the mostimportant important compound compound in this in thisclass class of natural of natural products products to toengineer engineer a heterologousa heterologous production production system system would would be be strictosidine strictosidine (11 (11),), because because it it is is the the universal universal intermediate intermediate for the biosynthesis of all MIAs. Below, Below, the bi biosyntheticosynthetic pathway leading to the formation of 11 and recent efforts in the engineered biosynthesisbiosynthesis ofof 11 usingusing aa yeastyeast hosthost systemsystem areare discussed.discussed. The general mechanism of the biosynthesis of 11 has been been reviewed reviewed elsewhere elsewhere [38]. [38]. Briefly, Briefly, 11 is formed with twotwo startingstarting substrates,substrates, geraniol geraniol and andL L-tryptophan.-tryptophan. The The first first starting starting substrate substrate geraniol geraniol is is initially converted into a dihydroxy intermediate, 8-hydroxygeraniol, by geraniol 8-hydroxylase (G8H). Then, both hydroxyl groups are oxidized to form 8-oxogeranial. Subsequent cyclization and oxidation of 8-oxogeranial yields loganic acid, and methylation of the carboxylic acid moiety of loganic acid results in the formation of loganin (12) (Figure 9). The following transformation of 12 into secologanin (13) is an interesting reaction that is catalyzed by secologanin synthase (SLS, a cytochrome P450). This step is thought to be the rate-limiting step in the biosynthetic pathway leading to the formation of 11 [44]. SLS is proposed to catalyze the opening of the 5-membered ring of 12 by

Molecules 2016, 21, 1078 8 of 19 initially converted into a dihydroxy intermediate, 8-hydroxygeraniol, by geraniol 8-hydroxylase (G8H). Then, both hydroxyl groups are oxidized to form 8-oxogeranial. Subsequent cyclization and oxidation of 8-oxogeranial yields loganic acid, and methylation of the carboxylic acid moiety of loganic acid results in the formation of loganin (12) (Figure9). The following transformation of 12 into secologanin (13) is an interesting reaction that is catalyzed by secologanin synthase (SLS, a cytochrome P450).

ThisMolecules step 2016 is, thought 21, 1078 to be the rate-limiting step in the biosynthetic pathway leading to the formation8 of of19 11 [44]. SLS is proposed to catalyze the opening of the 5-membered ring of 12 by a radical-mediated ahydroxylation, radical-mediated followed hydroxylation, by a dehydration followed toby yield a dehydration13 (Figure 9to)[ yield45]. 13 The (Figure other starting9) [45]. The substrate other startingL-tryptophan substrate is modified L-tryptophan through is modified a decarboxylation through stepa decarb to affordoxylation tryptamine, step to whichafford istryptamine, combined withwhich13 isto combined give 11 by with the 13 action to give of strictosidine 11 by the action synthase of strictosidine (STR, an amine synthase ), (STR, a key an enzymeamine lyase), in MIA a keybiosynthesis. enzyme in This MIA transformation biosynthesis. This would transformation be the most chemicallywould be the interesting most chemically step inthe interesting strictosidine step inbiosynthetic the strictosidine pathway; biosynthetic it is an another pathway; example it is an of another the Pictet-Spengler example of reactionthe Pictet-Spengler applied toward reaction the appliedbiosynthesis toward of alkaloids, the biosynthesis as it was of also alkaloids, mentioned as it during was also the discussionmentioned of during the saframycin the discussion biosynthesis, of the (Figuresaframycin9). As biosynthesis, to the formation (Figure of 9).11 ,As the to reaction the formation is proposed of 11 to, the occur reaction between is proposed the aldehyde to occur of 13 betweenand the aminethe aldehyde of tryptamine of 13 and to couplethe amine the of two tryptamine substrates to through couple the the two formation substrates of a throughβ-carboline the formationmoiety [46 of]. a β-carboline moiety [46].

Figure 9. Proposed biosynthetic pathway of strictosidine 11, the universal intermediate in the monoterpene Figure 9. Proposed biosynthetic pathway of strictosidine 11, the universal intermediate in the indolemonoterpene alkaloid indole biosynthesis alkaloid biosynthesis [38]. G8H, [38geraniol]. G8H, 8-hydroxylase; geraniol 8-hydroxylase; SLS, secologanin SLS, secologanin synthase; synthase; STR, strictosidineSTR, strictosidine synthase. synthase.

To develop a yeast strain that can biosynthesize 11 de novo, 21 gene additions and three gene To develop a yeast strain that can biosynthesize 11 de novo, 21 gene additions and three gene deletions were introduced into the yeast genome [47]. Fourteen of the added genes were known genes deletions were introduced into the yeast genome [47]. Fourteen of the added genes were known from a MIA producer plant [48]. Of the 14 genes, 11 constituted the actual strictosidine genes from a MIA producer plant Catharanthus roseus [48]. Of the 14 genes, 11 constituted the actual biosynthesis pathway and the other three were involved in enhancing the activity of P450s. Among strictosidine biosynthesis pathway and the other three were involved in enhancing the activity of the remaining seven added genes, five were existing yeast genes that were included as an extra copy P450s. Among the remaining seven added genes, five were existing yeast genes that were included of the genes in the genome. Incorporation of multiple copies of genes is frequently performed in as an extra copy of the genes in the genome. Incorporation of multiple copies of genes is frequently building a heterologous production system to increase the expression level of specific genes for performed in building a heterologous production system to increase the expression level of specific improved productivity of the biosynthetic pathway of interest [49]. This strategy was also applied to genes for improved productivity of the biosynthetic pathway of interest [49]. This strategy was also the strictosidine biosynthetic gene G8H, where four copies of the codon-optimized G8H gene were G8H G8H incorporatedapplied to the into strictosidine the yeast genome biosynthetic to allow gene production, where of 11 four without copies exogenous of the codon-optimized feeding of geraniol. gene were incorporated into the yeast genome to allow production of 11 without exogenous feeding Lastly, two more genes, AgGPPS2 and mFPS144, were taken from exogenous sources and incorporated of geraniol. Lastly, two more genes, AgGPPS2 and mFPS144, were taken from exogenous sources into the yeast genome to increase the cellular pool of geranyl pyrophosphate (GPP). AgGPPS2 is a GPPand incorporatedsynthase from into grand the yeastfir (Abies genome grandis to) increase that is known the cellular to produce pool of only geranyl GPPpyrophosphate and does not convert (GPP). Abies grandis AgGPPS2GPP further is ainto GPP farnesyl synthase pyrophosphate from grand fir (FPP) ( [50], and) thatmFPS144 is known is an to N114W produce mutant only GPP of avian and doesFPP synthasenot convert from GPP the further red junglefowl into farnesyl (Gallus pyrophosphate gallus) that produces (FPP) [50 GPP], and nearly mFPS144 exclusively is an N114W [51]. Those mutant two of genes were used to replace the yeast FPP synthase gene ERG20, which was one of the three yeast genes deleted from the genome for the biosynthesis of 11. The other two genes were ATF1 and OYE2, both of which are involved in metabolizing geraniol into other products. After those extensive modifications, the engineered yeast successfully produced 11 at a yield of approximately 0.5 mg/L of culture medium comprised of only simple carbon and nitrogen sources [47]. This yeast strain provides an important first step toward building a microbial system for the production of expensive, complex molecules that only plants produce in small amounts.

Molecules 2016, 21, 1078 9 of 19 avian FPP synthase from the red junglefowl (Gallus gallus) that produces GPP nearly exclusively [51]. Those two genes were used to replace the yeast FPP synthase gene ERG20, which was one of the three yeast genes deleted from the genome for the biosynthesis of 11. The other two genes were ATF1 and OYE2, both of which are involved in metabolizing geraniol into other products. After those extensive modifications, the engineered yeast successfully produced 11 at a yield of approximately 0.5 mg/L of culture medium comprised of only simple carbon and nitrogen sources [47]. This yeast strain provides an important first step toward building a microbial system for the production of expensive, complex Moleculesmolecules 2016 that, 21, 1078 only plants produce in small amounts. 9 of 19

