Microbial production of isoquinoline alkaloids as plant ......Review Microbial production of isoquinoline alkaloids as plant secondary metabolites based on metabolic engineering research

Review

Microbial production of isoquinoline alkaloids as plant secondarymetabolites based on metabolic engineering research

By Fumihiko SATO*1,† and Hidehiko KUMAGAI*2,†

(Communicated by Koichiro TSUNEWAKI, M.J.A.)

Abstract: Plants produce a variety of secondary metabolites that possess strongphysiological activities. Unfortunately, however, their production can suffer from a variety ofserious problems, including low levels of productivity and heterogeneous quality, as well as difficultyin raw material supply. In contrast, microorganisms can be used to produce their primary and someof their secondary metabolites in a controlled environment, thus assuring high levels of efficiencyand uniform quality. In an attempt to overcome the problems associated with secondary metaboliteproduction in plants, we developed a microbial platform for the production of plant isoquinolinealkaloids involving the unification of the microbial and plant metabolic pathways into a singlesystem. The potential applications of this system have also been discussed.

Keywords: aromatic amino acid metabolism, isoquinoline alkaloid biosynthesis, metabolicengineering, microbial production

Introduction

Higher plants produce a wide range of chemicals,with more than 25,000 terpenoids, 12,000 alkaloidsand 8,000 phenolic substances having been identifiedand reported in the literature to date.1) Thesechemicals serve a variety of different functions inplants, where they defend against herbivores andpathogens, aid in inter-plant competition and attractbeneficial organisms such as pollinators. They canalso have protective effects with regard to abioticstresses such as UV exposure, and changes in thetemperature, water status and mineral nutrientcomposition. In addition, many secondary metabo-lites produced in plants have been used by humans ina variety of different applications, including spices,dyes, fragrances, flavoring agents and pharmaceut-icals. Many of these chemicals have also beenreported to promote human health and can thereforeenrich our lives in a variety of different ways.2)

The use of plant metabolites as natural medi-cines has a long history that can be traced back overmore than 3,500 years.3) In the more recent years,research has focused predominantly on the identi-fication of the active ingredients. Following the firstidentification of morphine as the principal activeingredient in opium (1804), many compounds havebeen identified from plant materials and used as

*1 Division of Integrated Life Science, Graduate School ofBiostudies, Kyoto University, Kyoto, Japan.

*2 Ishikawa Prefectural University, Ishikawa, Japan.† Correspondence should be addressed: F. Sato, Division of

Integrated Life Science, Graduate School of Biostudies, KyotoUniversity, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan (e-mail:[email protected]). H. Kumagai, Ishikawa Prefectural Uni-versity, Ishikawa 921-8836, Japan (e-mail: [email protected]).

Abbreviations: AADC: aromatic amino acid decarboxy-lase; BBE: berberine bridge enzyme; bHLH: basic helix-loop-helix;Cen: central domain; CM/PD: chorismate mutase/prephenatedehydrogenase; CNMT: coclaurine N-methyltransferase; CoOMT:columbamine O-methyltransferase; CT: C-terminal domain;CYP80A1: berbamunine synthase; CYP80B1: (S)-N-methylcocla-urine 3B-hydroxylase; CYP719A1: canadine synthase; CYP719A2/A3: stylopine synthase; CYP719A5: cheilanthifoline synthase;CYP719B1: salutaridine synthase; CYP80G2: corytuberine syn-thase; DAHPS: 3-deoxy-d-arabino-heptulosonate-7-phosphate syn-thase; 3,4-DHPAA: 3,4-dihydroxyphenylacetaldehyde; DDC:DOPA decarboxylase; EST: expressed sequence tag; fbr: feed-back-inhibition-resistance; IQAs: isoquinoline alkaloids; MAO:monoamine oxidase; MSH: N-methylstylopine hydroxylase; NCS:norcoclaurine synthase; NT: N-terminal domain; 4BOMT: 3B-hydroxy-N-methylcoclaurine-4B-O-methyltransferase; 6OMT: nor-coclaurine 6-O-methyltransferase; 7OMT: (S)-reticuline 7-O-meth-yltransferase; PEP: phosphoenolpyruvate; PEPS: PEP synthetase;P6H: protopine 6-hydroxylase; PLP: pyridoxal phosphate; PR10:pathogenesis-related protein 10; RNAi: RNA interference; SAM: S-adenosylmethionine; SMT: (S)-scoulerine-9-O-methyltransferase;TAT: tyrosine aminotransferase; THB: tetrahydroberberine;THBO: THB oxidase; THC: tetrahydrocolumbamine; TKT:transketolase; TNMT: (S)-tetrahydroprotoberberine N-methyl-transferase; TPL: tyrosine phenol-lyase; TYR: tyrosinase; TYDC:tyrosine/DOPA decarboxylase.

Proc. Jpn. Acad., Ser. B 89 (2013)No. 5] 165

doi: 10.2183/pjab.89.165©2013 The Japan Academy

http://dx.doi.org/10.2183/pjab.89.165

medicines, including the pain-killing and fever-reducing salicylic acid from willow bark extract(1826), the anticancer agent paclitaxel from thePacific yew tree (1971), and the anti-malarial drugartemisinin from Artemisia annua (1972). Japanesescientists have also provided a significant contribu-tion to the isolation and identification of naturalproducts from medicinal plants. The isolations ofephedrine from Ephedra vulgaris and matrine fromSophora flavescens (1885) by Dr. Nagayoshi Nagaihighlight the early success of natural productresearch in Japan. Following on from this earlysuccess, many excellent works have been produced,including the structural determination studies ofsinomenine in Sinomenium acutum and its synthesisby Dr. Kakuji Goto.4)

The use of biochemical and molecular character-ization techniques in natural product research,however, has been largely limited, because of thelow levels of enzymatic activity often encounteredin the secondary metabolism of plants, especiallytowards the production of alkaloids. In the late 1970sand early 1980s, several groups developed cell culturesystems for the production of large quantities ofsecondary metabolites.5),6) Although the levels ofproductivity encountered were often not sufficient forthe industrial production of the desired metabolites,because of limitations regarding the market size anddifficulties associated with the use of these materialsas crude medicines, these materials have provideduseful information pertaining to the study of thebiosynthetic pathways associated with their produc-tion. Zenk et al. in particular studied a variety ofdifferent alkaloid pathways, including those of theisoquinoline alkaloids (IQAs).5)

IQAs are a large and diverse group of alkaloidsthat are derived from tyrosine with approximately2,500 defined structures, which all contain a hetero-cyclic isoquinoline backbone (Fig. 1).6) Some signifi-cant examples from this group of natural productsinclude the analgesic morphine from Papaver somni-ferum L., the anti-gout colchicine from Colchicumautumnale L., the emetic and antiamoebic emetinefrom Cephaelis ipecacuanha (Brot.) A. Rich., theskeletal muscle relaxant tubocurarine from Strychnostoxifera Bentham, and the antimicrobial compoundsberberine and sanguinarine from divergent plantspecies including Berberis spp. and Sanguinariaspp. It is noteworthy that many of these compoundsand their derivatives have been used as pharmaceut-ical agents. Sato et al. independently selected acultured cell line with a high level of IQA productiv-

ity for a particular medicinal herb known as theJapanese goldthread (Coptis japonica Makino) andcharacterized the molecular basis of the IQA path-way to consolidate the results of their biochemicalcharacterization.7),8)

In this review, we have provided a summaryof the progress that has been made towards thebiochemical and molecular biological characteriza-tion of the IQA biosynthetic pathways using culturedC. japonica and California poppy (Eschscholziacalifornica) cells. The application of this progress tothe metabolic engineering, and microbial productionof plant IQAs will also be discussed.

Clarification of the molecular basisof the IQA biosynthetic pathways

and metabolic engineering

IQA biosynthesis is generally considered to beginwith the conversion of tyrosine to both dopamine and4-hydroxyphenylacetaldehyde via a sequential decar-boxylation, ortho-hydroxylation and deaminationpathway (Fig. 1).6),9) From these early steps, onlythe tyrosine/DOPA decarboxylase (an aromatic L-amino acid decarboxylase; TYDC), which convertstyrosine and DOPA to their corresponding amines,and the aminotransferase have been purified andcharacterized.10),11) The general lack of a detailedunderstanding of the other early step enzymes has ledto the use of alternative techniques for the supply ofthe substrate via reconstituted pathways in microbialcells (this will be discussed in a later section).

