© 2015 Nature America, Inc. All rights reserved. P enable low-cost production ofmany high-value BIAs. work to improve titers and connect these steps with downstream pathway branches, already demonstrated in these technologiesthese revolutionizing pharmaceutical manufacturing yeast the in acid artemisinic ie a cmeca sae n ae nta etatd rm plants, ( poppyopium from the notably most extracted instead are and synthe scale commercial chemically at sized be cannot BIAs most complexity, structural production inyeast from glucose o aiy nierd n saal idsra pouto hosts production industrial scalable and engineered easily to chemical pathways in plants such that they can bionow be transplanted complex of elucidation the accelerated has sequencing DNA raim wt nw rpris osdrby atr cepr and cheapereasier faster, considerably properties new with organisms micro of engineering the made has cells manipulating genetically Meanwhile, the development of powerful synthetic biology tools for molecules have become high-value targets for production via via production for targets microbial fermentation. high-value become have molecules these biosynthesis, BIA of understanding our in advances recent crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate ( intermediate Saccharomyces pharmaceuticerevisiaeBIA . Tokey the aid the in this effort, traditionalof we developed productionto an enzyme-coupled include alternativebiosensor the for the demonstrateupstreaman intermediate we as that Here promise manufacturing. holds metabolites crop-based BIAs of plant-specialized synthesis Microbial derivatives. of their and family morphine and diverse codeine cals a are (BIAs) alkaloids Benzylisoquinoline William CDeLoache An enzyme-coupled biosensor enables ( nature CHEMIC Québec, Canada. 1 eiaie o te I itreit thebaine intermediate BIA suppressant the of derivatives cough the and noscapine. Several analgesics papaverine(for example, oxycodone) are synthetic and relaxants (+)-tubocurarine muscle codeine, the berberine, and and morphine sanguinarine antibiotics analgesics the opioid the includes pounds cycles developmentlong and tools genetic of lack plant metabolic engineering, namely complex pathway in regulation, a levels low in limitationsby hindered are improveyields to atefforts and plants, accumulate often metabolites Target synthesis. too are they because structurally complex to extracts be cost-effectively produced by plant total organic from manufactured still are from pathway seven-enzyme the reconstitute fully to pathwaymutant this best extendedWe our enzyme. for obtained titer in 7.4-foldimprovement a with yeast, in glucose from production dopamine of onstration byyields 2.8-fold mutagenesis.PCR via Coexpression DOPAof decarboxylase knowledgetoour is what enabled firstthe dem ( dihydroxyphenylalanine tural diversity of natural product derivatives struc potential the explore fully to challenging it made also have Vincent JMartin metabolites with a variety of therapeutic uses therapeutic of variety a with metabolites icvr poess o mn ntrl products natural many for processes discovery drug and manufacturing the transform to poised are and barriers epartment ofBioengineering,UniversityDepartment of California, Berkeley,Berkeley, California, USA. BIAs are a large family of family large a are BIAs Microbially based production systems overcome many of these these of manyovercome systems production based Microbially iy ht a gvn ie o hs o poiet pharmaceuti prominentcals of host a to rise given has that sity lant-specialized metabolites are a rich source of chemical diver 7,8 . Commercial production of the antimalarial drug precursor 1 . Despite. widespread their use AL BIOLOGY 3 C entre for Structural andFunctional Genomics,C

| Adv 2, 3 &JohnEDueber 1 L , ZacharyNRuss is a pioneeringcerevisiaea S. exampleis of ance online public -DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its improved and hydroxylase tyrosine active an identified we sensor, this -DOPA).Using L -tyrosine–derived plant-specialized -tyrosine–derived Papaversomniferum ) 2 , many, compounds of these 4 . 10 a 10 tion | . This class of com of class This . . Because of their their of Because . 5 Next-generation . 3 1 . Such problems Such . * 1 , Lauren Narcross www.nature.com/naturechemicalbiology 1 . In light of light In . oncordia University, Montréal, Québec, Canada.*e-mail: 9 6 ------. .

n h bacterium the in pathway. Despite high titers BIA the of branches major the between shared be to intermediate marily attributable to difficulties at the first biosynthetic step from step biosynthetic first the at difficulties attributableto marily by the synthesis of the key intermediatekey synthesisbyof the the ( ezpeatrdns (CYP82N4) and benzophenanthridines (CYP80B2) bisbenzylisoquinolines (CYP80G2), aporphines the generation in of new backbones such as morphinans (CYP719B1), especially diversification, and synthesis BIA in role important L tp t mgolrn ad scoulerine and magnoflorine to steps cocultured with reticuline-producing o te ontem tp o BA biosynthesis BIA of steps downstream endomembrane- the of for host expression suitable more a is functional therefore and P450s cytochrome the localized to amenable is dihydrosanguinarine. and oxycodone, (S)-tetrahydroberberine hydromorphone, hydrocodone, codeine, morphine, e nradnsln i yeast in early norlaudanosoline fed the given difficult unexpectedly in successes proven has cerevisiae S. monocultures over more complicated, multistrain strategies. thebaine such and intermediates norlaudanosoline cost-prohibitive as from albeit BIAs, valuable in stituted recon successfully been have steps downstream these of number in demonstrated been since have n h laboratory the in fermentations sequential and cocultures microbial establishing successes in recent been have there Although processes. mentation fer simplify and loss carbon minimize to microorganism single a intoconsolidated be steps all that necessitate likely most will BIAs synthase (NCS) h snhss f oluaooie r h ntrl I backbone BIA natural (S)-norcoclaurine the from or norlaudanosoline of synthesis the -tyrosine to -tyrosine A major achievement in microbial BIA production was marked was productionBIA microbial in achievementmajor A -eiuie rm eta mtblts in metabolites central from (S)-reticuline of Production published online:18ma 2, 3 , Andrew MGonzales 2 , leading to the recent production of many of production recent the to leading cerevisiae, S. epartment ofBiology,Department C . Although reticuline has been synthesized from synthesized been has reticuline Althoughcoli . E.

L- DOPA as well as poor activity of the norcoclaurine the of activity poor as well DOPAas 20 . Prior to this work, there were two known families 13,1819 shrci coli Escherichia idsra frettos rqety favor frequently fermentations industrial , L -tyrosine have remained elusive. This is pri 13 , no steps downstream of ( y 2015 | L -tyrosine to ( to -tyrosine 14 te ptem tp rqie for required steps upstream the , oncordia University, Montréal, S Hwvr a impressive an However, coli. E. )-reticuline from glucose in glucose from S)-reticuline 1 12

14–16 0 doi: 10.1038/nche mbio . Although yeast have been been have yeast Although . 1 )-Reticuline is the final final the is (S)-Reticuline . E. coli to catalyze downstream )-reticuline )-reticuline , 17 lresae rdcin of production large-scale , Tee rdcs include products These . [email protected] )-reticuline from glucose S)-reticuline article )-reticuline. Future S)-reticuline. S. cerevisiae, will 8 P5s ae an have P450s . . cerevisiae S. S)-reticuline L -DOPA .181 L -3,4- 6 - - - - - 1

© 2015 Nature America, Inc. All rights reserved. article Because a substantial fraction of betaxanthin remains intracellular,remainsbetaxanthin of fraction substantial a Because ( mechanisms unknown through cell the pellet. of out pumped cell the and water supernatantsoluble, accumulates in the yeast vacuole the in addition to getting both Fluorescence in microscopy indicated that betaxanthin, eye which is highly by detected DOD-expressing strain grown in 0.1–10,000 a for curve dose-response a generated fluorometer,we microplate ). Using this detection technique, we achieved we technique, detection this Using 1d). (Fig. cytometry flow via level single-cell at the detectablereadily wererescencealso excellenthigh-throughputanassay a variability,sample-to-sampleyielding low and range dynamic fold for biosensor enzyme is a cytochrome P450 from the sugar beet pigments called betaxanthins ( betaxanthins called pigments of three orders of magnitude from 2.5 Changes in betaxanthin fluorescence were detectable across a range xds atvte t produce to activities oxidase organisms many for by melanin used production, exhibit 1.14.18.1), both tyrosine (EC hydroxylase tyrosinases and containingDOPA etd with mented the most sensitive response observed between 25 between observed response sensitive most the iaii jalapa Mirabilis molecules of family entire this to collectively as betaxanthin. refer will we simplicity, For lhuh atra trsnss ae en sd for used been have tyrosinases bacterial Although production in production L L L achieve to inability long-standing A Development ofanenzyme-coupled RESULTS L require a cofactor (tetrahydrobiopterin) not Plants found hydroxylases. in yeast tyrosine 3-monooxygenases( tyrosine use animals and as to referred broadly enzymes of 2 ht converts that Caryophyllales order the of members in found enzyme plant a is (DOD) activity.DOPAdioxygenasefor hydroxylases tyrosine date intermediate pathway BIA the for biosensor enzyme-coupled an developed we S. cerevisiae remains achallenge. able to further improve ablefurther to mutagenesisPCR via itsactivity increaseto tyrosine hydroxylase that was activehighly in of of unwantedoxidationadditional an catalyzes enzyme this of version value BIAs at commercial scale. high- producing of capable microorganism a of development the demonstratedpreviously in been downstream the toofyeastofBIAsteps lism biosynthesis thathave ( and allowed for the synthesis of the BIA intermediatespoppy, opium( from NCS identified newly a to coupled when and, yeast in dopamine of production the enabledhydroxylase tyrosine mutantoff-pathwayactivity.suppresses Our this identified we that ecn pget betaxanthin pigment rescent upeetr Results (Supplementary DOD by produced acid betalamic and amines free between reaction neous sponta a of result the are properties, optical similar have which of isnhtc nye ht converts that enzyme biosynthetic P450 capable of capable P450 a of example known first the knowledge our to is what represents -DOPA levels can be quantified via cellular fluorescence. Using a Usingfluorescence. cellular via quantified be can -DOPAlevels ). The sensor demonstrated a 110- demonstrateda sensor The 2). -DOPAFig. (Supplementary enzyme-coupled an develop to us prompted yeast in -DOPA This 7.4-fold. by titer dopamine and 2.8-fold by titer -DOPA

