Review Article Engineering Microbial Cells for the...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 780145 13 pageshttpdxdoiorg1011552013780145

Review ArticleEngineering Microbial Cells for the Biosynthesis ofNatural Compounds of Pharmaceutical Significance

Philippe Jeandet1 Yann Vasserot1 Thomas Chastang2 and Eric Courot2

1 Laboratory of Enology and Applied Chemistry Research Unit on Vines and Wines in Champagne UPRES EA 4707Faculty of Sciences University of Reims PO Box 1039 51687 Reims Cedex 02 France

2 Laboratory of Stress Defenses and Plant Reproduction Research Unit on Vines and Wines in ChampagneUPRES EA 4707 Faculty of Sciences University of Reims PO Box 1039 51687 Reims Cedex 02 France

Correspondence should be addressed to Philippe Jeandet philippejeandetuniv-reimsfr

Received 13 February 2013 Accepted 30 March 2013

Academic Editor George Tsiamis

Copyright copy 2013 Philippe Jeandet et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Microbes constitute important platforms for the biosynthesis of numerous molecules of pharmaceutical interest such as antitumoranticancer antiviral antihypertensive antiparasitic antioxidant immunological agents and antibiotics as well as hormonesbelonging to various chemical families for instance terpenoids alkaloids polyphenols polyketides amines and proteinsEngineering microbial factories offers rich opportunities for the production of natural products that are too complex for cost-effective chemical synthesis and whose extraction from their originating plants needs the use of many solvents Recent progressesthat have beenmade since themillennium beginning withmetabolic engineering of microorganisms for the biosynthesis of naturalproducts of pharmaceutical significance will be reviewed

1 Introduction

Genetic engineering of cells particularly microorganismscan be successfully applied for the development of strainsdedicated to the overproduction of natural products In factmicrobes are widely used for the biosynthesis of numerousvaluable molecules such as antitumor anticancer antiviralantiparasitic antioxidant immunological agents antibioticsand hormones as well as biofuels [1ndash7] Biotechnology mightrepresent a powerful means for large-scale synthesis ofnatural compounds as the use of microorganisms allows forlow cost and rapid production of biologically activemoleculesthrough an environmentally benign route Such approachesavoid organic solvents heavy metal catalyzers and strongacids and bases that are currently employed upon syntheticchemistry-based routes Some natural compounds includingnicotianamine for instance can reach unrealistic prices ($350000 per gram) On the other hand extraction of valuablenatural products from native plant sources is difficult as thesecompounds generally accumulate in very small amounts alsoneeding the use of many solvents As an example the doses oftaxol needed for the treatment of a single patient requires the

sacrifice of two to four fully grown trees of Taxus brevifoliaFinally microbial fermentations are very easy to transpose atthe industrial level as previously reported for the productionof resveratrol [8]

Currently Escherichia coli on one hand and Saccha-romyces cerevisiae on the other hand are employed for themicrobial synthesis of almost all natural products of interestalthough new platform microorganisms are emerging [6]

This review summarizes without being exhaustive effortsthat have been made since the millennium beginning withthe metabolic engineering of microbes for the biosynthesisof natural products with a particular emphasis on com-pounds showing antibiotic antitumoral antiparasitic antihy-pertensive immunological hormonal and antioxidant activ-ities and belonging to various chemical families includingterpenoids polyphenols polyketides alkaloids stilbenoidsamines and proteins

2 Antibiotics

Among the alkaloid family which are nitrogen-containingmolecules of low molecular weight synthesized by numerous

2 BioMed Research International

plants fungi bacteria and animals benzylisoquinolinealkaloids constitute a remarkable group of pharmaceuticalmolecules including for example the narcotic analgesicmorphine and the antibacterial agents berberine scoulerinepalmatine and magnoflorine Besides these alkaloids havebeen reported such as anti-human immunodeficiency virus(HIV) and antioxidant and anticancer agents (see [3 4]and the references therein) Because these compounds aretoo complex for cost-effective chemical total synthesismicrobial systems for benzylisoquinoline-type alkaloidproduction were used [9 10] but attempts to reconstructthe entire metabolic pathway leading to (S)-reticuline andbeyond this branch-point biochemical intermediate tothe synthesis of many other related alkaloids have beenachieved recently by the group of Minami and coworkers[11] They established a microbial system combining twodifferent microorganisms together with microbial andplant-derived genes Reticuline production was achievedfirst in engineered Escherichia coli and the biosynthesisof specific alkaloids was terminated using a coculture ofengineered E coli and Saccharomyces cerevisiae [11] Thisstudy resulted in (i) the biosynthesis of (S)-reticuline thekey intermediate in the formation of benzylisoquinolinealkaloids only when using a methyl group donor and(ii) the production of specific alkaloids magnoflorine andcorytuberine on one hand and on the other hand scoulerinedepending on the targeted genes Reticuline in engineeredE coli was obtained as follows (Figure 1) dopaminewas converted first to 34-dihydroxyphenylacetaldehydeby a monoamine oxidase from Micrococcus luteus (S)-norlaudanosoline was synthesized by the condensationof dopamine and 34-dihydroxyphenylacetaldehyde bya norcoclaurine synthase from Coptis japonica thenleading to (S)-31015840-hydroxycoclaurine via a norcoclaurine6-O-methyltransferase from C japonica (S)-31015840-hydroxy-N-methylcoclaurine was obtained through the action ofcoclaurine-N-methyltransferase from C japonica leadingto (S)-reticuline via the 31015840-hydroxy-N-methylcoclaurine-41015840-O-methyltransferase from C Japonica When S-adenosyl-L-methionine (SAM) was used as a methyl group donorthe engineered biosynthesis pathway leads predominantlyto the stereospecific (S)-reticuline with a yield of 55mgLwithin 1 h S cerevisiae expressing heterologous geneswere added to E coli cells engineered with the reticulinebiosynthetic genes with 5mM dopamine in the medium aftera certain period of culture S cerevisiae was engineered toharbor the genes encoding the P450 enzyme corytuberinesynthase (CYP80G2) and a CNMT from C japonica(possibly a CNMT-like methyltransferase) leading tomagnoflorine at a yield of 72mgL within 72 h without anyaddition of SAM Otherwise the second alkaloid produced(S)-scoulerine was obtained with a S cerevisiae strainexpressing the berberine bridge enzyme (BBE) from Cjaponica at a yield of 83mgL within 48 h The conversionefficiencies of reticuline to magnoflorine or scoulerine inS cerevisiae cells were 655 and 755 respectively Thisunderlines that such a working system results in the efficientproduction of benzylisoquinoline alkaloids in engineeredmicrobes The system could be generalized and applied to

MAODopamine 34-dihydroxyphenyl

acetaldehyde

NCS

(119878)-norlaudanosoline60MT

-hydroxycoclaurine

CNMT(119878)-3998400-hydroxy-119873-methylcoclaurine

4998400OMT

BBE(119878)-scoulerine

CYP80G2CorytuberineBerberine

Corytuberine-119873-methyltransferase

(CNMT)Magnoflorine

(119878)-reticuline (119878)-reticuline

(119878)-3998400

Saccharomyces cerevisiae

Escherichia coli

Figure 1 Benzylisoquinoline alkaloid biosynthetic pathway recon-structed in Escherichia coli and Saccharomyces cerevisiae For in vivoproduction of (S)-reticuline transgenicE coli expresses biosyntheticgenes MAO NCS 6OMT CNMT and 41015840OMT For in vivo produc-tion of (S)-scoulerine and magnoflorine from reticuline originatingfrom engineered E coli transgenic S cerevisiae expresses respec-tively the genes BBE CYP80G2 andCNMT All genes are fromCop-tis japonica except the geneMAOwhich originates fromMicrococcusluteus MAO monoamine oxidase NCS norcoclaurine synthase6OMT norcoclaurine 6-O-methyltransferase CNMT coclaurine-N-methyltransferase 41015840OMT 31015840-hydroxy-N-methylcoclaurine-41015840-O-methyltransferase BBE berberine bridge enzyme CYP80G2P450 corytuberine synthase

the production of many other alkaloids of medical interest[11]