Dopamine and 4-hydroxyphenylacetaldehydeare condensed by norcoclaurine synthase (NCS; EC4.2.1.78) to yield (S)-norcoclaurine, which is thecentral precursor for the construction of all ofthe benzylisoquinoline alkaloids. NCS has beenpurified and characterized from cultured Thalictrumflavum spp., and a TfNCS cDNA belonging to thepathogenesis-related protein (PR)10 family wascloned.12),13) It is noteworthy that a novel dioxyge-nase (CjNCS) from cultured C. japonica cells wasalso shown to catalyze this NCS reaction.14)

(S)-Norcoclaurine is converted to coclaurine byS-adenosyl methionine (SAM)-dependent norcoclaur-ine 6-O-methyltransferase (6OMT; EC 2.1.1.128),15)

which is in turn converted to N-methylcoclaurineby SAM-dependent coclaurine N-methyltransferase(CNMT; EC 2.1.1.140),16) before being converted to3B-hydroxy-N-methyl coclaurine by P450 hydroxy-lase (CYP80B1; EC 1.14.13.71),17) and then finallyconverted to (S)-reticuline by SAM-dependent 3B-hydroxy N-methylcoclaurine 4B-O-methyltransferase

F. SATO and H. KUMAGAI [Vol. 89,166

(4BOMT; EC 2.1.1.116).15) Both of the enzymes wereco-purified by SDS-gel electrophoresis, because of thehigh level of similarity between the proteinaceousproperties of 6OMT to those of 4BOMT, to give afinal preparation containing homologous 40- and 41-kD polypeptides. To effectively discriminate the twoenzymes, the screening of a cDNA library of culturedC. japonica cells was carried out with oligonucleotideprobes designed on the basis of the internal aminoacid sequences of the two polypeptides. The twokinds of cDNA obtained in this way were expressedin Escherichia coli, and characterization of therecombinant proteins enabled us to confirm thatthe 40-kDa polypeptide corresponded to the 6OMTwhereas the 41-kD corresponded to the 4BOMT.Similarly, the cloning of the cDNA of CNMT wascarried out based on the amino acid sequence of thepurified polypeptide.16)

Detailed biochemical studies using recombinantenzymes have demonstrated the strict reactionspecificities of these enzymes, with the enzymesthemselves regulating the biosyntheses sequentiallyand in a coordinated manner. For example, CNMTprefers coclaurine to 6-O-methylnorlaudanosoline,whereas 4BOMT prefers an N-methylated sub-strate,15),16) which suggested that the pathway shownin Fig. 1 was preferred to a sequence consisting of N-methylation, hydroxylation and 4B-O-methylation.Current data also indicate that all of the enzymesinvolved in the early steps of (S)-reticuline biosyn-thesis from norcoclaurine, except for the membrane-bound P450 CYP80B1, are located in the cytosol.(S)-Reticuline, which is produced by reaction with4BOMT, is the central intermediate in the branchpathways leading to many divergent IQAs, includingthe aporphine alkaloids (e.g., magnoflorine), benzo-

Dopamine

4-Hydroxyphenyl-acetaldehyde

(S )-Norcoclaurine(S )-Coclaurine

(S )-N -Methylcoclaurine

(S )-3’-Hydroxy-N -methylcoclaurine

(S )-Reticuline

Magnoflorine

(S )-Scoulerine

Protopine Sanguinarine

(S )-Tetrahydrocolumbamine

Palmatine

Columbamine

(S )-Tetrahydroberberine

Berberine

Cj NCS Cj 6OMT

Cj CNMT

Cj CYP80B1

Cj 4’OMT

Cj BBECj SMT

Cj CYP719A1

Cj THBO

Cj THBOCj CoOMT

Cj CoOMT

Cj THBOCheilanthifoline

Stylopine

Ec CYP719A5

Ec CYP719A2/A3

N -Methylstylopine

EcTNMT

Ec P6H

Ec 7OMT

Cj CYP80G2

Corytuberine

(S )-7-O -Methyl reticuline

protoberberine pathway

benzophenanthridine pathway

morphinan pathway

aporphine pathway

NH2HO

HO

O

HHO

NHHO

HO

HO

HNH

HO

MeO

HO

H

NMeHO

MeO

HO

H

NMeHO

MeO

HO

HHO

NMeHO

MeO

MeO

HHO

NHO

MeO

MeO

HOMeMe+

NMeHO

MeO

MeO

HO

NMeMeO

MeO

MeO

HHO

N

O

OO

Me

O

O NO

O

Me+

OO

N

OMe

OMe

MeO

HOH

N

OH

OMe

MeO

HOH

N

OMe

OMe

O

OH

N

OMe

OMe

O

O+

N

OMe

OMe

MeO

MeO+

N

OMe

OMe

MeO

HO+

TYDC

TYDC

CYP719B1

SalATSalR

T6ODMCOR

CODM

Morphine

Isoquinoline

N

Fig. 1. Outline of isoquinoline alkaloid biosynthesis pathway. The cDNAs of the enzymes shown in red were first cloned and/orcharacterized in Sato’s laboratory, and those in blue were cloned as described in the literature, from cultured Coptis japonica (Cj) andCalifornia poppy (Eschscholzia californica: Es). Genes of the following enzymes have been also cloned from Papaver somniferum byother several groups;83) TYDC, CYP719B1, SalR (salutaridine reductase), SalAT (salutaridinol 7-O-acetyltransferase), T6ODM(thebaine 6-O-demethylase), CODM (codeinone O-demethylase) and COR (codeinone reductase).

Microbial production of plant isoquinoline alkaloidsNo. 5] 167

phenanthridine alkaloids (e.g., sanguinarine), mor-phinan alkaloids (e.g., morphine and thebaine) andprotoberberine alkaloids (e.g., berberine) (Fig. 1).

The next step in the biosynthesis of berberinefrom (S)-reticuline involves the conversion of the N-methyl group of (S)-reticuline to the methylenebridge moiety linked to (S)-scoulerine by the berber-ine bridge enzyme (reticuline oxidase, BBE; EC1.21.3.3).18) This unique enzyme is soluble andlocalized in the vesicles.19) (S)-Scoulerine is convertedto (S)-tetrahydrocolumbamine (THC) by the SAM-dependent scoulerine 9-O-methyltransferase (SMT;EC 2.1.1.117),20) and then to tetrahydroberberine(THB; canadine) by a P450-dependent canadinesynthase (CYP719A1; EC 1.14.21.5).21) The charac-terization of these enzymes expressed from thecorresponding cDNAs effectively confirmed that thebiosynthesis of berberine proceeds via canadine inCoptis cells and not via columbamine. Once again, theenzyme substrate specificity shows a clear preferencefor this pathway. Although the hydrophobic N-terminal region of SMT suggests that this enzymemay be targeted to the membrane fraction, itslocalization in the cytosol as well as within the lumenof alkaloid-specific vesicles has been reported.20),22)

(S)-Tetrahydroberberine oxidase (THBO orSTOX; EC 1.3.3.8) is the enzyme involved in thefinal step of the berberine biosynthesis.23)–25) A cDNAof THBO was cloned based on the partial amino acidsequence of the purified enzyme and its deducedamino acid sequence suggested that CjTHBO belongsto the FAD-containing BBE oxidoreductase family.As a consequence of the difficulties encounteredduring the attempted production of active recombi-nant CjTHBO in E. coli and Saccharomyces cerevi-siae cells, recombinant CjTHBO was heterologouslyexpressed in E. californica cells and its metabolicactivities subsequently characterized.26)

As well as the biosynthesis of berberine, thecDNAs of several other biosynthetic enzymes in-volved in the biosynthesis of related protoberberinealkaloids have been also cloned. For example,SAM-dependent columbamine O-methyltransferase(CoOMT; EC 2.1.1.118), which is involved in thebiosynthesis of palmatine, was cloned from culturedC. japonica cells using expressed sequence taginformation. Recombinant CjCoOMT clearly indi-cated that THC could be a used as a substrate for theformation of tetrahydropalmatine (Fig. 1),27) where-as columbamine was thought to be the exclusivesubstrate of CoOMT in Berberis plants based onthe substrate specificity of Berberis CoOMT.28)

Differences in substrate specificity have also beenreported for the CNMT of Coptis and Berberis, inthat the Coptis enzyme could N-methylate norlau-danosoline, whereas the Berberis enzyme couldnot.16) These results suggest that the pathwaysassociated with the secondary metabolism may varyconsiderably depending on the enzyme(s) that eachplant has acquired during its evolution.

The cDNA of a novel P450 enzyme involved inthe biosynthesis of aporphine-type alkaloids from (S)-reticuline was also cloned from cultured C. japonicacells.29) A P450 corytuberine synthase (CYP80G2;EC 1.14.21.-) was found to have a significant level ofamino acid sequence similarity to that of berbamu-nine synthase (CYP80A1; EC 1.14.21.3) in the bio-synthesis of bisbenzylisoquinoline alkaloids via a C–Ophenol-coupling reaction.30) Recombinant CYP80G2produced from yeast showed intramolecular C–Cphenol coupling activity to produce the aporphinealkaloid corytuberine from (S)-reticuline (Fig. 1). Incontrast, the cDNA of a native N-methyltransferasecapable of producing magnoflorine from corytuberinehas not yet been cloned, although the potentialcatalytic activity of CNMT towards corytuberinecould be used for the production of magnoflorinefrom (S)-reticuline in a microbial system.

The benzophenanthridine alkaloid sanguinarineis also produced from (S)-reticuline via (S)-scoulerine(Fig. 1). This conversion begins with the action ofthe P450-dependent oxidases, (S)-chelanthifolinesynthase (CYP719A5; EC 1.14.21.2) and (S)-stylo-pine synthase (CYP719A2/A3; EC 1.14.21.1), whichcatalyze the formation of a methylenedioxy-bridgeto produce (S)-stylopine from (S)-scoulerine. TheircDNAs were also cloned from cultured E. californicacells.31),32) (S)-Stylopine was then converted to cis-N-methylstylopine by (S)-tetrahydroprotoberberine N-methyltransferase (TNMT; EC 2.1.1.122),33) beforebeing converted to protopine by N-methylstylopinehydroxylase (MSH; EC 1.14.13.37), and then todihydrosanguinarine by protopine 6-hydroxylase(P6H; EC 1.14.13.55).34) Although the cDNA ofMSH was not yet been cloned, the cDNA of P6H(CYP82N2v2) has been cloned from E. californicacells, based on an integrated analysis of metaboliteaccumulation and the mRNA expression profile oftransgenic cells.35) The identification of CYP82N2v2as a protopine 6-hydroxylase for the production ofdihydrosanguinarine from protopine, as well as itsbroad range of reaction specificity in the conversionof corycavine to corynoloxine, indicate thatCYP82N2v2 plays an important role in the biosyn-


thesis of a diverse range of benzophenanthridinealkaloids.36)

In total, Sato et al. successfully cloned thecDNAs of 12 enzymes belonging to the IQAbiosynthetic pathways to provide a better under-standing of the molecular basis of these particularpathways. Furthermore, seven of these cDNAs weresubsequently used in the later work to construct amicrobial platform for the production of plant IQAs.

