Microbial Factories for the Production of Benzylisoquinoline Alkaloids

14 Pages·2016·2.21 MB·English

by  Lauren Narcross

Special Issue: Industrial Biotechnology

Review
Microbial Factories for the

Production of

Benzylisoquinoline Alkaloids

Lauren Narcross,1,2 Elena Fossati,1,2 Leanne Bourgeois,1,2

John E. Dueber,3 and Vincent J.J. Martin1,2,*

Benzylisoquinoline alkaloids (BIAs) are a family of (cid:1)2500 alkaloids with both
Trends
potentialandrealizedpharmaceuticalvalue,includingmostnotablytheopiates
Both Escherichia coli and Saccharo-
such as codeine and morphine. Only a few BIAs accumulate readily in plants,
myces cerevisiae have been engi-

whichlimitsthepharmaceuticalpotentialofthefamily.ShiftingBIAproduction neered to convert a simple carbon
to mic robial so urces could pro vide a sc al able and fl exible s ourc e of these source su ch as gl uco se to c omplex
BIAs.
compounds in the future. This review details the current status of microbial

BIA synthesis and derivatization, including rapid developments in the past 6 ThevarietyofBIAscaffoldssynthesized
inm icrobial ho sts continues toincrease,
monthsculminatinginthesynthesisofopioidsfromglucoseinamicrobialhost.
nowencompassingbenyliosquinolines,

aporphines, protoberberines, proto-

Microbial Synthesis of Pharmaceuticals To Enhance Drug Discovery pines,benzophenanthridines,pro-mor-
Plant secon dary metab olite s are a valuable sou rce of natural produc ts with pharmaceutical phinan s, and morphinans.

properties[1,2].Untilrecentlypharmaceuticalcompanieshadphasedoutscreeningofnatural
productsa spote ntial druglea dsinfavorofche micallysynt hesiz edlibra ries, citing‘poo ry ieldsof bKeeeyn cidheanlletinfiegdes, infoclru dfuintugrep awthowrka yhbaovte-

chemicalsynthesis’and‘impracticalityofscale-up’[3,4].Whilethesechallengesremain,therehas tlenecks and the generation of side-
products from pro miscuouse nz ymes.
been a resurgence of natural products in the drug discovery process owing to their potent

biologicalactivityanduntappedpotential,andrecentimprovementsinscreeningtechnology[4,5].

Biosynthesis of plant metabolites in microbes provides an opportunity to advance the drug

discovery process. Microbial production circumvents the need for the cultivation of source

plants[6].Notallproductsaccumulatetohighlevelsinplants,andhencemicrobesprovidea

waytoscaleupproductionoftheseinterestingcompoundstorelievesupplylimitations[7].The

successful industrial production of the antimalarial artemisinic acid in yeast highlights the

capability of microbes to act as natural product factories [8]. Finally, microbial production

facilitates the possibility to design new compounds with novel activity or improve the clinical

profileof existingdrugs through combinatorial chemistry [9].

1DepartmentofBiology,Concordia

Pharmaceutical Properties of Benzylisoquinoline Alkaloids University,Montréal,QuébecH4B
Benzylisoquinoline alkaloids (BIA s) a re a class of molecules that would benefit from microbial 21CRe6n, tCreanfoar dSatructura landFu nctional
synthesis.Withover2500familymembers,BIAsexhibitdiversepharmaceuticalproperties,with Genomi cs, Concordia Univ ersity,

ahistoryofhumanusedatingbackthousandsofyears[10].Inadditiontotheirprominentrolein Montréal,QuébecH4B1R6,Canada
3Departm entofBi oeng ineeri ng,
traditional medicine, BIAs have a wide variety of pharmacological applications, acting as
UniversityofCalifornia,Berkeley,
analgesics, antitussives, antimicrobials, and antispasmodics, and several members are on Berkeley, CA 94704,US A

theWorldHealthOrganizationlistofessentialmedicines(Table1).Recently,preliminarystudies

haveuncoverednewpotentialintreatingcancer,malaria,HIV,andpsychosis(Table1).Despite
*Correspondence:
theirclinicalapplications,itisarguedthatBIAs(amongotheralkaloids)arenotproportionately
[email protected]
represented in modern medicine and drug development, in large part owing to difficulties in (V.J.J.Martin).

228 TrendsinBiotechnology,March2016,Vol.34,No.3 http://dx.doi.org/10.1016/j.tibtech.2015.12.005

©2015ElsevierLtd.Allrightsreserved.
Table1.DiversityofBIAsinDrugDevelopment

BIASubfamily Compound Pharmaceutical ClinicalStatus Refs

Applications
Benzylisoquinoline Norcoclaurine CardiacStimulant Clinicaltrials [68–70]

(Phase1)

Drotaverine Antispasmodic Approved [68,71]

Papaverine Vasodilator Approved [68,71,72]

Smoothmuscle Approved

relaxant
Bisbenzylisoquinoline Atracuriuma Neuromuscular Approved [71,73]

blocker
Mivacuriuma Neuromuscular Approved [81,74]

blocker
Musclerelaxant Approved

Phthalidisoquinoline Noscapine Antitussive Approved [72,75];Drugs.com

(www.drugs.com/international/
Potential Clinicaltrials
noscapine.html)
Anticancer (Phase2)

