Microbial production of isoquinoline alkaloids as plant secondary

18 Pages·2013·1.22 MB·English

No.5] Proc.Jpn.Acad.,Ser.B89(2013) 165
Review
Microbial production of isoquinoline alkaloids as plant secondary
metabolites based on metabolic engineering research
By Fumihiko SATO*1,† and Hidehiko KUMAGAI*2,†
(CommunicatedbyKoichiroTSUNEWAKI,M.J.A.)
Abstract: Plants produce a variety of secondary metabolites that possess strong
physiological activities. Unfortunately, however, their production can suffer from a variety of
seriousproblems,includinglowlevelsofproductivityandheterogeneousquality,aswellasdifficulty
inrawmaterialsupply.Incontrast,microorganismscanbeusedtoproducetheirprimaryandsome
of their secondary metabolites in a controlled environment, thus assuring high levels of efficiency
anduniformquality.Inanattempttoovercometheproblemsassociatedwithsecondarymetabolite
production in plants, we developed a microbial platform for the production of plant isoquinoline
alkaloids involving the unification of the microbial and plant metabolic pathways into a single
system. The potential applications of this system have also been discussed.
Keywords: aromatic amino acid metabolism, isoquinoline alkaloid biosynthesis, metabolic
engineering, microbial production
*1 Division of Integrated Life Science, Graduate School of
Introduction
Biostudies,KyotoUniversity,Kyoto,Japan.
*2 IshikawaPrefecturalUniversity,Ishikawa,Japan. Higherplantsproduceawiderangeofchemicals,
† Correspondenceshouldbeaddressed:F.Sato,Divisionof
with more than 25,000 terpenoids, 12,000 alkaloids
Integrated Life Science, Graduate School of Biostudies, Kyoto
University,Kitashirakawa,Sakyo,Kyoto606-8502,Japan(e-mail: and 8,000 phenolic substances having been identified
[email protected]). H. Kumagai, Ishikawa Prefectural Uni- and reported in the literature to date.1) These
versity, Ishikawa 921-8836, Japan (e-mail: hidekuma@ishikawa- chemicals serve a variety of different functions in
pu.ac.jp).
plants, where they defend against herbivores and
Abbreviations: AADC: aromatic amino acid decarboxy-
lase;BBE:berberinebridgeenzyme;bHLH:basichelix-loop-helix; pathogens,aidininter-plantcompetitionandattract
Cen: central domain; CM/PD: chorismate mutase/prephenate beneficial organisms such as pollinators. They can
dehydrogenase;CNMT:coclaurineN-methyltransferase;CoOMT: also have protective effects with regard to abiotic
columbamine O-methyltransferase; CT: C-terminal domain;
stresses such as UV exposure, and changes in the
CYP80A1:berbamuninesynthase;CYP80B1:(S)-N-methylcocla-
urine3B-hydroxylase;CYP719A1:canadinesynthase;CYP719A2/ temperature, water status and mineral nutrient
A3: stylopine synthase; CYP719A5: cheilanthifoline synthase; composition. In addition, many secondary metabo-
CYP719B1: salutaridine synthase; CYP80G2: corytuberine syn-
litesproducedinplantshavebeenusedbyhumansin
thase;DAHPS:3-deoxy-d-arabino-heptulosonate-7-phosphatesyn-
a variety of different applications, including spices,
thase; 3,4-DHPAA: 3,4-dihydroxyphenylacetaldehyde; DDC:
DOPA decarboxylase; EST: expressed sequence tag; fbr: feed- dyes, fragrances, flavoring agents and pharmaceut-
back-inhibition-resistance; IQAs: isoquinoline alkaloids; MAO: icals. Many of these chemicals have also been
monoamine oxidase; MSH: N-methylstylopine hydroxylase; NCS:
reportedtopromotehumanhealthandcantherefore
norcoclaurine synthase; NT: N-terminal domain; 4BOMT: 3B-
hydroxy-N-methylcoclaurine-4B-O-methyltransferase; 6OMT: nor- enrich our lives in a variety of different ways.2)
coclaurine6-O-methyltransferase;7OMT:(S)-reticuline7-O-meth- The use of plant metabolites as natural medi-
yltransferase;PEP:phosphoenolpyruvate;PEPS:PEPsynthetase; cines has a long history that can be traced back over
P6H:protopine6-hydroxylase; PLP:pyridoxalphosphate;PR10:
more than 3,500 years.3) In the more recent years,
pathogenesis-relatedprotein10;RNAi:RNAinterference;SAM:S-
adenosylmethionine; SMT: (S)-scoulerine-9-O-methyltransferase; research has focused predominantly on the identi-
TAT: tyrosine aminotransferase; THB: tetrahydroberberine; fication of the active ingredients. Following the first
THBO: THB oxidase; THC: tetrahydrocolumbamine; TKT: identification of morphine as the principal active
transketolase; TNMT: (S)-tetrahydroprotoberberine N-methyl-
ingredient in opium (1804), many compounds have
transferase;TPL:tyrosinephenol-lyase;TYR:tyrosinase;TYDC:
tyrosine/DOPAdecarboxylase. been identified from plant materials and used as
doi:10.2183/pjab.89.165
©2013TheJapanAcademy
166 F.SATOandH.KUMAGAI [Vol.89,
medicines, including the pain-killing and fever- ity for a particular medicinal herb known as the
reducing salicylic acid from willow bark extract Japanese goldthread (Coptis japonica Makino) and
(1826), the anticancer agent paclitaxel from the characterized the molecular basis of the IQA path-
Pacific yew tree (1971), and the anti-malarial drug way to consolidate the results of their biochemical
artemisinin from Artemisia annua (1972). Japanese characterization.7),8)
scientists have also provided a significant contribu- In this review, we have provided a summary
tion to the isolation and identification of natural of the progress that has been made towards the
products from medicinal plants. The isolations of biochemical and molecular biological characteriza-
ephedrine from Ephedra vulgaris and matrine from tionoftheIQAbiosyntheticpathwaysusingcultured
Sophora flavescens (1885) by Dr. Nagayoshi Nagai C. japonica and California poppy (Eschscholzia
highlight the early success of natural product californica) cells. The application of this progress to
research in Japan. Following on from this early the metabolic engineering, and microbial production
success, many excellent works have been produced, of plant IQAs will also be discussed.