5. Ergotamine Ergotamine Ergot alkaloids form form another another fam familyily of of alkaloids alkaloids under under the the structurally structurally diverse diverse group group of of TIAs. TIAs. Unlike MIAs MIAs described described in inthe the previous previous section section that thatuse GPP use GPPand L and-tryptophanL-tryptophan as their as building their building blocks, ergotblocks, alkaloids ergot alkaloids are derived are derived from dime fromthylallyl dimethylallyl pyrophosphate pyrophosphate (DMAPP) (DMAPP) and L and-tryptophanL-tryptophan [52]. The [52]. ergotThe ergottype of type natural of natural products products exerts biological exerts biological activities by activities acting as by either acting an asagonist either or anan antagonist agonist or towardan antagonist various toward receptors various for a receptors number forof neurotrans a number ofmitters, neurotransmitters, such as dopamine such as, serotonin, dopamine, adrenaline serotonin, andadrenaline noradrenaline and noradrenaline [53]. Certain [53 ].members Certain membersof ergot al ofkaloids ergot alkaloids have also have been also shown been shownto exhibit to exhibit anti- tumoranti-tumor activities activities [54]. The [54]. potent The potent bioactivities bioactivities of ergot of alkaloids ergot alkaloids have led have people led to people use them to use in themvarious in medicalvarious medicalapplications applications since early since in the early human in the history, human with history, thewith oldest the record oldest dating record back dating to around back to 1000around B.C.E. 1000 [55]. B.C.E. The [55 ergot]. The alkaloids ergot alkaloids are produced are produced by fungi by and fungi plants, and but plants, the butClavicipitaceae the Clavicipitaceae family offamily fungi of is fungimost well is most known well for known ergot for prod ergotuction. production. Among the Among Clavicipitaceae the Clavicipitaceae family, Claviceps family, purpureaClaviceps, anpurpurea endophytic, an endophytic parasite parasitethat is also that known is also knownas the asergot the fungus ergot fungus of rye, of and rye, andNeotyphodiumNeotyphodium lolii,lolii an, endophytican endophytic symbiont, symbiont, are are the the two two most-studied most-studied er ergot-producinggot-producing fungi fungi [52]. [52]. Another Another fungus fungus known known forfor production production of of the the ergot ergot family family of of natural natural products, products, primarily primarily clavine-type clavine-type alkaloids, alkaloids, is is AspergillusAspergillus fumigatusfumigatus,, an an opportunistic opportunistic pathogen pathogen of of mammals mammals [56]. [56]. Below, Below, we we will will focus focus on the biosynthetic pathway of ergot alkaloids, and in particular discuss the mechanism involved in the formation of the interestinginteresting backbone backbone core core of of 6,8-dimethylergoline 6,8-dimethylergoline tetracyclic tetracyclic ring ring structure structure ( (1414)) that that is is shared shared by by all all ergot alkaloids (Figure 10).10).

Figure 10. ProposedProposed biosynthetic biosynthetic pathway pathway of of DD-lysergic-lysergic acid acid 2020 [53],[53], having having the the principal 6,8-dimethylergoline tetracyclic ring structure 14 of ergot alkaloids.

Biosynthesis of ergot alkaloids was initially investigated through feeding experiments that used Biosynthesis of ergot alkaloids was initially investigated through feeding experiments that used isotopically labeled substrates in the cultures of C. purpurea [53]. Based on those experiments, the isotopically labeled substrates in the cultures of C. purpurea [53]. Based on those experiments, the ergot biosynthetic pathway was determined to be initiated by the prenylation of L-tryptophan with ergot biosynthetic pathway was determined to be initiated by the prenylation of L-tryptophan with DMAPP that forms 4-(γ,γ-dimethylallyl) tryptophan (15) (Figure 10) [57,58]. The next step involves DMAPP that forms 4-(γ,γ-dimethylallyl) tryptophan (15) (Figure 10)[57,58]. The next step involves the N-methylation of 15 to yield 4-dimethyl-L-abrine (16) [59]. Subsequently, a series of successive the N-methylation of 15 to yield 4-dimethyl-L-abrine (16)[59]. Subsequently, a series of successive oxidation steps is proposed to take place, resulting in the intramolecular cyclization of the prenyl and oxidation steps is proposed to take place, resulting in the intramolecular cyclization of the prenyl indole moieties to form the tricyclic chanoclavine-I (17) along with ring C of the tetracyclic core of and indole moieties to form the tricyclic chanoclavine-I (17) along with ring C of the tetracyclic ergot alkaloids [60]. Compound 17 is then oxidized to form chanoclavine-I-aldehyde (18) from which ergot alkaloid biosynthesis can diverge. In one pathway, 18 can undergo intramolecular cyclization to form ring D to complete the tetracyclic ergoline core structure, yielding agroclavine (19). Subsequently, 19 is converted to D-lysergic acid (20) via an oxidation of a methyl group as illustrated in Figure 10. In another pathway, 18 undergoes a similar cyclization to form a closely related tetracyclic intermediate, festuclavine, which becomes the core molecule for the clavine-type fumigaclavines and related compounds produced by A. fumigatus. In this section, we will focus on the biosynthesis of ergotamine (21) from 20, which takes place in C. purpurea and N. lolii. Further steps in the biosynthesis of ergotamine (21) have been elucidated predominantly by Keller et al. [61–64]. Those steps mainly concern the formation and the modification of the peptide- derived segment of 21 (Figure 11). The peptide extension of 20 is initiated by LPS2 (lysergic peptide synthetase 2, NRPS). The adenylation domain of LPS2 accepts 20 as the starter unit, and the condensation

Molecules 2016, 21, 1078 10 of 19 core of ergot alkaloids [60]. Compound 17 is then oxidized to form chanoclavine-I-aldehyde (18) from which ergot alkaloid biosynthesis can diverge. In one pathway, 18 can undergo intramolecular cyclization to form ring D to complete the tetracyclic ergoline core structure, yielding agroclavine (19). Subsequently, 19 is converted to D-lysergic acid (20) via an oxidation of a methyl group as illustrated in Figure 10. In another pathway, 18 undergoes a similar cyclization to form a closely related tetracyclic intermediate, festuclavine, which becomes the core molecule for the clavine-type fumigaclavines and related compounds produced by A. fumigatus. In this section, we will focus on the biosynthesis of ergotamine (21) from 20, which takes place in C. purpurea and N. lolii. Further steps in the biosynthesis of ergotamine (21) have been elucidated predominantly by Keller et al. [61–64]. Those steps mainly concern the formation and the modification of the peptide-derivedMolecules 2016, 21, 1078 segment of 21 (Figure 11). The peptide extension of 20 is initiated by LPS2 (lysergic10 of 19 peptide synthetase 2, NRPS). The adenylation domain of LPS2 accepts 20 as the starter unit, and the condensationdomain couples domain it with couplesL-alanine, it withwhichL-alanine, is activate whichd by the is activatedfirst adenylation by the firstdomain adenylation of LPS1 (lysergic domain ofpeptide LPS1 synthetase (lysergic peptide 1, NRPS), synthetase through an1 ,amide NRPS), linkage through formation. an amide The linkage resultant formation. D-lysergyl-monopeptide The resultant intermediateD-lysergyl-monopeptide is extended intermediate further with isL-phenylalanine extended further and with L-prolineL-phenylalanine by LPS1 to and yieldL-proline the D-lysergyl- by LPS1 totripeptide, yield the whichD-lysergyl-tripeptide, remains tethered whichto the phosphopantetheinyl remains tethered to thearm phosphopantetheinyl of the third thiolation armdomain of the of thirdLPS1. thiolationThe terminal domain cyclization of LPS1. or Thelactamization terminal cyclizationdomain is orthought lactamization to catalyze domain the release is thought of the to catalyzeD-lysergyl-tripeptide the release of from the D LPS1-lysergyl-tripeptide as N-(D-lysergyl- fromL-Ala)- LPS1L-Phe- as N-(L-Pro-lactamD-lysergyl-L (-Ala)-L,L-ergotamam).L-Phe-L-Pro-lactam Finally, (LL,L,L-ergotamam-ergotamam). is converted Finally, L, Linto-ergotamam ergotamine is converted (21) by the into hydroxylation ergotamine (activity21) by the of hydroxylationthe non-heme 2+ activitydioxygenase of the EasH non-heme (Fe2+/2-ketoglutarate-dependent dioxygenase EasH (Fe /2-ketoglutarate-dependent dioxygenase) [64]. dioxygenase) [64].

Figure 11. Nonribosomal assembly of the peptide portion of ergotamine 21, initiatedinitiated withwith D-lysergic acid 2020 asas a primer a primer unit [64]. unit LPS, [64]. D-lysergyl LPS, D -lysergylpeptide synthetase; peptide synthetase;C, condensation C, condensationdomain; A, adenylation domain; A,domain; adenylation T, thiolation domain; domain; T, thiolation Cyc, cyclolization domain; Cyc, domain. cyclolization domain.

Very recently, heterologous partial reconstitution of the ergot alkaloid biosynthetic pathway was Very recently, heterologous partial reconstitution of the ergot alkaloid biosynthetic pathway accomplished in Aspergillus nidulans by introducing a segment of the A. fumigatus genome covering was accomplished in Aspergillus nidulans by introducing a segment of the A. fumigatus genome a portion of the ergot alkaloid biosynthetic gene cluster into the A. nidulans chromosome [65]. covering a portion of the ergot alkaloid biosynthetic gene cluster into the A. nidulans chromosome [65]. The integrated A. fumigatus genomic DNA segment contained four genes, dimethylallyltryptophan The integrated A. fumigatus genomic DNA segment contained four genes, dimethylallyltryptophan synthase-coding dmaW, dimethylallyltryptophan N-methyltransferase-coding easF, - synthase-coding dmaW, dimethylallyltryptophan N-methyltransferase-coding easF, oxidoreductase-like like protein-coding easE and catalase-like protein-coding easC. The transformed A. nidulans was able protein-coding easE and catalase-like protein-coding easC. The transformed A. nidulans was able to produce 16 and 17, early intermediates in the D-lysergic acid biosynthetic pathway (Figure 10). to produce 16 and 17, early intermediates in the D-lysergic acid biosynthetic pathway (Figure 10). Using this heterologous production system, gene deletion experiments were performed to determine Using this heterologous production system, gene deletion experiments were performed to determine for the first time that the four genes were sufficient for the biosynthesis of 17 [65]. This successful for the first time that the four genes were sufficient for the biosynthesis of 17 [65]. This successful production of ergot alkaloid biosynthetic intermediates in A. nidulans would facilitate the effort production of ergot alkaloid biosynthetic intermediates in A. nidulans would facilitate the effort toward toward achieving engineered biosynthesis of this class of natural products. Such a heterologous achieving engineered biosynthesis of this class of natural products. Such a heterologous biosynthetic biosynthetic platform would promote the study on the complex mechanism of ergot alkaloid biosynthesis and advance the drug discovery efforts that evolve around ergot-type natural products.