Antimalarial N/A

Aporphine Glaucine Antitussive Approved [68,76];Drugs.com

(www.drugs.com/international/
Anticancer N/A
glaucine.html)
Protoberberine Berberine HIVtreatment N/A [68,71,72,77–79]

Antibacterial N/A

Antiparasitic Experimental

Antifungal Experimental

Antidiarrheal Experimental

TypeIIdiabetes Clinicaltrials

(Phase3)

Stepholidine Psychosis N/A [80]

Benzophenanthridine Sanguinarine Antimicrobial Notapproved [68,81]

Morphinan Codeine Analgesic Approved [68,71,72]

Antitussive Approved

Morphine Analgesic Approved [68,71,72]

Morphinan Oxycodonea Analgesic Approved [50,68,71]

semisynthetic
Naloxonea Opioidantagonist Approved
derivatives
Naltrexonea Opioidantagonist Approved

aSyntheticorsemi-syntheticBIAs.

supply [11]. To overcome this barrier, several groups have been working to synthesize and

derivatize BIAs inthe microbialhosts Escherichiacoli andSaccharomyces cerevisiae.

BIA Diversity Is Derived from a Single Scaffold

BIA synthesis in plants begins with the condensation of dopamine and 4-hydroxyphenylace-

taldehyde(4-HPAA)toform(S)-norcoclaurine(Figure1A).(S)-norcoclaurineisthescaffoldfrom

whichover2500otherBIAscanbeproduced(examplesofthestructuraldiversityofBIAsare

providedinFigure1B)[12].Thismeansthatamicrobialplatformstraincapableofproducingthe

BIA scaffold from simple carbon sources could then be engineered to produce any BIA of

interest, providedthe necessary enzymes havebeen elucidated.

TrendsinBiotechnology,March2016,Vol.34,No.3 229
(A) OOHAAT COOH (B) PPaavviinnee BeHnOzylisoquinoline BBiissbbeennzzyylliissooqquuiinnoolliinnee
HO4-HPP O HOL-Tyrosine NH HO H NH HO
TYR AADC Cularine
HO COOH HO HO HO
AADC O
HOL-DOPA HOTyramine H
N
AADC TYR HO
HO HO O HO H
MAO
HO4-HPAA HODopamine H3O,4d-HPAA Pro-morphinan HO
Aporphine
NCS HO NCS HO
HO HO
HHOO H NH H HO H HO
O
HO
(S)-Norcoclaurine
(S)-Norlaudanosoline Protoberberine Phthalideisoquinoline
O
6OMT 6OMT
HO N O
CNMT CNMT H H OH O
NMCH HO O
4′OMT 4′OMT
Benzophenanthridine Protopine Rhoeadine
O O O
HO
HO H O O N O
O N O O O
(S)-Re(cid:2)culine O O O O
Figure1.MicrobialSynthesisofBIAsandBIAdiversity.(A)4-HPPisthemicrobialprecursortothesubstratesof

norcoclaurine/norlaudanosoline condensation. Dopamine and 4-HPAA condense to form norcoclaurine (black), while

dopamine and 3,4-dHPAA condense to form norlaudanosoline (red). The extra hydroxyl group on 3,4-dHPAA and

norlaudanosolineisindicatedinred,andreactionsthatacceptnorlaudanosolineareindicatedwithredarrows.Metabolite

abbreviations:4-HPP,4-hydroxyphenylpyruvate;4-HPAA,4-hydroxyphenylacetaldehyde;3,4-dHPAA,3,4-dihydroxyphe-

nylacetaldehyde.Enzymeabbreviations:AADC,aminoaciddecarboxylase;AAT,aminoacidtransferase;CNMT,coclaurine
N-methyltransfera se;NCS ,norcoclaurin esynth ase;N MCH ,N-methylcocla urine 30-O-h ydro xylase;40OM T,30-h ydroxy-N-
methylcoclaurine40-O -met hyltransferase; 6OMT,n orcoclau rine6-O-methyltran sferase;TYR,any enzyme whichhydro-

xylatestyrosine.(B)DiversityofBIAscaffolds.FeaturedBIAsareexamplesofindicatedscaffoldtypes.Thenumberofarrows

isnotindicativeofpathwaylength.Dashedlinesindicateunknownpathways;bluelinesindicateoneormorecytochrome

P450-catalyzedreactions.

Ten enzyme families add functional groups and catalyze the rearrangement of BIA scaffolds

[10,13].ManyoftheseenzymesarecytochromeP450sthatrequireendomembranesforoptimal

activity (Figure 1B, blue arrows). For this reason, S. cerevisiae has been used extensively for

heterologousBIApathwaydevelopment,althoughamplesuccesseshavebeenachievedinE.

coli.EnzymesinBIAsynthesistendtohavebroadsubstrateranges,allowingthesamereactions

to be performed even as BIAs diverge in structure (see [14–18] for examples). Although BIA

synthesispathwaysarecommonlydrawninstraightlines,thesubstrateacceptanceprofilesof

enzymesinasinglepathwaycanoverlap,meaningthatthesepathwaysareoftenmoreofaweb

resultinginamultitudeofproducts(anexampleisthemorphinepathway,showninFigure2).