including the structural determination studies of
Clarification of the molecular basis
sinomenine in Sinomenium acutum and its synthesis
of the IQA biosynthetic pathways
by Dr. Kakuji Goto.4)
and metabolic engineering
Theuseofbiochemicalandmolecularcharacter-
ization techniques in natural product research, IQAbiosynthesisisgenerallyconsideredtobegin
however, has been largely limited, because of the withtheconversionoftyrosinetobothdopamineand
low levels of enzymatic activity often encountered 4-hydroxyphenylacetaldehydeviaasequentialdecar-
in the secondary metabolism of plants, especially boxylation, ortho-hydroxylation and deamination
towardstheproductionofalkaloids.Inthelate1970s pathway (Fig. 1).6),9) From these early steps, only
andearly1980s,severalgroupsdevelopedcellculture the tyrosine/DOPA decarboxylase (an aromatic L-
systems for the production of large quantities of amino acid decarboxylase; TYDC), which converts
secondary metabolites.5),6) Although the levels of tyrosine and DOPA to their corresponding amines,
productivityencounteredwereoftennotsufficientfor and the aminotransferase have been purified and
the industrial production of the desired metabolites, characterized.10),11) The general lack of a detailed
because of limitations regarding the market size and understandingoftheotherearlystepenzymeshasled
difficulties associated with the use of these materials to the use of alternative techniques for the supply of
as crude medicines, these materials have provided thesubstrateviareconstitutedpathwaysinmicrobial
useful information pertaining to the study of the cells (this will be discussed in a later section).
biosynthetic pathways associated with their produc- Dopamine and 4-hydroxyphenylacetaldehyde
tion. Zenk et al. in particular studied a variety of are condensed by norcoclaurine synthase (NCS; EC
different alkaloid pathways, including those of the 4.2.1.78) to yield (S)-norcoclaurine, which is the
isoquinoline alkaloids (IQAs).5) central precursor for the construction of all of
IQAs are a large and diverse group of alkaloids the benzylisoquinoline alkaloids. NCS has been
that are derived from tyrosine with approximately purified and characterized from cultured Thalictrum
2,500 defined structures, which all contain a hetero- flavum spp., and a TfNCS cDNA belonging to the
cyclic isoquinoline backbone (Fig. 1).6) Some signifi- pathogenesis-related protein (PR)10 family was
cant examples from this group of natural products cloned.12),13) It is noteworthy that a novel dioxyge-
include the analgesic morphine from Papaver somni- nase (CjNCS) from cultured C. japonica cells was
ferum L., the anti-gout colchicine from Colchicum also shown to catalyze this NCS reaction.14)
autumnale L., the emetic and antiamoebic emetine (S)-Norcoclaurine is converted to coclaurine by
from Cephaelis ipecacuanha (Brot.) A. Rich., the S-adenosylmethionine(SAM)-dependentnorcoclaur-
skeletalmusclerelaxanttubocurarinefromStrychnos ine 6-O-methyltransferase (6OMT; EC 2.1.1.128),15)
toxifera Bentham, and the antimicrobial compounds which is in turn converted to N-methylcoclaurine
berberine and sanguinarine from divergent plant by SAM-dependent coclaurine N-methyltransferase
species including Berberis spp. and Sanguinaria (CNMT; EC 2.1.1.140),16) before being converted to
spp. It is noteworthy that many of these compounds 3B-hydroxy-N-methyl coclaurine by P450 hydroxy-
and their derivatives have been used as pharmaceut- lase (CYP80B1; EC 1.14.13.71),17) and then finally
ical agents. Sato et al. independently selected a converted to (S)-reticuline by SAM-dependent 3B-
cultured cell line with a high level of IQA productiv- hydroxy N-methylcoclaurine 4B-O-methyltransferase
No.5] Microbialproductionofplantisoquinolinealkaloids 167
TYDC
N HO HO MeO
Isoquinoline HODopamiNnHe2 Cj NCS HO H NH Cj 6OMT HO H NH
O
O TYDC HO H (S)-NHoOrcoclaurine HO (S )-Coclaurine
+ 4-Hydroxyphenyl- CjCNMT
O N protoberberine acetaldehyde
OMe
pathway MeO
MeO MeO
BerOberine COjM TeHBO aporphine HHOO +NMMee MHeOO H NMe HO H NM(Se )-N -Methyl
O H N pathway MeO MeO HO coclaurine
(S )-TetrahydroberbeOOrMMineee MagnCofoloryritnueberineMHHeOOO NMe (MrSeet )itc-h7uy-liOln e - Ec 7OMT MHeOO H NMCej CYP80B1
CjCYP719A1 HO
MHeOO N MHeOO H N MCejO CYP80G2 MHeOO CNj 4M’eOMT HO N(S - )m-3e’t-hHyyldcorocxlayu-rine
(S )-TetrahydrocolumbaminHe OMe Cj SMT OH Cj BBE HO H
CjC TjH CBoOOMT OMe Ec CYP719A5 (S )-SOMcoeulerine MeO (S )-Reticuline
MeO Cheilanthifoline
MeO N+ Cj THBO Ec CYP719A2/A3 CYP719B1 morphinan
OMe Stylopine SalR pathway
Palmatine SalAT
OMe MeO EcTNMT T6ODM
Cj CoOMT HO N+ OMe N -Methylstylboepinnzeophenanthridine pathway COCRODM
Columbamine
OMe
O O
Me
O O N OEc P6H N+ O
O Me
Protopine O O Sanguinarine Morphine
Fig. 1. Outline of isoquinoline alkaloid biosynthesis pathway. The cDNAs of the enzymes shown in red were first cloned and/or
characterizedinSato’slaboratory,andthoseinbluewereclonedasdescribedintheliterature,fromculturedCoptisjaponica(Cj)and
Californiapoppy(Eschscholziacalifornica:Es).GenesofthefollowingenzymeshavebeenalsoclonedfromPapaversomniferumby
other several groups;83) TYDC, CYP719B1, SalR (salutaridine reductase), SalAT (salutaridinol 7-O-acetyltransferase), T6ODM
(thebaine6-O-demethylase),CODM(codeinoneO-demethylase)andCOR(codeinonereductase).