-factor of 0.91, where a value between 0.5–1.0 typically denotestypically 0.5–1.0 between value a where 0.91, ofZ′-factor L We expressed in yeast a DOD variant from the flowering plant flowering the from variant DOD a yeast in Weexpressed o i i te erh o a es-cie yoie hydroxylase, tyrosine yeast-active a for search the in aid To DP into -DOPA )-reticuline from glucose. By connecting the centralmetabo the connectingfromByglucose. S)-reticuline L L L L DP. hs isno tks datg o a plant a of advantage takes biosensor This -DOPA. -DOPA produced enough betaxanthin to be easily easily be to betaxanthin enough produced -DOPA -DOPA that could be used to quickly screen candi screen quickly to used be could -DOPAthat E. coli E. DP it a elw hgl furset aiy of family fluorescent highly yellow, a into -DOPA 25 L n fud ht el gon n eim supple medium in grown cells that found and L -tyrosine hydroxylation. Although the wild-type wild-type the Although hydroxylation. -tyrosine dpqioe oe f h bnfca mutations beneficial the of one -dopaquinone, 12 , functional expression of these enzymes in enzymes these of expression functional , 24 Uig hs esr w ietfe a identified we sensor, this Using . L Fig. 1a) Fig. dpqioe from -dopaquinone , this workcerevisiaeaccelerates this S. , 28 and and L . Changes in betaxanthin fluo betaxanthin in Changes . μM to 2,500 - fluo yellow, the into -DOPA L 25,26 trsn hdoyain to hydroxylation -tyrosine upeetr Fg 1) Fig. Supplementary L . These betaxanthins, all all betaxanthins, These . -DOPA biosensor μM S. cerevisiae and were ), which which 1.14.16.2), EC L μM -DOPA (Fig. 1c). Beta vulgaris Beta S)-norcoclaurine M and 250 and μM L L -DOPA, with nature -tyrosine 21 . . Copper- Fig. 1b). Fig. L -DOPA chemic and and 22,23 μM 27 Nature chmical biolo gy - - - - - . .

al round of fluorescence-activated cell sorting (FACS) if given a in population the of rest the over 50-fold and 25- between enriched be to expected be would activity improved twofold with variant hydroxylase tyrosine a that indicated data cytometry flow ferences in cell size ( ferencessize cell in andnormalizing fluorescence by forward scatter accountto for dif single-cell for measurements,despite gating both cellsby forward and scatterside common is as observed variability, we cell-to-cell however, considerable sensor; the for range dynamic 145-fold a cific for cific proved technique sufficient for (Supplementary our Fig. purposes 5). this as libraries, hydroxylase tyrosine our of screening applica and tions, we elected to use colony-based colorimetric screensand fluorometric future enabling for promising is sort cell to duction fell in the linear range of the biosensor. Although the ability altering its substrate from specificity ofobjective the with selected wasit enzyme; ouslyfor reportedthis purposes color, beet determining in attractiveanit candidatemaking forour enzyme’srole this elucidate to demonstrated previously was yeast beets in analog) 1 Fig. betaxanthin (Supplementary (a betanidin pigment violet the of with relatively low DOPA oxidase activity oxidase DOPA low relatively with xds atvt i udsrbe s t diverts it as undesirable is activity oxidase DOPA secondary the BIAs, of production the exhibited For tyrosinases. activities by oxidase DOPA and hydroxylase tyrosine the yeast, this enzyme had over tenfold higher activity on (polyphenol oxidase 2 (AbPPO2)) has been successfully expressed in Although a tyrosinase from the button mushroom Supplementary Fig. 1). and tyrosine hydroxylase activity maximized (activity 1; 2; (activity are active in yeast. In this manner, undesired DOPA oxidase activity and and OA oxidase DOPA beet AbPPO2 screening: initial from for selected were enzymes candidate variants with higher activity thanin this those described work. Two identify even and class enzyme this into insights additional reveal might biosensor enzyme-coupled our with libraries such screen to date without resorting to these techniques. We note that future work candi- acceptable anfortuitously foundwe bioinformaticcuration, or cDNA from libraries variant large screening involve to search this a expected we Althoughmutagenesis. as enzyme for point use starting to DOD with expressed when betaxanthin of levels able Wehydroxylasetyrosine a measursoughtvariantfirst thatyielded Identification ofayeast-active tyrosine hydroxylase it did on did it uaeei t atr h rltv sbtae pcfct for specificity substrate relative the alter to mutagenesis enzyme alongside used be could assayproduction betaxanthin our rvos xeiet wt CP6D wr cnutd sn a a has which using DOD, vulgaris B. conducted from were CYP76AD1 DOD with a experiments used previous strains our While used. enzyme DOD the of identity the is results contradictory these for account bevbe betaxanthin observable no yielded and yeast in coexpressed were DOD a and CYP76AD1 which in work previous given unexpected particularly was result were clearly visible in colonies growing on agar plates that( betaxanthin of levels to leading activity,hydroxylase considerable tyrosine exhibited CYP76AD1 surprise, our To production. betaxanthin detectable, low,but extremely showed AbPPO2 work, previous from expected As DOD. with along expressed and yeast biology Notably, our enzyme-coupled biosensor is designed to be spe be to designed is biosensor enzyme-coupled Notably,our oh bP2 n CP6D wr cdn piie for optimized codon were CYP76AD1 and AbPPO2 Both L . The latter is a recently identified cytochrome P450 cytochromerecently identified a latter Theis vulgaris . B. -tyrosine in enzymes that preferentially act on act preferentially that enzymes in -tyrosine (discussed above) and CYP76AD1 from the sugarfrom the above)andCYP76AD1 bisporus(discussed A.

| Adv L 32 -DOPA as it should be capable of differentiating between differentiatingbetween of capable be should it-DOPA as L Ntby trsn hdoyae ciiy a nt previ not was activity hydroxylase tyrosine Notably, . -tyrosine and and 1a Fig. ance online public 32 Ti ezm i rsosbe o te production the for responsible is enzyme This . 30 SupplementaryFigs.3 . Given the existence of bacterial tyrosinases bacterial of existence the Given . ) could be minimized minimized be could 1) Fig.Supplementary 32 . ucinl xrsin f Y7A1 in CYP76AD1 of expression Functional ). Oe xeietl ifrne ht could that difference experimental One . a tion | K m of 7 mM 7 of www.nature.com/naturechemicalbiology L -DOPA to d o i 31 : 10 3 , we hypothesized that hypothesized we , and L , well above the levels the above well , DP t melanin to -DOPA . 10 4 L 38/n -tyrosine. Agaricus bisporus ). Analysisof). our L L c Fig. 2a). This L -DOPA than -DOPA pro- -DOPA and -DOPA h and Fig. 1a and . jalapa, M. e m L b -DOPA i o . 186 29 - - - - - .

© 2015 Nature America, Inc. All rights reserved. Nature chmical biolo gy DOD. CYP76AD1 yielded 1.3 mg l mg 1.3 yielded CYP76AD1 DOD. percent ofthemodeeachpopulation. the desired activity. However, many enzymesthatproduce See SupplementaryFigure 1for amore detailedpathway diagram. (b)Fluorescence microscopy activity. Red arrows denote enzymaticreactions; black arrows denote spontaneousreactions. (a) were predictive of predictive were production, as the betaxanthin levels from AbPPO2 and CYP76AD1 17 of the highest betaxanthin producers for sequence analysis and analysis sequence for producers betaxanthin highest the of 17 betaxanthin the amplify ( signal to fluorescence filter emission amber-green an and colonies epi light blue of aid between the with eye by detected be could differences obvious that large sufficiently was clones library between production betaxanthin in difference The an average mutation rate of approximately four mutations gene. per This mutant library contained approximately 200,000 members with DOD.expressing strain yeast a into transformed was thatmutants We performed error-prone PCR to generate a library of CYP76AD1 Improving tyrosine hydroxylase activity L for tested we CYP76AD1, oxidase activity of CYP76AD1. DOPA competing the given yeast in accumulate to likely are that to forward scatter to account for variation incell size. C scatter asindicated inSupplementaryFigure 3 supplemented witharange of histograms for DOD-expressing yeast cells (andawild-type control, orange) grown inmedium fluorometer. Error bars represent mean twofold increments. C supplemented witharange of curve for aDOD-expressing strain (blue)andawild-type control strain (red) grown inmedium N terminus, grown inmediumwithandwithout of yeast cells expressing DODwiththered fluorescent protein (RFP plants andwas usedinyeast asan pathway to melanin.DODconverts activity 1)alsohave asecond activity (DOP Figure 1|Development andcharacterization ofanenzyme-coupled c L ab nature CHEMIC activity and set outmutagenesis perform andtoset activity ourandwithscreening titers could improvedbe by abolishing CYP76AD1’s DOPA oxidase anthin biosensor would have utility in further optimizing further in utility have would biosensor anthin AbPPO2.betax resultourthatsuggestedobtainedwith This levels -Tyrosine -DOPA biosensor. Betaxanthin fluorescence fe aheig lal dtcal btxnhn ytei with synthesis betaxanthin detectable clearly achieving After L