Metabolicengineeringstrategieswillbenecessarytoovercomepromiscuityforthereconstitu-

tionof BIA synthesispathways in heterologoushosts (Box1).

Current Status of Microbial Aromatic Amino Acid (AA) Production

The (S)-norcoclaurine precursors 4-HPAA and dopamine are derived from the aromatic AA

pathway,andhencearomaticAAoverproductionisakeygoalforthedevelopmentofmicrobial

sourcesofBIAs.EntryofcarbonintothearomaticAApathwaybeginswiththecondensationof

the glycolysis intermediate phosphoenolpyruvate and the pentose phosphate pathway inter-

mediate erythrose-4-phosphate. This committed step is transcriptionally and allosterically

regulated, which is a common theme throughout the aromatic AA pathway, especially for

enzymes at key metabolic branch-points [19]. Strategies for improving flux towards tyrosine

in E. coli and S. cerevisiae are similar, but the highest published yields of aromatic AAs or

230 TrendsinBiotechnology,March2016,Vol.34,No.3
HO REP HO SAS HO
HO H HO H
CPR H
(S)-Re(cid:2)culine (R)-Re(cid:2)culine SalutaOridine
SAR
Sp
pH 6–7 HO
HO HO
H SAT
H
O H
HOH
O
Dibenz[d,f]azonine Sal-7-O-acetate Salutaridinol

Sp
pH 8–9
HO CODM T6ODM COR CODM HO
(morA)
O O O O O
H H H H H
O HO HO
Oripavine Thebaine Neopinone Neopine Neomorphine
T6ODM Sp
HO morB HO morB morA
O O O O O
H H H H H H H H H H
O O O O HO
Morphinone Hydromorphone Codeinone Hydrocodone Dihydrocodeine
COR COR
Sp
(morA) (morA)
HO
CODM morB
O O O O
H H H H HO H HO H
HO HO O O
Morphine Codeine 14-Hydroxycodeinone Oxycodone
COR
(morA)
O
HO H
HO
14-Hydroxycodeine
Figure2.SynthesisofMorphinanAlkaloidsandDerivativesinSaccharomycescerevisiae.Epimerizationof(S)-

reticulineto(R)-reticulineandspontaneousrearrangementofsalutaridinol-7-O-acetatetothebainemarkthestartingpoints

forthesynthesisofpro-morphinanandmorphinanalkaloids,respectively.Coloredlinesindicateoverlappingbiosynthetic

pathwaysleadingtoopiatesandopiatederivativeswithpharmaceuticalproperties.Pink,synthesisofmorphinefrom

thebaineviacodeine;purple,synthesisofmorphinefromthebaineviaoripavine;yellow,synthesisofhydromorphonevia

morphine;green,synthesisofhydromorphoneviaoripavine;blue,synthesisofhydrocodoneandoxycodonefromthebaine.

Promiscuousenzymesareindicatedincoloredboxes:thebaine6-O-demethylase(T6ODM,blue),codeinedemethylase

(CODM,red),Pseudomonasputidamorphinonereductase(morB,purple),codeinonereductase(COR),andP.putida

morphinedehydrogenase(morA),whichcatalyzemostofthesamereactions,areingreen.Spontaneous(Sp)rearrange-

ments,whichfavorsynthesisofadditionalsubstratesforpromiscuousenzymes,areindicatedinyellow.Otherabbrevia-

tions: CPR, cytochrome P450 reductase; REP, reticuline epimerase; SAS, salutaridine synthase (CYP719B1); SAR,

salutaridinereductase;SAT,salutardinol7-O-acetyltransferase.

TrendsinBiotechnology,March2016,Vol.34,No.3 231
Box1.StrategiesforReducingSide-ProductsinBIASynthesisPathways

Atleast37%ofE.colienzymescanacceptmorethanonesubstrate[82],andthispercentageincreasesforenzymes

involvedwithsecondarymetabolism[83].ThebroadsubstraterangeofenzymefamiliesinBIAsynthesishasbeen
demons trated invitro(exa mplesinclud e[14 –18] ).Cons equently,r econst itut ionofBIA synthe sis path wayshas resu ltedin

thegenerationofside-productsinmicrobialhosts(FigureIA)forboththemorphine[53]andsanguinarinepathways[61].

Promiscuitypresentsbothachallengeandanopportunity.Asapositive,promiscuousenzymescanbeusedtocatalyze
reactionsfo rwhicha ded ic atedenzy meh as notbeenid en tifi ed(revie wedin[84]). Forexam ple, a prom isc uousN-

methyltransferasewasusedtomethylateBIAstructuresotherthanthoseforwhichitwascharacterized[45].Whilenot

alwaysideal,initialsuccessescanbeastartingpointformutagenesisordirectedevolutiontopromotethedesiredactivity

[85,86].However,directedevolutionbeginswithaneffectivescreeningstrategy.Acolorimetricsensorhasbeenusedto
improve thesynthe sisofthe BIAprec ursorL -DOP A [38],buta nefficient screenre m ainstobede velope dfor down strea m

BIAderivatization.