(4BOMT; EC 2.1.1.116).15) Both of the enzymes were Detailed biochemical studies using recombinant
co-purifiedbySDS-gelelectrophoresis,becauseofthe enzymes have demonstrated the strict reaction
high level of similarity between the proteinaceous specificities of these enzymes, with the enzymes
properties of 6OMT to those of 4BOMT, to give a themselves regulating the biosyntheses sequentially
final preparation containing homologous 40- and 41- and in a coordinated manner. For example, CNMT
kD polypeptides. To effectively discriminate the two prefers coclaurine to 6-O-methylnorlaudanosoline,
enzymes,thescreeningofacDNAlibraryofcultured whereas 4BOMT prefers an N-methylated sub-
C. japonicacellswascarriedoutwitholigonucleotide strate,15),16)whichsuggestedthatthepathwayshown
probes designed on the basis of the internal amino inFig. 1waspreferredtoasequenceconsisting ofN-
acid sequences of the two polypeptides. The two methylation, hydroxylation and 4B-O-methylation.
kinds of cDNA obtained in this way were expressed Current data also indicate that all of the enzymes
in Escherichia coli, and characterization of the involved in the early steps of (S)-reticuline biosyn-
recombinant proteins enabled us to confirm that thesis from norcoclaurine, except for the membrane-
the 40-kDa polypeptide corresponded to the 6OMT bound P450 CYP80B1, are located in the cytosol.
whereas the 41-kD corresponded to the 4BOMT. (S)-Reticuline, which is produced by reaction with
Similarly, the cloning of the cDNA of CNMT was 4BOMT, is the central intermediate in the branch
carried out based on the amino acid sequence of the pathwaysleadingtomanydivergentIQAs,including
purified polypeptide.16) the aporphine alkaloids (e.g., magnoflorine), benzo-
168 F.SATOandH.KUMAGAI [Vol.89,
phenanthridine alkaloids (e.g., sanguinarine), mor- Differences in substrate specificity have also been
phinan alkaloids (e.g., morphine and thebaine) and reported for the CNMT of Coptis and Berberis, in
protoberberine alkaloids (e.g., berberine) (Fig. 1). that the Coptis enzyme could N-methylate norlau-
The next step in the biosynthesis of berberine danosoline, whereas the Berberis enzyme could
from (S)-reticuline involves the conversion of the N- not.16) These results suggest that the pathways
methyl group of (S)-reticuline to the methylene associated with the secondary metabolism may vary
bridge moiety linked to (S)-scoulerine by the berber- considerably depending on the enzyme(s) that each
ine bridge enzyme (reticuline oxidase, BBE; EC plant has acquired during its evolution.
1.21.3.3).18) This unique enzyme is soluble and The cDNA of a novel P450 enzyme involved in
localizedinthevesicles.19)(S)-Scoulerineisconverted thebiosynthesisofaporphine-typealkaloidsfrom(S)-
to (S)-tetrahydrocolumbamine (THC) by the SAM- reticuline was also cloned from cultured C. japonica
dependent scoulerine 9-O-methyltransferase (SMT; cells.29) A P450 corytuberine synthase (CYP80G2;
EC 2.1.1.117),20) and then to tetrahydroberberine EC1.14.21.-) was found tohavea significant level of
(THB; canadine) by a P450-dependent canadine amino acid sequence similarity to that of berbamu-
synthase (CYP719A1; EC 1.14.21.5).21) The charac- nine synthase (CYP80A1; EC 1.14.21.3) in the bio-
terization of these enzymes expressed from the synthesisofbisbenzylisoquinolinealkaloidsviaaC–O
corresponding cDNAs effectively confirmed that the phenol-coupling reaction.30) Recombinant CYP80G2
biosynthesis of berberine proceeds via canadine in produced from yeast showed intramolecular C–C
Coptiscellsandnotviacolumbamine.Onceagain,the phenol coupling activity to produce the aporphine
enzyme substrate specificity shows a clear preference alkaloid corytuberine from (S)-reticuline (Fig. 1). In
for this pathway. Although the hydrophobic N- contrast, the cDNA of a native N-methyltransferase
terminal region of SMT suggests that this enzyme capableofproducingmagnoflorinefromcorytuberine
may be targeted to the membrane fraction, its has not yet been cloned, although the potential
localizationinthecytosolaswellaswithinthelumen catalytic activity of CNMT towards corytuberine
of alkaloid-specific vesicles has been reported.20),22) could be used for the production of magnoflorine
(S)-Tetrahydroberberine oxidase (THBO or from (S)-reticuline in a microbial system.