6. Opiates The last section will look at opiates, which is a group of alkaloids that has been known to humans for about 6000 years and played a significant role as analgesic therapeutics and as abused recreational substances [66]. Opiates are naturally occurring members of opioids that can be isolated from the latex collected from opium poppy Papaver somniferum. The representative examples of opiates include reticuline, thebaine, codeine and (Figures 12–15). Opioids also include semi-synthetic and synthetic compounds, for example oxycodone and fentanyl, that interact with opioid receptors to effect physiological responses, such as analgesia, euphoria and respiratory depression [67]. Currently, the pharmaceutical industry relies heavily on plants as the predominant source of opiates and opiate-derived compounds. However, plant cultivation is labor intensive, difficult to scale up and hard to maintain yield consistency. While metabolic engineering of plants has been attempted to improve growth parameters and opiate productivity, the efforts have generally been met with difficulties thus far [68].

Molecules 2016, 21, 1078 11 of 19

platform would promote the study on the complex mechanism of ergot alkaloid biosynthesis and advance the drug discovery efforts that evolve around ergot-type natural products.

6. Opiates The last section will look at opiates, which is a group of alkaloids that has been known to humans for about 6000 years and played a significant role as analgesic therapeutics and as abused recreational substances [66]. Opiates are naturally occurring members of opioids that can be isolated from the latex collected from opium poppy Papaver somniferum. The representative examples of opiates include reticuline, thebaine, codeine and morphine (Figures 12–15). Opioids also include semi-synthetic and synthetic compounds, for example oxycodone and fentanyl, that interact with opioid receptors to effect physiological responses, such as analgesia, euphoria and respiratory depression [67]. Currently, the pharmaceutical industry relies heavily on plants as the predominant source of opiates and opiate-derived compounds. However, plant cultivation is labor intensive, difficult to scale up and hard Moleculesto maintain 2016, yield21, 1078 consistency. While metabolic engineering of plants has been attempted to improve11 of 19 Molecules 2016, 21, 1078 11 of 19 growth parameters and opiate productivity, the efforts have generally been met with difficulties thus MorefarMore [68 recently,].recently, More recently, metabolicmetabolic metabolic engineeringengineering engineering ofof microbesmicrobes of microbes hashas made hasmade made aa significantsignificant a significant progressprogress progress andand and isis is quicklyquickly becomingbecoming a a viable viable alternative alternative strategy strategy for for improving improving the the opiate opiate production production process. process. NotNot only only microbe microbe cultivationcultivation isis lowerlower inin cost cost and and easier easier to to scale scale up up thanthan plantplant cultivation,cultivation, the the product product can can be be purified purifiedpurified moremore easily easily from from microbial microbial extracts extracts than than from from plant plant extracts extracts duedue toto lacklack ofof similarsimilar metabolitesmetabolites [47,69,70].[47,69,70]. [47,69,70]. Below,Below, wewewe will willwill discuss discussdiscuss recent recentrecent successes successessuccesses in developing inin devedevelopingloping microbe-based microbe-basedmicrobe-based biosynthetic biosyntheticbiosynthetic systems systemssystems that produce thatthat produceaproduce type of aa opiates typetype ofof called opiatesopiates benzylisoquinoline calledcalled benzylisoquinolinebenzylisoquinoline alkaloids alkaloidsalkaloids (BIAs). (BIAs).(BIAs).

Figure 12. Heterologous biosynthetic pathway constructed in yeast (Saccharomyces cerevisiae) for the Figure 12. HeterologousHeterologous biosynthetic pathwaypathway constructed in yeast ((Saccharomyces cerevisiae)) for thethe production of (R,S)-reticuline from the exogenously provided substrate, (R,S)-tetrahydropapaveroline production of (R,,S)-reticuline)-reticuline from thethe exogenouslyexogenously providedprovided substrate,substrate, ((RR,,SS)-tetrahydropapaveroline)-tetrahydropapaveroline (THP, also known as norlaudanosoline) [71]. 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, (THP, alsoalso knownknown asas norlaudanosoline)norlaudanosoline) [[71].71]. 6OMT, norcoclaurinenorcoclaurine 6-O-methyltransferase; CNMT, CNMT, coclaurine N-methyltransferase; 4′0OMT, 3′0-hydroxy-N-methylcoclaurine 4′0-O-methyltransferase. coclaurine N-methyltransferase; 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase.

Figure 13. Extension of the Saccharomyces cerevisiae heterologous reticuline biosynthetic pathway for the Figure 13. Extension of the Saccharomyces cerevisiae heterologous reticuline biosynthetic pathway for the productionFigure 13. Extensionof precursors of theof theSaccharomyces berberine- and cerevisiae sanguinarine-typeheterologous benzylisoquino reticuline biosyntheticline alkaloids pathway (in black) for production of precursors of the berberine- and sanguinarine-type benzylisoquinoline alkaloids (in black) andthe productionthe morphinan of precursors alkaloids (in of red). the berberine- Compounds and that sanguinarine-type are actually biosynthesized benzylisoquinoline by the engineered alkaloids and the morphinan alkaloids (in red). Compounds that are actually biosynthesized by the engineered yeast(in black) strains and [71,72] the morphinanare shown in alkaloids solid, whereas (in red). downstream Compounds products that areare actuallyshown in biosynthesized semi-transparent. by yeast strains [71,72] are shown in solid, whereas downstream products are shown in semi-transparent. BBE,the engineered berberine bridge yeast strainsenzyme; [71 SMT,,72] are(S)-scoulerine shown in solid,9-O-methyltransferase; whereas downstream CYP719A, products canadine are shownsynthase in BBE, berberine bridge enzyme; SMT, (S)-scoulerine 9-O-methyltransferase;S O CYP719A, canadine synthase cytochromesemi-transparent. P450 719A; BBE, berberineCOX, canadine bridge oxidas enzyme;e; CYP2D6, SMT, ( )-scoulerine human cytochrome 9- -methyltransferase; P450 2D6. CYP719A, canadinecytochrome synthase P450 719A; cytochrome COX, canadine P450 719A; oxidas COX,e; CYP2D6, canadine human oxidase; cytochrome CYP2D6, P450 human 2D6. cytochrome P450 2D6.

Figure 14. Heterologous biosynthetic pathway constructed in Saccharomyces cerevisiae for the production Figure 14. Heterologous biosynthetic pathway constructed in Saccharomyces cerevisiae for the production of morphinan alkaloids and related semi-synthetic opioids from thebaine, the exogenously provided of morphinan alkaloids and related semi-synthetic opioids from thebaine, the exogenously provided starting material [73]. The biosynthetic pathway leading to the formation of morphine, the prominent starting material [73]. The biosynthetic pathway leading to the formation of morphine, the prominent member of the morphinan alkaloids, is shown in black. The biosynthetic pathways leading to the member of the morphinan alkaloids, is shown in black. The biosynthetic pathways leading to the formation of semi-synthetic opioids, hydromorphone, hydrocodone and oxycodone, are shown in formation of semi-synthetic opioids, hydromorphone, hydrocodone and oxycodone, are shown in red. T6ODM, thebaine 6-O-demethylase; COR, codeinone reductase; CODM, codeine O-demethylase; red. T6ODM, thebaine 6-O-demethylase; COR, codeinone reductase; CODM, codeine O-demethylase; MorA, morphine dehydrogenase; MorB, morphinone reductase. MorA, morphine dehydrogenase; MorB, morphinone reductase.

Molecules 2016, 21, 1078 11 of 19

More recently, metabolic engineering of microbes has made a significant progress and is quickly becoming a viable alternative strategy for improving the opiate production process. Not only microbe cultivation is lower in cost and easier to scale up than plant cultivation, the product can be purified more easily from microbial extracts than from plant extracts due to lack of similar metabolites [47,69,70]. Below, we will discuss recent successes in developing microbe-based biosynthetic systems that produce a type of opiates called benzylisoquinoline alkaloids (BIAs).

Figure 12. Heterologous biosynthetic pathway constructed in yeast (Saccharomyces cerevisiae) for the production of (R,S)-reticuline from the exogenously provided substrate, (R,S)-tetrahydropapaveroline (THP, also known as norlaudanosoline) [71]. 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; 4′OMT, 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase.