Theeffectsofpromiscuitycanbemediatedbyimprovingfluxthroughtheappropriatepathway,orthroughspatialor
tem poralse qu estrationofe nzy mes awayfrom p athwayint erme diates.G ene ralfluximpr ovements tra tegies(re viewed in

[87]),suchasmodulationofgenecopynumber[46,53]andpromoterstrength(FigureIB)[48,58],haveimprovedyieldin
BIAs ynthe sis pathways. En zyme scaffo ldingco uldalso pus hfluxthro ughthe intende dp athway (Figur eIC).Seq uestr a-

tionofpromiscuousenzymesintootherorganelles[53]orseparateengineeredmicrobes[37]hasimprovedsynthesisof

BIAs(FigureIE).Alternatively,temporalcontrolattheleveloftranscriptionalregulationcouldallowbuildupofthedesired

intermediatebeforetheexpressionofapromiscuousenzyme(FigureIE).

Re-engineeringthepathwayitselfcanalsobeusedtoavoidpromiscuousside-reactions.Forexample,non-productiveside-

productscouldbebroughtbackintothemainpathwaythroughtheco-expressionofotherpromiscuousenzymes(FigureIF).
Alternativ ely,the su bstrate accep tan cep rofile sofeach enzyme cou ldbematched to avoid thegeneratio nofside- produc ts,

recentlydemonstratedbytheheterologoussynthesisof>90%uniquecarotenoidsusingonlypromiscuousenzymes[88].

Finally,theuseofenzymestoprotectfunctionalgroupsfromunwantedside-activitieshasrecentlybeendescribedinopium

poppy[62].Thisraisesthepossibilityofre-engineeringpathwaystoincludeblockingstepstoavoidpromiscuousenzymes,

followedbylaterremovalofthegroup(FigureIF),amethodcommonlyusedinsyntheticorganicchemistry.

(A) (B) (C)
Pathway with Modula(cid:2)on of Scaffolding of
side-products enzyme levels upstream enzymes
(D) (E) (F)
– ind. + ind.
Spa(cid:2)al separa(cid:2)on of Temporal separa(cid:2)on of Pathway re-design to
promiscuous enzyme promiscuous enzyme avoid side-products
Key:
Pathway intermediate Reac(cid:2)on
Side-product Promiscuous reac(cid:2)on
Absence of intermediate Absence of reac(cid:2)on
FigureI.StrategiesfortheReductionofSide-ProductsinPathwayswithPromiscuousEnzymes.(A)Aninitial

pathwaythatresultsintheaccumulationofside-products.(B)DNAcopynumber,ribosomebindingsite(RBS)strength,
andprom ote rstreng th can beadjusted to improvefluxb ym odula tinge nzymee xpression (thinner pip e,less enzyme;

fatterpipe,moreenzyme;smallreddot,lessside-product).(C)Enzymescaffolding(blacklineandshorterpipes)can

preventaccessofintermediatestopromiscuousenzymes.(D)Enzymescanbephysicallyseparatedintosubcellular

compartmentsorbetweenmicrobialstrains(blackboxes).(E)Expressionofapromiscuousenzymecanbedelayed
((cid:3)ind)untilan in ducerisa ddedor growth condit ionsres ult intranscript ion ( +ind).(F)Alte rnativee nzym es canbe

expressedtoadjustthepathwaysuchthatside-productsarenotproduced.

232 TrendsinBiotechnology,March2016,Vol.34,No.3
aromaticAA-derivedcompoundsarecurrentlyanorderofmagnitude higherinE.colithanin

yeast, both in shake-flask ((cid:1)300mg/l vs 2g/l) [20–22] and fermentation (2g/l vs 55g/l)

conditions [23,24]. Notably, the shift from laboratory shake-flask to industrial fermentation

can improve total titers, although not necessarily yield, by one to two orders of magnitude.

A striking example of this is the development of fermentation conditions for a yeast strain to

synthesize artemisinic acid, in which titers of the intermediate amorpha-4,11-diene increased

from160mg/lto40g/l[25].AlthoughBIAtitersreportedthroughoutthisreviewarelow,muchof

theworkwasdoneinshakeflasks,suggestingthatoptimizedfermentationconditionscombined

with the latestdevelopments instrain engineering will easilyimproveupon these titers.

Aldehyde Scavenging Limits the Available 4-HPAA Pool

Becausethealdehyde4-HPAAisasubstrateofnorcoclaurinesynthesis,itsinvivostabilityis

necessaryforBIAoverproductioninmicrobes.However,aldehydesarerapidlyscavengedto

limitcellulartoxicity[26].Aldehydescanbereducedoroxidizeddependingontheredoxstatusof

thecell[27].Nolessthansixaldehydedehydrogenases(ALDs)and16alcoholdehydrogenases

(ADHs)canparticipateinaldehydereduction/oxidationinyeast,withsignificantredundancy[27].

E. coli also harbors multiple ALDs and ADHs, whose knockout has improved heterologous

productionofaldehydes[28,29].PreliminarydatasuggeststhatALDandADHknockoutsmay

improve denovoBIA production inyeast [30].