STOX; EC 1.3.3.8) is the enzyme involved in the The benzophenanthridine alkaloid sanguinarine
finalstepoftheberberinebiosynthesis.23)–25)AcDNA isalsoproducedfrom(S)-reticulinevia(S)-scoulerine
ofTHBOwasclonedbasedonthepartialaminoacid (Fig. 1). This conversion begins with the action of
sequence of the purified enzyme and its deduced the P450-dependent oxidases, (S)-chelanthifoline
aminoacidsequencesuggestedthatCjTHBObelongs synthase (CYP719A5; EC 1.14.21.2) and (S)-stylo-
to the FAD-containing BBE oxidoreductase family. pinesynthase(CYP719A2/A3; EC1.14.21.1),which
As a consequence of the difficulties encountered catalyze the formation of a methylenedioxy-bridge
during the attempted production of active recombi- to produce (S)-stylopine from (S)-scoulerine. Their
nant CjTHBO in E. coli and Saccharomyces cerevi- cDNAs werealso cloned from culturedE. californica
siae cells, recombinant CjTHBO was heterologously cells.31),32)(S)-Stylopinewasthenconvertedtocis-N-
expressed in E. californica cells and its metabolic methylstylopine by (S)-tetrahydroprotoberberine N-
activities subsequently characterized.26) methyltransferase (TNMT; EC 2.1.1.122),33) before
As well as the biosynthesis of berberine, the being converted to protopine by N-methylstylopine
cDNAs of several other biosynthetic enzymes in- hydroxylase (MSH; EC 1.14.13.37), and then to
volved in the biosynthesis of related protoberberine dihydrosanguinarine by protopine 6-hydroxylase
alkaloids have been also cloned. For example, (P6H; EC 1.14.13.55).34) Although the cDNA of
SAM-dependent columbamine O-methyltransferase MSH was not yet been cloned, the cDNA of P6H
(CoOMT; EC 2.1.1.118), which is involved in the (CYP82N2v2) has been cloned from E. californica
biosynthesis of palmatine, was cloned from cultured cells, based on an integrated analysis of metabolite
C. japonica cells using expressed sequence tag accumulation and the mRNA expression profile of
information. Recombinant CjCoOMT clearly indi- transgenic cells.35) The identification of CYP82N2v2
catedthatTHCcouldbeausedasasubstrateforthe as a protopine 6-hydroxylase for the production of
formation of tetrahydropalmatine (Fig. 1),27) where- dihydrosanguinarine from protopine, as well as its
as columbamine was thought to be the exclusive broad range of reaction specificity in the conversion
substrate of CoOMT in Berberis plants based on of corycavine to corynoloxine, indicate that
the substrate specificity of Berberis CoOMT.28) CYP82N2v2 plays an important role in the biosyn-
No.5] Microbialproductionofplantisoquinolinealkaloids 169
thesis of a diverse range of benzophenanthridine
alkaloids.36)
MeO O
In total, Sato et al. successfully cloned the HO N Cj CYP719A1 O N
cDNAs of 12 enzymes belonging to the IQA H H
OMe OMe
biosynthetic pathways to provide a better under-
standing of the molecular basis of these particular OMe OMe
pathways. Furthermore, seven of these cDNAs were (S )-Tetrahydrocolumbamine (S )-Tet(r(aSh)y-dCraonbaedrbineer)ine

subsequently used in the later work to construct a
microbial platform for the production of plant IQAs. MeO MeO
Unique properties of the biosynthetic en- N N
zymes of the IQA pathways. The cloning of the HHOO H Me Cj CYP80G2 HHOO Me
cDNAs of the biosynthetic enzymes from the IQA
biosynthetic pathways enabled the detailed charac- MeO MeO
terization of their enzymatic properties. Of the (S )-Reticuline (S )-Corytuberine
enzymes characterized from the IQA biosynthetic
pathway, CYP719A1 and CYP80G2 provided the MeO MeO
A
first molecular basis for methylenedioxy ring for- N N
HO Me HO Me
mation and C-C phenol coupling reactions in MeO 3’ H Cj CYP80G2 MeO H
eukaryotic cells.21),29) Interestingly, the CYP719 C
family of enzymes was not found in Arabidopsis MeO 4’ HO
(R,S)-Codamine (R,S)-Orientaline
andrice,withthesemembersofthecytochromeP450
super-family being unique to benzylisoquinoline Fig. 2. SomeuniquereactionscatalyzedbyP450enzymesfound
alkaloid biosynthesis.37) intheIQApathway.CYP719A1fromC.japonicacatalyzesthe
oxidative cyclization of an ortho-hydroxymethoxy-substituted
Although the oxidative cyclization of an ortho-
aromatic ring (i.e. methylenedioxy bridge formation) and
hydroxymethoxy-substituted aromatic ring (i.e.