Figure 13. Extension of the Saccharomyces cerevisiae heterologous reticuline biosynthetic pathway for the production of precursors of the berberine- and sanguinarine-type benzylisoquinoline alkaloids (in black) and the morphinan alkaloids (in red). Compounds that are actually biosynthesized by the engineered yeast strains [71,72] are shown in solid, whereas downstream products are shown in semi-transparent. MoleculesBBE,2016 berberine, 21, 1078 bridge enzyme; SMT, (S)-scoulerine 9-O-methyltransferase; CYP719A, canadine synthase12 of 19 cytochrome P450 719A; COX, canadine oxidase; CYP2D6, human cytochrome P450 2D6.

Figure 14. Heterologous biosynthetic pathway constructed in Saccharomyces cerevisiae for the production Figure 14. Heterologous biosynthetic pathway constructed in Saccharomyces cerevisiae for the production of morphinan alkaloids and related semi-synthetic opioids from thebaine, the exogenously provided of morphinan alkaloids and related semi-synthetic opioids from thebaine, the exogenously provided starting material [73]. The biosynthetic pathway leading to the formation of morphine, the prominent starting material [73]. The biosynthetic pathway leading to the formation of morphine, the prominent member of thethe morphinanmorphinan alkaloids,alkaloids, isis shownshown inin black.black. The biosynthetic pathways leading to thethe formationformation ofof semi-syntheticsemi-synthetic opioids,opioids, hydromorphone,hydromorphone, hydrocodone and oxycodone, are shown in red.red. T6ODM, thebaine 6- O-demethylase; COR, codeinone reductase; CODM, codeine O-demethylase;-demethylase; MorA, morphine dehydrogenase;dehydrogenase; MorB,MorB, morphinonemorphinone reductase.reductase.

In one approach toward developing microbes that can biosynthesize opiates, Smolke et al. engineered yeast S. cerevisiae to produce a series of BIAs. First, they developed a yeast strain harboring a set of biosynthetic genes that successfully produced (R,S)-reticuline upon feeding of a racemic mixture of tetrahydropapaveroline (THP), also known as norlaudanosoline (Figure 12)[71]. To produce reticuline, the key starting intermediate in the BIA biosynthetic pathway, a pair of plasmids carrying three biosynthetic genes encoding norcoclaurine 6-O-methyltransferase (6OMT), coclaurine-N-methyltransferase (CNMT) and 30-hydroxy-N-methylcoclaurine 40-O-methyltransferase (40OMT) from Thalictrum flavum (yellow meadow-rue) or P. somniferum were introduced to S. cerevisiae. All three enzymes were known to accept both (R)-and (S)-enantiomers of their substrates [74,75]. The yeast strain carrying those three genes was able to biotransform the fed (R,S)-THP into (R,S)-reticuline (Figure 12). Then, a plasmid-born biosynthetic pathway comprised of the P. somniferum berberine bridge enzyme gene (BBE), the T. flavum (S)-scoulerine 9-O-methyltransferase gene (SMT) and the T. flavum canadine synthase gene (CYP719A1, a P450 gene) was established in the reticuline-producing yeast to produce BIA products found along the sanguinarine and berberine biosynthetic pathways. This yeast strain was able to produce (S)-scoulerine (sanguinarine precursor), (S)-tetrahydrocolumbamine and (S)-tetrahydroberberine (also known as canadine, a berberine precursor) (Figure 13, in black) [71]. Subsequently, incorporation of the canadine oxidase (COX) from Berberis wilsonae [76] and optimization of the berberine biosynthetic pathway in yeast led to successful production of berberine from fed (R,S)-THP [72]. Furthermore, a successful production of the first intermediate of the morphinan alkaloid biosynthesis pathway, salutaridine, was also accomplished by introducing a human P450 gene CYP2D6 into the reticuline-producing yeast strain (Figure 13, in red). Related to this work, another group also established a biosynthetic pathway in yeast that converted fed (R,S)-THP into dihydrosanguinarine, an immediate precursor of sanguinarine [77], through the formation of (R,S)-reticuline and (S)-scoulerine using a biosynthetic scheme that was practically identical to that developed by Smolke et al. [71]. Molecules 2016, 21, 1078 12 of 19

In one approach toward developing microbes that can biosynthesize opiates, Smolke et al. engineered yeast S. cerevisiae to produce a series of BIAs. First, they developed a yeast strain harboring a set of biosynthetic genes that successfully produced (R,S)-reticuline upon feeding of a racemic mixture of tetrahydropapaveroline (THP), also known as norlaudanosoline (Figure 12) [71]. To produce reticuline, the key starting intermediate in the BIA biosynthetic pathway, a pair of plasmids carrying three biosynthetic genes encoding norcoclaurine 6-O-methyltransferase (6OMT), coclaurine-N- methyltransferase (CNMT) and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) from Thalictrum flavum (yellow meadow-rue) or P. somniferum were introduced to S. cerevisiae. All three enzymes were known to accept both (R)-and (S)-enantiomers of their substrates [74,75]. The yeast strain carrying those three genes was able to biotransform the fed (R,S)-THP into (R,S)-reticuline (Figure 12). Then, a plasmid-born biosynthetic pathway comprised of the P. somniferum berberine bridge enzyme gene (BBE), the T. flavum (S)-scoulerine 9-O-methyltransferase gene (SMT) and the T. flavum canadine synthase gene (CYP719A1, a P450 gene) was established in the reticuline-producing yeast to produce BIA products found along the sanguinarine and berberine biosynthetic pathways. This yeast strain was able to produce (S)-scoulerine (sanguinarine precursor), (S)-tetrahydrocolumbamine and (S)-tetrahydroberberine (also known as canadine, a berberine precursor) (Figure 13, in black) [71]. Subsequently, incorporation of the canadine oxidase (COX) from Berberis wilsonae [76] and optimization of the berberine biosynthetic pathway in yeast led to successful production of berberine from fed (R,S)-THP [72]. Furthermore, a successful production of the first intermediate of the morphinan alkaloid biosynthesis pathway, salutaridine, was also accomplished by introducing a human P450 gene CYP2D6 into the reticuline-producing yeast strain (Figure 13, in red). Related to this work, another group also established a biosynthetic pathway in yeast that converted fed (R,S)-THP into dihydrosanguinarine, Moleculesan immediate2016, 21, 1078 precursor of sanguinarine [77], through the formation of (R,S)-reticuline and (S)-scoulerine13 of 19 using a biosynthetic scheme that was practically identical to that developed by Smolke et al. [71].

Figure 15. Heterologous biosynthetic pathway installed in Saccharomyces cerevisiae for de novo Figure 15. Heterologous biosynthetic pathway installed in Saccharomyces cerevisiae for de novo production of morphinan alkaloids and their semi-synthetic compounds from simple sugar [78]. Only production of morphinan alkaloids and their semi-synthetic compounds from simple sugar [78]. the pathway leading to the formation of the key biosynthetic intermediate thebaine is shown. For the Onlybiosynthetic the pathway pathway leading leading to the to formation the formation of theof morphine key biosynthetic and semi-synthetic intermediate morphinan thebaine opioids, is shown. For thesee biosyntheticFigure 14. 4-HPP, pathway 4-hydroxyphenylpyruv leading to the formationate; 4-HPAA, of 4-hydroxyphenylacetaldehyde; morphine and semi-synthetic L-DOPA, morphinan opioids,L-3,4-dihydroxyphenylalanine; see Figure 14. 4-HPP, 4-hydroxyphenylpyruvate; Aro8, S. cerevisiae aromatic 4-HPAA,aminotransferase 4-hydroxyphenylacetaldehyde; I; Aro9, S. cerevisiae L-DOPA,aromaticL-3,4-dihydroxyphenylalanine; aminotransferase II; TyrH, tyrosine Aro8, S. cerevisiaehydroxylase;aromatic DODC, aminotransferase L-DOPA decarboxylase; I; Aro9, Aro10,S. cerevisiae aromaticS. cerevisiae aminotransferase phenylpyruvate II; decarboxylase; TyrH, tyrosine NCS, hydroxylase; norcoclaurine DODC, synthase;L CYP80B,-DOPA N decarboxylase;-methylcoclaurine Aro10, S. cerevisiae3′-hydroxylase;phenylpyruvate DRS–DRR, decarboxylase; 1,2-dehydroreticuline NCS, norcoclaurine synthase–1,2-dehydroreticu synthase; CYP80B,line reductase;N-methylcoclaurine SalSyn, 30-hydroxylase;salutaridine DRS–DRR,synthase; SalR, 1,2-dehydroreticuline salutaridine reductase; synthase–1,2-dehydroreticuline SalAT, 7-O-acetyltransferase. reductase; SalSyn, salutaridine synthase; SalR, salutaridine reductase; SalAT, salutaridinol 7-O-acetyltransferase.