Recently an important modification of the pathwayleading to (S)-reticuline was reported by the same group [12]An E coli strain that over produces L-tyrosine by amino-acidfermentation was first generated This was accomplished bythree steps of genetic engineering Production of dopaminewas then obtained via the introduction in E coli of a tyrosi-nase and its adaptor protein from Ralstonia solanacearumable to convert L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) along with a L-DOPA decarboxylase from Pseu-domonas putida leading to L-dopamine This short pathwayclosed the gap between L-tyrosine and the synthetic pathwayto reticuline via the monoamine oxidase from M luteusand leading to the 34-dihydroxyphenylacetaldehyde (see theprevious section) In the final step of the fermentative pro-duction of (S)-reticuline the dopamine-producing pathwaydescribed above was combined with the synthetic pathwayfrom dopamine to (S)-reticuline

Engineering microbes also offers great opportunitiesfor the biosynthesis of plant polyketides of medical interestincluding antibiotics (erythromycin rifamycin) anticancerdrugs (tetracyclines anthracyclines and epothilones)antiparasitic agents (avermectin) cholesterol-lowering

BioMed Research International 3

SCoA SCoA

O OHCO2H

Propionyl-CoA primer unit

DBES 1 DBES 2 DBES 3

Loading Module 1 Module 2 Module 3Module 4 Module 5 Module 6 Release

AT ACP KS AT KR ACP KS AT KR ACP KS KSAT ACP KS AT DH ER KR ACP AT KR ACP KS AT KR ACP TE

Erythromycin A

6-deoxyerythronolide B

= 4998400-phosphopantetheine cofactor

methylmalonyl-CoA extender units

SSSSSSSO

O

OH

OH

OH

OH

O

O

O

OO

O

O

O

O

OOH

OH

OHOH

OHOH

HOHO

N(CH3)2

OCH3

O

O

OH

OH

O

O

OH

OH

O

O

OH

OH

OH

O

OH

OH

OOOH

6x

Figure 2 Biosynthesis of deoxyerythronolide B in the route to erythromycin A (adapted from [19]) Deoxyerythronolide B synthases (DEBS)catalyze iterative claisen-type condensations by progressively associating one propionyl-CoA primer unit with six methylmalonyl-CoAextender units Each DEBS molecule contains two modules these modules representing a physical location for a claisen-type condensationOnce loaded onto the ketosynthase (KS) of module 1 by the loading acyl transferase (AT) and the acyl carrier protein (ACP) domainspropionyl-CoA condenses with a methylmalonyl unit loaded onto module 1 ACP by the module 1 AT domain After that ketoreductases(KR) specific for each module reduce the resulting ketone group The chain then passes in a progressive manner from module 1 ACP (viaa phosphopantetheine cofactor) to the KS domain of the next module (module 2) Following these iterative condensations the polyketidechain grows before being released and cyclized by a thioesteraseclaisen cyclase (TE) Other abbreviations ER are enoyl reductase and DHdehydratase

agents (lovastatin) and immunosuppressants (rapamycin)[13] They are synthesized from acyl-CoA precursors bypolyketide synthases (PKSs) [14] enzymes that are groupedin three different classes (i) type I refers to large andmultifunctional enzymes (ii) type II refers dissociablecomplexes usually composed of monofunctional enzymesfound in bacteria [15] (iii) type III consists of homodimericenzymes of relatively small size found in plants [16] as wellas in bacteria [17] and fungi [18] Type III PKSs catalyzeiterative condensations of malonyl-CoA units with a greatvariety of CoA-linked starter substrates (see below)

The macrocyclic core of erythromycin 6-deoxyerythronolide B (6-dEB) a broad spectrum antibioticsynthesized by the soil bacterium Saccharopolysporaerythraea is a prototype of polyketides As mentionedabove polyketides are obtained from single building blocksacetyl-CoA propionyl-CoA and methylmalonyl-CoA(Figure 2) [19] requiring the production of the last twocompounds reengineering of E coli metabolism due tolimited capabilities for molecular biological manipulationof microorganisms especially actinomycetes [20] For anefficient 6-dEB production the intracellular activity of the


deoxyerythronolide synthase (DEBS) catalyzing formationof the macrocyclic core of erythromycin has to be wellsynchronized with precursor biosynthesis

First attempts for engineering this polyketide pathway inE coli addressed expression of the genes encoding DEBSEach of the three genes encoding for DEBS 1 DEBS 2 andDEBS 3 from S erythraea was cloned into E coli BL21 (DE3) along with the sfp phosphopantetheinyl transferase genefrom Bacillus subtilis the plasmid-based coexpression of thisgene facilitating posttranslational modification of the DEBSprotein in E coli [20] A single copy of the sfp gene underthe control of the T7 RNA polymerase promoter was thusintegrated in the prp operon of strain BL21 (DE 3) yielding Ecoli strain BAP1This sitewas chosen because it suppresses thepossibility that the bacterium cell uses propionate a startingblock for 6-dEB biosynthesis as a source for catabolismand anabolism Secondly along with the sfp gene the prpEgene of the strain BAP1 was placed under control of anIPTG inducible T7 promoter PrpE is thought to convertpropionate into propionyl-CoA thus allowing accumulationof this precursor inside the cell that is well synchronizedwith DEBS expression Finally a suitable pathway for thebiosynthesis of (2S)-methylmalonyl-CoA was engineered inE coli The strain BAP1 was thus transformed with plasmidsharboring the propionyl-CoA carboxylase genes pccA andpccB from Streptomyces coelicolor Coexpression of the birAbiotin ligase from the bacterium was shown to enhance theactivity of the biotinylated subunit pccA [20]

To resume engineering of E coli to produce 6-dEBincluded introduction of the DEBS genes from S erythraeaintroduction of a propionyl-CoA carboxylase fromB subtilisintroduction of a propionyl-CoA carboxylase from S coeli-color for (2S)-methylmalonyl-CoA biosynthesis and deletionof the endogenous prpRBCD genes to avoid utilization ofpropionate as a carbon source for catabolism and anabolismof the cell along with overexpression of the endogenousprpE and birA genes (to ensure conversion of propionateto propionyl-CoA and to enhance conversion of propionyl-CoA to (2S)-methylmalonyl-CoA resp) A major challengeinmetabolic engineering is indeed to achieve an optimal flowthrough a given heterologous metabolic pathway for highyield and productivity The best performing system led to acalculated specific productivity of 01mmol that is 386mgof 6-dEBg cellular proteinday

Production of 6-dEB in E coli was improved further bythe same group [21] by cloning an S-adenosylmethionine(SAM) synthase gene (metK) from Streptomyces spectabilisinto an expression plasmid under the control of an inducibleT7 promoter according to the assumption that the pro-duction of several antibiotics (for instance streptomycinand erythromycin) is improved by increasing the intra-cellular concentration of SAM The same observation wasmade with the production of nicotianamine by Wadaand coworkers [22] indicating that increased SAM levelsmay be a generally useful tool for improving the produc-tion of various natural products This plasmid was intro-duced into the engineered BAP1 engineered E coli straindescribed above [20] improving the specific productiv-ity of 6-dEB from 1086mgLOD

600to 2008mgLOD

600

without or with heterologous metK expression within 5 days[21]