converts only tetrahydrocolumbamineto (S)-tetrahydroberber-
methylenedioxy bridge formation) is a P450-depend- ine. CjCYP80G2 not only catalyzes the intramolecular C-C
ent reaction, this reaction is not a simple mono- phenol coupling reaction and converts (S)-reticuline to (S)-
oxygenation reaction as demonstrated by the lack corytuberine, but also 4-O-demethylates codamine to form
the demethylated product. The reaction mechanism has been
of oxygen insertion into the products. The P450-
discussedinthetext.
dependent hydroxylation of the methoxy group
yields an intermediate corresponding to the hemi-
acetal of formaldehyde that can cyclize to form a initially with the C-ring and not the A-ring during
methylenedioxy bridge via an ionic mechanism the 4-O-demethylation of codamine. The product
involvingthemethyleneoxoniumionintermediate.38) obtained from the reaction of codamine with
All of the methylenedioxy bridge-forming reactions CjCYP80G2 suggested that hydrogen abstraction
involved in IQA biosynthesis have been found to be occurred as the first step from the 4-methoxy group
catalyzed by members of the CYP719A subfamily, oftheC-ring,followedbyhydroxyradicalreboundto
which exhibit rather strict levels of substrate spec- the same position of the substrate, with the eventual
ificity. CYP719A1 from C. japonica has been iden- release of formaldehyde to form the demethylated
tified as a canadine synthase and can only convert product (Fig. 2). Similarly, when CYP80G2 was
(S)-THC to (S)-THB (Fig. 2).21) E. californica reactedwith(S)-reticuline,theinitialhydrogenatom
CYP719A5 can convert (S)-scoulerine to (S)-chei- abstraction step occurred at the 3-hydroxy group of
lanthifoline, whereas CYP719A2 and CYP719A3 theC-ringtogenerateaphenoxyradical,followedby
have been identified as stylopine synthases respon- subsequent oxidation at the 7-hydroxy group of the
sibleforcatalyzingtheconversionof(S)-cheilanthifo- A-ring to generate a second radical. The terminal
line to (S)-stylopine. CYP719A9 from E. californica reaction was then achieved by the coupling of the
can convert (R,S)-reticuline to the corresponding diradical species (i.e. bond formation between the
products containing a methylenedioxy ring.31),32) C8 and C2 positions of (S)-reticuline). The reaction
DetailedcharacterizationofCjCYP80G2,which mechanism of CjCYP80G2 is therefore best ex-
catalyzes the intramolecular C-C phenol coupling plained by the diradical process.
reaction for the conversion of (S)-reticuline to (S)- Metabolic engineering. The isolation of the
corytuberine,29) indicated that CjCYP80G2 reacts genes of biosynthetic enzymes has enabled the
170 F.SATOandH.KUMAGAI [Vol.89,
Cj6OMT MeO

HO N
H
OH
OMe
(S)-Norcoclaurine (S)-Coclaurine (S)-Scoulerine Sanguinarine

CjSMT

EcP6H etc EcCYP719A3/

EcTNMT/EcMSH
10-Hydroxychelerythrine Allocryptopine (S )-Tetrahydrocolumbamine Columbamine
EcBBE RNAi

(S)-Norcoclaurine (S)-Reticuline (S)-Scoulerine

Fig. 3. OutlineofmetabolicengineeringintransgenicE.californicacells.(A)Overexpressionoftherate-limitingenzyme,norcoclaurine
6-O-methyltransferase(6OMT),usingC.japonicacDNAinCaliforniapoppycellsledtoanincreaseintheaccumulationofthemain
alkaloid of the cells, sanguinarine, as shown by the upward arrow. (B) Introduction of a branch pathway with scoulerine 9-O-
methyltransferase(SMT)alsoinducedamarkedshiftinthemetabolicflow,asshownbythearrows.SMToverexpressionledtoa
significantreductionintheaccumulationofsanguinarine-typealkaloidswithtwomethylenedioxyringstructures,butinducedthe
production of protoberberine-type alkaloids, such as berberine and columbamine, and of allocryptopine- and chelerythrine-type
alkaloidswithonemethylenedioxyringandtwomethoxygroups.(C)Theshutdownoftheberberinebridgeenzyme(BBE)expression
with an RNAi vector clearly downregulated the accumulation of sanguinarine and the intermediate scoulerine, as shown by the
downwardarrow.Incontrast,RNAiofBBEinducedamarkedaccumulationofreticuline,whichisasubstrateofBBE,asshownby
theupwardarrow.