Multiple Strategies for Dopamine Synthesis in Microbial Hosts

Theformationofdopaminefromtyrosinerequiresonehydroxylationandonedecarboxylation

event(Figure1A).Dependingonenzymespecificity,thesereactionscouldoccurineitherorder;

whendecarboxylationoccursfirsttheintermediateistyramine,whileifhydroxylationoccursfirst

the intermediate is L-DOPA. Thus far, the decarboxylation-first pathway has been avoided

through the use of a decarboxylase that has a strong preference for L-DOPA [31]. This is

historically because production of L-DOPA, as opposed to tyramine, made downstream

pathway engineering easier in E. coli [31]. L-DOPA has remained the intermediate of choice

as denovoBIA synthesishasbeen introduced toyeast.

Many optionsforheterologousL-DOPAsynthesisexist, withnooneenzymestandingoutas

being clearly superior to other enzymes. Currently, enzyme selection for L-DOPA synthesis

requireschoosingbetweenundesiredside-activitiesandtherequirementforacofactor.There

are two types of side-activities relating to L-DOPA synthesis: broad substrate range and

L-DOPAoxidationtodopaquinone(diphenolaseactivity).Tyrosinases(TYR)andhydroxyphenyl-

acetic acid hydroxylases (HPAH) have broad substrate ranges [32,33], while TYRs and the

cytochromeP450hydroxylaseCYP76AD1havediphenolaseactivityonL-DOPA[34,35].While

catalyzingtwotypesofsideactivity,TYRhasthelowestcofactorrequirement(somerequireonly

inorganiccopper).Bycontrast,tyrosinehydroxylase(TH)hasthelowestamountofside-activity

butthehighestcofactorrequirementsbecauseitusesthecofactortetrahydrobiopterin(BH ).
4

BH isnotnativetoE.coliandS.cerevisiae,andhencethefunctionalexpressionofTHalso
4

requirestheheterologousexpressionofaBH synthesisandregenerationpathway.THshave
4

an additional disadvantage in that they are heavily regulated by allosteric inhibition and post-

translationalmodificationreflectingtheirroleastherate-limitingstepofcatecholaminesynthesis

in neurons [36].

Thedisadvantagesofvariousenzymefamilieshavebeenaddressedduringtheintroductionof

L-DOPA synthesis in both E. coli and S. cerevisiae. A TYR with an unusually low level of

diphenolaseactivity(RsTYR)wasexpressedinE.coliforheterologousBIAsynthesis,resultingin

the production of 2.5g/l dopamine [37]. However, RsTYR still possessed some diphenolase

activityanditsbroadsubstraterangeallowedittooxidizedownstreamBIAs.Thishassincebeen

addressed by using multiple strains of E. coli to sequester RsTYR from downstream BIA

TrendsinBiotechnology,March2016,Vol.34,No.3 233
synthesis enzymes [37]. However, this multistrain system is not without drawbacks (Box 2).

WhileHPAHalsohasabroadsubstraterange,itdoesnotoxidizeitsproducts,andhenceHPAH

may bea betteroption than RsTYRforL-DOPAsynthesisin E.coli.

HPAHhasnotbeendemonstratedtobefunctionalinyeast,andTYRactivityinyeastislow[38],

pointingtowardsTHandCYP76AD1asbetteroptionsforL-DOPAproductioninS.cerevisiae.The

side-activityofCYP76AD1wasreducedbysubjectingittomutagenesisfollowedbyscreeningwith

a color-based biosensor that can detect and distinguish between L-DOPA and dopaquinone

synthesis[38].Alternatively,theBH synthesispathwayhasnowbeenintroducedintoS.cerevisiae
4

[39,40],permittingfunctionalTHexpressionforBIAsynthesis[40].Theuseofeitherenzymeto

producedopamine,regardlessofstrainengineeringstrategy,currentlyresultsinyieldsof(cid:1)10–

25mg/l. These titers are lower than the 2.5g/l dopamine produced in E. coli, but are not

uncommonforheterologousproductsderivedfromaromaticAAsinyeast[22,41],whichpoints

tothenecessitytoimprovethesynthesisofprecursorstoachievehigherdopaminelevels.Recent

titersof2and3g/lofaromaticAApathwayderivativesinyeastindicatethatengineeringstrategies

arebeingdevelopedthatshouldfurtherimproveBIAyields[24,42].

BIA Scaffolds Can Be Synthesized De Novo from Simple Sugars

Pictet–Spenglercondensationofanamineandaldehydeisareactionmechanismcommonto

the committed step of several alkaloid families [43]. Spontaneous condensation generates

racemic (R,S) mixtures, whereas enzymatic condensation is enantio-specific. The committed

step of BIA synthesis is enzymatically catalyzed by norcoclaurine synthase (NCS), which

condenses dopamine and 4-HPAA to generate (S)-norcoclaurine. Because BIAs are derived

from(S)-norcoclaurine,thesynthesisof(R)-norcoclaurineisunproductive,andhenceenzymatic

condensation is preferable to spontaneous condensation for microbial production of BIA

scaffolds. NCS has a broad substrate range for aldehydes. In addition to 4-HPAA, NCS can

acceptthedouble-hydroxylated3,4-dHPAA,whichwhencondensedwithdopaminegenerates

(S)-norlaudanosoline(Figure1A).NorlaudanosolinehasbeenusedextensivelyforBIAderivati-

zation in E. coli because the extra hydroxyl group on 3,4-dHPAA negates the need for later

cytochromeP450-catalyzedhydroxylationoftheBIAscaffold(Figure1A)[44].Bothnorcoclaur-

ine and norlaudanosoline are unstable end-products because they are subject to enzymatic