(fold over background) 100 -DOP 10 1 10 –1 A canbeacted onby three distinctenzymes.For theproduction ofBIAs,DODCis 1 L 10 -Dopaquinone – DOD + DOD Dopamine 0 AL BIOLOGY L -DOPA L DODC -DOPA (µM) 10 2 L 1 ellular betaxanthin fluorescence was measured onamicroplate ). We hypothesized that hypothesizedWe b). 2 (Fig. titers -DOPA DOD ). We visually selected selected visually We 5). Fig. Supplementary 10

| Adv 2 L L -DOP -DOP L DP pouto i te bec of absence the in production -DOPA ance online public (e.g., morphine) 10 Betaxanthin 3 L Melanin -DOP L -DOP A concentrations. C A concentrations ranging from 10,000 BIAs 10 ± s.d.oftwelve biologicalreplicates. (d)Flow cytometry −1 4 A biosensorto optimize tyrosine hydroxylase

A into thefluorescent, yellow pigmentbetaxanthin in L A oxidase, activity 2)thatintroduces acompeting -DOPA, almost 20 times the the times 20 -DOPA,almost . Betaxanthin fluorescence was normalized L L -DOPA (µM) -DOP d d o a Normalized count L-DOPA tion | i 100 : 10 A. Scalebars, 10 40 80 60 20 DOD 1 mM 0 mM ell counts were normalized asa 0 ells were gated by forward andside 10 . 10 –3 DICR L www.nature.com/naturechemicalbiology 5,000 -DOP -illumination 38/n Betaxanthin fluorescence/forwardscatter A (tyrosine hydroxylases, L 05 c 10 -DOPA PDDBtxnhnMerge Betaxanthin FP-DOD ) mKate2 fusedto the h –2 L e -DOPA biosensor. m μm. (c b i - o 07 1 ,5 5,000 1,250 312 78 20

μM to 0.15 μMin 10 . 186 ) Dose-response –1 aminel mgtiterof 1.5 in demonstratedpreviously been not has knowledge our to that thing single strain, both activities resulted in dopamine production, some measurements for F309Lmutant. the and W13L F309L mutations improved upon the the upon improved mutations F309L W13L and F309L W13L, the withDOD, ofCYP76AD1 absence Inthe 8). Fig. double mutant compared to wild-type CYP76AD1 (Supplementary improvement4.3-foldable,showing a forthe fluorescence mean in cytometry,flow via mutant the populations were distinguish easily - F309L single mutant. When betaxanthin fluorescence was compared fold over wild-type CYP76AD1 with a Z-score of 10.0 relative2.7- to the of fluorescence betaxanthin in increase net a showed mutant on a microplate fluorometer ( eaati fursec ad nrclual produced intracellularly and fluorescence between correlation linearbetaxanthin strong a was there Overall, 2b). (Fig. wild-type CYP76AD1 by 1.9-fold, 1.4-fold and 2.8-fold, respectively (R highly highly specific for from (DODC) DOPA decarboxylase a of the downstream BIA intermediate dopamine when expressed with as decarboxylationof as yaie n rdc fu t dopamine to flux reduce and tyramine 2 Uig idtp CP6D, e civd dop- a achieved we CYP76AD1, wild-type Using cerevisiae. S. We next tested the ability of CYP76AD1 to catalyze the synthesis = 0.92), although there was a slight discrepancy between the two

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+++++++– 10 1 L -DOPA on betaxanthin and betaxanthin on effects their analyzed we mutations, best two W13L for convenience. as to This referred be will mutation.mutation double linked W13L the involving - experi ments future in also included was and (9T>C) for enriched W13L to linked was that mutation silent additional An multiplicative. gesting that the effects of these mutations were the incorporated sug - F309L), and (W13L mutations two had same 20 library, shuffling DNA the from sequenced we that producers format microplate (SupplementaryFig.Outhighestof 7). 28 the a in done was screening visually liquidin medium, fluorescence and secondary We 67 high-fluorescenceselected colonies to grow measurements. plate-based and fluorescence screening visual combi- of a nation through production betaxanthin for screened and DOD expressing yeast into transformed again mutations. was library resulting beneficial The potentially randomly combine to library shuffling DNA a struct con to used were mutants six These 6). Fig. mrvmn i btxnhn production the into betaxanthin retransformation and 1.5-foldisolation upon a in than less improvement showed that proteins removingduplicate genotypesand any mutant distinct two genotypes. were there mutation, this with those amongmutation; missense F309L same enteen clones that we sequenced contained microplate the sev using the of Six shown). not (data fluorometry activity of confirmation 62 n 2., epciey we measured when respectively) 21.0, and of 16.2 (Z-scores respectively betaxanthin, more 1.9-fold and 1.7-fold yielded mutants single F309L and W13L the to CYP76AD1, wild-type Compared combination. in and vidually L Supplementary (Supplementary strain base sensor -DOPA L −1 -tyrosine would introduce a side pathwayside to introducewoulda -tyrosine ( Because Because W13L and F309L seemed to be our e arwd u st f is o i by six to hits of set our narrowed We ). Both the W13L and F309L single andF309L W13L the Both 2c). Fig. 34 . The specificity of DODC is important, Fig. 2b). When combined, doublethe Pseudomonas putida Pseudomonas 12 L We cmie it a into combined When . -DOPAindi- production article L DP ttr of titer -DOPA L -DOPA that is is that 3 - - -

© 2015 Nature America, Inc. All rights reserved. article e netgtd oeta cags n aayi atvt resulting activity catalytic from mutation. this in changes potential investigated we Therefore, abundance. protein in increase 1.6-fold a fully by be explained cannot mutation F309L the by caused titer dopamine in production caused by the W13L mutation, the 3.8-fold improvement level of dopamine production to an unexpectedly high 10.8 mg l mg 10.8 high unexpectedly an to productiondopamine of level tively). The W13L F309L double mutant of CYP76AD1 increased the increase intracellular tyrosine levels (Aro4 to known Aro4p, of mutant resistant feedback overexpressinga strain A +. a with indicated A. bisporus fluorescence onamicroplate fluorometer. Fluorescence values were normalized to the complete medium with(Fig. 4%glucose 2c). isolated with the betaxanthin biosensor screen. Yellow bars represent betaxanthin fluorescence Yellow fluorescence screen. betaxanthin biosensor represent bars betaxanthin the with isolated complete medium.(b ees I ti sri bcgon, e civd fnl dopamine final l a mg 23.8 of titer achieved we background, strain this In levels. dopamine in increase 2.2-fold additional an in resulted so Doing CYP76AD1 a level 7.4-fold greater thanenzyme. that wild-type inthe increase the intracellular the increase sufficient to explain the improvements in improvements the explain to sufficient reticu endoplasmic 11 Fig. the (Supplementary to lum CYP76AD1 of localization affected mutation neither that showedproteins fusion these of microscopy ( fusion protein fluorescent wereconsistentmeasurements proteinwith of expressionvia made and 3a (Fig. expression in increase 1.6-fold a to led mutation F309L the whereas protein, blot analysis showed that the W13L mutation yielded 2.8-fold more Westernexpression. enzyme in changes at looking by started and activity hydroxylase tyrosine increased to led mutants CYP76AD1 our which by mechanism the characterizing Wein interestedwere Characterization ofreduced DOPA oxidase activity for observed been had than largerconsiderably was titer dopamine mutanton F309L the of effect the fact, in production; dopamine improvedmutants Error bars indicate mean standard curve. T coexpressing mean ± substitutions are listed for enzymemutants(76AD1 represents C measured by LC/MS ofculture supernatantsandcomparison to astandard curve. Aminoacid L hydroxylase. tyrosine no expressed that strain control a of autofluorescence with variant hydroxylase tyrosine a coexpressing cells in expressing DODaloneorwithacandidate tyrosine hydroxylase (either AbPPO2from Figure 2|Isolationandimprovement ofatyrosine hydroxylase inyeast. 4 hrfr, e vrxrse a edakisniie uat of mutant feedback-insensitive the a overexpressed we Therefore, strain. our in production dopamine limiting be might availability ab -

DOP bP2CYP76AD1 AbPPO2 Given the high tyrosine hydroxylase activity of the the of activity hydroxylase tyrosine high the Given L -tyrosine pathway enzyme Aro4p that is known to markedly to known is that Aro4p enzyme pathway -tyrosine A titer after 48 h of growth for strains expressing a candidate tyrosine hydroxylase as as hydroxylase tyrosine candidate a expressing strains for growth of h 48 after titer A s.d.offour biologicalreplicates. (c None orC W13L F309L DODC YP76AD1 from he presence oftheW13Land/or F309Lmutationinagiven variant is −1 and a a and when cells were grown for 48 h in 2× synthetic 2× in h 48 for grown were cells when ) Quantification of tyrosine hydroxylase activity for enzyme variants variants enzyme for activity hydroxylase tyrosine of Quantification ) double mutant, we hypothesized that L -DOPA titer (3.8-fold versus 1.4-fold, respec 1.4-fold, versus (3.8-fold titer -DOPA ± s.d.offour biologicalreplicates. C Betaxanthin fluorescence L Y . hs changes These 9). Fig. Supplementary -tyrosine concentration-tyrosinein