Unique properties of the biosynthetic en-zymes of the IQA pathways. The cloning of thecDNAs of the biosynthetic enzymes from the IQAbiosynthetic pathways enabled the detailed charac-terization of their enzymatic properties. Of theenzymes characterized from the IQA biosyntheticpathway, CYP719A1 and CYP80G2 provided thefirst molecular basis for methylenedioxy ring for-mation and C-C phenol coupling reactions ineukaryotic cells.21),29) Interestingly, the CYP719family of enzymes was not found in Arabidopsisand rice, with these members of the cytochrome P450super-family being unique to benzylisoquinolinealkaloid biosynthesis.37)

Although the oxidative cyclization of an ortho-hydroxymethoxy-substituted aromatic ring (i.e.methylenedioxy bridge formation) is a P450-depend-ent reaction, this reaction is not a simple mono-oxygenation reaction as demonstrated by the lackof oxygen insertion into the products. The P450-dependent hydroxylation of the methoxy groupyields an intermediate corresponding to the hemi-acetal of formaldehyde that can cyclize to form amethylenedioxy bridge via an ionic mechanisminvolving the methylene oxonium ion intermediate.38)

All of the methylenedioxy bridge-forming reactionsinvolved in IQA biosynthesis have been found to becatalyzed by members of the CYP719A subfamily,which exhibit rather strict levels of substrate spec-ificity. CYP719A1 from C. japonica has been iden-tified as a canadine synthase and can only convert(S)-THC to (S)-THB (Fig. 2).21) E. californicaCYP719A5 can convert (S)-scoulerine to (S)-chei-lanthifoline, whereas CYP719A2 and CYP719A3have been identified as stylopine synthases respon-sible for catalyzing the conversion of (S)-cheilanthifo-line to (S)-stylopine. CYP719A9 from E. californicacan convert (R,S)-reticuline to the correspondingproducts containing a methylenedioxy ring.31),32)

Detailed characterization of CjCYP80G2, whichcatalyzes the intramolecular C-C phenol couplingreaction for the conversion of (S)-reticuline to (S)-corytuberine,29) indicated that CjCYP80G2 reacts

initially with the C-ring and not the A-ring duringthe 4-O-demethylation of codamine. The productobtained from the reaction of codamine withCjCYP80G2 suggested that hydrogen abstractionoccurred as the first step from the 4-methoxy groupof the C-ring, followed by hydroxy radical rebound tothe same position of the substrate, with the eventualrelease of formaldehyde to form the demethylatedproduct (Fig. 2). Similarly, when CYP80G2 wasreacted with (S)-reticuline, the initial hydrogen atomabstraction step occurred at the 3-hydroxy group ofthe C-ring to generate a phenoxy radical, followed bysubsequent oxidation at the 7-hydroxy group of theA-ring to generate a second radical. The terminalreaction was then achieved by the coupling of thediradical species (i.e. bond formation between theC8 and C2 positions of (S)-reticuline). The reactionmechanism of CjCYP80G2 is therefore best ex-plained by the diradical process.

Metabolic engineering. The isolation of thegenes of biosynthetic enzymes has enabled the

NHO

MeO

MeO

HOMe

NHO

MeO

MeO

HMeO

Me

(S )-Tetrahydrocolumbamine (S )-Tetrahydroberberine((S )-Canadine)

Cj CYP719A1

Cj CYP80G2

(S )-Reticuline (S )-Corytuberine

Cj CYP80G2

(R,S )-Codamine (R,S )-Orientaline

A

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OMe

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O

OH

Fig. 2. Some unique reactions catalyzed by P450 enzymes foundin the IQA pathway. CYP719A1 from C. japonica catalyzes theoxidative cyclization of an ortho-hydroxymethoxy-substitutedaromatic ring (i.e. methylenedioxy bridge formation) andconverts only tetrahydrocolumbamine to (S)-tetrahydroberber-ine. CjCYP80G2 not only catalyzes the intramolecular C-Cphenol coupling reaction and converts (S)-reticuline to (S)-corytuberine, but also 4-O-demethylates codamine to formthe demethylated product. The reaction mechanism has beendiscussed in the text.


molecular design of the metabolic flow of plant cells(Fig. 3). Using the Agrobacterium-mediated trans-formation technique, Sato et al.8) reported themolecular engineering of IQA biosynthesis in C.japonica and E. californica cells with C. japonicagenes, and demonstrated the high level of flexibilityof the IQA biosynthetic pathway towards its geneticalteration.

Attempts have been made to overexpress thegenes responsible for producing the enzymes involvedin the rate-limiting steps of these processes with theaim of improving the yields of the associated IQAs.For example, the overexpression of C. japonica SMT

and 4BOMT using a strong constitutive promoter,Cauliflower mosaic virus 35S, led to an increase inberberine production in C. japonica cells and plants,respectively.39),40) Similarly, heterologous overexpres-sion of C. japonica 6OMT improved the level ofalkaloid productivity in E. californica cells.41) It isimportant to note, however, that the overexpressionof a biosynthetic enzyme does not always result in asuccessful increase in the levels of alkaloid produc-tion. For example, the overexpression of Cj4BOMT inE. californica cells only provided a marginal increasein alkaloid production.41) The bottleneck step(s)associated with the IQA biosynthetic pathways can

(S )-Norcoclaurine (S )-Coclaurine

(S )-Reticuline (S )-Scoulerine

Sanguinarine

(S )-Tetrahydrocolumbamine Columbamine

Cj 6OMT

Ec BBE RNAi

Cj SMT

10-Hydroxychelerythrine Allocryptopine

EcCYP719A3/EcTNMT/EcMSH

Ec P6H etc

(S )-Norcoclaurine

N

OH

OMe

MeO

HOH

(S )-Scoulerine

Fig. 3. Outline of metabolic engineering in transgenic E. californica cells. (A) Overexpression of the rate-limiting enzyme, norcoclaurine6-O-methyltransferase (6OMT), using C. japonica cDNA in California poppy cells led to an increase in the accumulation of the mainalkaloid of the cells, sanguinarine, as shown by the upward arrow. (B) Introduction of a branch pathway with scoulerine 9-O-methyltransferase (SMT) also induced a marked shift in the metabolic flow, as shown by the arrows. SMT overexpression led to asignificant reduction in the accumulation of sanguinarine-type alkaloids with two methylenedioxy ring structures, but induced theproduction of protoberberine-type alkaloids, such as berberine and columbamine, and of allocryptopine- and chelerythrine-typealkaloids with one methylenedioxy ring and two methoxy groups. (C) The shutdown of the berberine bridge enzyme (BBE) expressionwith an RNAi vector clearly downregulated the accumulation of sanguinarine and the intermediate scoulerine, as shown by thedownward arrow. In contrast, RNAi of BBE induced a marked accumulation of reticuline, which is a substrate of BBE, as shown bythe upward arrow.


vary across different plant species and isolated celllines because of variations in the expression levels ofthe genes required to produce the enzymes. Further-more, changes in the bottleneck step(s) may alsooccur as a consequence of modifications to theenzyme expression level resulting from metabolicengineering.

Given that there may be multiple bottlenecksteps involved in a particular biosynthetic process,the overall regulation of the expression levels ofbiosynthetic genes in the pathway would provide amore effective means of control, and further studiesare currently underway to isolate general tran-scription factors. The recent identification of thetranscription factors CjWRKY1 and CjbHLH1 aspossible general regulators in IQA biosynthesis usinga transient RNAi screening system could represent asignificant discovery in the field and provide signifi-cant improvements in the yields of IQAs by enablingthe regulation of the transcription processes associ-ated with their production.42)–44)

As well as providing improvements in the yield,improvements in the quality of the IQAs are perhapsmore desirable, in that although increases in meta-bolic diversity would be useful for drug discovery, thereduction of the metabolic diversity is critical for thefacile preparation of pure compounds. The introduc-tion of a novel branch pathway to a biosyntheticpathway represents one of the most useful ap-proaches for increasing the level of metabolicdiversity resulting from the pathway. We haveectopically expressed CjSMT in E. californica cells(Fig. 3).39) CjSMT catalyzes the O-methylation ofscoulerine to produce THC in the berberine biosyn-thesis and is not involved in the benzophenanthridinealkaloid biosynthesis. These transgenic E. californicacells with CjSMT expression not only producedcolumbamine (i.e. the oxidized product of THC),39)

but also provided novel products, including allocryp-topine and 10-hydroxychelerythrine, which wereproduced from the CjSMT reaction products follow-ing their reaction with other endogenous enzymesin the benzophenanthridine alkaloid biosyntheticpathway, effectively demonstrating the enhanceddiversification of the alkaloid profile in transgeniccells.35)

Modifications to the metabolic flow leading to areduction in the levels of enzymes associated withundesired pathways represents another promisingapproach to increase the product levels of the desiredmetabolites, while reducing unwanted byproducts.The combination of this approach with the intro-

duction of a new pathway allows for the productionof novel products from transgenic cells. Of themethods used to reduce gene expression (e.g.,antisense, RNA interference (RNAi), knockout withT-DNA insertion and mutation), RNA silencing withdouble-stranded RNA (i.e. RNAi with siRNA ormiRNA) is the most powerful and reliable of thesetechniques.45) The versatility of RNAi for controllingthe multiple genes responsible for metabolite pro-duction across a variety of different tissues anddevelopmental steps has been well recognized. Forexample, in 2007, Fujii et al.46) reported the success-ful accumulation of reticuline, which is a directsubstrate of BBE, by the use of an RNAi of BBE inE. californica cells (Fig. 3), whereas little accumu-lation of reticuline was reported by Park et al.47)

using the antisense method.