One unique strategy developed by Smolke et al. for optimizing the performance of a heterologous biosynthetic pathway in yeast was to use a GAL1-10 promoter system that is tunable with galactose concentration. Using this tunable expression system, Smolke et al. determined the optimal expression level of each of the heterologous genes for the production of desired products in yeast. Once the optimal expression level for each of the genes was determined, promoter of appropriate strength was selected for each of the genes to fine-tune the overall performance of the biosynthetic pathway. This type of optimization may be crucial in this type of study, where genes from diverse sources are assembled together to form a cooperative metabolic pathway. Streamlining the identification of the bottleneck in a newly installed heterologous metabolic pathway can also be accomplished by using a systems biological approach to illuminate the source of stress that is imposed on the host cell by expressing a foreign gene. In one example, effect of overproducing a foreign P450 in yeast was analyzed by DNA microarray analyses to reveal that transcription factors associated with iron starvation and heme-dependent gene regulation were overrepresented when the foreign P450 gene was expressed. This result indicated heme depletion as a possible stress in the system, and the activity of the target P450 was elevated by engineering the host0s heme biosynthesis to increase the intracellular heme level [79]. A similar approach can be taken to identify the source of stress in an engineered strain so that necessary measures can be implemented to eliminate the bottleneck in the exogenous biosynthetic pathway of interest. The Smolke group also developed engineered yeast strains that can convert fed thebaine into codeine, morphine, hydromorphone, hydrocodone and oxycodone (Figure 14)[73]. To establish the biosynthetic pathway that converts thebaine into morphine, three P. somniferum genes, 6-O-demethylase (T6ODM), codeinone reductase isoform 1.3 (COR1.3) and codeine O-demethylase (CODM), were assembled into a single yeast artificial chromosome (YAC) and introduced into yeast. In one pathway, thebaine is converted into codeinone by T6ODM via the formation of neopinone. Then, COR reduces Molecules 2016, 21, 1078 14 of 19 the carbonyl of codeinone to yield codeine. Finally, codeine is demethylated to morphine by CODM (Figure 14, in black). With the feeding of thebaine, the yeast strain carrying the YAC was able to produce codeine and morphine successfully. However, the pathway performance was improved by supplementing the culture medium with 2-oxoglutarate, a key co-substrate of dioxygenases T6ODM and CODM, as well as the precursors of 2-oxoglutarate, L-glutamine and L-glutamic acid. The copy number of the three genes was also optimized, where the ratio of 2:1:3 for T6ODM:COR1.3:CODM copy number was determined to give the best result with 5.2 mg/L of morphine being produced. The morphine-producing yeast was further engineered with two genes, morphine dehydrogenase (morA) and morphinone reductase (morB) from Pseudomonas putida M10 to biosynthetically prepare semi-synthetic morphine derivatives, hydromorphone, hydrocodone and oxycodone (Figure 14, in red). Most recently, the yeast engineering effort was able to develop a strain capable of producing thebaine and hydrocodone from simple sugar [78]. To eliminate the need for supplementation of THP or thebaine, steps leading to the formation of (S)-reticuline were established by incorporating five plant-derived genes, including 6OMT, CNMT and 40OMT from P. somniferum that were also used in the previous study [71]. In this system, dopamine, which is formed from L-tyrosine by the combined action of a mammalian and a bacterial enzyme, and 4-hydroxyphenylacetaldehyde (4-HPAA), an intermediate in the aromatic amine degradation pathway [80], were combined together by Coptis japonica (Japanese goldthread) norcoclaurine synthase (NCS) to form (S)-norcoclaurine (Figure 15). Compared to THP, norcoclaurine lacks one hydroxyl group. Thus, CYP80B, another gene from the Eschscholzia californica (California poppy) N-methylcoclaurine 30-hydroxylase, also had to be included in the pathway to produce (S)-reticuline. Once (S)-reticuline became available, it was converted into (R)-reticuline by introducing 1,2-dehydroreticuline synthase–1,2-dehydroreticuline reductase (DRS–DRR), a natural fusion protein from Papaver bracteatum (Iranian poppy), into the system to initiate the morphinan alkaloid biosynthesis. Unlike the previous study that used the human P450 CYP2D6 to convert (R)-reticuline into salutaridine (Figure 13, in red) [71], this study employed an engineered P. bracteatum salutaridine synthase (SalSyn) that was fused to another plant P450 cheilanthifoline synthase to drive proper folding and glycosylation of P. bracteatum SalSyn in yeast. This fusion enzyme attained an approximately six-fold greater activity than the native P. bracteatum SalSyn [78]. Subsequently, salutaridine was reduced by P. bracteatum salutaridine reductase (SalR) to yield salutaridinol, which was then acetylated by P. somniferum salutaridinol 7-O-acetyltransferase (SalAT) to form 7-O-acetylsalutaridinol. Finally, 7-O-acetylsalutaridinol was spontaneously converted into thebaine, completing the de novo biosynthesis of the key intermediate for morphine biosynthesis in yeast. Conversion of thebaine to hydrocodone was achieved by essentially the same approach used in the previous study (Figure 14)[73]. Overall, the complete biosynthesis of hydrocodone from simple sugar required introduction and engineering of a total of 23 genes from plants (11 genes from four plant species, including one extensively engineered SalSyn gene), mammal (one engineered gene from rat), bacteria (two genes from P. putida) and yeast (nine genes, of which four were modified). In another approach, Sato et al. focused their efforts on establishing a BIA biosynthetic pathway in E. coli and successfully developed production systems capable of biosynthesizing (R,S)-THP, (R,S)-reticuline, thebaine and hydrocodone (Figure 16)[70,81–83]. Their strategy relied on supplying the key intermediate reticuline as a racemic mixture using an artificial biosynthetic pathway comprised of , L-3,4-dihydroxyphenylalanine (L-DOPA) decarboxylase and monoamine oxidase (MAO) obtained from various bacterial sources. These three enzymes work together to convert L-tyrosine into dopamine and 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) [81]. Initially, an NCS gene was introduced to the system to combine dopamine and 3,4-DHPAA into (S)-THP for subsequent conversion into (S)-reticuline [70,81]. However, NCS was later eliminated to rely on the spontaneous formation of a racemic mixture of THP from dopamine and 3,4-DHPAA via a Pictet-Spengler reaction inside the cells [82]. Then, (R,S)-THP was converted into (R,S)-reticuline by 6OMT, CNMT and 40OMT [83]. This was the same set of three enzymes that was used by Smolke et al. to prepare (S)-reticuline from (R,S)-THP (Figure 12)[71], except all of the genes used by Sato et al. originated Molecules 2016, 21, 1078 14 of 19

salutaridine was reduced by P. bracteatum salutaridine reductase (SalR) to yield salutaridinol, which was then acetylated by P. somniferum salutaridinol 7-O-acetyltransferase (SalAT) to form 7-O-acetylsalutaridinol. Finally, 7-O-acetylsalutaridinol was spontaneously converted into thebaine, completing the de novo biosynthesis of the key intermediate for morphine biosynthesis in yeast. Conversion of thebaine to hydrocodone was achieved by essentially the same approach used in the previous study (Figure 14) [73]. Overall, the complete biosynthesis of hydrocodone from simple sugar required introduction and engineering of a total of 23 genes from plants (11 genes from four plant species, including one extensively engineered SalSyn gene), mammal (one engineered gene from rat), bacteria (two genes from P. putida) and yeast (nine genes, of which four were modified). In another approach, Sato et al. focused their efforts on establishing a BIA biosynthetic pathway in E. coli and successfully developed production systems capable of biosynthesizing (R,S)-THP, (R,S)- reticuline, thebaine and hydrocodone (Figure 16) [70,81–83]. Their strategy relied on supplying the key intermediate reticuline as a racemic mixture using an artificial biosynthetic pathway comprised of tyrosinase, L-3,4-dihydroxyphenylalanine (L-DOPA) decarboxylase and monoamine oxidase (MAO) obtained from various bacterial sources. These three enzymes work together to convert L-tyrosine into dopamine and 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) [81]. Initially, an NCS gene was introduced to the system to combine dopamine and 3,4-DHPAA into (S)-THP for subsequent conversion into (S)-reticuline [70,81]. However, NCS was later eliminated to rely on the spontaneous Moleculesformation2016, 21 ,of 1078 a racemic mixture of THP from dopamine and 3,4-DHPAA via a Pictet-Spengler reaction15 of 19 inside the cells [82]. Then, (R,S)-THP was converted into (R,S)-reticuline by 6OMT, CNMT and 4′OMT [83]. This was the same set of three enzymes that was used by Smolke et al. to prepare (S)-reticuline fromfromC. japonica (R,S)-THP. Once (FigureE. coli 12) strains[71], except for theall of production the genes used of reticulineby Sato et al. were originated established, from C.P. japonica somniferum. genesOnce that E. code coli forstrains SalR, for SalAT the production and engineered of reticuline SalSyn were having established, its N-terminal P. somniferum transmembrane genes that code region removedfor SalR, were SalAT added and to engin the systemeered SalSyn to produce having thebaine its N-terminal [83]. Lastly,transmembrane addition region of P. somniferum removed were T6ODM and P.added putida to the morB systemgenes to produce accomplished thebaine the[83]. biosynthesis Lastly, addition of of hydrocodone P. somniferum T6ODM [83]. This and E.P. putida coli-based morB genes accomplished the biosynthesis of hydrocodone [83]. This E. coli-based biosynthetic system biosynthetic system developed by Sato et al. employed a stepwise fermentation technique to avoid developed by Sato et al. employed a stepwise fermentation technique to avoid tyrosinase from tyrosinase from degrading the early intermediate THP [82]. This method also helped minimize product degrading the early intermediate THP [82]. This method also helped minimize product inhibition of inhibitionpathway of pathwayenzymes and enzymes propagation and propagation of undesired ofbypr undesiredoducts through byproducts the biosynthetic through thesteps. biosynthetic Those steps.factors Those are factors thought are to thought have contri to havebuted contributed to the superior to theproduct superior yield productobtained yield with obtainedtheir multi-step with their multi-stepE. coli fermentationE. coli fermentation strategy strategyfor the opioid for the biosynthesis. opioid biosynthesis.