3 Antitumor Antibiotics

Important human antibiotics and antitumor antibiotics suchas tetracyclines and anthracyclines are of bacterial originThese compounds are synthesized by bacterial type II polyke-tide synthases (PKSs) The main limitation for reconstitutingthe biosynthesis of aromatic polyketides in E coli consists inthe inability to generate the elongated poly-120573-ketone back-bone from malonyl-CoA the starting block of the synthesispathway [24] This requires a minimal PKS comprising aketosynthase- (KS-) chain length factor (CLF) and an acyl-carrier protein (ACP) In fungi PKSs are megasynthases inwhich unlike bacterial aromatic PKSs the enzymatic com-ponents are not dissociated Megasynthases include a starter-unit acyltransferase (SAT) a KS a malonyl-CoAacyl carrierprotein (ACP) transacylase (MAT) a product template (PT)an acyl-carrier protein (ACP) and a thioesteraseclaisen-cyclase (TECLC) (Figure 2) (see [24] and the referencestherein) A fungal megasynthase the PKS4 from Gibberellafujikuroi can be expressed by Ecoli and the biosynthesisof aromatic polyketides can aslo be reconstituted in Ecoli [25] In order to produce polyketides with cyclizationregioselectivities not observed among fungal polyketidesthe PKS4 KS MAT didomain and the ACP domain wereextracted as stand-alone proteins leading to a dissociatedPKS4 minimal PKS Such an approach mimics the dissoci-ation of the components that are observed in bacterial typeII minimal polyketide synthases [24] In a second approachTang and coworkers [24] used a compact synthetic polyketidemegasynthase PKS WJ consisting of KS MAT and ACP on asingle polypeptide Two plasmids encoding the PKS WJ weretransformed into the E coli strain BAP1 Under fed-batchfermentation high cell density cultures of engineered E colilead to the production of 3mgL of the aromatic polyketideanthraquinone within 60 h after addition of isopropyl 120573-D-1-thiogalactopyranoside In contrast no polyketide productionwas observed in the dissociated PKS4 minimal PKS Usingengineered fungal PKSs in bacteria thus paves the way fora more general approach towards the production of variousaromatic polyketides

4 Anticancer Drugs

Taxol (paclitaxel) and its structural analogs are potentand commercially successful anticancer drugs [26 27]Taxol is a diterpene originating from the isoprenoid (ter-penoid) pathway Opportunities for biosynthesis of natu-ral terpenoid drugs using engineered microorganisms havebeen reviewed elsewhere [28ndash30] Terpenoids are synthe-sized from the condensation of two C

5units respec-

tively isopentenyl-pyrophosphate (IPP) and its isomerdimethylallyl-pyrophosphate (DMAPP) as starting blocksboth originating from either the mevalonate pathway orthe 2C-methyl-D-erythritol-4-phosphate pathway (Figure 3)[31ndash35] Condensation of these two C

5units and larger


Sugars

PyruvateG3P

Methylerythritol-phosphate pathway(119889119909119904 119894119889119894 119894119904119901119863 and 119894119904119901119865 genes)

FPPsynthase

GPPsynthase

FPP GPP IPP DMAPPGGPP

synthase

Taxadienesynthase

BaccatinIII

Taxol

Taxadiene5120572-

hydroxylase

Taxadiene5120572-OL

Taxa-4(5)11(12)

-diene

Geranyl-geranyl PP

Figure 3 Pathway for in vivo production of taxadiene and 5-120572-hydroxytaxadiene in the route to taxol via the methylerythri-tol phosphate pathway in E coli (adapted from [23]) Genesdxs idi ispD and ispF respectively encoding for 1-deoxy-D-xylulose-5-phosphate synthase isopentenyl diphosphate isomeraseisoprenoid synthase domain and 2C-methyl-D-erythritol-24-cyclodiphosphate synthase IPP isopentenyl diphosphate DMAPPdimethylallyl diphosphate GPP geranyl diphosphate FPP farnesyldiphosphate GGPP geranylgeranyl diphosphate

IPP- and DMAPP-derived building blocks such as the C10

unit geranyl pyrophosphate (GPP) the C15

unit farnesylpyrophosphate (FPP) and the C

20unit geranylgeranylpy-

rophosphate (GGPP) yields monoterpenes sesquiterpenes(for example artemisin see below) triterpenes (steroids)tetraterpenes (carotenoids) and diterpenes such as taxol

Taxol has been isolated from Taxus brevifolia (Pacific yewtree) (see [23] and the references therein) Unfortunatelysufficient taxol dosage for one patient requires sacrificingtwo to four fully grown trees of this species Moreover thechemical synthesis of this compound is extremely complex(requiring 35 to 51 steps) with a highest total yield ofthe best synthesis of 04 (see [23] and the referencestherein) Although a semisynthetic route using baccatin III(an intermediate in the taxol biosynthetic pathway isolatedfrom plant sources) (Figure 3) was described limitationswere encountered in terms of productivity and scalability (see[23] and the references therein) Metabolic engineering ofmicrobes thus appears alongwith plant cell cultures as a valu-able alternative for the production of such compounds whicheliminates reliance on yew tree plantations As the biosynthe-sis of taxol comprises many enzyme-catalyzed steps researchwas limited to attempts made towards the production oftaxadiene the first committed taxol intermediate Metabolicengineering of taxadiene production as an essential steptowards taxol production was first described in yeast Scerevisiae [38] In the best producing system heterologous

expression of the Sulfolobus acidocaldarius geranylgeranyl-diphosphate synthase along with the codon-optimized taxa-diene synthase from Taxus chinensis a truncated version ofyeast 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMG-CoA reductase) (which was also shown to increase produc-tion of artemisinic acid in recombinant yeast see the nextsection and [36]) and the UPC2-1 transcription factor geneleads to taxadiene levels of 87mgL [38]

To go farther in the production of taxol Stephanopoulosand co-workers designed a multivariate modular approachand succeeded in increasing the titer of taxadiene up to1 gL [23] The multivariate modular pathway engineeringwas composed of two modules separated at the level ofisopentenyl pyrophosphate (IPP)dimethylallyl pyrophos-phate (DMAPP) The first module comprised an eight-gene upstream methylerythritol-phosphate (MEP) pathwaynative to E coli the pathway in which the expression ofonly four genes (dxs idi ispD and ispF) that deemed to berate-limiting was modulated To channel the overflow fluxfrom the starting blocks IPP and DMAPP towards taxolproduction a second module comprised a two-gene (encod-ing for the geranyl geranyldiphosphate synthase GGPPS andthe taxadiene synthase TS resp) downstream heterologouspathway to taxadiene Using this modular approach taxa-diene production exhibited a 15000-fold increase over thecontrol yielding 102 gL in defined media with controlledglycerol feedingThis has to be compared with themilligramsof taxadiene obtained in previous studies [38]

As the cyclic olefin taxadiene undergoes multiple steps ofstereospecific oxidations (including oxygenation at seven dif-ferent positions mediated by CYP450-dependent monooxy-genases) [39] acylations and benzoylation to form baccatinIII the next step in taxol biosynthesis leading to taxadien-5120572-ol was engineered The first oxygenation step is the hydroxy-lation of the C5 position catalyzed by a CYP450 taxadien-5120572-hydroxylase (Figure 3) [40] To succeed in the functionalexpression of plant CYP450 in E coli engineering taxolP450 oxidation chemistry in the bacterium was performedby the construction of a chimera protein from taxadien-5120572-hydroxylase with its redox partner Taxus cytochromeP450 reductase One of the chimera enzymes generatedAt24T5120572OH-tTCPR carried out the first hydroxylation stepwith a yield higher than 98 leading to taxadien-5120572-ol alongwith a byproduct

Owing to the fact that there are still six more hydroxy-lation steps (and probably some other modifications of thetaxane core structure that have not yet been identified) untilreaching the suitable taxol precursor baccatin III a lot ofwork remains for engineering the complete pathway to taxolin microbes

The epothilones are potential anticancer agents usefulparticularly in case of paclitaxel- (taxol-) resistant tumor celllines [41] Epothilones A and B are synthesized by the actionof a hybrid nonribosomal peptide synthetase (NRPS)typeI polyketide synthase (PKS) together with a P450 epoxi-dase that converts desoxyepothilone into epothilone by themyxobacterium Sorangium cellulosum [42] Inactivation ofthe epoxidase or deletion of the gene encoding for thisenzyme yields epothilones C and D as direct products of the