P (fold over background) ), streaked on an agar plate with synthetic synthetic with plate agar an streakedon B. vulgaris), 40 20 30 Supplementary Fig. 10 Fig. Supplementary 25 35 10 76A 15 0 5 . lhuh nrae epeso is expression increased Although ). bP27A176AD1 76AD1 AbPPO2 D 1 mutant as measured by by measured as mutant 1 L Betaxanthin -DOPA ) ) FBR D ), was alsotested withselectC opamine titers in the culture supernatant of cells cells of supernatant culture the in titers opamine W13L L -DOPA and dopamine and -DOPA 76AD1 F309L DOD 76AD1 F309L ). Fluorescence ). W13L S. cerevisiae S. nature as measured by cellular cellular by measured as LC 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 L

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a al YP76AD1 variants. B ) Yeast cells –+ – lue bars show show bars lue rz ad oe t voe color violet its lose and erize F309L mutation would have similar effects in these orthologs, we orthologs, these in effects similar have would mutation F309L conserved ( is residue each this in that showed which CYP76AD4), of and alignment protein a performed CYP76AD3 itsknowntothreeorthologs(CYP76AD2, CYP76AD1 we activity, oxidase DOPA a downstream enzyme. amine titers than direct measurement of dop of predictive more were titers betaxanthin fact, In 15). Fig. amine levels measured in measured levels amine dop the to correlation linear strong a showed mutations the by quantifiedLC/MS,changes the via tyrosine-betaxanthinin caused When production. tyrosine-betaxanthin in increase 3.7-fold a by CYP76AD1 ( levels decreased by 80% in the F309L mutant, compared 14 Fig.to (Supplementary acid wild-type betalamic with amine an of condensation spontaneous the from form can betaxanthins different many as species betaxanthin the of memberrepresentative a as selected LC/MS. Tyrosine-betaxanthinwas using betaxanthin and betanidin measured we natants, reduces DOPA oxidase activity. F309L To W13L better quantify the the culture for double super mutant. observed This change demonstrates also that the F309L mutation was which yellow, to from violet color in shift a produced CYP76AD1 into mutation point change not ( did color violet overall the supernatant, culture the in pigments of concentration the increased mutation W13L the ( DOPAactivity oxidasehigh indicating violet, was tested in this betaxanthin-betanidin assay, the supernatant was CYP76AD1 wild-type when Asacid. expected, andbetalamic thin generate a yellow supernatant that is composed mostly of - betaxan will activity oxidase DOPA low with enzymes whereas betanidin, of levels high containing supernatant violet a produce should ity - activ oxidase DOPA high with hydroxylases tyrosine candidate biology ). In contrast, incorporation of the F309L contrast, the incorporationInof 13). Fig. Supplementary +– ––F309L o ute ivsiae h iprac o te 39 eiu on residue F309 the of importance the investigate further To

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). This decrease in betanidin was accompanied dation, which causes the pigment to polym to pigment the causes which dation, reducing a oxi betanidin as spontaneous prevent to agent acid ascorbic with medium required Doingso supplementing growth the yeast. in activity oxidase DOPA of indicator and form a violet pigment called betanidin ( the the with react can acid betalamic however, dase, catalytic activity. As stated previously, the the previously, from pathway stated As activity. catalytic CYP76AD1’sin changeapparent this analyze to pathway betaxanthin the to turned again hc udros pnaeu condensa 1 Fig. (Supplementary amines spontaneouswith tion acid, undergoes betalamic which intermediate the through uniiain f eai ad t precursor, its and melanin of quantification Because of challenges associated with accurate by generated CYP76AD1’s DOPA oxidase activity ( melanin of accumulation the to due likely most was color brown this that ( incorporating mutation F309L CYP76AD1 the of version a ing were brown in color compared to mutantcells express single W13L the or CYP76AD1 type feedback-insensitive wild- expressing cells that noticed we Aro4p, overexpressing tures L L -dopaquinone is generated via a DOPA oxiDOPA a via generated is -dopaquinone we ), 12 Fig. (Supplementary -dopaquinone e sd eaii pouto a an as production betanidin used We While pelleting pelleting While L Supplementary Fig. 1). -dopaquinone derivative cyclo-DOPA tocyclo-DOPA derivative -dopaquinone Figure 2c Figure a ). Our results showed that betanidin thatshowed results Our ). tion | 3 6 We cepesd ih DOD, with coexpressed When . www.nature.com/naturechemicalbiology L ( -DOPA to betaxanthin goes goes betaxanthin to -DOPA R d 2 = 0.999; = o L L -DOPA in the absence of i - cul -DOPA–producing : 10 ). We suspected suspected We 3b). Fig. . 10 Fig. 3d Fig. 38/n Supplementary Supplementary c h ). Although ). e m Fig. 3c). b Fig. 3c i o . 186 ). If ). ------

© 2015 Nature America, Inc. All rights reserved. Nature chmical biolo gy the reducing agent ascorbic acid. Relative levels of betanidin production production Relativebetanidin levels of acid. ascorbic agent reducing the curves generated by serialdilutionof thesamplesto obtainlevels of diagram. (d variants.pathway SeeSupplementaryFiguremore detailed a for 1 in thesupernatantsfrom d levels (violet) (yellow) betanidin and tyrosine-betaxanthin of analysis to produce betaxanthins (a yellow family of pigments) or with the the with or pigments) of yellow family (a produce betaxanthins to the intermediate betalamicacidthatcaneitherreact withfree amines cated tyrosine hydroxylase for betanidin synthesis betanidin for hydroxylase tyrosine cated dedi a ofpresence the suggestingproduction persists, betaxanthin inactivation of CYP76AD1 abolishes betanidin production, whereas predicted active site. enzyme’s the from Å 14 was residue the as CYP76AD1’sbehavior mutation’sF309L on the effect of mechanism the into insight little fied fied through PCR mutagenesis ( identi had we that substitution F309L same the contained seven all that found and genome beet sequenced recently the from logs para closest seven its to sequence protein CYP76AD1 the aligned from supernatantsyellow( to violet shift a produced mutation the of introduction cases, the of all assay.In betaxanthin-betanidin the in mutants F309L and type wild- testedand yeast expressionfor genesin the codon-optimized sample. Error bars represent mean wild-type relativethe to tyrosine-betaxanthin and betanidin supernatant. culture the in exist species different many as betaxanthins the of representativemember acid. Negative control indicates noC a and comparewere to used pigment). L control denotes astrain notexpressing C cells expressing C intensity, normalized to wild-type (WT)C anti-GA to intensity anti-Flag ratioof representsexpression the anti-GA An of C DOPA reduced of Characterization in Figure| 3 activity oxidase nature CHEMIC human CYP1A2 ( CYP1A2 human but a homology model was generated by threading CYP76AD1 onto unsuccessful, were CYP76AD1 for structure crystal a determine to Efforts shown). not (data production betaxanthin no observed but two closest paralogs for tyrosine hydroxylase activity in -dopaquinone derivative cyclo- derivative -dopaquinone a c CYP76AD1 YP76AD1 mutants. expression C Relative GAPDH Y P C 76A Y Betaxanthin L Betalamic -Tyrosine L P -DOPA acid 76A B oto TW3 F309L W13L WT control D PD ) ) .×10 .×16 3.2× 1.6× 2.8× 1.0× 0.0× etanidin is labile but can be stabilized with the addition of of addition the with stabilized be can but labile is etanidin Neg. DOD 1 mutants in yeast. yeast. in mutants 1 C 1 H antibody was used as a loading control.Relative loading a as used was antibody H D ulture supernatant from strains coexpressingfrom strains supernatant ulture AL BIOLOGY 1 mutant grown in minimal medium with ascorbic ascorbic with medium grown minimal in mutant 1 2 YP76AD1 mutants(andAro4 Supplementary Fig. 19 Fig. Supplementary ( L a -Dopaquinone DOP Cyclo-DOPA ) Western blotcomparing expression level Betanidin Melanin

. . | Adv A oxidase activity (activity 2) in in 2) (activity activity oxidase A T yrosine-betaxanthin was selected as a as selected was yrosine-betaxanthin DOP E ance online public P nzymes contain a a contain nzymes eak areaswererelative to standard eak fit Supplementary Fig. 17 Fig. Supplementary F309L W13L ± Supplementary Fig. 18 s.d.offour biologicalreplicates. A to produce betanidin (a violet violet (a produce betanidin to A YP76AD1 expression. ( YP76AD1. ( YP76AD1. ( d b e

Production relative to WT control control FBR ) 0 4 8 6 2 5 7 3 1 38 d ). ). . The model provided model The . C TW3 W13L W13L WT o a T WTNeg. c tion | -terminal Flag tag. tag. Flag -terminal he negative ( negative he i b ) ) : 10 Betanidin Tyr-betaxanthin ) ) DOD P 32 W13LWTNeg. . W13L elleted yeast elleted 10 . We tested the the Wetested . www.nature.com/naturechemicalbiology e C DOD C 38/n ). We). further generates ) ) Y Y F309L ) S. cerevisiae F309LW LC F309LW 37 P P PD 76A . In beets, 76A /MS c