FigureFigure 16. 16.Heterologous Heterologous biosynthetic biosynthetic pathwaypathway established established in inE. E.coli coli forfor de denovo novo production production of of (R,S)-reticuline(R,S)-reticuline from from simple simple sugar sugar using using a three-step a three-step fermentation fermentation method [81–83]. method The three [81– fermentation83]. The three steps are designated by three different colors: black for the dopamine production step, red for the fermentation steps are designated by three different colors: black for the dopamine production step, (R,S)-THP production step and blue for the (R,S)-reticuline production step. The initial attempt to use red for the (R,S)-THP production step and blue for the (R,S)-reticuline production step. The initial the norcoclaurine synthase (NCS) for the production of (S)-THP for the preparation of (S)-reticuline attempt(shown to use in thegreen) norcoclaurine was replaced synthase by the use (NCS) of a forspontaneous the production intracellular of (S)-THP Pictet-Spengler for the preparation reaction of (S)-reticuline(shown in (shown red) that in resulted green) in was the replaced eventual byformation the use of of (R a,S spontaneous)-reticuline (shown intracellular in blue). Subsequent Pictet-Spengler reactionbiosynthesis (shown of in thebaine red) that and resulted hydrocodone in the followed eventual the same formation biosynthetic of (R ,schemeS)-reticuline shown (shownin Figures in 15 blue). Subsequentand 14, biosynthesisrespectively. MA of thebaineO, monoamine and hydrocodoneoxidase. followed the same biosynthetic scheme shown in Figures 14 and 15, respectively. MAO, monoamine oxidase.

7. Conclusions Over the past several decades, elucidation of metabolic pathways leading to the biosynthesis of various alkaloids has seen a tremendous advancement. Those efforts have been facilitated greatly by the rapid accumulation of genomic sequence information and the use of heterologous host organisms that allow efficient reconstitution of the biosynthetic pathway being investigated. For the heterologous host, E. coli, S. cerevisiae and A. nidulans are used most commonly. Those hosts are particularly attractive because of their fast and robust growth and ease of genetic manipulation. They also have a metabolic background that imposes low interference to the alkaloid biosynthesis being studied. In addition to those in vivo analyses, in vitro characterizations of the biosynthetic enzymes also provided deep insight into the mechanisms involved in the generation of representative members of alkaloids. Biosynthesis of some of those compounds, such as diketopiperazine and tetrahydroisoquinoline natural products, employ the use of NRPSs, while a great number of alkaloids rely on oxidoreductases, primarily cytochrome P450s and flavin-containing monooxygenases, for extensive modifications of the core scaffolds that are typically constructed from aromatic amino acids and isoprenes. Combining the current efforts in the discovery of alkaloid biosynthetic pathways with the knowledge and experiences acquired from previous successful incorporation and engineering of NRPSs, oxidoreductases and other auxiliary enzymes in bacteria, yeast and filamentous fungi could further generalize our approach for comprehensive characterizations of alkaloid biosynthetic pathways and mass-production of clinically and industrially valuable alkaloids that are typically produced in limited quantity by the original producers. Detailed knowledge of biosynthesis of those compounds will also facilitate application Molecules 2016, 21, 1078 16 of 19 of synthetic biological approaches in developing efficient and sustainable platforms for preparing semi-synthetic analogs of alkaloids.

Acknowledgments: We wish to thank the financial support from the Japan Society for the Promotion of Science (JSPS) Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (No. G2604) (K.W.). This work was also supported in part by the Japan Society for the Promotion of Science (JSPS) (K.W., 15KT0068, and 26560450), the Takeda Science Foundation (K.W.), the Institution of Fermentation at Osaka (K.W.), the Japan Antibiotics Research Association (K.W.), the Uehara Memorial Foundation (K.W.), and the Tokyo Biochemical Research Foundation (K.W.). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Aniszewski, T. Definition, typology, and occurrence of alkaloids. In Alkaloids, 2nd ed.; Elsevier: Boston, MA, USA, 2015; Chapter 1; pp. 1–97. 2. Rathbone, D.A.; Bruce, N.C. Microbial transformation of alkaloids. Curr. Opin. Microbiol. 2002, 5, 274–281. [CrossRef] 3. Diamond, A.; Desgagné-Penix, I. Metabolic engineering for the production of plant isoquinoline alkaloids. Plant Biotechnol. J. 2016, 14, 1319–1328. [CrossRef][PubMed] 4. Srivastava, S.; Srivastava, A.K. Biotechnology and genetic engineering for alkaloid production. In Natural Products: Phytochemistry, Botany and Metabolism of Alkaloids, Phenolics and Terpenes; Ramawat, G.K., Mérillon, J.-M., Eds.; Springer-Verlag Berlin Heidelberg: Berlin, Germany, 2013; pp. 213–250. 5. Springer, J.P.; Büchi, G.; Kobbe, B.; Demain, A.L.; Clardy, J. The structure of ditryptophenaline—A new metabolite of Aspergillus flavus. Tetrahedron Lett. 1977, 18, 2403–2406. [CrossRef] 6. Voloshchuk, T.; Farina, N.S.; Wauchope, O.R.; Kiprowska, M.; Haberfield, P.; Greer, A. Molecular bilateral symmetry of natural products: Prediction of selectivity of dimeric molecules by density functional theory and semiempirical calculations. J. Nat. Prod. 2004, 67, 1141–1146. [CrossRef][PubMed] 7. Barrow, C.J.; Sedlock, D.M. 10-(2-Phenyl-ethylene)-ditryptophenaline, a new dimeric diketopiperazine from Aspergillus flavus. J. Nat. Prod. 1994, 57, 1239–1244. [CrossRef][PubMed] 8. Barrow, C.J.; Cai, P.; Snyder, J.K.; Sedlock, D.M.; Sun, H.H.; Cooper, R. WIN 64821, a new competitive antagonist to substance P,isolated from an Aspergillus species: Structure determination and solution conformation. J. Org. Chem. 1993, 58, 6016–6021. [CrossRef] 9. Saruwatari, T.; Yagishita, F.; Mino, T.; Noguchi, H.; Hotta, K.; Watanabe, K. Cytochrome P450 as dimerization catalyst in diketopiperazine alkaloid biosynthesis. Chembiochem 2014, 15, 656–659. [CrossRef][PubMed] 10. Payne, G.A.; Nierman, W.C.; Wortman, J.R.; Pritchard, B.L.; Brown, D.; Dean, R.A.; Bhatnagar, D.; Cleveland, T.E.; Machida, M.; Yu, J. Whole genome comparison of Aspergillus flavus and A. oryzae. Med. Mycol. 2006, 44, 9–11. [CrossRef] 11. Chang, P.K.; Scharfenstein, L.L.; Wei, Q.; Bhatnagar, D. Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus. J. Microbiol. Methods 2010, 81, 240–246. [CrossRef][PubMed] 12. Tsunematsu, Y.; Ishikawa, N.; Wakana, D.; Goda, Y.; Noguchi, H.; Moriya, H.; Hotta, K.; Watanabe, K. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk. Nat. Chem. Biol. 2013, 9, 818–825. [CrossRef][PubMed] 13. Maiya, S.; Grundmann, A.; Li, S.M.; Turner, G. The fumitremorgin gene cluster of Aspergillus fumigatus: Identification of a gene encoding brevianamide F synthetase. Chembiochem 2006, 7, 1062–1069. [CrossRef] [PubMed] 14. Saruwatari, T.; Watanabe, K. Additional unnatural diketopiperazine heterodimers produced by cytochrome P450 DtpC. 2014, unpublished results. 15. Raju, R.; Piggott, A.M.; Conte, M.; Aalbersberg, W.G.; Feussner, K.; Capon, R.J. Naseseazines A and B: A new dimeric diketopiperazine framework from a marine-derived actinomycete, Streptomyces sp. Org. Lett. 2009, 11, 3862–3865. [CrossRef][PubMed] 16. Li, X.B.; Li, Y.L.; Zhou, J.C.; Yuan, H.Q.; Wang, X.N.; Lou, H.X. A new diketopiperazine heterodimer from an endophytic fungus Aspergillus niger. J. Asian. Nat. Prod. Res. 2015, 17, 182–187. [CrossRef][PubMed] 17. Ding, G.; Jiang, L.; Guo, L.; Chen, X.; Zhang, H.; Che, Y. Pestalazines and pestalamides, bioactive metabolites from the plant pathogenic fungus Pestalotiopsis theae. J. Nat. Prod. 2008, 71, 1861–1865. [CrossRef][PubMed] Molecules 2016, 21, 1078 17 of 19