Simple sugarAcetyl-CoA

Acetoacetyl-CoA

Mevalonate pathway

Ergosterol IPP DMAPPGPP

synthaseERG20

FPPsynthaseERG20

Squalene GPP

FPPGossypolArtemisinin

Semisynthesis

Successivehydroxylationsthrough a P450monooxygenase

(CYP71AV1 gene) ADS CDS CAH

Artemisinicacid

Cadinene8-hydroxy-cadineneAmorpha-

411-diene

Figure 4 Engineering the pathways for the biosynthesis of artemisinic acid and 8-hdroxycadinene via the mevalonate pathway (adaptedfrom [36 37]) ADS amorphadiene synthase CDS cadinene synthase CAH cadinene hydroxylase For other abbreviations see legend ofFigure 3

NRPSPKS Heterologous expression of an entire epothilonegene cluster composed of six open reading frames (ORFs)epoA epoB epoC epoD epoE and epoF encoding a sin-gle NRPS module eight PKS modules and a C-terminalthioesterase has already been described in Streptomycescoelicolor but with a low yield of epothilones [43] andMyxococcus xanthus the growth of which is slow comparedto E coli [44] In a more recent work [42] the genesencoding the entire cluster epoA epoB epoC epoD epoEand epoF were redesigned and synthesized to allow forexpression into two polypeptides with compatible modulelinkers Splitting this large protein into two polypeptides wasnecessary to obtain its expression in a soluble form alongwith the lowering of the incubation temperature (15∘C) coexpression of molecular chaperones and switching from aT7 promoter to an arabinose-inducible PBAD promoter Theentire cluster was expressed in the modified E coli K207-3which is a derivative of BL21 (DE3) the so-called strain BAP1already mentioned in the production of the macrocyclic coreof erythromycin 6-deoxyerythronolide B [20] This in turnresulted in the complete biosynthesis of epothilones C and Din E coli as clearly evidenced by the LCMSMS analysis ofE coli extracts The design of the synthetic epothilone genestogetherwithE coli expression provides an excellent platformfor the production of epothilone analogues according toprotein engineering strategies to redesign a large gene cluster

5 Antiparasitic Agents

Terpenoids and their derivatives together with their roles inrespiration electron transport photosynthesis and hormonesignaling may serve as antiparasitic agents One of the

most successful examples of terpenoid biosynthetic path-way engineering is the production of artemisinic acid theprecursor for the antimalarial agent artemisinin in yeast[36] Engineering of the pathway leading to artemisinic acidwas achieved in yeast S cerevisiae in three steps (Figure 4)(1) increasing farnesyl pyrophosphate (FPP) production (2)conversion of FPP to amorpha-411-diene and (3) three-stepoxidation of amorphadiene to artemisinic acid Combina-tion on one hand of the overexpression of a truncatedsoluble form of 3-hydroxy-3-methylglutaryl-CoA reductase(tHMGR gene) along with the down regulation of ERG9which encodes squalene synthase (leading to the productionof sterols) increased amorphadiene production fivefold andtwofold respectively in yeast Downregulating ERG9 andoverexpressing upc2-1 which enhances the activity of UPC2(a global transcription factor regulating the biosynthesisof sterols in yeast) were combined with integration of anadditional copy of tHMGR into the chromosome Intro-ducing the amorphadiene synthase gene from Artemisiaannua to this high FPP engineered producer resulted inan increased production of amorphadiene up to 149mgLFinally overexpression of the gene encoding FPP synthase(ERG20) enhanced amorphadiene production by ca 10 thatis 153mgL in the resulting engineered yeast strain EPY224(about 500-fold the production previously reported [45])

This transgenic yeast strainwas transformedwith a vectorharboring a cytochrome P450 monooxygenase and its redoxpartnerCYP71AV1CPR fromA annua that performs a three-step oxidation of amorphadiene to artemisinic acid underthe control of galactose-inducible promoters The producedacid was efficiently transported out of the cell making itspurification relatively easy In a one-litre aerated bioreactor


115mg of artemisinic acid was produced of which 76mg wasrecovered following a one-step silica gel column chromato-graphic separation of ether extracts from the washed buffersArtemisinic acid is produced by the engineered S cerevisiaestrain EPY224 at a biomass fraction comparable to thatproduced by the plant but within few days for the yeast versusseveral months in A annua Artemisinin is then obtainedfrom artemisinic acid by hemisynthesis [46] Further experi-ments achieved by the same group have improved productionof artemisinic acid in a transgenic yeast transformed withan amorphadiene synthase an amorphadiene oxidase and acytochrome P450 reductase expressed from a single plasmidModulating the selection markers and using appropriatemedium formulation production of artemisinic acid in theengineered yeast reached 250 120583gmL in shake-flask culturesand 1 gL in bioreactors [47]

More recently a novel semibiosynthetic route towardsartemisinin has been reported using the long-chain fatty acidP450BM3 hydroxylase from Bacillus megaterium capable offorming artemisinic minus11S 12-epoxide from amorphadienethis epoxide being an interesting intermediate to form dihy-droartemisinic alcohol [48] An engineered E coli expressingthis particular P450 hydroxylase yielded high titers of theepoxide (250mgL) Finally engineering of the mevalonatepathway in Seo E coli with equivalent genes from Staphy-lococcus aureus yielded an average amount of 227 gL ofamorphadiene in the culture medium [49]

Difficulties in efficient translations and functional expres-sion of key enzymes including plant P450 enzymes areencountered in E coli To overcome this limitation theproduction of functionalized terpenoids in E coli such as theconversion of cadinene to 8-hydroxycadinene a precursorfor the phytoalexin gossypol was achieved by Chang andco-workers by cloning a cadinene hydroxylase along with acytochrome P450 reductase (Figure 4) [37]

6 Antihypertensive Agents

Nicotianamine (NA) is an ubiquitous compound in plantsthought to play a role as a metal chelator in the internaltransport of metal nutrients as well as to be a precursor ofmugineic acids secreted from the roots and facilitating ironsolubilization in the soil Interestingly NA possesses anti-hypertensive effects via the inhibition of the angiotensin I-converting enzyme which catalyzes hydrolysis of angiotensinI to the potent vasoconstrictor angiotensin II [50] NAbiosynthesis is quite simple as it is obtained from the conden-sation of three S-adenosylmethionine (SAM) units by nico-tianamine synthase (NAS) Large amounts of NA (766 120583ggwet weight) were obtained following the introduction ofthe Arabidopsis AtNAS2 gene into the yeast Saccharomycescerevisiae strain SCY4 [22] Two vectors for NAS expressionin S cerevisiae were constructed the first one containing anintegrative vector pINNAS (strain SCY4-pINNAS) and thesecond one a high-copy episomal vector pEPNAS (strainSCY4-pEPNAS) Both contained the AtNAS2 ORF fromArabidopsis under the control of the GAPDH promoter toinduce constitutive and strong expression of AtNAS2 NA

production in the engineered SCY4-pINNAS strain reachedup to 328 120583gg wet weight within 48 h when the growthmediumwas supplementedwith glycine and formate (leadingto a 100-fold increased accumulation of the usual level ofthe precursor SAM) The intracellular concentration of NAreached 766120583gg wet weight within 60 h when the transgenewas introduced with the high-copy episomal vector withoutany noticeable effect on yeast growth (strain SCY4-pEPNAS)As the chemical synthesis of NA for commercial use as anantihypertensive agent is very expensive ($ 350mg) thismakes its production through a relatively simple fermentativeprocess very efficient [22]

7 Hormones and Immunological Agents

Engineering microbes for the production of hormonesand immunological agents has also been described Mazorand coworkers have reported facile isolation of full-lengthimmunoglobulin G antibodies from combinatorial librariesexpressed inE coli [51] Full-length heavy and light chains aresecreted into the periplasmic space where they assemble intoaglycosylated IgGs that are captured by an Fc-binding proteinanchored in the inner membrane of the bacterium