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b i - - - o . 186 was the (S AB strain by producednorcoclaurine the of all that confirmed ysis and and was also clearly detected in the supernatant of strain ( ABC levels were also measured using extraction with methanol. Less Less methanol. (S with total of 3% than extraction using product measured Intracellular also were saturation. levels cell after increases substantial from from ( poppy californica California the from isolated been however, commerciallyavailable,ishas, expensive. NMCH which albeit line, norlaudanosonorcoclaurine, of derivativehydroxylated a feeding in reticuline synthesize to work Previous CYP80B1). as known also (NMCH; hydroxylase The remaining enzyme is the cytochrome P450 Having achieved efficient dopamine production from production dopamine efficient achieved Having Production of(S)-reticuline from glucose ( code accession (GenBank a to homology with variants NCS four code accession from NCS an included B Module levels. P. somniferum have been demonstrated to function in methyltransferase (4′OMT). Variants of each methyltransferase from ( branchespathway BIA major the ( produce to needed 4-hydroxyphenylacetate (4-HPA) by ahost of redundant enzymes. and transamination orbroken intotyrosol downbefore being decarboxylationreactions sequential through 4-HPAA to converted is u sris i oeepes edakisniie r4, which Aro4p, feedback-insensitive increases flux through 4-HPAA in addition overexpress to increasing did strains our although production, 4-HPAA for enzymes yeast endogenous on NMCH from 4 and CNMT 6OMT, including ( of conversion the for required ( (CYP76AD1 aha fr mn ai catabolism acid amino for pathway in endogenously ( NCS by catalyzed is that reaction a (4-HPAA), 4-hydroxyphenylacetaldehyde and dopamine of sation ( molecule backbone the offormation BIA intermediates. The first committed step in BIA biosynthesisdownstream towardis pathway the the extend to sought we cerevisiae, S. be be stereoselective, producing only ( to known is NCS racemic, is norcoclaurine formed spontaneously ee 104.6 were ( for titers Maximum 23). Fig. fermentation in shake flasks for strains AB and ABC ( ( ( to from efficiency conversion the that ( indicating detectable no had strain ferase (6OMT), coclaurine 6-O-methyltransmethylationreactions: - catalyze enzymes these of systems A, norcoclaurine synthesis (which can occur spontaneously in other Given that this product was not observed in the supernatant of strain when grown in synthetic complete medium with glucose ( a on maintained or B) and low-copy plasmid (module C). A (modules chromosome are the into strains these in genes integrated either are heterologous promotersand high-strength using expressed All ABC). and AB (A, express they that modules pathway the by here to refer we which Supplementary Fig. 21) Fig.Supplementary Fig. 4a rm ( From e iie te ( the divided We tan B rdcd aiy eetbe ees f norcoclaurine of levels detectable easily produced AB Strain S )-reticuline was high. To quantify titers, we performed a 96-h 96-h a performed we titers,To quantify high. was )-reticuline upeetr Fg 22d Fig. Supplementary Spodopterafrugiperda 1 ). Module A combines our best tyrosine hydroxylase mutant 3 ) seemed to be dependent on NCS expression. Although expression. NCS on dependent be to seemed ) )-enantiomer (Supplementary Fig. 22a – ) and successfully expressed and purified heterologously purified and expressed successfully and ) -occarn, or diinl nyai ses are steps enzymatic additional four S)-norcoclaurine, μ W13L F309L W13L

l g E. E. californica −1 KP262411 n 80.6 and as an intermediate of the Ehrlich Ehrlich the of intermediate an as cerevisiae S. )-norcoclaurine and (S and)-norcoclaurine ) and DODC to produce dopamine. Wedopamine.produce relied to DODC and ) -eiuie te at hrd nemdae of intermediate shared last the S)-reticuline, S -eiuie aha it tre modules three into pathway )-reticuline circumvented this enzyme by enzyme this circumvented cerevisiae S. 4 1 . We constructed three strains for testing, ACO9 . Module C consists of the fourenzymes consists Modulethe ofC . N-methyltransferase (CNMT) and 4 , hc ws eetd fe screening after selected was which ), Sf9 cells μ S -occarn i te supernatant, the in )-norcoclaurine l g S – S −1 )-norcoclaurine and ( and )-norcoclaurine 0248.1 f )-norcoclaurine to ( to )-norcoclaurine ′ . h (S The ). rsetvl, n dd o show not did and respectively, , M from OMT S Supplementary Fig. 20 Fig. Supplementary )-norococlaurine 40 3 Fig. 4a Fig. 9 . S I ti pathway, this In . )-norcoclaurine via conden via )-norcoclaurine ) for activity in activity for ) )-reticuline was observed observed was )-reticuline P. somniferum P. hlcrm flavum Thalictrum ). 4-HPAA is produced 4-HPAAis ). )-reticuline–producing N-methylcoclaurine . somniferum P. article c S ). (S )-norcoclaurine Supplementary Supplementary 4 S. cerevisiae 2 S . Chiral anal L S Eschscholzia )-Reticuline )-Reticuline )-reticuline, S. cerevisiae S. -tyrosine in in -tyrosine )-reticuline )-reticuline (GenBank (GenBank Fig. 4b, L L -tyrosine -tyrosine Fig. 4d, ). Three ). NCS NCS and and ′-O c 14 ). ). e 5 - - - .

© 2015 Nature America, Inc. All rights reserved. article ecule, ecule, betaxanthin, that is easy to detect. This tool helped us identify (c,d technology fortechnology production. industrial-scale of discovery in the new enzymes and pathways and the commercial accelerating deployment platform of this synthesized, be microbial can BIAs a all establishes which advance This cerevisiae. the S. of biosynthesis demonstrated we ( point branchBIA major NCS, identified newly a into medium. the isms tools and techniques for controlling the metabolism of microorgan new developmentof the spur to likely is that challenge engineering complexity of the BIA pathway also presents a formidable metabolic potential to exhibit new pharmacological properties. The length and and use widespread their given target engineering attractive an are to promises discovery.manufacturingandpharmaceutical BIAs transformboth therapeutics plant-derived of production Microbial DISCUSSION escape readily products these that indicating pellet, cell the in in comparison to chemicalstandards. P norcoclaurine from strain AB( reticuline. T Strain ABisengineered to produce norcoclaurine, andstrain ABCisengineered to produce expressing 48hofgrowth modulesA,ABandBCafter insynthetic mediumwith4%glucose. See SupplementaryFigure 20 not heterologously expressed. C divided into three modulesto facilitate analysis. Because4-HP Figure 4|Production of( e b a 6 plant enzyme to convert by a new strategy for intracellular monitoring of future dispensing of for need the large-scale dedicated crop cultivation. for alterna an point therebyBIAs, high-value to producing for method lead cost-effective starting tive, eventually to a expect marks we which strain optimization, generation first this ( intermediatekey the synthesizingstrain yeast developmenta of the in S.cerevisiae ‘Std’ denotes a5-μMchemicalstandard, whichwas normalized separately. (b,e)MS/MS of HO HO HO S Relative Relative )-reticuline from glucose. Although initial titers are low (80.6 Thus, by combining our engineered tyrosine hydroxylase with hydroxylase tyrosine engineered our combining by Thus, (

) LC/MS analysis ofnorcoclaurine ( ion abundance ion abundance S 1 )-Reticuline production from central metabolites was enabled was metabolites central fromproduction )-Reticuline 6 L . We report a major breakthrough in microbial BIA production: -Tyrosine L -DOPA 137.1 137.1 107.0 107.0 CYP76AD1 races are normalized to themaximumpeakheightacross allthree samples. 123.0 123.0 NH NH (unlabeledarrows), enzymescatalyzing4-HP 175.1 175.1 2 2 COOH COOH 192.1 192.1 Module A 161.1 161.1 DODC m/z m/z S)-reticuline from glucose. (a HO HO L 4-Hydroxyphenyl- HO Norcoclaurine standard -DOPA into a highly fluorescent small mol- acetaldehyde )-reticuline in the industrial workhorse industrial the in S)-reticuline b Dopamine for amore detailedpathway diagram ofmoduleC. Reticuline standard ) andreticuline from strain ABC( YP76AD1, tyrosine hydroxylase (W13LF309doublemutant). 299.1 299.1 + 255.1 255.1 330.2 330.2 272.1 272.1 NH arent ionsare marked withafilleddiamond. c) andreticuline (d)inthesupernatantofstrains ABC 2 NCS AB O c HO HO HO Module B

Normalized intensity (S)-Norcoclaurine 0.4 0.8 0.6 0.2 1.0 0 91 Retention time L Norcoclaurine -DOPA that used a ) T H (min) 01 he ( AA synthesis from 11 nature NH AA isproduced endogenously S)-reticuline pathway was 2 6OMT e) confirm theiridentity A AB CNMT ABC Std chemic μ NMCH g/l), g/l), 4′OMT Nature chmical biolo gy d - -

Normalized intensity Module C 0.4 0.8 0.6 0.2 al 1.0 0 213 12 L optimization. endogenous factors that limit flux will be important for future strain (S of yields increased v the in available are references associated any and Methods Methods published online18May 2015 R in ucts tyrosol and 4-HPA and tyrosol ucts 4-HPAA, both of which are derived from derived are which of both 4-HPAA, trans yet-to-be-identified porter.Finally, productioncarefullybalancing the of dopamine and a by secreted is intermediate this that suggesting supernatant, the in found was strain our by produced oe de i pof Drn te eiin f hs manuscript, this of revision the During al. et Fossati proof: in added Note minimizing potential the for abuse.while benefits considerable the garner can we that so established be strains such controlling for policies appropriate that it critical derivatives, is its and morphine including products, these illicit of for use potential the of Because accelerate. will feedstocks able strain capable of high-yielding BIA fermentation from readily avail intermediate key improve,continuetotools progress towardbiology a synthetic and the of production (S)-reticuline to linking yeast central metabolism. As metabolic engineering by synthesis BIA biology Retention time -tyrosine were MeO MeO er eceived 11March 2015; accepted 9April2015; HO HO S.cerevisiae.

ee e ae ecie a onainl dac i microbial in advance foundational a described have we Here Reticuline sio (min) (S)-Reticuline n o 14