molecular design of the metabolic flow of plant cells and 4BOMT using a strong constitutive promoter,
(Fig. 3). Using the Agrobacterium-mediated trans- Cauliflower mosaic virus 35S, led to an increase in
formation technique, Sato et al.8) reported the berberine production in C. japonica cells and plants,
molecular engineering of IQA biosynthesis in C. respectively.39),40)Similarly,heterologousoverexpres-
japonica and E. californica cells with C. japonica sion of C. japonica 6OMT improved the level of
genes, and demonstrated the high level of flexibility alkaloid productivity in E. californica cells.41) It is
of the IQA biosynthetic pathway towards its genetic important to note, however, that the overexpression
alteration. of a biosynthetic enzyme does not always result in a
Attempts have been made to overexpress the successful increase in the levels of alkaloid produc-
genesresponsibleforproducingtheenzymesinvolved tion.Forexample,theoverexpressionofCj4BOMTin
in the rate-limiting steps of these processes with the E. californicacellsonlyprovidedamarginalincrease
aim of improving the yields of the associated IQAs. in alkaloid production.41) The bottleneck step(s)
Forexample,theoverexpression ofC. japonicaSMT associated with the IQA biosynthetic pathways can
No.5] Microbialproductionofplantisoquinolinealkaloids 171
vary across different plant species and isolated cell duction of a new pathway allows for the production
lines because of variations in the expression levels of of novel products from transgenic cells. Of the
the genes required to produce the enzymes. Further- methods used to reduce gene expression (e.g.,
more, changes in the bottleneck step(s) may also antisense, RNA interference (RNAi), knockout with
occur as a consequence of modifications to the T-DNAinsertionandmutation),RNAsilencingwith
enzyme expression level resulting from metabolic double-stranded RNA (i.e. RNAi with siRNA or
engineering. miRNA) is the most powerful and reliable of these
Given that there may be multiple bottleneck techniques.45)The versatilityof RNAiforcontrolling
steps involved in a particular biosynthetic process, the multiple genes responsible for metabolite pro-
the overall regulation of the expression levels of duction across a variety of different tissues and
biosynthetic genes in the pathway would provide a developmental steps has been well recognized. For
more effective means of control, and further studies example, in 2007, Fujii et al.46) reported the success-
are currently underway to isolate general tran- ful accumulation of reticuline, which is a direct
scription factors. The recent identification of the substrate of BBE, by the use of an RNAi of BBE in
transcription factors CjWRKY1 and CjbHLH1 as E. californica cells (Fig. 3), whereas little accumu-
possible general regulators in IQA biosynthesis using lation of reticuline was reported by Park et al.47)
a transient RNAi screening system could represent a using the antisense method.
significant discovery in the field and provide signifi-
Characterization of aromatic amino acid
cantimprovements intheyields of IQAs byenabling
metabolizing enzymes in microorganisms
the regulation of the transcription processes associ-
ated with their production.42)–44) Kumagai et al. have been working on microbial
As well as providing improvements in the yield, enzymes capable of metabolizing aromatic amino
improvementsinthequalityoftheIQAsareperhaps acids and aromatic amines, such as L-tyrosine, L-
more desirable, in that although increases in meta- DOPAandtheircorrespondingamines.Theenzymes
bolicdiversitywouldbeusefulfordrugdiscovery,the used in their work include amino acid-lyases,48)–57)
reductionof the metabolic diversity iscritical for the aromaticaminoaciddecarboxylases,58)–64)andamine
facile preparation of pure compounds. The introduc- oxidases.65)–72) When they started their work,
tion of a novel branch pathway to a biosynthetic enzymes were not routinely purified or particularly
pathway represents one of the most useful ap- well characterized. Consequently, the bacterial
proaches for increasing the level of metabolic strains which provided the highest levels of enzyme
diversity resulting from the pathway. We have activity from a number of microbial strains were
ectopically expressed CjSMT in E. californica cells screened first. The culture conditions were then
(Fig. 3).39) CjSMT catalyzes the O-methylation of investigated extensively, with particular emphasis
scoulerine to produce THC in the berberine biosyn- on the induction conditions of the enzyme biosyn-
thesisandisnotinvolvedinthebenzophenanthridine thesistoobtainthehighestenzymeactivity,withthe
alkaloidbiosynthesis.ThesetransgenicE. californica investigation ultimately contributing to the isolation
cells with CjSMT expression not only produced of a pure enzyme fraction as crystals. The character-
columbamine (i.e. the oxidized product of THC),39) ization of these aromatic amino acid metabolizing
butalso providednovelproducts,including allocryp- enzymes and the elucidation of the control mech-
topine and 10-hydroxychelerythrine, which were anisms associated with the enzyme biosynthetic
produced from the CjSMT reaction products follow- pathways in the microorganisms were indispensable
ing their reaction with other endogenous enzymes fortheconstructionofthemicrobialIQAproduction
in the benzophenanthridine alkaloid biosynthetic platform. This paragraph describes the synthesis of
pathway, effectively demonstrating the enhanced L-DOPA by tyrosine phenol-lyase from Erwinia
diversification of the alkaloid profile in transgenic herbicola, aromatic amino acid decarboxylase from
cells.35) Pseudomonas putida, and monoamine oxidase (tyr-
Modifications to the metabolic flow leading to a amine oxidase) from Micrococcus luteus.
reduction in the levels of enzymes associated with L-DOPA synthesis by tyrosine phenol-lyase
undesired pathways represents another promising and tyrosinase. L-DOPA, which is otherwise
approachtoincreasetheproductlevelsofthedesired known as 3,4-dihydroxy-L-phenylalanine, and dop-
metabolites, while reducing unwanted byproducts. amine are the starting materials required of IQA
The combination of this approach with the intro- production in microorganisms. Although tyrosine
172 F.SATOandH.KUMAGAI [Vol.89,
COOH
NH2 + H2O + COOH+ NH3 (a)
HO Tyrosine HO O
L-Tyrosine phenol-lyase Phenol Pyruvic acid
HO COOH HO
NH2 + H2O + COOH+ NH3 (b)
HO Tyrosine HO O
L-DOPA phenol-lyase pyrocatechol Pyruvic acid
COOH HO COOH
+ (c)
NH2 1/2O2 NH2
HO Tyrosinase HO
L-Tyrosine L-DOPA
Fig. 4. Reactioncatalyzedbytyrosinephenol-lyaseandtyrosinase.