The use of Bacillus subtilis for recombinant proteinexpression has been well established Advantageously thisbacterium is able to secrete functional extracellular proteinsdirectly into the culture medium making their purificationeasy it is not pathogenic for humans and lacks endotoxinsin the cell wall B subtilis was used for establishing a systemfor the suitable production of human interleukin-3 (hIL-3) which is a cytokine that regulates blood-cell productionby controlling the production differentiation and functionof granulocytes and macrophages [52] To facilitate optimalsecretion of hIL-3 by B subtilis three signal peptides weretested the modified AmyL Pel and SacB signal peptidesUse of the modified AmyL signal peptide resulted in areproducibly high secretion from the bacterial cell The othersignal peptides used (SacB and Pel resp) did not result inproductive secretion of hIL-3 or led to plasmid instabilitysuch that it is impossible to predict which signal is optimalfor the secretion of a particular heterologous protein suchas hIL-3 The hIL-3 purified showed full biological activitythat is it was able to specifically induce proliferation ofthe hIL-3-dependent leukemia cell line MO7e The bestproduction system was reported for the B subtilis WB800strain in combination with the pP43LatIL3 vector containingthe AmyL signal peptide yielding 100mgL hIL-3 within 24 hof culture

As B subtilis E coli is a common host used for over-expression of proteins that do not require glycosylation Thisis due to the fact that recombinant proteins can easily betransported in and easily released from the periplasmic spaceby osmotic shock and that there are fewer proteases in theperiplasm compared to the cytoplasm According to thisperiplasmic production of human interferon-120574 (hINF-120574) butnot of human interleukin-2 (hIL-2) by the Tat (twin argininetranslocation) pathway in E coli strain BL21-SI was described[53] The Tat system was identified as a transport mechanismfor a specific group of periplasmic proteins [54] Expression


of recombinant proteins was obtained using the pEMR vectorcontaining the Tat-dependent modified penicillin acylasesignal peptide (mSPpac) driven by the T7 promoter

E coli was also engineered for the expression of arecombinant gonadotropin-releasing hormone (GnRH) vac-cine that could be used as a vaccine for the control offertility and hormone-dependent diseases [55] To this aima highly effective expression plasmid pED-GNRH3 wasconstructed to express GnRH3-hinge-MVP in the form ofa fusion protein The recombinant gene encoding GnRH3-hinge-MVP which contains three repeated GnRH units afragment of hinge region and aT-cell epitope ofmeasles virusprotein was cloned into E coli A crucial point was to insureplasmid stability during the fermentation process affectingthe production of the recombinant proteinThis was achievedby the optimization of many culture conditions includingfermentation temperature and the culture medium Both thestability of the plasmid and the expression of GnRH3-hinge-MVP are of real concern for the industrial production of theengineered strain

8 Antioxidant and Anticancer Agents

Hydroxystilbenes including resveratrol and its derivativespossess a large spectrum of biological activities in humanhealth as cardioprotective antitumor and antioxidant agentsEngineering bacteria or yeast for resveratrol might representa valuable means for its production in large quantities [8 56ndash59] At the same time the potential role of resveratrol asan antimicrobial compound acting as an allelochemical or aphytoalexin and protecting plants from fungal attacks [60ndash62] or increasing their antioxidant capabilities has led tonumerous studies concerning molecular engineering of thiscompound in plants [59 63ndash66]

Resveratrol is obtained through the universal phenyl-propanoic acid pathway beginning with phenylalanine (viathe phenylalanine ammonia lyase PAL) or with tyrosine (viathe tyrosine ammonia lyase TAL) (Figure 5) The obtainedphenylpropanoic acids are then activated by ligation tocoenzyme A by coumarateCoA ligase (CL) often actingonly in the presence of a 4-hydroxyphenyl group and thustermed 4-coumaroylCoA ligase (4CL) Hydroxystilbenes arethen synthesized by type III polyketide synthases stilbenesynthases (STS) which compete with chalcone synthasesimplied in the biosynthesis of the C

6-C3-C6flavonoid ring

[67 68] PKS act to condense successive units of malonyl-CoA with p-coumaroyl-CoA forming a linear polyketidemolecule before cyclization

Two main strategies have already been used in theengineering of resveratrol in bacteria or yeast (i) introduc-ing the entire biosynthetic pathway using aromatic aminoacids as substrates (L-phenylalanine or L-tyrosine) and (ii)introducing specific genes such as 4CL and STS startingwithpara-coumaric acid as a substrate

Engineering the entire pathway consists in the productionof resveratrol from its precursor phenylalanine or tyrosinethrough the introduction of the genes encoding for PAL orTAL the cinnamate-4-hydroxylase leading from cinnamate

Shikimatepathway

Phenylalanine

PALTyrosine

Cinnamic acidTAL

4CH

4CL

+

Sugarmetabolism

Malonyl-coenzyme A

STS CHS

Resveratrol Naringeninchalcone

119901-coumaric acid

119901-coumaroyl-coenzyme A

Figure 5 In vivo biosynthesis of resveratrol from phenylala-nine or tyrosine via the phenylpropanoidpolymalonate pathwayPALTAL phenylalaninetyrosine ammonia lyase C4H cinnamate-4-hydroxylase C4L coumaratecoenzyme A ligase STS stilbene(resveratrol) synthase CHS chalcone synthase

to p-coumarate (4CH) 4CL and STS activities The biosyn-thetic pathway has been introduced successfully into theoleaginous yeast Yarrowia lipolytica completed by constitu-tive expression ofmalonyl-CoA and resulting in a resveratrolproduction of 146mgL [69] The entire resveratrol pathwayhas been introduced in other microorganisms such as thebaker yeast S cerevisiae molds such as Aspergillus niger andA oryzae and bacteria such as Lactococcus lactis and Ecoli In all cases pathway expression started with PAL exceptfor E coli in which TAL was the first enzyme introduced[70] Resveratrol synthesis was only reported in E coli thatexpressed the TAL gene upon feeding with p-coumarate Thenecessary addition of p-coumarate to the culturemedium hasthe consequence that the TAL or PAL activities remained lowin the bacterium As a result much of the ongoing effortsare dedicated to increasing these activities in bacteria forexample by aiming to develop novel TAL or PAL enzymes[71 72] Two more recent works constructed the completeresveratrol biosynthetic pathway in S cerevisiae to pro-duce resveratrol from phenylalanine Trantas and coworkersreported a resveratrol production of 029mgL within 120 hof cultivation after feeding with 10mM of phenylalanine[73] Finally Shin and coworkers engineered the S cerevisiaestrain (W303-1A) with four heterologous genes the PALgene from Rhodosporidium toruloides the C4H and the 4CL1genes from A thaliana and the STS gene from Arachishypogea [74] Overexpression of the acetyl-CoA carboxylase(ACC1) gene for increasing the pool of malonyl-CoA resultedin an engineered strain termed W303-1AACC1 harboringthe p425PAL p423C4H and pESCTRP4CL1-STS plasmidscapable of synthesizing 58mgL resveratrol upon feedingwith 12mM tyrosine in YPmedium containing 2 galactose


Introducing selective genes is an alternative strategydirected towards transforming microorganisms with onlytwo genes (4CL and STS) under utilization of p-coumaricacid In fact p-coumaric acid can be used advantageously as aprecursor for the bioproduction of resveratrol in engineeredbacteria rather than the basic amino-acid phenylalanineThisamino-acid has indeed to be converted to p-coumaric acidfrom cinnamic acid via the P450 enzyme C4H HoweverP450 monooxygenases failed to be effectively expressed in EcoliAdditionally p-coumarate is a relatively cheap precursoruseful for bioproduction systems [75]