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h A AB

44 ance online public e p ABC NMe described the synthesis of codeine from ( from codeine of synthesis the described Std a p er . n neial sd ptwy o eai, we our used melanin, to pathway side undesirable an second activity a (DOPA oxidase) had that introduces CYP76AD1 wild-type Although yeast. a in biosynthesis BIA filled early in thatgap longstanding (CYP76AD1) beets from P450 previouslya unknowncytochrome a in activity example, (for interest of metabolites other of duction pro the to By fluorescence FACS. betaxanthin linking to amenable are screening that techniques high-throughput new of opment studying transport or as a model pathway for the devel for tool valuable subcellular enzyme a localization and be metabolite could way path betaxanthin fluorescent, the product, water-soluble its of Because useful. generally more prove could pathway this that believe we activity, hydroxylase tyrosine optimize to way as an enzyme-coupled and increases toward flux (S mutation that suppresses DOPA oxidase activity (104.6 228-fold lower production of (S aha ezms ol b cnrbtn to contributingprod side the 4-HPAAintoof breakdown the be could Ehrlich enzymes potential pathway 20 than More step. this at inefficiencies for blame to largely are enzymes metabolite biosensors. robust by enabled is that progress rapid the of example an is work This addi molecules. tional for screens high-throughput develop dpmn ttr f 38 g l mg 23.8 of titer dopamine a achieved we Although feasible. is BIAs stream down of production before required be will )-norcoclaurine. Addressing these and other and these Addressing )-norcoclaurine. lhuh e sd h btxnhn path betaxanthin the used we Although ute otmzto o ( of optimization Further 4 3 . Additionally, the majority of dopamine of majority Additionally, the . μ g l g L a L -DOPA biosensor to identify a point a -DOPA identify to biosensor −1 tion | trsn) i mgt e osbe to possible be might it -tyrosine), ). It is likely that yeast’s endogenous www.nature.com/naturechemicalbiology d o i : 10 L -tyrosine, may lead to lead may -tyrosine, . 10 L )-reticuline. 38/n -DOPA biosensor S -eiuie titer )-reticuline −1 )-norcoclaurine , we observed observed we , c R)-reticuline h e m b i o o n . 186 lin e ------

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. 186 39. 38. 37. 36. 35. 34. 33. 32. 30. 29. 44. 43. 42. 41. 40. 31. Supplementary information is available o inthe A o The authors declare competing interests: financial details accompany the Competing financialinterests help from and L.N. Z.N.R. research. the W.C.D.,supervised V.J.J.M. and J.E.D. wrote manuscript the with editing preliminary studies. W.C.D., and L.N.analyzed Z.N.R. results. the V.J.J.M. and J.E.D. experiments, the performed and L.N.conducted chiral analysis. A.M.G.assisted in W.C.D., L.N.,V.J.J.M. Z.N.R., and J.E.D. research. the designed W.C.D. and Z.N.R. A Chair (V.J.J.M.) Research Canada a and Québec Genome Canada, by Genome supported financially was lab Martin the in Research Z.N.R.). to (fellowship ofDefense Department US W.C.D.)Foundation to J.E.D.),(fellowship to (grant National US Science the the and award DE-SC0008084 number under EnvironmentalResearch) and ofBiological (Office Program Research Career Early ofScience Office ofEnergy Department US bythe supported was biosensor enzyme-coupled an work onengineering The yeast. in active enzymes NCS identifying contributionin forJ. their and Bourgeois Scrivens L. and manuscript; preparation ofthe the in and project the throughout feedback for valuable Lee, M. particular in Labs, Dueber and Martin ofthe members training; for LC/MS Bauer S. experiments; preliminary Wewith for assistance Lee H. thank A permissions information is available online at h h f t t m dditional information uthor contributions cknowledgments h 257–270 (2006). 257–270 ratio. oxidase hydroxylase/dopa tyrosine high abnormally an tyrosol by tyrosol (2007). betanidin. on tyrosinase of activity the betalains. of biosynthesis the 4,5-DOPA-extradiol-dioxygenasein vulgaris active Beta recombinant (2008). 1190–1197 for morphinan alkaloids in alkaloids morphinan on research of century a production: alcohol fusel for pathway Ehrlich The J.R. (2007). in synthase norcoclaurine (1998). 793–801 biosynthesis. alkaloid benzylisoquinoline of mono-oxygenase P-450–dependentjasmonate-induciblecytochrome methyl new a (CYP80B1), 3′-hydroxylase(S)-N-methylcoclaurine encoding alleles two of expression 2259–2266 (2008). 2259–2266 impact. metabolic of quantification biosynthesis: acid amino aromaticcerevisiae models and associated resources. associated and models required for red betalain production. betalain red for required interest. biotechnological of metabolites specialized producingplants non-model of Beta vulgaris). (Beta beet Microbiology rhizosphere. the in allelochemical 3,4-dihydroxyphenyl-an l-alanine, colonizer root the from Sentheshanmuganathan, S. & Elsden, S.R. The mechanism of the formation of formation the of mechanism The S.R. Elsden, & Sentheshanmuganathan,S. U.Pieper, J.C. Dohm, F.of García-Carmona,Characterization & Gandía-Herrero,J.F., Escribano, M.A.H. Luttik, T.Koyanagi, recombinantF.of García-Carmona,CharacterizationGandía-Herrero,F. & G.J.Hatlestad, ofF. Grieco,Production & C. Perrotta, S., Spagnolo, G., Bleve, C., Lezzi, screen high-throughput Melanin-based G. Stephanopoulos, & C.N.S. Santos, Fossati, E., Narcross, L., Ekins, A., Falgueyret, J.P. & Martin, V.J.J.J.P.Martin,Falgueyret, & ofA., Synthesis Ekins, L., Narcross, E., Fossati, J.T.Dickinson, Pronk,A.J.A., & Maris, van J.-M., Daran, L.A., Hazelwood, of analysisF. Sato,Functional & K. Iwasa, E., Dubouzet, H., Minami, M. Xiao, heterologous functional and cloningKutchan,T.M. Molecular & H.H. Pauli, Hernández-Romero, D., Sanchez-Amat, A. & Solano, F. A tyrosinase with tyrosinase F.Solano,A & A. Sanchez-Amat,Hernández-Romero,D., J. Ind. Microbiol.Biotechnol.Ind.J. (2015). l e p . Correspondence and requests for materials should addressed to be J.E.D. a Saccharomycesmetabolism. cerevisiae -tyrosine production in production l-tyrosine p er . Metab.Eng. J. Biotechnol.J. Transcriptome analysis based on next-generation sequencing next-generation on Transcriptomebased al.analysis et ModBase,databaseal. annotateda ofet comparative protein structure Planta Saccharomycescerevisiae. The genome of the recently domesticated crop plant sugar plant crop domesticated recently the of genome The al. et

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n 7 © 2015 Nature America, Inc. All rights reserved. elct clne wr pce drcl fo ti tasomto pae for plate transformation analysis. further this from directly picked were colonies Replicate marker. auxotrophic YIP’s the to corresponding plates dropout onto plated weretransformation. acetateCells lithiumstandard a using yeastinto directly micrograms of plasmid was linearized by digestion with NotI and transformed included regions of homology to either the URA3 or LEU2 locus. Five hundred butreplication of origin yeast a lacked that constructed were(YIPs) plasmids Yeastintegration integration. genomic via made were yeast to modifications tan ws rw wt o wtot mM 1 without or with grown was 2 Strain ( betaxanthin intracellular of analysis microscopy.For Fluorescence FlowJo (http://flowjo.c om package software the using performed was analysis data and prepared, were normalized to forward scatter to account for differences in side cell size. Histograms and forward for in gated shown were as scatter Cells 20. of setting gain a with nm 510 of emission and nm 488 ofexcitation an at fluorescence betaxanthin quantify to used Millipore)was (EMD cytometer flow easyCyteguavacytometry. Flow A or below). aflowcytometer (see fluorometermicroplate a either on quantified was betaxanthin and 7.4), (pH 0.15 to sd fo mtn lbais n te reticuline the and 33; (Strain ABC strain production libraries mutant from Aside construction. strain Yeast Tables 1 depositedand in GenBank, the accession are codes listed in been have work this in used plasmids and strains All locus. LEU2 or URA3 into integration direct the yeast genome via homologous recombination at the for and were of designed replication no origin yeast contained plasmids other All C). (modulepathway enzymesreticuline the forexpressionplasmid the did as transformations, library high-efficiency enable to replication of origin were removed to facilitate plasmid construction. were derived from the yeast genome. Unique restriction sites (BsaI and BsmBI) mids. Promoter and terminator sequences for heterologous enzyme expression L ( BY4741 was article this base The media. growth and Strains ONLINE METHODS nature Gate Assembly Gate vectors. expression Yeast on Lysogeny Broth (LB) containing the antibiotics ampicillin or kanamycin. EPI300 (Epicentre) electrocompetent petent listed inSupplementary Table 1. (US Biological), 20 g/l dextrose). ofAll the strains constructed in this work are out Mix Synthetic minus appropriate amino acids without YeastDrop- Nitrogen Base g/l 2 Chemical), (Spectrum Difco acids amino g/l without (6.7 Base YeastNitrogen medium complete synthetic in performed LEU2 was HIS3) (URA3, and/or markers auxotrophic of Selection dextrose). g/l Bacto g/l 20 20 peptone, Extract, Yeast Bacto g/l (10 YPD in grown were cultures yeast hkn a 3 ° fr 4 h. 24 for °C 30 at shaking on a spinning disk confocal microscope. Strains 41–44 were grown to saturation the protein yellow fluorescent Venus to variant each and enzyme visualization excitation at 500/20nmand emission at 535/30nmfor betaxanthin 560/40 nm and emission at 630/75 nm for mKate2 and filter set 46 (Zeiss) with at excitation with (Zeiss), 45 set filter and (Excelitas) lamp 120 Series X-Cite an using taken were images Fluorescence auto-exposure. using C11440) no. (Hamamatsu camera 4.0 Orca-flash a using captured were DICImages objective.100× a using (Zeiss) microscope D1 Observer an with examined be to slides glass ontoplainspotted and 7.4), (pH PBS in washedcentrifugation, by medium (minus uracil) with 2% glucose overnight. Cultures were concentrated with glucose. supplemented medium fresh 2% into 100× with back-diluted were cultures uracil) Saturated (minus medium complete synthetic in overnight -DOPA titrations for betaxanthin production. betaxanthin for titrations-DOPA Error-prone PCR library plasmids included a CEN6/ARS4 low copy number Golden Golden gate assembly reactions were transformed in TG1 chemically com Intracellular CYP76AD1 localization was analyzed via C-terminal fusion of L -DOPA (Sigma no. D9628) and grown in 24-well or 96-well blocks with blocks 96-wellor -DOPA24-well grownin andno. D9628) (Sigma . Error-prone PCR libraries were transformed in TransforMaxin transformed were librariesError-prone coli PCR . E. M in twofold increments. Cells were spun down and washed in PBS in washed and down spun were Cells increments.twofold in μM chemic and 2 . 45 al . Vector sequences were derived from the pRS series of plas of series pRS the from derived were Vectorsequences . biology Supplementary Figure 3 Figure Supplementary /). MATa Yeast expression vectors were built using Golden Golden using built were vectors expression Yeast L DP cnetain rne fo 10,000 from ranged concentrations-DOPA his3 Supplementary TableSupplementary 1 Δ strain for all experiments in experiments all forstrain cerevisiae S. E. coli 1 leu2 1 . Transformed cells were selected Δ L 0 met15 0 . Betaxanthin fluorescence was fluorescence Betaxanthin . DP i snhtc complete synthetic in -DOPA Strains 1 and 3 were grown were 3 and 1 Strains Δ 0 ura3 0 ), all of the genetic the of all ), Supplementary Supplementary Δ ). Wild-type 0). 24 . Fig. 1b), Fig. μM - -