Box2.FermentationConditionsforBIASynthesis

ThepHoffermentationaffectsBIAyields,bothsynthesisandderivatization,inE.coliandinyeast.E.colifermentations
are usu ally performeda tpH7, wh ichiss ufficie ntlyalkalin efo rthenon-prod u ctiv esp ont an eouso xid atio nofL-DOPA,

dopamine,andnorlaudanosoline[31].pH6wasfoundtobethebestcompromisebetweenE.coligrowthanddenovo

BIAsynthesis[46].However,thecontinueddisappearanceofnorlaudanosolineinsupernatantindicatesthatthese

conditionsarestillnotoptimal[37].

ThepHofyeastcultures(3–6)islowerthanE.colicultures,whichreducesspontaneousoxidationofnorlaudanosoline

andprecursorsinsupernatant.However,BIA(andotheralkaloid)[39]derivatizationfromsupplementedprecursorsis
mor eefficientat hi gherpHs.As pHisincre ased from 3to8 ,agreat erfra ctionofsupp lemen tedBIAsareas sociatedwi th

cellextracts,andconversiontodownstreamproductsishigher[89].Whilesupplementedprecursorsrepresentan
inte rmediate step toafinalp rod uction strain ,endogen ou slypro duce ddop amine,norcocl aurine,and reticuline are
primarilyfoun din yea st sup ernatant[38 ],and typicallydonot re-enterc ellsefficient lyonceoutside [40] .Knockou tof

transporters to prevent secretion is an alternative method for controlling ratios of BIA fractionation [90]. However,

secretionofend-productswouldmakedownstreamindustrialprocessingeasier.Importantly,thefractionationofBIAsas
wellasth e rateoffluxthr oughB IAsy nthesispathw aysmus tbebalanc edfor thegreatest pat hwayefficien cy .

Groups working with E. coli and yeast have explored other fermentation conditions for de novo synthesis and

derivatizationofBIAsinvivo.GrowthatlowertemperaturesimprovesBIAconversioninbothspecies[46,48].Alternative

carbonsourcescanimprovetheproductionofprecursors[31]andderivatizationofdownstreamBIAs[48].Conditions

suchaspH,temperature,andcarbonsourcecanbeadjustedthroughoutfermentativeproduction.Aninitialaccumula-

tionofbiomassbeforeheterologouscompoundproduction,suchashasbeendoneforproductionof1,3-propanediolin

E.coli[91],hasalsoimprovedboththesynthesisofaromaticAApathwayderivatives[42]aswellasthederivatizationof

BIAs[48,56,61].

234 TrendsinBiotechnology,March2016,Vol.34,No.3
oxidation as well as spontaneous oxidation at alkaline pHs. Therefore, the key branch-point

intermediate reticuline (Figure 1B), derived from norcoclaurine/norlaudanosoline (Figure 1A), is

frequentlyusedasareadoutfordenovosynthesisofnorcoclaurine/norlaudanosoline.

Norlaudanosoline Synthesis inE.coli

3,4-dHPAAcanbegeneratedfromdopamineviamonoamineoxidase(MAO),makingdopamine

the source of both amine and aldehyde for norlaudanosoline synthesis (Figure 1A). As a first

proof of concept for the synthesis of BIAs in a microbial host, reticuline was produced from

supplementeddopamineinE.coli[45].Initially,1.3%ofsupplementeddopaminewasconverted

toreticuline,whichwasimprovedinlaterstudiesafteroptimizingbothfermentationconditions

andgenecopy-number(Box2,Table2)[46].Thefurtherintroductionofendogenousdopamine

synthesistoE.colienabledfermentationofreticulinefromglycerol,withaninitialdopamine-to-

reticuline conversionof 4%(Table2)[31].

Enzymaticoxidationofnorlaudanosolinewasidentifiedasaside-activityofTYRthatreduced

conversion of dopamine to reticuline. To prevent TYR activity on norlaudanosoline, norlauda-

nosoline synthesis was divided between two strains of E. coli (glycerol-to-dopamine and

dopamine-to-norlaudanosoline), which were cultured sequentially (Box 1). Preventing diphe-

nolase activity on norlaudanosoline improved its accumulation 300-fold, resulting in 16%

conversionfromdopamine.However,theadditionofathirdstraintoconvertnorlaudanosoline

to reticuline did not improve total dopamine-to-reticuline yields compared to previous results

(Table2).Itispossiblethatthisdiscrepancyisduetotheextendedtimethatnorlaudanosoline

wasexposedtoculturesupernatant,whichpromotesspontaneousoxidation(Box2).Norlau-

danosolineoxidationinsupernatantmaybeavoidedinthefuturebyre-engineeringthemulti-

strainsystemsuchthatdopamineproducedbythefirststrainisconverteddirectlytoreticuline

by a secondstrain expressing MAOas wellasthe reticulinesynthesispathway.