Recombinant E coli strains able to produce resveratrolwere obtained [76ndash78] An accumulation of 36mgL resver-atrol was reported in the E coli BL21 (DE3) engineered strainthrough an increase in malonate transport andmalonyl-CoAsynthase activity [78] Transformation of the BL21 E colistrain with the 4CL gene from tobacco and the grapevineSTS gene resulted in a resveratrol production of 16mgL [76]Similarly a metabolically engineered E coli strain which wastransformed with the 4CL gene from A thaliana and a STSgene from A hypogea resulted in a final high titer gt100mgLof resveratrol [77] Replacement of the 4CL gene from Athaliana by the 4CL gene from Lithospermum erythrorhizonin the same JM109 E coli strain leads to a resveratrolproduction of 171mgL after feeding with p-coumaroyl-CoA[79] When modified cinnamoyl-CoA was used unnaturalstilbenes were formed suggesting that engineering microbespaves the way for the biosynthesis of novel compounds withpotent biological activities [79]

An original approach was developedmore recently by thegroup of Lim and coworkers in which two different E colistrains (BL21 Star and BW27784) two different promoters(the strong T7 promoter and the constitutive promoter of theglyceraldehyde-3-phosphate dehydrogenase) and a library oftwo 4CLs from Petroselinum crispum and A thaliana andnine STS (frompine Polygonum PsilotumVitis andArachis)in different combinations were systematically examined tooptimize resveratrol production from p-coumaric acid in Ecoli [75] Both STS from Vitis vinifera (VvSTS) gene and fromA hypogea (AhSTS) gene were more efficient in producingresveratrol when coupled with the 4-CL from A thaliana(At4CL) gene than with the 4CL from Petroselinum crispum(Pc4CL) gene The best performing system was observedwith the BW27784 pUCo-VvSTS-At4CL strain at which005mM cerulenin was added to the culture medium forimproving the malonyl-CoA pool Resveratrol productionfrom p-coumaric acid reached the impressive value of 23 gLDue to the prohibitive cost of cerulenin the simultaneousoverexpression of both the acetyl-CoA carboxylase (ACC)gene and the biotin ligase (BirA) gene from Photorhabdusluminescens was obtained allowing acetyl-CoA to be moreefficiently converted to malonyl-CoA

There are many reports of selective genes being intro-duced in S cerevisiae resulting in various productions ofresveratrol ranging from 065 to 58mgL [76 80 81] Anengineered yeast strain W303-1A containing a 4CL gene(4CL1 gene) from A thaliana and as STS gene from Ahypogea produced unglycosylated resveratrol at a titer of31mgL after feeding with 153mgL p-coumaric acid that is

144mol yield [82] When the 4CL and the STS-encodinggenes added to the yeast genome were submitted to proteinfusion expression of the 4CLSTS fusion protein increasedresveratrol production in yeast by up to 10-fold (525mgLresveratrol) This can be compared to the coexpression of4CL and STS without any protein fusion (065mgL) [81]Works also clearly demonstrated that synthetic scaffoldscan optimize engineered metabolic pathways Use of a syn-thetic scaffold to recruit the 4CL1 and STS enzymes of theresveratrol pathway improved resveratrol production in Scerevisiae resulting in a 5-fold improvement of the resveratrolproduction over the nonscaffolded control (144mgL) [83]Finally engineered S cerevisiae yeast cells expressing 4CL andSTS genes along with the araE gene encoding for a high-capacity E coli transporter exhibited a resveratrol productionof 31mgL [84]

The efficacy of recombinant microorganisms for resver-atrol production depends on various factors such as thespecies and the strain the origin of the transferred genesand culture conditions as well as other parameters such asplasmids or precursors used A recent study has underlinedthe crucial role played by the culture medium [85] Resver-atrol production by an industrial Brazilian sugar cane yeastexpressing the 4CL1 gene from A thaliana and the grapevineSTS gene in a rich medium reached up to 391mgL

9 Conclusions

There is an increasing number of examples demonstratingthat engineering microbial cells for the production of naturalcompounds of pharmaceutical interest is a successful methodto gain access to some bioactive substances only accumu-lating in low amounts in plants and whose total chemicalsynthesis is too complex Moreover recovery of unnaturalcompounds with significant biological properties has alsobeen reported through metabolic engineering of microbes[79]

Some particularly interesting results have been obtainedfor engineered systems where relatively simple genetic con-structs are needed Extraordinary progresses have also beendone in the engineering of very complex biosynthetic path-ways such as benzylisoquinoline alkaloids artemisinic acidsor taxol pathways Stephanopoulos and co-workers recentlyachieved the production of taxadiene a key intermediate inthe route for taxol at low cost in E coli [23] However stillwork has to be done before optimizing taxol production bymicroorganisms as many steps remain to be engineered untilreaching taxol

Moreover successful endeavors in the future for optimiz-ing microbial production of natural compounds will requirebesides the functional integration of a complete or a partialheterologous pathway in a given microbe adaptation ofthe host organism to its environment to improve producttiters For example resveratrol production by a recombinantindustrial yeast strain has been shown to be considerablyenhanced according to the culture medium used [85]

New techniques for metabolic engineering will beexploited in the next years to come Namely methodologiesfor generating high-quality libraries of enzyme variants [86]


and novel high-throughput screening (HTS) technologies[87] will open the way for the engineering of enzymes forthe biosynthesis of various compounds with potent biologicalactivities Specifically HTS technologies can rapidly leadto the identification of genes which modulate a particularbiosynthesis pathway Gathering all genes encoding for abiomolecular pathway will allow the assembly of geneticconstructs for the synthesis of a given product Newmethodsfor the rapid cloning of single genes together with theavailability of synthetic operons such as bacterial operons(commonly used in the biosynthesis of many medically andpharmaceutically important compounds) have acceleratedthe construction of synthetic multigene pathways [88 89]A very recent application was the coordinated inductionof a five-gene pathway leading to zeaxanthin production intailored S cerevisiae [90]

A major challenge in metabolic engineering is to achievean optimal flow through a given heterologous metabolicpathway for high yield and productivity A simple method tofine tune metabolic pathways named customized optimiza-tion of metabolic pathways by combinatorial transcriptionalengineering (COMPACTER) was described for rapid tuningof gene expression in a heterologous pathway under differentmetabolic backgrounds This was achieved by the creation ofa library of mutant pathways using de novo assembly of pro-moter mutants for each pathway gene in a target microorgan-ism followed by high-throughput screeningselection [91]

Finally systems biology metabolic engineering andldquoomicsrdquo technologies (genomics functional genomics andmetagenomics) have opened the way for protein andbiomolecular pathway engineering Production of novelsmall molecules of pharmaceutical interest by designingnovel proteins and pathways is also under progress [92]These new methodologies will thus pave the way for veryimportant advances in metabolic engineering of microbialcell factories

References

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[2] N Ferrer-Miralles J Domingo-Espın J Corchero E Vazquezand A Villaverde ldquoMicrobial factories for recombinant phar-maceuticalsrdquoMicrobial Cell Factories vol 8 article 17 2009

[3] S Y Lee H U Kim J H Park J M Park and T Y KimldquoMetabolic engineering of microorganisms general strategiesand drug productionrdquoDrug Discovery Today vol 14 no 1-2 pp78ndash88 2009

[4] J Du Z Shao and H Zhao ldquoEngineering microbial factoriesfor synthesis of value-added productsrdquo Journal of IndustrialMicrobiology and Biotechnology vol 38 pp 873ndash890 2011

[5] M Papagianni ldquoRecent advances in engineering the central car-bon metabolism of industrially important bacteriardquo MicrobialCell Factories vol 11 article 50 2012

[6] J Marienhagen and M Bott ldquoMetabolic engineering ofmicroorganisms for the synthesis of plant natural productsrdquoJournal of Biotechnology vol 163 pp 166ndash178 2013

[7] P Xu N Bhan and M A G Koffas ldquoEngineering plantmetabolism into microbes from systems biology to synthetic

biologyrdquo Current Opinion in Biotechnology vol 24 pp 291ndash2992013

[8] D Donnez P Jeandet C Clement and E Courot ldquoBioproduc-tion of resveratrol and stilbene derivatives by plant cells andmicroorganismsrdquo Trends in Biotechnology vol 27 no 12 pp706ndash713 2009