18. Pérez-Balado, C.; de Lera, A.R. Concise total synthesis and structural revision of (+)-pestalazine B. Org. Biomol. Chem. 2010, 8, 5179–5186. [CrossRef][PubMed] 19. Varoglu, M.; Corbett, T.H.; Valeriote, F.A.; Crews, P. Asperazine, a selective cytotoxic alkaloid from a sponge-derived culture of Aspergillus niger. J. Org. Chem. 1997, 62, 7078–7079. [CrossRef][PubMed] 20. Loach, R.P.; Fenton, O.S.; Movassaghi, M. Concise total synthesis of (+)-asperazine, (+)-pestalazine A, and (+)-iso-pestalazine A. Structure revision of (+)-pestalazine A. J. Am. Chem. Soc. 2016, 138, 1057–1064. [CrossRef][PubMed] 21. Arai, T.; Takahashi, K.; Kubo, A. New antibiotics saframycins A, B, C, D and E. J. Antibiot. 1977, 30, 1015–1018. [PubMed] 22. Scott, J.D.; Williams, R.M. Chemistry and biology of the tetrahydroisoquinoline antitumor antibiotics. Chem. Rev. 2002, 102, 1669–1730. [CrossRef][PubMed] 23. Rao, K.E.; Lown, J.W. Mode of action of saframycin antitumor antibiotics: Sequence selectivities in the covalent binding of saframycins A and S to deoxyribonucleic acid. Chem. Res. Toxicol. 1990, 3, 262–267. [CrossRef] 24. Corey, E.J.; Gin, D.Y.; Kania, R.S. Enantioselective total synthesis of Ecteinascidin 743. J. Am. Chem. Soc. 1996, 118, 9202–9203. [CrossRef] 25. Jin, S.; Gorfajn, B.; Faircloth, G.; Scotto, K.W. Ecteinascidin 743, a transcription-targeted chemotherapeutic that inhibits MDR1 activation. Proc. Natl. Acad. Sci. USA 2000, 97, 6775–6779. [CrossRef][PubMed] 26. Cuevas, C.; Francesch, A. Development of Yondelis (trabectedin, ET-743). A semisynthetic process solves the supply problem. Nat. Prod. Rep. 2009, 26, 322–337. [CrossRef][PubMed] 27. Cuevas, C.; Perez, M.; Martin, M.J.; Chicharro, J.L.; Fernandez-Rivas, C.; Flores, M.; Francesch, A.; Gallego, P.; Zarzuelo, M.; de la Calle, F.; et al. Synthesis of ecteinascidin ET-743 and phthalascidin Pt-650 from cyanosafracin B. Org. Lett. 2000, 2, 2545–2548. [CrossRef][PubMed] 28. Pospiech, A.; Bietenhader, J.; Schupp, T. Two multifunctional peptide synthetases and an O-methyltransferase are involved in the biosynthesis of the DNA-binding antibiotic and antitumour agent saframycin Mx1 from Myxococcus xanthus. Microbiology 1996, 142, 741–746. [CrossRef][PubMed] 29. Velasco, A.; Acebo, P.; Gomez, A.; Schleissner, C.; Rodriguez, P.; Aparicio, T.; Conde, S.; Munoz, R.; de la Calle, F.; Garcia, J.L.; et al. Molecular characterization of the safracin biosynthetic pathway from Pseudomonas fluorescens A2-2: Designing new cytotoxic compounds. Mol. Microbiol. 2005, 56, 144–154. [CrossRef] [PubMed] 30. Li, L.; Deng, W.; Song, J.; Ding, W.; Zhao, Q.F.; Peng, C.; Song, W.W.; Tang, G.L.; Liu, W. Characterization of the saframycin A gene cluster from Streptomyces lavendulae NRRL 11002 revealing a nonribosomal peptide synthetase system for assembling the unusual tetrapeptidyl skeleton in an iterative manner. J. Bacteriol. 2008, 190, 251–263. [CrossRef][PubMed] 31. Mikami, Y.; Takahashi, K.; Yazawa, K.; Arai, T.; Namikoshi, M.; Iwasaki, S.; Okuda, S. Biosynthetic studies on saframycin A, a quinone antitumor antibiotic produced by Streptomyces lavendulae. J. Biol. Chem. 1985, 260, 344–348. [PubMed] 32. Arai, T.; Yazawa, K.; Takahashi, K.; Maeda, A.; Mikami, Y. Directed biosynthesis of new saframycin derivatives with resting cells of Streptomyces lavendulae. Antimicrob. Agents Chemother. 1985, 28, 5–11. [CrossRef][PubMed] 33. Nelson, J.T.; Lee, J.; Sims, J.W.; Schmidt, E.W. Characterization of SafC, a catechol 4-O-methyltransferase involved in saframycin biosynthesis. Appl. Environ. Microbiol. 2007, 73, 3575–3580. [CrossRef][PubMed] 34. Schmidt, E.W.; Nelson, J.T.; Fillmore, J.P. Synthesis of tyrosine derivatives for saframycin MX1 biosynthetic studies. Tetrahedron Lett. 2004, 45, 3921–3924. [CrossRef] 35. Koketsu, K.; Watanabe, K.; Suda, H.; Oguri, H.; Oikawa, H. Reconstruction of the saframycin core scaffold defines dual Pictet-Spengler mechanisms. Nat. Chem. Biol. 2010, 6, 408–410. [CrossRef][PubMed] 36. Hansen, D.B.; Bumpus, S.B.; Aron, Z.D.; Kelleher, N.L.; Walsh, C.T. The loading module of mycosubtilin: An adenylation domain with fatty acid selectivity. J. Am. Chem. Soc. 2007, 129, 6366–6367. [CrossRef][PubMed] 37. Baltz, R.H.; Miao, V.; Wrigley, S.K. Natural products to drugs: Daptomycin and related lipopeptide antibiotics. Nat. Prod. Rep. 2005, 22, 717–741. [CrossRef][PubMed] 38. O’Connor, S.E.; Maresh, J.J. Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat. Prod. Rep. 2006, 23, 532–547. [CrossRef][PubMed] 39. De Luca, V.; Salim, V.; Thamm, A.; Masada, S.A.; Yu, F. Making iridoids/secoiridoids and monoterpenoid indole alkaloids: Progress on pathway elucidation. Curr. Opin. Plant Biol. 2014, 19, 35–42. [CrossRef][PubMed] 40. Slim, H.B.; Black, H.R.; Thompson, P.D. Older blood pressure medications—Do they still have a place? Am. J. Cardiol. 2011, 108, 308–316. [CrossRef][PubMed] Molecules 2016, 21, 1078 18 of 19