phenol-lyase (TPL; EC 4.1.99.2) is not directly used fied a second-site suppressor mutations (N324D or
in the microbial IQA production platform, work A503T) that rescued the inability of TyrRE275Q to
focused on the enzymatic properties of TPL and the activate the tpl promoter. N324 is located in the
control mechanisms associated with the biosynthesis centraldomainofTyrRandisthoughttobeinvolved
of TPL has contributed greatly to the industrial intheformationofaTyRoligomer,whichisrequired
production of L-DOPA. for tpl-activation. Amino acid replacement of the
L-DOPA,isusedinthetreatmentofParkinson’s residue (N324D) compensated for the impaired
diseases. Approximately 250 tons of L-DOPA is ability of TyrRE275Q to form an oligomer.56) The
producedaroundtheworldeveryyear.Nearlyhalfof amino acid substitutions that lead to the enhanced
this material is synthesized according to an enzy- TPL expression were scattered into three domains,
matic method involving TPL.48)–50) TPL normally including: (1) V67A, Y72C and E201G in the N-
catalyzes the ,,O-elimination reaction of L-tyrosine terminal domain (NT); (2) N324D in the central
toproducespyruvate,ammoniaandphenol(Fig. 4a). domain (Cen); and (3) A503T in the C-terminal
This reaction is reversible, and when catechol is domain (CT). These mutations were genetically
substituted for phenol, the process produces L- combined by the domains, and the resulting tyrR
DOPA (Fig. 4b).51) TPL is inducible by tyrosine alleleswereinsertedintoalow-copy-numberplasmid.
and, from a practically perspective, E. herbicola These plasmids were then introduced to tyrR-
cells with a high level of TPL activity are prepared deficient E. herbicola ("tyrR::kanD) cells, and the
by cultivation in a medium supplemented with L- TPL activities of the cell-free extracts of the
tyrosine before being harvested and transferred to recombinant strains were measured. The strain
the reactor together with the substrates, pyruvate, carrying TyrRNTCenCT exhibited its highest level of
ammonia and catechol. Bai et al.73) and Kumagai L-DOPA productivity when cultivated under the
et al.52),53) elucidated the regulatory mechanism non-induced conditions, and its level of productivity
underlying the L-tyrosine-mediated induction of was30-foldgreaterthanthatofthetyrosine-induced
TPL, and identified TyrR as the transcriptional wild-type cells that are currently utilized in the
activator of tpl. The tyrR gene was subsequently industrial production of L-DOPA (11.1g/L/h vs.
cloned from E. herbicola cells and randomly muta- 0.375g/L/h).57)
genized to obtain a mutant protein with an TPLrequireshighconcentrationsofitpyruvate,
enhanced ability to activate tpl. One such mutant, ammonia and catechol substrates to carry out an L-
TyrRV67AY72CE201G, was found toeffectively increase DOPAproducingreaction,andtheconcentrationsof
TPL activity expression.54) Furthermore, when the thesesubstratescanbesohighthattheycanbecome
mutant gene was introduced to E. herbicola, the toxic to bacterial cells. The construction of an IQA
resulting strain showed improved levels of L-DOPA producing bacterial platform therefore requires the
production.55) Kumagai et al.56) subsequently identi- use of Ralstonia solanacearum tyrosinase (TYR; EC
No.5] Microbialproductionofplantisoquinolinealkaloids 173
COOH NH2
+
NH2 CO2 (a)
HO Tyrosine HO
L-Tyrosine Tyramine
decarboxylase
HO COOH HO NH2
+
NH2 CO2 (b)
HO DOPA HO
L-DOPA Dopamine
decarboxylase
Fig. 5. Reactionscatalyzedbyaromaticaminoaciddecarboxylase.
1.14.18.1, monophenol, L-DOPA: oxygen oxidore- treus AJ1065 was selected as a promising source of
ductase) to produce L-DOPA from L-tyrosine. TYR aromatic L-amino acid decarboxylase. M. percitreus
is a copper containing monophenol monooxygenase was found to constitutively produce an enzyme that
that catalyzes the oxidation of the 3B-position of exhibited decarboxylase activity towards L-trypto-
the L-tyrosine phenol-ring (Fig. 4c). This particular phan, and cultivation conditions to produce high
reaction mechanism was developed on the basis of levels of decarboxylase activity were subsequently
anX-raycrystallographicinvestigationconductedby examined.58)
Matoba et al.74) R. solanacearum TYR has low o- TheAADCfromM. percitreuswaspurifiedand
diphenolase activity,75) and its introduction to the successfully crystallized for the first time, allowing
IQA producing bacterial platform was considered to for its complete characterization.59),60) The enzyme
increasetheamountofaccumulationofL-tyrosinein that found to require pyridoxal phosphate (PLP) as
the bacterial cells,76) as will be described in a later a cofactor and to be deactivated by the addition of
section. L-DOPA to the reaction mixture as a substrate.61)
Aromatic amino acid decarboxylases from The mechanism of the inactivation was investigated
bacteria. Aromatic amino acid decarboxylases by enzyme- and organic-chemical methods, which
(AADCs; EC 4.1.1.28) catalyze the decarboxylation revealed that L-DOPA reacted slowly with the PLP
reactions of aromatic amino acids (Fig. 5) and are cofactor resulting in the formation of the so-called
found in a variety of different organisms, where they tetrahydroisoquinoline adduct compound during the
play distinct physiological roles.AADCs fromhigher reaction.62) Inhibition of the enzyme was also found
eukaryotes have been well studied because they are to occur when ,-methyl aromatic amino acids were
involved in the synthesis of biologically important used as substrates, and analysis of this inhibition
moleculessuchasneurotransmittersandalkaloids.In mechanism resulted in the proposal of a reaction