Characterization of aromatic amino acidmetabolizing enzymes in microorganisms

Kumagai et al. have been working on microbialenzymes capable of metabolizing aromatic aminoacids and aromatic amines, such as L-tyrosine, L-DOPA and their corresponding amines. The enzymesused in their work include amino acid-lyases,48)–57)

aromatic amino acid decarboxylases,58)–64) and amineoxidases.65)–72) When they started their work,enzymes were not routinely purified or particularlywell characterized. Consequently, the bacterialstrains which provided the highest levels of enzymeactivity from a number of microbial strains werescreened first. The culture conditions were theninvestigated extensively, with particular emphasison the induction conditions of the enzyme biosyn-thesis to obtain the highest enzyme activity, with theinvestigation ultimately contributing to the isolationof a pure enzyme fraction as crystals. The character-ization of these aromatic amino acid metabolizingenzymes and the elucidation of the control mech-anisms associated with the enzyme biosyntheticpathways in the microorganisms were indispensablefor the construction of the microbial IQA productionplatform. This paragraph describes the synthesis ofL-DOPA by tyrosine phenol-lyase from Erwiniaherbicola, aromatic amino acid decarboxylase fromPseudomonas putida, and monoamine oxidase (tyr-amine oxidase) from Micrococcus luteus.

L-DOPA synthesis by tyrosine phenol-lyaseand tyrosinase. L-DOPA, which is otherwiseknown as 3,4-dihydroxy-L-phenylalanine, and dop-amine are the starting materials required of IQAproduction in microorganisms. Although tyrosine


phenol-lyase (TPL; EC 4.1.99.2) is not directly usedin the microbial IQA production platform, workfocused on the enzymatic properties of TPL and thecontrol mechanisms associated with the biosynthesisof TPL has contributed greatly to the industrialproduction of L-DOPA.

L-DOPA, is used in the treatment of Parkinson’sdiseases. Approximately 250 tons of L-DOPA isproduced around the world every year. Nearly half ofthis material is synthesized according to an enzy-matic method involving TPL.48)–50) TPL normallycatalyzes the ,,O-elimination reaction of L-tyrosineto produces pyruvate, ammonia and phenol (Fig. 4a).This reaction is reversible, and when catechol issubstituted for phenol, the process produces L-DOPA (Fig. 4b).51) TPL is inducible by tyrosineand, from a practically perspective, E. herbicolacells with a high level of TPL activity are preparedby cultivation in a medium supplemented with L-tyrosine before being harvested and transferred tothe reactor together with the substrates, pyruvate,ammonia and catechol. Bai et al.73) and Kumagaiet al.52),53) elucidated the regulatory mechanismunderlying the L-tyrosine-mediated induction ofTPL, and identified TyrR as the transcriptionalactivator of tpl. The tyrR gene was subsequentlycloned from E. herbicola cells and randomly muta-genized to obtain a mutant protein with anenhanced ability to activate tpl. One such mutant,TyrRV67A Y72C E201G, was found to effectively increaseTPL activity expression.54) Furthermore, when themutant gene was introduced to E. herbicola, theresulting strain showed improved levels of L-DOPAproduction.55) Kumagai et al.56) subsequently identi-

fied a second-site suppressor mutations (N324D orA503T) that rescued the inability of TyrRE275Q toactivate the tpl promoter. N324 is located in thecentral domain of TyrR and is thought to be involvedin the formation of a TyR oligomer, which is requiredfor tpl-activation. Amino acid replacement of theresidue (N324D) compensated for the impairedability of TyrRE275Q to form an oligomer.56) Theamino acid substitutions that lead to the enhancedTPL expression were scattered into three domains,including: (1) V67A, Y72C and E201G in the N-terminal domain (NT); (2) N324D in the centraldomain (Cen); and (3) A503T in the C-terminaldomain (CT). These mutations were geneticallycombined by the domains, and the resulting tyrRalleles were inserted into a low-copy-number plasmid.These plasmids were then introduced to tyrR-deficient E. herbicola ("tyrR::kanD) cells, and theTPL activities of the cell-free extracts of therecombinant strains were measured. The straincarrying TyrRNT Cen CT exhibited its highest level ofL-DOPA productivity when cultivated under thenon-induced conditions, and its level of productivitywas 30-fold greater than that of the tyrosine-inducedwild-type cells that are currently utilized in theindustrial production of L-DOPA (11.1 g/L/h vs.0.375 g/L/h).57)

TPL requires high concentrations of it pyruvate,ammonia and catechol substrates to carry out an L-DOPA producing reaction, and the concentrations ofthese substrates can be so high that they can becometoxic to bacterial cells. The construction of an IQAproducing bacterial platform therefore requires theuse of Ralstonia solanacearum tyrosinase (TYR; EC

HO

COOH

NH2

L-TyrosineHO

Phenol

Tyrosine phenol-lyase

+ H2O + COOH

O+ NH3

Pyruvic acid

(a)

HO

COOH

NH2

L-DOPAHOpyrocatechol

Tyrosine phenol-lyase

+ H2O + COOH

O+ NH3

Pyruvic acid

(b)HOHO

HO

COOH

NH2

L-Tyrosine

Tyrosinase + 1/2O2 (c)

HONH2

L-DOPA

HO COOH

Fig. 4. Reaction catalyzed by tyrosine phenol-lyase and tyrosinase.


1.14.18.1, monophenol, L-DOPA: oxygen oxidore-ductase) to produce L-DOPA from L-tyrosine. TYRis a copper containing monophenol monooxygenasethat catalyzes the oxidation of the 3B-position ofthe L-tyrosine phenol-ring (Fig. 4c). This particularreaction mechanism was developed on the basis ofan X-ray crystallographic investigation conducted byMatoba et al.74) R. solanacearum TYR has low o-diphenolase activity,75) and its introduction to theIQA producing bacterial platform was considered toincrease the amount of accumulation of L-tyrosine inthe bacterial cells,76) as will be described in a latersection.

Aromatic amino acid decarboxylases frombacteria. Aromatic amino acid decarboxylases(AADCs; EC 4.1.1.28) catalyze the decarboxylationreactions of aromatic amino acids (Fig. 5) and arefound in a variety of different organisms, where theyplay distinct physiological roles. AADCs from highereukaryotes have been well studied because they areinvolved in the synthesis of biologically importantmolecules such as neurotransmitters and alkaloids. Incontrast, bacterial AADCs have received less atten-tion because of the simplicities associated with theirphysiology and target substrate (tyrosine) (Fig. 5a).Although the existence of strongly active AADCs hasbeen reported in some lactic acid bacteria,77) theseAADCs have never been purified and characterizedas pure preparations because of their instability.

The formation of aromatic L-amino acid decar-boxylase in bacteria was studied with intact cells in areaction mixture containing the aromatic amino acid.The activity was found to be widely distributedin such genera as Achromobacter, Micrococcus,Staphylococcus and Sarcina. Strains belonging toMicrococcus showed especially high levels of decar-boxylase activity towards L-tryptophan, 5-hydroxy-L-tryptophan and phenylalanine. Micrococcus perci-

treus AJ1065 was selected as a promising source ofaromatic L-amino acid decarboxylase. M. percitreuswas found to constitutively produce an enzyme thatexhibited decarboxylase activity towards L-trypto-phan, and cultivation conditions to produce highlevels of decarboxylase activity were subsequentlyexamined.58)

The AADC from M. percitreus was purified andsuccessfully crystallized for the first time, allowingfor its complete characterization.59),60) The enzymethat found to require pyridoxal phosphate (PLP) asa cofactor and to be deactivated by the addition ofL-DOPA to the reaction mixture as a substrate.61)

The mechanism of the inactivation was investigatedby enzyme- and organic-chemical methods, whichrevealed that L-DOPA reacted slowly with the PLPcofactor resulting in the formation of the so-calledtetrahydroisoquinoline adduct compound during thereaction.62) Inhibition of the enzyme was also foundto occur when ,-methyl aromatic amino acids wereused as substrates, and analysis of this inhibitionmechanism resulted in the proposal of a reactionmechanism for the AADC from M. percitreus.63)

A eukaryotic-type AADC exhibiting a high levelof specificity for L-DOPA (Fig. 5b) has recently beenfound and its genetic and enzymatic characteristicsdetermined.64) The purified enzyme converted L-DOPA to dopamine with Km and Kcat values of0.092mM and 1.8 s!1, respectively. The enzyme wasinactive towards other aromatic amino acids such as5-hydroxy-L-tryptophan (HTP), L-phenylalanine, L-tryptophan and L-tyrosine (Table 1). The observedstrict substrate specificity was distinct from that ofany other AADC characterized to date. The nameDOPA decarboxylase (DDC) was subsequently pro-posed for the enzyme. Quantitative RT-PCR experi-ments revealed that the expression of this gene wasinduced by L-DOPA. DDC was found to be encoded

+ CO2

L-DOPA DopamineHO

NH2HOCOOH

HONH2

HO

+ CO2

L-Tyrosine TyramineHO

NH2COOH

HONH2

Tyrosinedecarboxylase

(a)