Table2.SynthesisofReticulineinMicrobialHosts

Strain Dopamine Norlaudanosoline Reticuline %Yielda Comments Ref.

E.coli Supplement,5mM (cid:3) 33mM 1.3 Firstsynthesisof [45]

reticulineinamicrobial

host
E.coli Supplement,3mM (cid:3) 165mM 11 Improvedfermentation [46]

conditions
E.coli Denovo,7mMb (cid:3) 140mM 4 FirstdenovoBIA [31]

synthesis.Fed-batch

E.coli Denovo,14mMb (cid:3) 145mM 2 Three-stepfermentation. [37]

Fed-batch
S.cerevisiae (cid:3) Supplement, 455mMc 10 Firstderivatizationof [58]

4mM norlaudanosolinein

S.cerevisiae

S.cerevisiae (cid:3) Supplement, 2mM 20 Improvedyield [61]

0.01mM

S.cerevisiae Denovo,0.155mMb (cid:3) 0.2mM 0.13 FirstdenovoBIA [38]

synthesisinS.cerevisiae.
Shake-fla sk
S.cerevisiae Denovo,0.065mM (cid:3) 0.2mM 0.31 Improvedyield. [52]
Shake-flas k
aMolaryield.
bDopam inequantifiedinsupernatantofstrainsexpressingnodownstreamenzymes.
cReticuline concentrat ion estimatedb as edonc losestavail able standard.

TrendsinBiotechnology,March2016,Vol.34,No.3 235
Norcoclaurine Synthesisin S.cerevisiae

Thisyear,twogroupshaveachievedBIAsynthesisfromsimplecarbonsourcesinyeast.Neither

group used MAO to generate 3,4-dHPAA from dopamine. Instead, endogenous cytosolic

4-HPAA was the source of the aldehyde. Both groups reported comparable reticuline yields

severalordersofmagnitudelowerthanthemostrecentyieldspublishedforE.coli(Table2).In

particular, the drop in titers from dopamine ((cid:1)10–25mg/l) to norcoclaurine ((cid:1)80–100mg/l)

indicatesthatnorcoclaurinesynthaseisakeybottleneckinthedenovosynthesisofBIAsinyeast

[38].
Supplementation of dopamine for spontaneous condensation to norcoclaurine in yeast was

muchlessefficientthaninE.coli(0.0025%vs16%)[40].Endogenouslyproduceddopamine

wasalsoconvertedtoreticulineatlowerlevelsinyeastthaninE.coli(0.3%vs4%)(Table2).

Considering that endogenously produced dopamine is found in the supernatant [38], low

norcoclaurine yields could in part be due to dopamine secretion occurring more readily than

norcoclaurine synthesiscanoccur.The K fordopaminefortheNCSofThalictrum flavum is
M

25mM[47],whichisapproximatelydoubletheconcentrationofdopamineproducedinE.coli,

but170-foldofthatcurrentlyproducedinyeast.Itispossiblethatdopaminevaluesinyeastare

currentlytoolowforefficientsynthesisofnorcoclaurine.Localdopamineconcentrationscould

beimprovedfurtherviastrainengineeringtoimprovetiters,reduceefflux,orsequesterdopa-

mine in subcellularcompartments.

Currently,asapercentageofdopamine,reticulineyieldsfromgroupsworkinginS.cerevisiaeare

similar to the first numbers published in E. coli (Table 2). Increases in E. coli yields were

incremental, and required optimization of strain design and fermentation conditions

[31,37,45,46]. Fermentation conditions developed for BIA derivatization in yeast will likely

improve de novo synthesis as well (Box 2) [48]. Further strain engineering will be necessary

forcompetitive titers ofdenovo BIA synthesisin yeast.

Synthesis and Derivatization of Morphinan Alkaloids in S. cerevisiae

Synthesisofnaturally-occurringmorphinanalkaloidsproceedsthrough(R)-reticuline(Figure2)

[49,50].Therecentidentificationoftheepimerasethatcatalyzestheconversionof(S)-reticuline

to (R)-reticuline [18,51] marks the complete characterization of all the genes involved in

morphinan biosynthesis in planta since the first isolation of morphine from opium poppy in

1806[10].PartsofthemorphinepathwayhavebeenreconstitutedinScerevisiaebysupple-

mentationofintermediates.Thisyear,thesynthesisofopioidsfromsimplecarbonsourcesina

microbial system wasachieved[52].

Derivatization of MorphinansfromSupplemented Precursors

Thebaineis aprimaryfeedstockforthechemicalsynthesisofnaturally-occurringandsemi-

syntheticopioids[50].Asaproofofconcept,S.cerevisiaeexpressingmorphinansynthesis

geneshasbeenusedtoprovideamicrobialalternativetochemicalderivatizationofthebaine

[53]. Synthesis of any single morphinan, and in particular morphine, from thebaine is an

engineeringchallengeowingtothecomplexarrayofproductsthatcanbegeneratedfroma

small number of enzymes. Morphine synthesis from thebaine requires the co-expression of

three enzymes (Figure 2). Each enzyme has broad substrate specificity [14,54,55] and the

reactionscanoccurinmultipleorders.Twooftheseorderscanresultinmorphinesynthesis

(pink and purple pathways in Figure 2) while other orders result in the synthesis of side-

products (e.g., neopine and 14-hydroxycodeine). Spontaneous rearrangements (within

thepinkpathwayinFigure2)addanotherlevelofcomplexitybecausesomeareproductive

for morphine synthesis while others are not. In short, the array of morphinans produced

is entirely dependent on the relative rates of enzyme activities and spontaneous reactions

(Box 1).