a NuPAGE Novex 4–12% Bis-Tris gel (Life Technologies) and run for 2.5 h at 80 V. rdcin sa for assay Production fluorescence average the incontrol to observed Strain 4. normalizing by calculated was measurements sn Gle Gt Shuffling shuffled Gate were Golden mutants CYP76AD1 using construction. library shuffling DNA using a standard LiOAc transformation. screening for 3 Strain into transformed was DNA resulting all and prepped, plusLB kanamycin overnight. Two millilitersofsaturated culture mini was were in grown to 500 ml cells the of saturation a After 1-h rescue, cells. petent transformed in its entirety into TransforMax EPI300 (Epicentre) electrocom 10 up,in elutedcleaned again was reaction This 2. Plasmid and product PCR the both of fmol 40 using run was reaction Assembly Gate Golden BsaI A Research). (Zymo kit Concentrator and Clean DNA a using GeneMorph manual, IIuser using an annealing temperature of 45°C. to achieve the desired error rate. The PCR reaction was run as suggested in the Plasmid template was added to the PCR reaction at a concentration1; of 40 ng/(Plasmid PCR for template the of off sequence coding CYP76AD1 the amplify to used Tablewere (Supplementary 3 ) 2 and Technologies).(Agilent1 Mutagenesis KitOligos Random II GeneMorph the Error-prone PCR library construction. 500-cm multiple on plated were transformations yeast mutants,hydroxylase tyrosine of screening For production. betaxanthin improved for screening Library construction.PCR library preppedabove the in for on error-pronescreening section library as described as templates. These PCRs shuffled. were mixed in a Golden Gate Assembly reaction and being mutations plasmids mutant the using of fragments amplify PCR to set used were 5–14 Oligos the distributed equally that regions four ie va CM i te ae of case the in LC/MS for via measured titer were supernatants culture and pelleted, were Cultures °C. 30 at h 48 for Resources) Technical (Appropriate shaker Multitron a in grown were cultures The medium. fresh with blocks 24-deep-well into 50× diluted glucose.withAfter4% overnight uracil) growth, saturated(minus cultures were back- medium complete synthetic 2× of ml 2.5 into picked were Colonies Samples were diluted tenfold in PAGE pelleted, sample buffer, and again8 were Cells NaOH min. 50 in mM resuspended 5 for 200 temperature in room resuspended at water, incubated in and washed pelleted, at were cells °C, OD 30 at 2.5 shaking with growth of h 6 After medium. fresh into 50× overnightback-dilutedand glucose 2% with complete (minusuracil) medium synthetic in saturation to grown were 22–25 and 1 StrainsWestern blotting. production, were pellets photographed directly. growth the to melanin added of case the In product.unstable this was ofpreventoxidation to medium acid ascorbic mM 10 production, dopaquinone n Sfr2 irpae edr TCN (xiain 455 m emission: (ref. 120) nm, gain: 485/5 nm, (excitation: 505/5 (TECAN) reader microplate Safire2 a in were transferred to glass-bottomed microplates and measured for fluorescence cells The 7.4). (pH PBSand resuspendedwashedin were pelleted, Cells °C. 30 at Resources) Technical (Appropriate shaker Multitron a in h 48 for grown cultures Saturated Cultureswereblocks. overnight. 96-deep-well in media freshinto 50× back-dilutedwere grown and glucose 2% with media complete Betaxanthinfluorescencequantification. applied to plates the and through viewed an amber emission high-pass filter. epi light LED blue nm 470 colonies,high-producing of tion most the °C, 30 at growth of intenselyyellow colonies were analysis.isolated To for h further selec the aidin 72 After plate. per colonies 50,000 mately camera (Photometrics) and analyzed using Fiji ( 488-nm excitation laser. Images were taken using a QuantEM 512SC EMCCD confocal microscope (Yokogawa) using a 100× bright-field objective and a and objective bright-field 100× a using (Yokogawa) microscope confocal disk spinning CSU-X1 a with examined be to slides glass plain onto spotted and 7.4) (pH PBS in washed by centrifugation, concentrated were cells °C, 30 in SD-uracil and back-diluted 50× into fresh medium. After 6 h of growth at The PCR was incubated with DpnI for DpnI with 1 up h at cleaned The was incubated PCR 37 being °C before 2 agar plates (with synthetic dropout medium) at a density of approxiof density a at dropoutmedium) synthetic (with platesagar l of PAGE sample buffer and boiled at 95 °C for 5 min. 5 for °C 95 at boiled and buffer PAGEsample of μl L DP, oaie dpqioe n melanin. and dopaquinone dopamine, -DOPA, . od hne vr akrud fluorescence background over change Fold 24). 46 Te oig eune a dvdd into divided was sequence coding The . L DP, oaie n dpqioe For dopaquinone. and dopamine -DOPA, Error-prone PCR was performed using Colonieswere picked into synthetic http://fiji.sc doi:10.1038/nchembio.1816 Supplementary Table 2). Table Supplementary μl was ontoloaded illumination was -illumination / ). μ l of water andwater of l μl - - - -