(b)DOPAdecarboxylase

Fig. 5. Reactions catalyzed by aromatic amino acid decarboxylase.


in a cluster together with a LYSR-type (LysineR-type) transcriptional regulator and a major facilitatorsuperfamily transporter. This genetic organizationwas found to be conserved among all of the sequencedP. putida strains that inhabit the rhizosphere envi-ronment, where DOPA acts as a strong allelochem-ical. These findings suggest the possible involvementof this enzyme in the detoxification of the allelochem-ical in the rhizosphere, and potential occurrence of ahorizontal gene transfer event between the pseudo-monad and its host organism.64)

DDC from P. putida is particularly interestingbecause its physiological role is related to that of thehost plant. In addition, its strict specificity for L-DOPA could be useful for the construction of amicrobial platform for plant IQA production becauseit actually plays an important role in this context,and this will be discussed in more detail in a latersection.76)

Aromatic amine oxidases from bacteria.Amine oxidases catalyze the oxidative deaminationof amines to produce the corresponding aldehydes,together with ammonia and hydrogen peroxide(Fig. 6a). The Gram-positive bacterium Sarcina lutea(M. luteus, later) contains an amine oxidase specificfor tyramine when it grows on nutrient medium. Itwas purified as a crystalline preparation and charac-terized. This enzyme is a monoamine oxidase (MAO;EC 1.4.3.4) and uses FAD as a cofactor.65)–67) WhenM. luteus was grown on synthetic medium containingtyramine as a sole nitrogen source, its level of MAOincreased extensively to give 20- to 30-fold morecrystalline MAO than that obtained under theformer cultivation conditions in a nutrient medium.68)

The mechanism of the reaction catalyzed by theMAO was studied based on the overall reactionkinetics as well as by the stopped flow.69)

The enzyme was found to oxidize tyramine anddopamine at a similar rate, but other monoamines,diamines and polyamines were not oxidized at all.

The aldehyde formed during the reaction withdopamine was found to condense with unchangeddopamine to yield the IQA compound norlaudanoso-line (i.e. tetrahydropapaveroline) (Fig. 6b).70) Thisfinding suggested the idea of the microbial produc-tion of isoquinoline compounds, with MAO and itsgene playing important roles in the construction ofmicrobial platform for the production of IQAs.

Kumagai et al.71) have completed the genecloning and three-dimensional structure predictionexperiments for M. luteus MAO. The amine oxidaseof the fungi Aspergillus niger has also been reportedto be inactivated during the reaction with an analogof the substrate (bromo-ethylamine), and its reactionmechanism was elucidated by analysis of thematerials resulting from its suicide inactivation.72)

Construction of the microbial platformfor plant IQA production

First-generation microbial platforms forplant IQA production. Following an increase inthe availability of molecular biological tools, thereconstitution of an entire metabolic pathway becamefeasible in microorganisms.78)–80) Microorganismshave a simpler internal structure than plants becauseof their smaller genome size, and this effectivelysimplifies metabolite transport between the enzy-matic steps.81) The reconstitution of a secondarymetabolite pathway in an engineered microbial hostoffers several advantages over other methods directedtowards native plant hosts including the specificproduction of key intermediate molecules, such asreticuline, the rapid accumulation and facile purifi-cation of the target IQA, and the availability ofgenetic tools for further engineering and pathwayoptimization of the IQA production.82) A combina-tion of microbial and plant-derived genes offerssome advantages for the establishment of efficientand productive systems for the entry compound.79)

Unfortunately, however, microbial systems also

Table 1. Substrate specificity of P. putida DDC

Substrate Km (mM) kcat (s!1) kcat/Km (mM!1 s!1)

DOPA 9.2 (’1.9) # 10!2 1.8 (’0.1) # 100 2.1 (’0.4) # 101

5-HTP 9.3 (’2.3) # 10!1 9.5 (’0.6) # 10!2 1.0 (’0.2) # 10!1

Phe 8.8 (’1.9) # 100 2.3 (’0.2) # 10!1 2.7 (’0.4) # 10!2

Trp 8.8 (’2.1) # 100 4.4 (’0.4) # 10!3 5.1 (’0.8) # 10!4

Tyr 1.1 (’0.1) # 10!3

3-Methoxy-Tyr 6.3 (’0.3) # 10!4

The kcat values were calculated based on the reported Vmax values. The estimated molecular mass was 54 kDa.


possess disadvantages, such as a limited substrateavailability for plant metabolites, although thislimitation is currently being challenged (see below).

The first reported example of a reconstitutionwas performed in yeast cells for the production ofartemisinic acid according to an engineered isopre-noid pathway.80) The microbial production of alka-loids, however, was not reported in the literatureuntil the pathway from dopamine to reticuline wasreconstituted in E. coli. The first artificial pathwayfor the production of reticuline to be reported in theliterature was assembled in E. coli via the introduc-tion of genes for MAO together with C. japonicaNCS, 6OMT, CNMT and 4BOMT to a plasmid-basedexpression systems (Fig. 7).79) The use of microbialMAO allowed for the incorporation of the hydroxygroup early in the reticuline pathway via thesynthesis of 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) from dopamine, thereby avoiding the needto express the plant cytochrome P450 hydroxylase(CYP80B1) in the bacterium, which can often beproblematic in a microbial expression. Followingthe induction of enzyme expression and dopaminesupplementation, the final yield of (S)-reticulinesynthesized in vitro by the crude enzymes fromrecombinant E. coli was 55mgL!1 within 1 hour.In a separate report, Hawkins and Smolke82) usedS. cerevisiae as the sole host organism for theassembly of an artificial IQA pathway. In thisparticular case, three enzymes were combined from

plant sources (i.e. 6OMT, CNMT and 4BOMTderived from either T. flavum or P. somniferum)and a human P450 enzyme, and (R,S)-norlaudanoso-line was used as a substrate.82) Two systems aretherefore now available for the production of plantIQAs, with one of these systems requiring dopamineand the other requiring norlaudanosoline.

Second-generation platform for microbialalkaloid production. The reconstitution of alkaloidbiosynthesis in microbial systems has shown prom-ising results, as described above. Unfortunately,however, the existing need to exogenously supplyexpensive intermediate precursors has limited thecommercial application of this technique. An incom-plete understanding of the complex alkaloid biosyn-thetic networks, especially during the early steps, haseffectively hindered progress towards the synthesisof alkaloids from simple precursors. In addition, thecytotoxicity of alkaloids in yeast may also posea similar problem in other microbial systems. Thedevelopment of practical and effective strategiesto minimize toxicity is therefore needed to createalkaloid-overproducing microbes, such as the engi-neering of transcription factors to increase microbialtolerance towards toxic metabolites. Further at-tempts have enabled the successful production of(S)-reticuline from simple carbon sources withoutthe need for an additional substrate in an E. colifermentation system (Figs. 8 and 9). Nakagawaet al.76) produced tyrosine-overproducing E. coli via

4-Hydroxyphenyl- acetaldehyde

HO

NH2

Tyramine

H2O O2 NH3 HO

O

Monoamineoxidase

H2O2

DHPAADopamineHO

NH2HO

HO

OHONH3, H2O2H2O, O2

Monoamineoxidase

Norlaudanosoline

(b)

(a)

Fig. 6. Reaction catalyzed by monoamine oxidase (a) and formation of the IQA norlaudanosoline from the reaction of MAO withdopamine (b).


Fig. 8. Construction of a platform for the production of IQA precursors from glucose in E. coli. The “universal aromatic amino acidsynthetic module” was prepared by genetic modifications in E. coli to facilitate overproduction of L-tyrosine from glucose as the solecarbon source. The “tailor-made biosynthetic module” was also assembled and optimized for the production of the two IQA precursors,dopamine and 3, 4-DHPAA, from L-tyrosine. E4P, erythrose-4-phosphate; DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate;HPP, para-hydroxyphenylpyruvate

Fig. 7. Production of IQA from dopamine in E. coli. The “fundamental IQA synthetic module” for the production of (S)-reticuline, whichis the key substance in the IQA synthetic pathway, was constructed in E. coli with the genes of four enzymes (NCS, 6OMT, CNMTand 4BOMT) cloned from C. japonica and a microbial enzyme (MAO).


the overexpression of biosynthetic enzymes (TKT,transketolase; PEPS, phosphoenolpyruvate syn-thetase; fbr-DAHPS, feedback-inhibition-resistant3-deoxy-d-arabino-heptulosonate-7-phosphate syn-thase; fbr-CM/PD, feedback-inhibition-resistantchorismate mutase/prephenate dehydrogenase anddeletion of the repressor of aromatic amino acidbiosynthesis; tyrR), and then modified the pathwayto produce dopamine from tyrosine with the intro-duction of pathway-specific tyrosinase (TYR) ofR. solanacearum and DDC of Pseudomonas.62) Thecombination of these modifications with a previouslydeveloped alkaloid-production module enabled re-ticuline production from glucose or glycerol. Thisachievement could potential lead to the industrial-scale production of alkaloids in the near future.