41. Orhan, I.E.; Senol, F.S. Alkaloids and inhibitory effects against enzymes linked to neurodegenerative diseases (physostigmine, galanthamine, huperzine, etc.). In Natural Products: Phytochemistry, Botany and Metabolism of Alkaloids, Phenolics and Terpenes; Ramawat, G.K., Mérillon, J.-M., Eds.; Springer-Verlag: Berlin, Germany, 2013; pp. 1525–1539. 42. Li, J.; Wang, T.; Yu, P.; Peterson, A.; Weber, R.; Soerens, D.; Grubisha, D.; Bennett, D.; Cook, J.M. General approach for the synthesis of ajmaline/sarpagine indole alkaloids: Enantiospecific total synthesis of (+)-ajmaline, alkaloid G, and norsuaveoline via the asymmetric Pictet-Spengler reaction. J. Am. Chem. Soc. 1999, 121, 6998–7010. [CrossRef] 43. Ishikawa, H.; Colby, D.A.; Boger, D.L. Direct coupling of catharanthine and vindoline to provide vinblastine: Total synthesis of (+)- and ent-(−)-vinblastine. J. Am. Chem. Soc. 2008, 130, 420–421. [CrossRef][PubMed] 44. Irmler, S.; Schroder, G.; St-Pierre, B.; Crouch, N.P.; Hotze, M.; Schmidt, J.; Strack, D.; Matern, U.; Schroder, J. Indole alkaloid biosynthesis in Catharanthus roseus: New enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase. Plant J. 2000, 24, 797–804. [CrossRef][PubMed] 45. Yamamoto, H.; Katano, N.; Ooi, A.; Inoue, K. Secologanin synthase which catalyzes the oxidative cleavage of loganin into secologanin is a cytochrome P450. Phytochemistry 2000, 53, 7–12. [CrossRef] 46. Maresh, J.J.; Giddings, L.A.; Friedrich, A.; Loris, E.A.; Panjikar, S.; Trout, B.L.; Stöckigt, J.; Peters, B.; O0Connor, S.E. Strictosidine synthase: Mechanism of a Pictet-Spengler catalyzing enzyme. J. Am. Chem. Soc. 2008, 130, 710–723. [CrossRef][PubMed] 47. Brown, S.; Clastre, M.; Courdavault, V.; O’Connor, S.E. De novo production of the plant-derived alkaloid strictosidine in yeast. Proc. Natl. Acad. Sci. USA 2015, 112, 3205–3210. [CrossRef][PubMed] 48. Miettinen, K.; Dong, L.; Navrot, N.; Schneider, T.; Burlat, V.; Pollier, J.; Woittiez, L.; van der Krol, S.; Lugan, R.; Ilc, T.; et al. The seco-iridoid pathway from Catharanthus roseus. Nat. Commun. 2014, 5, 3606. [CrossRef][PubMed] 49. Nevoigt, E. Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 2008, 72, 379–412. [CrossRef][PubMed] 50. Burke, C.; Croteau, R. Geranyl diphosphate synthase from Abies grandis: cDNA isolation, functional expression, and characterization. Arch. Biochem. Biophys. 2002, 405, 130–136. [CrossRef] 51. Stanley Fernandez, S.M.; Kellogg, B.A.; Poulter, C.D. Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity. Biochemistry 2000, 39, 15316–15321. [CrossRef][PubMed] 52. Schardl, C.L.; Panaccione, D.G.; Tudzynski, P. Ergot alkaloids—Biology and molecular biology. Alkaloids Chem. Biol. 2006, 63, 45–86. [PubMed] 53. Gröcer, D.; Floss, H.G. Biochemistry of ergot alkaloids—Achievements and challenges. Alkaloids Chem. Biol. 1998, 50, 171–218. 54. Mrusek, M.; Seo, E.J.; Greten, H.J.; Simon, M.; Efferth, T. Identification of cellular and molecular factors determining the response of cancer cells to six ergot alkaloids. Investig. New Drugs 2015, 33, 32–44. [CrossRef] [PubMed] 55. Schiff, P.L. Ergot and its alkaloids. Am. J. Pharm. Educ. 2006, 70, Z1. [CrossRef] 56. Coyle, C.M.; Panaccione, D.G. An ergot alkaloid biosynthesis gene and clustered hypothetical genes from Aspergillus fumigatus. Appl. Environ. Microbiol. 2005, 71, 3112–3118. [CrossRef][PubMed] 57. Gebler, J.C.; Poulter, C.D. Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea. Arch. Biochem. Biophys. 1992, 296, 308–313. [CrossRef] 58. Gebler, J.C.; Woodside, A.B.; Poulter, C.D. Dimethylallyltryptophan synthase. An enzyme-catalyzed electrophilic aromatic substitution. J. Am. Chem. Soc. 1992, 114, 7354–7360. [CrossRef] 59. Otsuka, H.; Quigley, F.R.; Groeger, D.; Anderson, J.A.; Floss, H.G. D-Lysergyl peptide synthetase from the ergot fungus Claviceps purpurea. Planta Med. 1980, 40, 109–119. [CrossRef] 60. Kozikowski, A.P.; Wu, J.P.; Shibuya, M.; Floss, H.G. Probing ergot alkaloid biosynthesis: Identification of advanced intermediates along the biosynthetic pathway. J. Am. Chem. Soc. 1988, 110, 1970–1971. [CrossRef] 61. Riederer, B.; Han, M.; Keller, U. D-Lysergyl peptide synthetase from the ergot fungus Claviceps purpurea. J. Biol. Chem. 1996, 271, 27524–27530. [CrossRef][PubMed] 62. Walzel, B.; Riederer, B.; Keller, U. Mechanism of alkaloid cyclopeptide synthesis in the ergot fungus Claviceps purpurea. Chem. Biol. 1997, 4, 223–230. [CrossRef] 63. Ortel, I.; Keller, U. Combinatorial assembly of simple and complex D-lysergic acid alkaloid peptide classes in the ergot fungus Claviceps purpurea. J. Biol. Chem. 2009, 284, 6650–6660. [CrossRef][PubMed] Molecules 2016, 21, 1078 19 of 19

64. Havemann, J.; Vogel, D.; Loll, B.; Keller, U. Cyclolization of D-lysergic acid alkaloid peptides. Chem. Biol. 2014, 21, 146–155. [CrossRef][PubMed] 65. Ryan, K.L.; Moore, C.T.; Panaccione, D.G. Partial reconstruction of the ergot alkaloid pathway by heterologous gene expression in Aspergillus nidulans. Toxins 2013, 5, 445–455. [CrossRef][PubMed] 66. Wink, M. A Short History of Alkaloids. In Alkaloids: Biochemistry, Ecology, and Medicinal Applications; Roberts, M.F., Wink, M., Eds.; Springer US: Boston, MA, USA, 1998; pp. 11–44. 67. Waldhoer, M.; Bartlett, S.E.; Whistler, J.L. Opioid receptors. Annu. Rev. Biochem. 2004, 73, 953–990. [CrossRef] [PubMed] 68. Sato, F.; Inui, T.; Takemura, T. Metabolic engineering in isoquinoline alkaloid biosynthesis. Curr. Pharm. Biotechnol. 2007, 8, 211–218. [CrossRef][PubMed] 69. Chang, M.C.; Keasling, J.D. Production of isoprenoid pharmaceuticals by engineered microbes. Nat. Chem. Biol. 2006, 2, 674–681. [CrossRef][PubMed] 70. Minami, H.; Kim, J.S.; Ikezawa, N.; Takemura, T.; Katayama, T.; Kumagai, H.; Sato, F. Microbial production of plant benzylisoquinoline alkaloids. Proc. Natl. Acad. Sci. USA 2008, 105, 7393–7398. [CrossRef][PubMed] 71. Hawkins, K.M.; Smolke, C.D. Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat. Chem. Biol. 2008, 4, 564–573. [CrossRef][PubMed] 72. Galanie, S.; Smolke, C.D. Optimization of yeast-based production of medicinal protoberberine alkaloids. Microb. Cell Fact. 2015, 14.[CrossRef][PubMed] 73. Thodey, K.; Galanie, S.; Smolke, C.D. A microbial biomanufacturing platform for natural and semisynthetic opioids. Nat. Chem. Biol. 2014, 10, 837–844. [CrossRef][PubMed] 74. Sato, F.; Tsujita, T.; Katagiri, Y.; Yoshida, S.; Yamada, Y. Purification and characterization of S-adenosyl-L-methionine: Norcoclaurine 6-O-methyltransferase from cultured Coptis japonica cells. Eur. J. Biochem. 1994, 225, 125–131. [CrossRef][PubMed] 75. Ounaroon, A.; Decker, G.; Schmidt, J.; Lottspeich, F.; Kutchan, T.M. (R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum—cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J. 2003, 36, 808–819. [CrossRef] [PubMed] 76. Gesell, A.; Chavez, M.L.; Kramell, R.; Piotrowski, M.; Macheroux, P.; Kutchan, T.M. Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Argemone mexicana and Berberis wilsoniae in insect cells. Planta 2011, 233, 1185–1197. [CrossRef][PubMed] 77. Fossati, E.; Ekins, A.; Narcross, L.; Zhu, Y.; Falgueyret, J.P.; Beaudoin, G.A.; Facchini, P.J.; Martin, V.J. Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae. Nat. Commun. 2014, 5, 3283. [CrossRef][PubMed] 78. Galanie, S.; Thodey, K.; Trenchard, I.J.; Filsinger Interrante, M.; Smolke, C.D. Complete biosynthesis of opioids in yeast. Science 2015, 349, 1095–1100. [CrossRef][PubMed] 79. Michener, J.K.; Nielsen, J.; Smolke, C.D. Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases. Proc. Natl. Acad. Sci. USA 2012, 109, 19504–19509. [CrossRef][PubMed] 80. Hazelwood, L.A.; Daran, J.M.; van Maris, A.J.; Pronk, J.T.; Dickinson, J.R. The Ehrlich pathway for fusel alcohol production: A century of research on Saccharomyces cerevisiae metabolism. Appl. Environ. Microbiol. 2008, 74, 2259–2266. [CrossRef][PubMed] 81. Nakagawa, A.; Minami, H.; Kim, J.S.; Koyanagi, T.; Katayama, T.; Sato, F.; Kumagai, H. A bacterial platform for fermentative production of plant alkaloids. Nat. Commun. 2011, 2, 326. [CrossRef][PubMed] 82. Nakagawa, A.; Matsuzaki, C.; Matsumura, E.; Koyanagi, T.; Katayama, T.; Yamamoto, K.; Sato, F.; Kumagai, H.; Minami, H. (R,S)-tetrahydropapaveroline production by stepwise fermentation using engineered Escherichia coli. Sci. Rep. 2014, 4, 6695. [CrossRef][PubMed] 83. Nakagawa, A.; Matsumura, E.; Koyanagi, T.; Katayama, T.; Kawano, N.; Yoshimatsu, K.; Yamamoto, K.; Kumagai, H.; Sato, F.; Minami, H. Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli. Nat. Commun. 2016, 7, 10390. [CrossRef][PubMed]

© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).