[9] D A Rathbone and N C Bruce ldquoMicrobial transformation ofalkaloidsrdquo Current Opinion in Microbiology vol 5 no 3 pp274ndash281 2002

[10] J Keasling ldquoFrom yeast to alkaloidsrdquo Nature Chemical Biologyvol 4 no 9 pp 524ndash525 2008

[11] HMinami J S KimN Ikezawa et al ldquoMicrobial production ofplant benzylisoquinoline alkaloidsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 105 no21 pp 7393ndash7398 2008

[12] A Nakagawa H Minami J S Kim et al ldquoA bacterial platformfor fermentative production of plant alkaloidsrdquoNature Commu-nications vol 2 no 1 article 326 2011

[13] S Horinouchi ldquoCombinatorial biosynthesis of plant medicinalpolyketides by microorganismsrdquo Current Opinion in ChemicalBiology vol 13 no 2 pp 197ndash204 2009

[14] C Hertweck ldquoThe biosynthetic logic of polyketide diversityrdquoAngewandte Chemie vol 48 no 26 pp 4688ndash4716 2009

[15] B Shen ldquoPolyketide biosynthesis beyond the type I II and IIIpolyketide synthase paradigmsrdquo Current Opinion in ChemicalBiology vol 7 no 2 pp 285ndash295 2003

[16] I Abe and H Morita ldquoStructure and function of the chalconesynthase superfamily of plant type III polyketide synthasesrdquoNatural Product Reports vol 27 no 6 pp 809ndash838 2010

[17] N Funa Y Ohnishi I Fujli M Shibuya Y Ebizuka andS Horinouchi ldquoA new pathway for polyketide synthesis inmicroorganismsrdquo Nature vol 400 no 6747 pp 897ndash899 1999

[18] Y Seshime P R Juvvadi I Fujii and K Kitamoto ldquoDiscov-ery of a novel superfamily of type III polyketide synthasesin Aspergillus oryzaerdquo Biochemical and Biophysical ResearchCommunications vol 331 no 1 pp 253ndash260 2005

[19] Y A Chan A M Podevels B M Kevany and M G ThomasldquoBiosynthesis of polyketide synthase extender unitsrdquo NaturalProduct Reports vol 26 no 1 pp 90ndash114 2009

[20] B A Pfeifer S J Admiraal H Gramajo D E Cane and CKhosla ldquoBiosynthesis of complex polyketides in ametabolicallyengineered strain of E colirdquo Science vol 291 no 5509 pp 1790ndash1792 2001

[21] Y Wang B A Boghigian and B A Pfeifer ldquoImproving het-erologous polyketide production in Escherichia coli by overex-pression of an S-adenosylmethionine synthetase generdquo AppliedMicrobiology and Biotechnology vol 77 no 2 pp 367ndash373 2007

[22] Y Wada T Kobayashi M Takahashi H Nakanishi S Moriand N K Nishizawa ldquoMetabolic engineering of Saccharomycescerevisiae producing nicotianamine potential for industrialbiosynthesis of a novel antihypertensive substraterdquo BioscienceBiotechnology and Biochemistry vol 70 no 6 pp 1408ndash14152006

[23] P K Ajikumar W H Xiao K E J Tyo et al ldquoIsoprenoidpathway optimization for Taxol precursor overproduction inEscherichia colirdquo Science vol 330 no 6000 pp 70ndash74 2010

[24] W Zhang Y Li and Y Tang ldquoEngineered biosynthesis ofbacterial aromatic polyketides inEscherichia colirdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 105 no 52 pp 20683ndash20688 2008


[25] S M Ma J Zhan K Watanabe et al ldquoEnzymatic synthesisof aromatic polyketides using PKS4 from Gibberella fujikuroirdquoJournal of the American Chemical Society vol 129 no 35 pp10642ndash10643 2007

[26] M C Wani H L Taylor M E Wall P Coggon and A TMcPhail ldquoPlant antitumor agents VI The isolation and struc-ture of taxol a novel antileukemic and antitumor agent fromTaxus brevifoliardquo Journal of the American Chemical Society vol93 no 9 pp 2325ndash2327 1971

[27] D G I Kingston ldquoThe shape of things to come structural andsynthetic studies of taxol and related compoundsrdquo Phytochem-istry vol 68 no 14 pp 1844ndash1854 2007

[28] D Klein-Marcuschamer P K Ajikumar and G Stephanopou-los ldquoEngineeringmicrobial cell factories for biosynthesis of iso-prenoid molecules beyond lycopenerdquo Trends in Biotechnologyvol 25 no 9 pp 417ndash424 2007

[29] P K Ajikumar K Tyo S Carlsen O Mucha T H Phon andG Stephanopoulos ldquoTerpenoids opportunities for biosynthesisof natural product drugs using engineered microorganismsrdquoMolecular Pharmaceutics vol 5 no 2 pp 167ndash190 2008

[30] J Kirby and J D Keasling ldquoBiosynthesis of plant isoprenoidsperspectives for microbial engineeringrdquoAnnual Review of PlantBiology vol 60 pp 335ndash355 2009

[31] M Rohmer M Knani P Simonin B Sutter and H SahmldquoIsoprenoid biosynthesis in bacteria a novel pathway for theearly steps leading to isopentenyl diphosphaterdquo BiochemicalJournal vol 295 no 2 pp 517ndash524 1993

[32] T Kuzuyama and H Seto ldquoDiversity of the biosynthesis of theisoprene unitsrdquo Natural Product Reports vol 20 no 2 pp 171ndash183 2003

[33] V J J Martin D J Piteral S T Withers J D Newman and JD Keasling ldquoEngineering a mevalonate pathway in Escherichiacoli for production of terpenoidsrdquoNature Biotechnology vol 21no 7 pp 796ndash802 2003

[34] D J Pitera C J Paddon J D Newman and J D KeaslingldquoBalancing a heterologous mevalonate pathway for improvedisoprenoid production in Escherichia colirdquo Metabolic Engineer-ing vol 9 no 2 pp 193ndash207 2007

[35] Y Zhao J Yang B Qin et al ldquoBiosynthesis of isoprene inEscherichia coli via methylerythritol phosphate (MEP) path-wayrdquo Applied Microbiology and Biotechnology vol 90 no 6 pp1915ndash1922 2011

[36] D K Ro E M Paradise M Quellet et al ldquoProduction ofthe antimalarial drug precursor artemisinic acid in engineeredyeastrdquo Nature vol 440 no 7086 pp 940ndash943 2006

[37] M C Y Chang R A Eachus W Trieu D K Ro and JD Keasling ldquoEngineering Escherichia coli for production offunctionalized terpenoids using plant P450srdquo Nature ChemicalBiology vol 3 no 5 pp 274ndash277 2007

[38] B Engels P Dahm and S Jennewein ldquoMetabolic engineeringof taxadiene biosynthesis in yeast as a first step towards Taxol(Paclitaxel) productionrdquoMetabolic Engineering vol 10 no 3-4pp 201ndash206 2008

[39] R Kaspera and R Croteau ldquoCytochrome P450 oxygenases oftaxol biosynthesisrdquo Phytochemistry Reviews vol 5 no 2-3 pp433ndash444 2006

[40] S Jennewein R M Long R M Williams and R CroteauldquoCytochrome P450 taxadiene 5120572-hydroxylase a mechanisti-cally unusual monooxygenase catalyzing the first oxygenationstep of taxol biosynthesisrdquo Chemistry and Biology vol 11 no 3pp 379ndash387 2004

[41] R J Kowalski P Giannakakou and E Hamel ldquoActivities ofthe microtubule-stabilizing agents epothilones A and B withpurified tubulin and in cells resistant to paclitaxel (Taxol)rdquoJournal of Biological Chemistry vol 272 no 4 pp 2534ndash25411997

[42] S C Mutka J R Carney Y Liu and J Kennedy ldquoHeterologousproduction of epothilone C and D in Escherichia colirdquo Biochem-istry vol 45 no 4 pp 1321ndash1330 2006