Production of more complex alkaloids. Moresophisticated systems are required for the productionof more complex alkaloids. Although synthetic bio-logical approaches may in principle only require asingle host system such as E. coli or S. cerevisiae,or a combination of the two, one of the maindisadvantages of the E. coli system is that it lacksan internal membrane system. In 2008, Minamiet al.79) examined a combination system of E. coliand S. cerevisiae to overcome these problems(Fig. 9). When reticuline-producing E. coli was co-cultured with S. cerevisiae expressing C. japonicaBBE or CYP80G2 in the presence of dopamine,(S)-scoulerine and magnoflorine were detected at 8.3and 7.2mgL!1, respectively, following an incubationperiod of 48 to 72 hours. Although the use of two

E. coli

Yeast

Fig. 9. Platform to produce more complex IQAs. The “ad hoc IQA synthetic module” was prepared in S. cerevisiae to produce differentIQAs from (S)-reticuline. Co-cultures of IQA producing E. coli and yeast cells efficiently produced (S)-reticuline and other IQAs.Further potential products are also shown with gene names. Broken lines indicate enzyme steps for which the genes have not yet beenidentified. The abbreviations used are as in Fig. 1.


microbial systems for pathway construction shouldbe useful for establishing different biosyntheticmodules, as a strategy it could reduce the efficiencyof alkaloid synthesis because of the need formetabolite transport between the cell membranes ofthe two microorganisms. As shown in Fig. 1, moregenes are currently available. The possibility ofproducing a more divergent array of chemicals istherefore growing exponentially.83)

In summary, we have successfully constructedfor the first time a microbial platform for plant IQAproduction. The platform consists of four modules,including (1) a “universal aromatic amino acidsynthetic module” prepared through genetic modifi-cations of the E. coli metabolic system to facilitateoverproduction of L-tyrosine from glucose as asole carbon source; (2) a “tailor-made biosyntheticmodule” that has been optimized for the productionof the two IQA precursors dopamine and 3,4-DHPAA, from L-tyrosine using three microbial genes(Fig. 8); (3) a “fundamental IQA synthetic module”for the production of (S)-reticuline, which is the keysubstance in the IQA synthetic pathway (Fig. 7),from four genes cloned from C. japonica; and (4)an “ad hoc IQA synthetic module” consisting ofthree C. japonica genes that is capable of producingdifferent IQAs from (S)-reticuline. The first threemodules were installed in E. coli cells, whereas thelast one was prepared with budding yeast. Co-cultures of these E. coli and yeast cells efficientlyproduced (S)-reticuline and other IQAs, demonstrat-ing the overall utility of this constructed microbialplatform for plant IQA production (Fig. 9).

The present investigation has opened up a newavenue for the microbial production of plant secon-dary metabolites, using IQAs as the model. Themicrobial platform that we have constructed will beuseful for the production of phenylpropanoids andother phenylalanine- or tyrosine-derived plant me-tabolites and contribute to the development of newresearch areas in both synthetic biology and meta-bolic engineering.83)

Acknowledgments

The authors are deeply grateful for the contin-uous guidance and encouragement received fromProf. Emeritus Yasuyuki Yamada and HideakiYamada, as well as collaborations with previousand current colleagues and students at the GraduateSchool of Biostudies and the Faculty of Agricultureat Kyoto University, and at the Research Institutefor Bioresources and Biotechnology at Ishikawa

Prefectural University. The names of these peoplehave been provided in the references. The authorsalso appreciate the financial support offered by theMinistry of Education, Culture, Sports, Scienceand Technology of Japan, and the Japan Societyfor the Promotion of Science, Future Program GrantJSPS-RFTF00L01606, New Energy and IndustrialTechnology Development Organization (NEDO),and the Program for the Promotion of Basic andApplied researches for Innovations in Bio-orientedIndustry (Brain).

Note in proof

Very recently, cDNA of N-methylstylopinehydroxylase in sanguinarine biosynthesis was clonedfrom opium poppy.84)

References

1) Croteau, R., Kutchan, T.M. and Lewis, N.G. (2000)Natural products (Secondary metabolites). InBiochemistry and Molecular Biology of Plants(eds. Buchanan, B.B., Gruissem, W. and Jones,R.L.). Am. Soc. Plant Physiol., Maryland,pp. 1250–1318.

2) Briskin, D.P. (2000) Medicinal plants and phytome-dicines. Linking plant biochemistry and physiologyto human health. Plant Physiol. 124, 507–514.

3) Raskin, I., Ribnicky, D.M., Komarnytsky, S., Ilic, N.,Poulev, A., Borisjuk, N., Brinker, A., Moreno,D.A., Ripoll, C., Yakoby, N., O’Neal, J.M.,Cornwell, T., Pastor, I. and Fridlender, B. (2002)Plants and human health in the twenty-firstcentury. Trends Biotechnol. 20, 522–531.

4) Goto, K. (1964) Sinomenine: An Optical Antipode ofMorphine Alkaloids. Kitasato Institute, Tokyo.

5) Zenk, M.H. (1991) Chasing the enzymes of secondarymetabolism: Plant cell cultures as a pot of gold.Phytochemistry 30, 3861–3863.

6) Sato, F. and Yamada, Y. (2008) Engineeringformation of medicinal compounds in cell cultures.In Advances in Plant Biochemistry and MolecularBiology, Vol. 1. (eds. Bohnert, H.J., Nguyen, H.and Lewis, N.G.). Elsevier Ltd., Amsterdam,pp. 311–345.

7) Sato, F. and Yamada, Y. (1984) High berberine-producing cultures of Coptis japonica cells.Phytochemistry 23, 281–285.

8) Sato, F., Inai, K. and Hashimoto, T. (2007)Metabolic engineering in alkaloid biosynthesis:case studies in tyrosine- and putrescine-derivedalkaloids. In Applications of Plant MetabolicEngineering (eds. Verpoorte, R., Alfermann,A.W. and Johnson, T.S.). Springer, New York,pp. 145–173.

9) Facchini, P. (2001) Alkaloid biosynthesis in plants:biochemistry, cell biology, molecular regulation,and metabolic engineering applications. Annu.Rev. Plant Physiol. Plant Mol. Biol. 52, 29–66.


10) Facchini, P.J. and Deluca, V. (1994) Differential andtissue-specific expression of a gene family fortyrosine/dopa decarboxylase in opium poppy. J.Biol. Chem. 269, 26684–26690.

11) Lee, E.J. and Facchini, P.J. (2011) Tyrosine amino-transferase contributes to benzylisoquinoline alka-loid biosynthesis in opium poppy. Plant Physiol.157, 1067–1078.

12) Samanani, N. and Facchini, P.J. (2002) Purificationand characterization of norcoclaurine synthase. J.Biol. Chem. 277, 33878–33883.

13) Samanani, N., Liscombe, D.K. and Facchini, P.J.(2004) Molecular cloning and characterization ofnorcoclaurine synthase, an enzyme catalyzing thefirst committed step in benzylisoquinoline alkaloidbiosynthesis. Plant J. 40, 302–313.

14) Minami, H., Dubouzet, E., Iwasa, K. and Sato, F.(2007) Functional analysis of norcoclaurinesynthase in Coptis japonica. J. Biol. Chem. 282,6274–6282.

15) Morishige, T., Tsujita, T., Yamada, Y. and Sato, F.(2000) Molecular characterization of the S-adeno-syl-L-methionine: 3B-hydroxyl-N-methylcoclaurine4B-O-methyltransferase involved in isoquinolinealkaloid biosynthesis in Coptis japonica. J. Biol.Chem. 275, 23398–23405.

16) Choi, K.-B., Morishige, T., Shitan, N., Yazaki, K.and Sato, F. (2002) Molecular cloning andcharacterization of coclaurine N-methyltransferasefrom cultured cells of Coptis japonica. J. Biol.Chem. 277, 830–835.

17) Pauli, H.H. and Kutchan, T.M. (1998) Molecularcloning and functional heterologous expression oftwo alleles encoding (S)-N-methylcoclaurine 3B-hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome P-450-dependent mono-oxy-genase of benzylisoquinoline alkaloid biosynthesis.Plant J. 13, 793–801.

18) Dittrich, H. and Kutchan, T.M. (1991) Molecularcloning, expression, and induction of berberinebridge enzyme, an enzyme essential to the for-mation of benzophenanthridine alkaloids in theresponse of plants to pathogen attack. Proc. Natl.Acad. Sci. U.S.A. 88, 9969–9973.

19) Bock, A., Wanner, G. and Zenk, M.H. (2002)Immunocytological localization of two enzymesinvolved in berberine biosynthesis. Planta 216,57–63.

20) Takeshita, N., Fujiwara, H., Mimura, H., Fitchen,J.H., Yamada, Y. and Sato, F. (1995) Molecularcloning and characterization of S-adenosyl-L-methionine: scoulerine-9-O-methyltransferase fromcultured cells of Coptis japonica. Plant CellPhysiol. 36, 29–36.

21) Ikezawa, N., Tanaka, M., Nagayoshi, M., Shinkyo,R., Sasaki, T., Inoue, K. and Sato, F. (2003)Molecular cloning and characterization ofCYP719, a methylenedioxy bridge-forming enzymethat belongs to a novel P450 family, from culturedCoptis japonica cells. J. Biol. Chem. 278, 38557–38565.

22) Muemmler, S., Rueffer, M., Nagakura, N. and Zenk,

M.H. (1985) S-Adenosyl-L-methionine: (S)-scou-lerine 9-O-methyltransferase, a highly stereo- andregio-specific enzyme in tertrahydroprotoberberinebiosynthesis. Plant Cell Rep. 4, 36–39.

23) Galneder, E., Rueffer, M., Wanner, G., Tabata, M.and Zenk, M.H. (1988) Alternative final steps inberberine biosynthesis in Coptis japonica cellcultures. Plant Cell Rep. 7, 1–4.

24) Okada, N., Shinmyo, A., Okada, H. and Yamada, Y.(1988) Purification and characterization of (S)-tetrahydroberberine oxidase from cultured Coptisjaponica cells. Phytochemistry 27, 979–982.