236 TrendsinBiotechnology,March2016,Vol.34,No.3
Conversionofthebainetomorphinebyyeastexpressingthenecessaryenzymeswaslimitedto

1.5%, with another 12.7% of thebaine being converted to intermediates and side-products,

mainlyneopineand14-hydroxycodeine(Table3)[53].Iffluxtravelsthroughneopinone,these

side-productswillbedifficulttoavoidbecauseneopineisgeneratedwhencodeinonereductase

(COR) activity outcompetes a spontaneous reaction, while 14-hydroxycodeine is generated

whenspontaneousreactionsoutcompeteCORactivity(Figure2).Favoringsynthesisthrough

oripavine(purplepathwayinFigure2)islikelytoreducethenumberofside-productsinmorphine

synthesis (Box 1). To achieve the reported 1.5% conversion of thebaine to morphine, some

metabolicengineeringstrategiestofavorsynthesisofcodeinonehavealreadybeenemployed.

High-yieldsynthesisofmorphineinmicrobes,althoughpossible,willrequirethedevelopmentof

innovative strategies(see Box1forexamples) tofunnelthe carbonflux towardsmorphine.

Bycontrast,therightcombinationofenzymesandspontaneousreactionscanprovetobevery

effective at generating a particular morphinan of interest. For example, a pathway to hydro-

codoneandoxycodonewasdevelopedusinggenesisolatedfromasoilbacteriumgrowingon

industrialpoppywaste(Figure2,bluepathway)[53].WithoutCORexpression,manyoftheside-

productsofmorphinesynthesiswereavoided,andalmosthalfofsupplementedthebainewas

convertedtotheintendedproducts(Table3).Avoidingparticularenzymeswithhighpromiscuity,

oridentifying/engineeringenzymeswithdesiredsubstratespecificities,mayprovetobeamore

general strategy for morphinan and/or BIA synthesis in vivo. For example, hydromorphone

synthesisthroughneopinone(yellowpathwayinFigure2)resultedinonly0.4%hydromorphone

(Table3),whereasahypotheticalpathwaythroughoripavine(indicatedingreeninFigure2)could

reduce side-products substantially because it avoidsCOR (Figure2).

Thebaineitselfhasbeenderivatizedfromtheupstreampathwayintermediate(R)-reticulineinS.

cerevisiaeexpressingtheappropriateenzymes(Figure2)[52,56].Anotherspontaneousreaction

presents an engineering problem unique to this portion of the pathway: the intermediate

salutaridinol-7-O-acetate spontaneously rearranges to thebaine at pH 8–9, but rearranges to

an undesired side-product at pH 6–7 (Figure 2) [57]. To address this issue, a two-step

fermentation system was used in which yeast biomass was allowed to accumulate and

subsequently switched to pH-buffered media supplemented with either (R)-reticuline or salu-

taridine.ThehighestproductionofthebainewasobservedatalkalinepHs(pH8.5–9)[56].This

study highlights pH-adaptable fermentation conditions as an additional challenge for BIA

synthesis inyeast (Box2).

Discovery of ReticulineEpimerase Enables DeNovoSynthesis ofOpiates

Until recently the fermentation of opiates from simple sugars was not possible owing to the

inability toproduce (R)-reticuline in vivo. Initially, the in vivoproduction of (R,S)-reticuline from

spontaneously condensed (R,S)-norlaudanosoline was proposed as a source of (R)-reticuline

[45,58].However,morerecentreportsdemonstratethatonly(S)enantiomerscanbeaccepted

by enzymes of the norlaudanosoline-to-reticuline pathway that have been assayed thus far

[31,56].Whileenzymescapableofaccepting(R)enantiomersmayexist[59],epimerizationof(S)-

reticulineto(R)-reticulineiscurrentlycrucialforopiatebiosynthesis.Theenzymecatalyzingthis

stereochemicalconversionisreticulineepimerase(REP),acytochromeP450-reductasefusion

proteindiscoveredthisyear[18,51,60].REPactivityhasbeendemonstratedinvitro[18,51]and

invivo,bridgingtheupperglucose-to-(S)-reticulineandlower(R)-reticuline-to-opiatesectionsof

the pathway [38,40,52,56]. Yeast strains capable of converting supplemented (S)-norlauda-

nosoline or endogenously synthesized (S)-norcoclaurine to thebaine and hydrocodone have

nowbeenengineered.Whileyieldsarecurrentlylow(Table3),areasofpathwayimprovement

havebeenhighlighted.Inyeastcapableofconverting(S)-norlaudanosolineto(R)-reticuline,total

reticulinelevelswere100-foldlowerthanyieldsinotherengineeredS.cerevisiaestrainslacking

REP (Table 3) [58,61]. This could be due to the promiscuity of REP, which has been

TrendsinBiotechnology,March2016,Vol.34,No.3 237