contrast, bacterial AADCs have received less atten- mechanism for the AADC from M. percitreus.63)
tion because of the simplicities associated with their Aeukaryotic-typeAADCexhibitingahighlevel
physiology and target substrate (tyrosine) (Fig. 5a). ofspecificityforL-DOPA(Fig. 5b)hasrecentlybeen
AlthoughtheexistenceofstronglyactiveAADCshas found and its genetic and enzymatic characteristics
been reported in some lactic acid bacteria,77) these determined.64) The purified enzyme converted L-
AADCs have never been purified and characterized DOPA to dopamine with Km and Kcat values of
as pure preparations because of their instability. 0.092mM and 1.8s!1, respectively. The enzyme was
The formation of aromatic L-amino acid decar- inactive towards other aromatic amino acids such as
boxylaseinbacteriawasstudiedwithintactcellsina 5-hydroxy-L-tryptophan(HTP),L-phenylalanine,L-
reactionmixturecontainingthearomaticaminoacid. tryptophan and L-tyrosine (Table 1). The observed
The activity was found to be widely distributed strict substrate specificity was distinct from that of
in such genera as Achromobacter, Micrococcus, any other AADC characterized to date. The name
Staphylococcus and Sarcina. Strains belonging to DOPA decarboxylase (DDC) was subsequently pro-
Micrococcus showed especially high levels of decar- posed for the enzyme. Quantitative RT-PCR experi-
boxylase activity towards L-tryptophan, 5-hydroxy- ments revealed that the expression of this gene was
L-tryptophanandphenylalanine.Micrococcusperci- induced by L-DOPA. DDC was found to be encoded
174 F.SATOandH.KUMAGAI [Vol.89,
Table 1. SubstratespecificityofP.putidaDDC
Substrate K (mM) k (s!1) k /K (mM!1s!1)
m cat cat m
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
Thek valueswerecalculatedbasedonthereportedV values.Theestimatedmolecularmasswas54kDa.
cat max
in a cluster together with a LYSR-type (LysineR- The aldehyde formed during the reaction with
type)transcriptionalregulatorandamajorfacilitator dopamine was found to condense with unchanged
superfamily transporter. This genetic organization dopamine to yield the IQA compound norlaudanoso-
wasfoundtobeconservedamongallofthesequenced line (i.e. tetrahydropapaveroline) (Fig. 6b).70) This
P. putida strains that inhabit the rhizosphere envi- finding suggested the idea of the microbial produc-
ronment, where DOPA acts as a strong allelochem- tion of isoquinoline compounds, with MAO and its
ical. These findings suggest the possible involvement gene playing important roles in the construction of
ofthisenzymeinthedetoxificationoftheallelochem- microbial platform for the production of IQAs.
ical in the rhizosphere, and potential occurrence of a Kumagai et al.71) have completed the gene
horizontal gene transfer event between the pseudo- cloning and three-dimensional structure prediction
monad and its host organism.64) experiments for M. luteus MAO. The amine oxidase
DDC from P. putida is particularly interesting of the fungi Aspergillus niger has also been reported
because its physiological role is related to that of the to be inactivated during the reaction with an analog
host plant. In addition, its strict specificity for L- ofthesubstrate(bromo-ethylamine),anditsreaction
DOPA could be useful for the construction of a mechanism was elucidated by analysis of the
microbialplatformforplantIQAproductionbecause materials resulting from its suicide inactivation.72)
it actually plays an important role in this context,
Construction of the microbial platform
and this will be discussed in more detail in a later
for plant IQA production
section.76)
Aromatic amine oxidases from bacteria. First-generation microbial platforms for
Amine oxidases catalyze the oxidative deamination plant IQA production. Following an increase in
of amines to produce the corresponding aldehydes, the availability of molecular biological tools, the
together with ammonia and hydrogen peroxide reconstitutionofanentiremetabolicpathwaybecame
(Fig. 6a).TheGram-positivebacteriumSarcinalutea feasible in microorganisms.78)–80) Microorganisms
(M. luteus, later) contains an amine oxidase specific haveasimplerinternalstructurethanplantsbecause
for tyramine when it grows on nutrient medium. It of their smaller genome size, and this effectively
was purified as a crystalline preparation and charac- simplifies metabolite transport between the enzy-
terized.Thisenzymeisamonoamineoxidase(MAO; matic steps.81) The reconstitution of a secondary
EC 1.4.3.4) and uses FAD as a cofactor.65)–67) When metabolite pathway in an engineered microbial host
M. luteuswasgrownonsyntheticmediumcontaining offersseveraladvantagesoverothermethodsdirected
tyramine as a sole nitrogen source, its level of MAO towards native plant hosts including the specific
increased extensively to give 20- to 30-fold more production of key intermediate molecules, such as
crystalline MAO than that obtained under the reticuline, the rapid accumulation and facile purifi-
formercultivationconditionsinanutrientmedium.68) cation of the target IQA, and the availability of
The mechanism of the reaction catalyzed by the genetic tools for further engineering and pathway
MAO was studied based on the overall reaction optimization of the IQA production.82) A combina-
kinetics as well as by the stopped flow.69) tion of microbial and plant-derived genes offers
The enzyme was found to oxidize tyramine and some advantages for the establishment of efficient
dopamine at a similar rate, but other monoamines, and productive systems for the entry compound.79)
diamines and polyamines were not oxidized at all. Unfortunately, however, microbial systems also