25) Amman, M., Nagakura, N. and Zenk, M.H. (1988)Purification and properties of (S)-tetrahydropro-toberberine oxidase from suspension-cultured cellsof Berberis wilsoniae. Eur. J. Biochem. 175, 17–25.

26) Matsushima, Y., Minami, H., Hori, K. and Sato, F.(2012) Pathway engineering of benzylisoquinolinealkaloid biosynthesis in transgenic Californiapoppy cells with ectopic expression of tetrahydro-berberine oxidase from Coptis japonica. PlantBiotechnol. 29, 473–481.

27) Morishige, T., Dubouzet, E., Choi, K.B., Yazaki, K.and Sato, F. (2002) Molecular cloning of columb-amine O-methyltransferase from cultured Coptisjaponica cells. Eur. J. Biochem. 269, 5659–5667.

28) Rueffer, M., Amann, M. and Zenk, M.H. (1986) S-Adenosyl-L-methionine: columbamine-O-methyl-transferase, a compartmentalized enzyme in pro-toberberine biosynthesis. Plant Cell Rep. 3, 182–185.

29) Ikezawa, N., Iwasa, K. and Sato, F. (2008) Molecularcloning and characterization of CYP80G2, acytochrome P450 that catalyzes an intramolecularC-C phenol coupling of (S)-reticuline in magno-florine biosynthesis, from cultured Coptis japonicacells. J. Biol. Chem. 283, 8810–8821.

30) Krause, P.F.X. and Kutchen, T.M. (1995) Molecularcloning and heterologous expression of a cDNAencoding berbamunine synthase, a C-O-phenol-coupling cytochrome-P450 from the higher-plantBerberis stolonifera. Proc. Natl. Acad. Sci. U.S.A.92, 2071–2075.

31) Ikezawa, N., Iwasa, K. and Sato, F. (2007) Molecularcloning and characterization of methylenedioxybridge-forming enzymes involved in stylopinebiosynthesis in Eschscholzia californica. FEBS J.274, 1019–1035.

32) Ikezawa, N., Iwasa, K. and Sato, F. (2009) CYP719Asubfamily of cytochrome P450 oxygenases andisoquinoline alkaloid biosynthesis in Eschscholziacalifornica. Plant Cell Rep. 28, 123–133.

33) Liscombe, D.K. and Facchini, P.J. (2007) Molecularcloning and characterization of tetrahydroproto-berberine cis-N-methyltransferase, an enzyme in-volved in alkaloid biosynthesis in opium poppy. J.Biol. Chem. 282, 14741–14751.

34) Tanahashi, T. and Zenk, M.H. (1990) Elicitorinduction and characterization of microsomalprotopine-6-hydroxylase, the central enzyme inbenzophenanthridine alkaloid biosynthesis. Phyto-


chemistry 29, 1113–1122.35) Takemura, T., Ikezawa, N., Iwasa, K. and Sato, F.

(2010) Metabolic diversification of benzylisoquino-line alkaloid biosynthesis through the introductionof a branch pathway in Eschscholzia californica.Plant Cell Physiol. 51, 949–959.

36) Takemura, T., Ikezawa, N., Iwasa, K. and Sato, F.(2012) Molecular cloning and characterization ofa cytochrome P450 in sanguinarine biosynthesisfrom Eschscholzia californica cells. Phytochemis-try (in press). doi:10.1016/j.phytochem.2012.02.013.

37) Nelson, D.R., Schuler, M.A., Paquette, S.M., Werck-Reichhart, D. and Bak, S. (2004) Comparativegenomics of rice and Arabidopsis. Analysis of 727cytochrome P450 genes and pseudogenes from amonocot and a dicot. Plant Physiol. 135, 756–772.

38) Mizutani, M. and Sato, F. (2011) Unusual P450reactions in plant secondary metabolism. Arch.Biochem. Biophys. 507, 194–203.

39) Sato, F., Hashimoto, T., Hachiya, A., Tamura, K.,Choi, K.-B., Morishige, T., Fujimoto, H. andYamada, Y. (2001) Metabolic engineering of plantalkaloid biosynthesis. Proc. Natl. Acad. Sci. U.S.A.98, 367–372.

40) Inui, T., Kawano, N., Shitan, N., Yazaki, K., Kiuchi,F., Kawahara, N., Sato, F. and Yoshimatsu, K.(2012) Improvement of benzylisoquinoline alkaloidproductivity by overexpression of 3B-hydroxy-N-methylcoclaurine 4B-O-methyltransferase in trans-genic Coptis japonica plants. Biol. Pharm. Bull.35, 650–659.

41) Inui, T., Tamura, K., Fujii, N., Morishige, T. andSato, F. (2007) Overexpression of Coptis japonicanorcoclaurine 6-O-methyltransferase overcomesthe rate-limiting step in benzylisoquinolinealkaloid biosynthesis in cultured Eschscholziacalifornica. Plant Cell Physiol. 48, 252–262.

42) Dubouzet, J.G., Morishige, T., Fujii, N., An, C.I.,Fukusaki, E., Ifuku, K. and Sato, F. (2005)Transient RNA silencing of scoulerine 9-O-meth-yltransferase expression by double stranded RNAin Coptis japonica protoplasts. Biosci. Biotechnol.Biochem. 69, 63–70.

43) Kato, N., Dubouzet, E., Kokabu, Y., Yoshida, S.,Dubouzet, J., Yazaki, K. and Sato, F. (2007)Identification of a WRKY protein as a transcrip-tional regulator of benzylisoquinoline alkaloidbiosynthesis in Coptis japonica. Plant Cell Physiol.48, 8–18.

44) Yamada, Y., Kokabu, Y., Chaki, K., Yoshimoto, T.,Ohgaki, M., Yoshida, S., Kato, N., Koyama, T.and Sato, F. (2011) Isoquinoline alkaloid biosyn-thesis is regulated by a unique bHLH-type tran-scription factor in Coptis japonica. Plant CellPhysiol. 52, 1131–1141.

45) Sato, F. (2005) RNAi and functional genomics. PlantBiotechnol. 22, 431–442.

46) Fujii, N., Inui, T., Iwasa, K., Morishige, T. and Sato,F. (2007) Knockdown of berberine bridge enzymeby RNAi accumulates (S)-reticuline and activatesa silent pathway in cultured California poppy cells.

Transgenic Res. 16, 363–375.47) Park, S.U., Yu, M. and Facchini, P.J. (2002)

Antisense RNA-mediated suppression of benzo-phenanthridine alkaloid biosynthesis in transgeniccell cultures of California poppy. Plant Physiol.128, 696–706.

48) Kumagai, H., Yamada, H., Matsui, H., Ohkishi, H.and Ogata, K. (1970) Tyrosine phenol-lyase. I.Purification, crystallization, and properties. J.Biol. Chem. 245, 1767–1772.

49) Katayama, T. and Kumagai, H. (2010) TyrosinePhenol-Lyase. In The Encyclopedia of IndustrialBiotechnology 7 (ed. Flickinger, M.C.). John Wiley& Sons, Inc., U.S.A., pp. 4752–4757.

50) Katayama, T. and Kumagai, H. (2010) L-DOPA,Microbial Production. In The Encyclopedia ofIndustrial Biotechnology 3 (ed. Flickinger, M.C.).John Wiley & Sons, Inc., U.S.A., pp. 1937–1940.

51) Yamada, H., Kumagai, H., Kashima, N., Torii, H.,Enei, H. and Okumura, S. (1972) Synthesis ofL-tyrosine from pyruvate, ammonia and phenolby crystalline tyrosine phenol-lyase. Biochem.Biophys. Res. Commun. 46, 370–374.

52) Katayama, T., Suzuki, H., Yamamoto, K. andKumagai, H. (1999) Transcriptional regulation oftyrosine phenol-lyase gene mediated through TyrRand cAMP receptor protein. Biosci. Biotechnol.Biochem. 63, 1823–1827.

53) Suzuki, H., Katayama, T., Yamamoto, K. andKumagai, H. (1995) Transcriptional regulation oftyrosine phenol-lyase gene of Erwinia herbicolaAJ2985. Biosci. Biotechnol. Biochem. 59, 2339–2341.

54) Katayama, T., Suzuki, H., Koyanagi, T. andKumagai, H. (2000) Cloning and random muta-genesis of the Erwinia herbicola tyrR gene for high-level expression of tyrosine phenol-lyase. Appl.Environ. Microbiol. 66, 4764–4771.

55) Koyanagi, T., Katayama, T., Suzuki, H., Nakazawa,H., Yokozeki, K. and Kumagai, H. (2005) Effectiveproduction of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) with Erwinia herbicola cells carryinga mutant transcriptional regulator TyrR. J.Biotechnol. 115, 303–306.

56) Koyanagi, T., Katayama, T., Suzuki, H. andKumagai, H. (2008) Altered oligomerization prop-erties of N316 mutants of Escherichia coli TyrR.J. Bacteriol. 190, 8238–8243.

57) Koyanagi, T., Katayama, T., Suzuki, H., Onishi, A.,Yokozeki, K. and Kumagai, H. (2009) Hyper-production of 3,4-dihydroxyphenyl-L-alanine (L-Dopa) using Erwinia herbicola cells carrying amutant transcriptional regulator TyrR. Biosci.Biotechnol. Biochem. 73, 1221–1223.

58) Nakazawa, H., Sano, K., Kumagai, H. and Yamada,H. (1977) Distribution and formation of aromaticL-amino acid decarboxylase in bacteria. Agric.Biol. Chem. 41, 2241–2247.