© 2015 Nature America, Inc. All rights reserved. yoiebtxnhnpruaaati I ( II tyrosine-betaxanthin/portulacaxanthin were taken from the cultureswerefrom the measure totaken OD aliquots points, time designated At (Eppendorf). incubator 44 Innova an in h r.p.m.96 220 forat shaking with °C 30 at grownwere cultures The glucose. 4% with medium synthetic selective 2× fresh of ml containing baffled 50 flasks shake ml 250 into 50× back-diluted were cultures saturated growth, night over After glucose. 2% with medium synthetic selective of ml 2.5 intopicked reticuline. and norcoclaurine for fermentations Shake-flask reader (TECAN). microplateLC/MS analysis was performed as below.described Safire2 a using acquired were supernatants culture of 24 h. were and Cells pelleted, supernatants were Absorbance analyzed. spectra Multitrona in °C atblocks30 shaker(Appropriate Technical Resources)for and oxidation 1 Cultures were mM betanidin tyrosine. grown in 24-deep-well custom our minimal into medium, 50× which wasback-diluted supplementedthen were with They 10 mM glucose. ascorbic 2% acidwith to preventuracil) mg/l 400 HCL, pyridoxin mg/l 400 thiamine HCL, 20 g/l dextrose). inositol, g/l 2 pantothenate, calcium 76 mg/l methionine, 76 mg/l tryptophan,histidine, mg/l 76 380(Difco), vitamins mg/lor acids leucine,amino without Base Nitrogen2 mg/l biotin, 400 mg/l to changes visualize formation. inbetanidin betaxanthin had similar absorbance properties to betanidin, making it difficult neous condensation with betalamic acid to produce PABA-betaxanthin. PABA- componentmedium supernatants.cultureWe in betanidin standardof duction the discoveredthat pro the observe to required was medium assay. Special production Betalain fluorescence values to average the incontrol fluorescence observed Strain 1. Fold changes over background measurements were obtained by normalizing all Safire2 a microplate in reader (TECAN; excitation fluorescence 516/5nm, emission 530/5nm, gain for 100). measured and microplate glass-bottomed a to 100 °C, 30 at shaking with growth of h 24 After medium. fresh into back-diluted and saturation to uracil minus media completesynthetic in blocks 96-deep-well in grownwere 41–44 and 1 Strains of Colonies fusion. protein fluorescent via quantification level Expression XRS imager and (Bio-Rad), blots the was analyzed using Fiji (http://fiji.sc/). ChemiDoc a using chemiluminescence by detected were antibodies HRP the dilutions of 1:5,000 and 1:10,000, respectively. After six 5-min washes in min TBST, 5 for TBST anti-GAPDH (Fisher no. Scientific MA515738HRP) monoclonal antibodies at with twice washed and A8592) no. (Sigma conjugatedanti-Flag HRP with was h 1 for incubated and membrane The milk. 5% with Tween20) (0.05% TBST in overnight blocked Technologies)and (Life buffer Proteinsweretransferredonto transferPVDFmembrane NuPAGE in transfer doi:10.1038/nchembio.1816 masses between 100 Da and 700 Da. Peak identification was additionally additionally was ( identification acid betalamic of Peak extraction ion by assisted Da. 700 and Da 100 between masses electrosprayfragmentorat100-V positive)mode (API-ES voltage, scanning ionization–positive pressure atmospheric in Technologies) (Agilent LC/MS with 4-nm bandwidth. MS was carried out using an attached 6120 Quadrupole (betanidin) nm 536 and (betaxanthin) nm 480 acid), (betalamic nm 405 at detector array a diode using measured was Absorbance min. of 15 course the plus 0.1% formic acid to 65% water/35% acetonitrile plus 0.1% formicacetonitrile acid water/0% over 100% from gradient linear a with eluted were Samples phase column (Agilent Technologies) (Agilent at ~20 °C using a 0.5 ml/min flow System rate. LC Quaternary Infinity Technologies) with 1260 a Zorbax Eclipse a Plus C18 4.6 × on 100 mm-3.5 separated was extract its one-tenth to vacuum volume.original under concentrated was supernatant the Finally, precipitate.remove to RCF 14,000 at centrifuged and min 1 for vortexed was precipitatemixtureand protein.The cells disrupt to added was acetonitrile of 500 incubation, After yeast. the spheroplast to h 1 for °C 37 at incubated 500 resuspendedagainandin centrifuged 7.4), (pH PBSof ml 1 with washed then were cells The cells. collect to min 5 for RCF 3,000 at centrifuged was culture analysis. LC/MS −20 °Cfor later analysis by LC/MS. Cells were first grown to saturation in synthetic complete medium (minus (minus medium complete synthetic in saturation to grown first were Cells Yeast g/l (7.6 PABA lacking medium minimal prepared therefore We HPLC absorbance traces absorbance HPLC l of PBS containing 35 U of zymolyase (Zymo Research). This was then then was This Research). (Zymo zymolyase of U 35 containing PBS of μl Pellet extraction using acetonitrileusingextractionPellet para -aminobenzoic acid (PABA) was capable of sponta- of capable(PABA) was acid -aminobenzoic . Ten microliters of each culture supernatant or or supernatant culture each of microliters Ten . l of each culture was transferred was culture each of μl 600 m m , and the medium was storedmediumwas and the , at / z / 7.1 and 375.119 z 212.055 and 212.055 . One milliliter of each cell cell each of milliliter One . oois were Colonies R μ R t m reversed- 09 min), 10.9 t 11.0 min), 11.0 μl - -

309.110 and 309.110 n rtcln ( reticuline and (iso)leucine-betaxanthin ( Other Other species targeted were eight-point norcoclaurine ( an against quantified and dopamine calibration curve ranging from integrated 0.004 mM to 1 mM in twofold extracted, steps. were min 3.0 identified by exact mass ( mass exact by identified was Norcoclaurine Scientific). (Thermo instrument ICR FT 7T-LTQ a into injected was eluent separation, Following acid. acetonitrile/methanol/acetic 250 μ following the of rate B at 5% a flow min using B, 38–45 95% min B, 5% 35–38 min 0–35 separated gradient: and column the on loaded was supernatant of microliters Ten Chromatography). (FUTECS column CDBS-453 ORpak a Shodex with equipped (PerkinElmer) Micropump 200 a Series using HPLC ( acid ( V. 23 voltage ofbetalamic quantificationofFor energy collision a MS/MS, forfragmentor and, 100-V a with mode (ESI+) electrospray positive in run (Agilent Technologies) for column, fragmentation and mass same detection. The system was the LC/MS with Q-TOF Accurate-Mass 6520 a using but above as collected gradient and pump were data MS/MS and MS Quantitative as ( ion mass exact by identified was Reticuline Scientific). (Thermo instrument ICR eaati (m betaxanthin ( were smoothed with a seven-point boxcar average. were used as standards to determine retention time of each enantiomer. Curves Chemicals Inc.) and ( and and at extraction ion by quantified was Dopaquinone betaxanthin). (tyrosine- 9 Strain or (betanidin) 8 Strain from supernatant concentrated threefold of dilutions twofold repeated by generated curves standard relative eight-point against normalized and integrated were counts ion extracted ChemStation (Agilent Technologies). L ( centrated 10× by SpeedVac, resuspended 1:2 in MeOH and centrifuged at centrifuged and 21,000 MeOH in 1:2 resuspended SpeedVac, by 10× centrated ( MzMine2 and Technologies) (Agilent MassHunter using quantified and standards (Toronto Inc.). TracesChemicals Research were extracted analyzed, ( generatedwereusing curves calibration These respectively. and reticuline, for steps twofold norcoclaurine covering0.078–2.50 six-point calibrationcurves comparedintegratedagainstcountswereand ion extracted these purposes, 46. 45. in H in betanidin betanidin (m dopamine, ion counts with counts ion dopamine, mixture of 72:28:0.001 hexane/isopropanol/diethylamine at a flow rate of of rate flow a ml/min 0.55 at hexane/isopropanol/diethylamine 72:28:0.001 of isocratic an mixture using separated and column the on loaded was supernatant of microliters to Five 40 °C. heated Industries) Chemical (Daicel column OD-H 200 Micropump (PerkinElmer) equipped with a 4.6 × 250 mm CHIRALCEL Reticuline enantiomers were 21,000 analyzed by at standard-phase HPLC centrifuged using a and Series MeOH in Corporation) 1:7 (Waters resuspended then cartridge and C18 Light SepPak a using 200× centrated 33 supernatant was performed by HPLC-FT-MS. Yeast supernatant was con were Curves enantiomer. each of smoothed with time a seven-point boxcar average. retention determine to used were m http://mzm m R,S -dopamine (Sigma) and 4-HPAA (gift from P. Facchini, University of Calgary) LC/MS quantificationof LC/MS Norcoclaurine chiral Norcoclaurine analysis Reticuline chiral analysis / / ONE enzymes. restriction IIs type on based method shuffling DNA one-pot a (2011). e16765 6, constructs. multigene of assembly standardized for system cloning Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: gate Golden S. Marillonnet, & R. Kandzia, R., Gruetzner, C., Engler, modular A S. Marillonnet, Werner,& R., Gruetzner,S. Weber, C., Engler, E., z z )-norcoclaurine, generated through resuspension of an equal ratio of ratio equal an of resuspension through generated )-norcoclaurine, R 2 375.119 and 398.1357 and 398.1357 O, and ( O,and m l/min, where Solvent A was 0.1% acetic acid and Solvent B was 80:20:1 80:20:1 was B Solvent and acid acetic 0.1% was A Solvent where l/min, t 2.8 min; peaks were integrated and reported. For quantification of of quantification For reported. and integrated were peaks min; 2.8 g m / for 30 s. Norcoclaurine enantiomers were analyzed by reverse-phase reverse-phase by analyzed were enantiomers Norcoclaurine s. 30 for z , e5553 (2009). e5553 4, / 212.055 and z 3.7 [M+H] 330.17 ine.sourceforge.net 1 R / 2 S z . Following separation, eluent was injected into a 7T-LTQ FT FT 7T-LTQ a into injected was eluent separation, Following . t 8.8 min), valine-betaxanthin ( valine-betaxanthin min), 8.8 )-norcoclaurine standard (Toronto Research Chemicals Inc.) Inc.) Chemicals (TorontoResearch standard )-norcoclaurine 389.098 and / m z 5.2 and 359.123 R / z t R 10.9 min) and ( betanidin 3.7 [M+H] 330.17 S t 14.5 min). Traces were collected and analyzed using analyzed and collected Traceswere min). 14.5 )-reticuline (gift from P. Facchini, University of Calgary) R t 11.0 min), tyrosine-betaxanthin/portulacaxanthin II m/z 325.139 and m . Determination . of Determination enantiomers reticuline in Strain +

. uhni (R Authentic ). L m R / -DOPA and derivativesand -DOPA z t / 10.3 min) as well as proline-betaxanthin ( S 272.128 [M+H] 272.128 z . Culture supernatant from Strain 32 was con )-norcoclaurine( and / of 154.086 [M+H] 154.086 of ). R t 37 i) n tryptophan-betaxanthin and min) 13.7 + and and m R / R t z 13.1 or 13.4 min), phenylalanine- -eiuie Trno Research (Toronto )-reticuline t 272.121 [M+H] 33 i) Fr quantification For min). 13.3 m m + ). Spontaneously condensed condensed Spontaneously ). / / z z nature CHEMIC μ 311.125 and 311.125 389.098 and M and 0.039–1.25 and M + and a retention time of of time retention a and -DOPA and derivativesand -DOPA R )-reticuline authentic authentic )-reticuline + and and R R t t R AL BIOLOGY m 11.3 min), 11.3 10.3 min), g PLoS ONE PLoS t for 30 s. s. 30 for 10.7 min) / z 196.06 196.06 μ PLoS M in M m / z - - .