59) Nakazawa, H., Kumagai, H. and Yamada, H. (1974)Constitutive aromatic L-amino acid decarboxylasefrom Micrococcus percitreus. Biochem. Biophys.Res. Commun. 61, 75–82.


60) Nakazawa, H., Kumagai, H. and Yamada, H. (1981)Aromatic L-amino acid decarboxylase from Micro-coccus percitreus. Purification, crystallization andproperties. Agric. Biol. Chem. 45, 2543–2552.

61) Nakazawa, H., Kumagai, H. and Yamada, H. (1983)Cofacter requirement of constitutive aromatic L-amino acid decarboxylase from Micrococcus perci-treus. Agric. Biol. Chem. 47, 1605–1609.

62) Nakazawa, H., Kumagai, H. and Yamada, H. (1987)Inhibition of aromatic L-amino acid decarboxylasefrom Micrococcus percitreus by substrate analogs.Agric. Biol. Chem. 51, 2531–2537.

63) Nakazawa, H., Kumagai, H. and Yamada, H. (1985)Decarboxylation reaction of ,-methyl amino acidcatalyzed by aromatic L-amino acid decarboxylasefrom Micrococcus percitreus. Agric. Biol. Chem.49, 159–165.

64) Koyanagi, T., Nakagawa, A., Sakurama, H.,Yamamoto, K., Sakurai, N., Takagi, Y., Minami,H., Katayama, T. and Kumagai, H. (2012)Eukaryotic-type aromatic amino acid decarboxy-lase from the root colonizer Pseudomonas putida ishighly specific for 3,4-dihydroxyphenyl-L-alanine,an allelochemical in the rhizosphere. Microbiology158, 2965–2974.

65) Yamada, H., Uwajima, T., Kumagai, H., Watanabe,M. and Ogata, K. (1967) Crystalline tyramineoxidase from Sarcina lutea. Biochem. Biophys.Res. Commun. 27, 350–355.

66) Yamada, H., Uwajima, T., Kumagai, H., Watanabe,M. and Ogata, K. (1967) Bacterial amine oxidasesPart I. Purification and crystallization of tyramineoxidase of Sarcina lutea. Agric. Biol. Chem. 31,890–896.

67) Yamada, H., Kumagai, H., Uwajima, T. and Ogata,K. (1967) Bacterial amine oxidases Part II.Substrate and inhibitor specificities of tyramineoxidase of Sarcina lutea. Agric. Biol. Chem. 31,897–901.

68) Kumagai, H., Matsui, H., Ogata, K. and Yamada, H.(1969) Properties of crystalline tyramine oxidasefrom Sarcina lutea. Biochim. Biophys. Acta 171,1–8.

69) Kumagai, H., Yamada, H., Suzuki, H. and Ogura, Y.(1971) Action mechanism of tyramine oxidase fromSarcina lutea. J. Biochem. 69, 137–144.

70) Kumagai, H., Matsui, H., Ogata, K., Yamada, H.and Fukami, H. (1968) Oxidation of dopamine bycrystalline tyramine oxidase from Sarcina lutea.Memoirs of the Research Institute for FoodScience, Kyoto University 29, 69–71.

71) Roh, J.H., Wouters, E.D., Yukawa, H., Inui, M.,Minami, H., Suzuki, H. and Kumagai, H. (2000)Purification, cloning, and three dimensional struc-ture prediction of Micrococcus luteus FAD-con-taining tyramine oxidase. Biochem. Biophys. Res.Commun. 268, 293–297.

72) Kumagai, H., Uchida, H. and Yamada, H. (1979)Reaction of fungal amine oxidase with O-bromo-ethylamine. J. Biol. Chem. 254, 10913–10919.

73) Bai, Q. and Somerville, R.L. (1998) Integration ofhost factor and cAMP receptor protein arerequired for TyrR-mediated activation of tpl inCitrobacter freundii. J. Bacteriol. 180, 6173–6186.

74) Matoba, Y., Kumagai, T., Yamamoto, A., Yoshitu,H. and Sugiyama, M. (2006) Crystallographicevidence that the dinuclear copper center oftyrosinase is flexible during catalysis. J. Biol.Chem. 281, 8981–8990.

75) Hernández-Romero, D., Sanchez-Amat, A. andSolano, F. (2006) A tyrosinase with an abnormallyhigh tyrosine hydroxylase/dopa oxidase ratio.FEBS J. 273, 257–270.

76) Nakagawa, A., Minami, H., Kim, J.S., Koyanagi, T.,Katayama, T., Sato, F. and Kumagai, H. (2011) Abacterial platform for fermentative production ofplant alkaloids. Nature Comm. 2, Article number:326, doi:10.1038/ncomms1327

77) Epps, H.M. (1944) Studies on bacterial amino-aciddecarboxylases. Biochem. J. 38, 242–248.

78) Ajikumar, P.K., Xiao, W.-H., Tyo, K.E.J., Wang,Y., Simeon, F., Leonard, E., Mucha, O., Phon,T.H., Pfeifer, B. and Stephanopoulos, G. (2010)Isoprenoid pathway optimization for taxol precur-sor overproduction in Escherichia coli. Science330, 70–74.

79) Minami, H., Kim, J.-S., Ikezawa, N., Takemura, T.,Katayama, T., Kumagai, H. and Sato, F. (2008)Microbial production of plant benzylisoquinolinealkaloids. Proc. Natl. Acad. Sci. U.S.A. 105, 7393–7398.

80) Ro, D.K., Paradise, E.M., Ouellet, M., Fisher, K.J.,Newman, K.L., Ndungu, J.M., Ho, K.A., Eachus,R.A., Ham, T.S., Kirby, J., Chang, M.C.Y.,Withers, S.T., Shiba, Y., Sarpong, R. andKeasling, J.D. (2006) Production of the antima-larial drug precursor artemisinic acid in engineeredyeast. Nature 440, 940–943.

81) Leonard, E., Runguphan, W., O’Connor, S. andPrather, K.J. (2009) Opportunities in metabolicengineering to facilitate scalable alkaloid produc-tion. Nat. Chem. Biol. 5, 291–300.

82) Hawkins, K. and Smolke, C. (2008) Production ofbenzylisoquinoline alkaloids in Saccharomycescerevisiae. Nat. Chem. Biol. 4, 564–573.

83) Chow, Y.L. and Sato, F. (2012) Metabolic engineer-ing and synthetic biology for the production ofisoquinoline alkaloids. In Biotechnology for Medic-inal Plants; Micropropagation and Improvement.(eds. Chandra, S., Lata, H. and Varma, A).Springer-Verlag, Berlin, pp. 327–343.

84) Beaudoin, G.A. and Facchini, P.J. (2013) Isolationand characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opiumpoppy, a key enzyme in sanguinarine biosynthesis.Biochem. Biophys. Res. Commun. 431, 597–603.

(Received Jan. 31, 2013; accepted Mar. 25, 2013)


Profile

Fumihiko Sato was born in 1953, graduated from Kyoto University, and started hisresearch career in 1975 by studying the characterization of functional differentiation inplant cells at the Faculty of Agriculture at Kyoto University. He performed pioneeringwork on the selection of photoautotrophically cultured cells of higher plants in 1978 andof stable medicinal plant cell lines that produced desired natural products in high yield in1984; this was the first demonstration of somatic heterogeneity in cultured plant cells.These cell cultures enabled extensive studies on biochemical reactions, especially in thepathways for plant isoquinoline alkaloids, as well as the discovery of many importantenzymes and genes for the production of these alkaloids. Further metabolic engineeringtechnologies provided a platform for the production of valuable alkaloids in high yield intransgenic medicinal plant cells or in a novel combinatorial microbial platform. He was promoted to Professor atthe Faculty of Agriculture at Kyoto University in 1995, and moved to the Graduate School of Biostudies at KyotoUniversity in 1999, where he taught students about the cellular and molecular biology of plants. He was awardedthe Scientific Contribution Award (Ichimura Foundation) in 2003, the Prize for Science and Technology (ResearchCategory) from The Ministry of Education, Culture, Sports, Science and Technology, Japan in 2009, the JapanSociety for Bioscience, Biotechnology and Agrochemistry Award in 2011, and the Japan Academy Prize in 2012.

Profile

Hidehiko Kumagai was born in 1940 and started his research career in 1964 underthe guidance of Prof. Hideaki Yamada at the Institute for Food Science, KyotoUniversity where he worked on microbial enzymes capable of metabolizing amino acidsand amines. He obtained his PhD degree for studies on a PLP-dependent multi-functional enzyme, tyrosine phenol-lyase in 1969. He was appointed as an AssistantProfessor of the Institute for Food Science, Kyoto University in 1969. He moved toFaculty of Agriculture, Kyoto University in 1977 as an Associate Professor at theDepartment of Food Technology and was promoted to Professor in 1991. He moved toGraduate School of Biostudies, Kyoto University in 1999. After retirement of KyotoUniversity in 2004, he moved to Ishikawa Prefectural University as a Professor and nowhe is the President of the University. He served as a president of Japan Society for Bioscience, Biotechnology andAgrochemistry for 2003–2005. He is an emeritus member of Japan Society for Bioscience, Biotechnology andAgrochemistry and The Vitamin Society of Japan.

He received the Award of Vitamin Society of Japan in 1994 and the Japan Society for Bioscience,Biotechnology and Agrochemistry Award in 2001, and the Japan Academy Prize in 2012.


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Microbial production of isoquinoline alkaloids as plant ......Review Microbial production of isoquinoline alkaloids as plant secondary metabolites based on metabolic engineering research