[43] L Tang S Shah L Chung et al ldquoCloning and heterologousexpression of the epothilone gene clusterrdquo Science vol 287 no5453 pp 640ndash642 2000

[44] L Tang L Chung J R Carney C M Starks P Licari and LKatz ldquoGeneration of new epothilones by genetic engineeringof a polyketide synthase in Myxococcus xanthusrdquo Journal ofAntibiotics vol 58 no 3 pp 178ndash184 2005

[45] B E Jackson E A Hart-Wells and S P T Matsuda ldquoMetabolicengineering to produce sesquiterpenes in yeastrdquo Organic Let-ters vol 5 no 10 pp 1629ndash1632 2003

[46] N Acton and R J Roth ldquoOn the conversion of dihy-droartemisinic acid into artemisininrdquo Journal of Organic Chem-istry vol 57 no 13 pp 3610ndash3614 1992

[47] DK RoMOuellet EM Paradise et al ldquoInduction ofmultiplepleiotropic drug resistance genes in yeast engineered to producean increased level of anti-malarial drug precursor artemisinicacidrdquo BMC Biotechnology vol 8 article 83 2008

[48] J A Dietrich Y Yoshikuni K J Fisher et al ldquoA novel semi-biosynthetic route for artemisinin production using engineeredsubstrate-promiscuous P450BM3rdquo ACS Chemical Biology vol4 no 4 pp 261ndash267 2009

[49] H Tsuruta C J Paddon D Eng et al ldquoHigh-level productionof amorpha-4 11-diene a precursor of the antimalarial agentartemisinin in Escherichia colirdquo PLoS ONE vol 4 no 2 ArticleID e4489 2009

[50] A Hayashi and K Kimoto ldquoStudies on the mechanism of anti-hypertensive action by nicotianaminerdquo Journal of NutritionalScience and Vitaminology vol 56 no 4 pp 242ndash246 2010

[51] Y Mazor T V Blarcom R Mabry B L Iverson and GGeorgiou ldquoIsolation of engineered full-length antibodies fromlibraries expressed in Escherichia colirdquo Nature Biotechnologyvol 25 no 5 pp 563ndash565 2007

[52] L Westers D S Dijkstra H Westers J M van Dijl and WJ Quax ldquoSecretion of functional human interleukin-3 fromBacillus subtilisrdquo Journal of Biotechnology vol 123 no 2 pp 211ndash224 2006

[53] E Medina-Rivero V E Balderas-Hernandez L G Ordonez-Acevedo L M T Paz-Maldonado A P Barba-De La Rosaand A De Leon-Rodrıguez ldquoModified penicillin acylase signalpeptide allows the periplasmic production of soluble humaninterferon-120574 but not of soluble human interleukin-2 by the Tatpathway in Escherichia colirdquo Biotechnology Letters vol 29 no 9pp 1369ndash1374 2007

[54] T Palmer F Sargent and B C Berks ldquoExport of complexcofactor-containing proteins by the bacterial Tat pathwayrdquoTrends in Microbiology vol 13 no 4 pp 175ndash180 2005

[55] J Xu W Li J Wu et al ldquoStability of plasmid and expressionof a recombinant gonadotropin-releasing hormone (GnRH)vaccine in Escherichia colirdquo Applied Microbiology and Biotech-nology vol 73 no 4 pp 780ndash788 2006

[56] YWang H Chen and O Yu ldquoMetabolic engineering of resver-atrol and other longevity boosting compoundsrdquo BioFactors vol36 no 5 pp 394ndash400 2010


[57] K V Kiselev ldquoPerspectives for production and application ofresveratrolrdquoAppliedMicrobiology andBiotechnology vol 90 no2 pp 417ndash425 2011

[58] Y Wang S Chen and O Yu ldquoMetabolic engineering offlavonoids in plants andmicroorganismsrdquoAppliedMicrobiologyand Biotechnology vol 91 pp 949ndash956 2011

[59] P Jeandet B Delaunois A Aziz et al ldquoMetabolic engineeringof yeast and plants for the production of the biologicallyactive hydroxystilbene resveratrolrdquo Journal of Biomedicine andBiotechnology vol 2012 Article ID 579089 14 pages 2012

[60] P Langcake ldquoDisease resistance ofVitis spp and the productionof the stress metabolites resveratrol 120576-viniferin 120572-viniferin andpterostilbenerdquo Physiological Plant Pathology vol 18 pp 213ndash226 1981

[61] M Adrian P Jeandet J Veneau L A Weston and R BessisldquoBiological activity of resveratrol a stilbenic compound fromgrapevines against Botrytis cinerea the causal agent for graymoldrdquo Journal of Chemical Ecology vol 23 no 7 pp 1689ndash17021997

[62] M Adrian and P Jeandet ldquoEffects of resveratrol on the ultra-structure of Botrytis cinerea conidia and biological significancein plantpathogen interactionsrdquo Fitoterapia vol 83 pp 1345ndash1350 2012

[63] P Jeandet A CDouillet-Breuil SDebordM Sbaghi R Bessisand M Adrian ldquoPhytoalexins from the vitaceae biosynthesisphytoalexin gene expression in transgenic plants antifungalactivity and metabolismrdquo Journal of Agricultural and FoodChemistry vol 50 no 10 pp 2731ndash2741 2002

[64] C Halls and O Yu ldquoPotential for metabolic engineering ofresveratrol biosynthesisrdquo Trends in Biotechnology vol 26 no 2pp 77ndash81 2008

[65] B Delaunois S Cordelier A Conreux C Clement and PJeandet ldquoMolecular engineering of resveratrol in plantsrdquo PlantBiotechnology Journal vol 7 no 1 pp 2ndash12 2009

[66] P Jeandet B Delaunois A Conreux et al ldquoBiosynthesismetabolismmolecular engineering and biological functions ofstilbene phytoalexins in plantsrdquo BioFactors vol 36 no 5 pp331ndash341 2010

[67] M B Austin and J P Noel ldquoThe chalcone synthase superfamilyof type III polyketide synthasesrdquo Natural Product Reports vol20 no 1 pp 79ndash110 2003

[68] O Yu and J M Jez ldquoNaturersquos assembly line biosynthesis ofsimple phenylpropanoids and polyketidesrdquo Plant Journal vol54 no 4 pp 750ndash762 2008

[69] L L Huang Z Xue andQ Q Zhu ldquoMethod for the productionof resveratrol in a recombinant oleaginous microorganismrdquo USpatent 20090082286A1 2009

[70] M Katz J Forster H David et al ldquoFluxome Sciences ASMetabolically engineered cells for the production of resveratrolor an oligomeric or glycosidically-bound derivative thereofrdquo USPatent 20080286844A1 2008

[71] T Vannelli Z Xue S Breinig W W Qi and F S SariaslanildquoFunctional expression in Escherichia coli of the tyrosine-inducible tyrosine ammonia-lyase enzyme from yeast Tri-chosporon cutaneum for production of p-hydroxycinnamicacidrdquo Enzyme and Microbial Technology vol 41 no 4 pp 413ndash422 2007

[72] Z Xue M McCluskey K Cantera A Ben-Bassat F SSariaslani and L Huang ldquoImproved production of p-hydroxycinnamic acid from tyrosine using a novel thermostablephenylalaninetyrosine ammonia lyase enzymerdquo Enzyme andMicrobial Technology vol 42 no 1 pp 58ndash64 2007

[73] E Trantas N Panopoulos and F Ververidis ldquoMetabolicengineering of the complete pathway leading to heterologousbiosynthesis of various flavonoids and stilbenoids in Saccha-romyces cerevisiaerdquoMetabolic Engineering vol 11 no 6 pp 355ndash366 2009

[74] S Y Shin S M Jung M D Kim N S Han and J HSeo ldquoProduction of resveratrol from tyrosine in metabolically-engineered Saccharomyces cerevisiaerdquo Enzyme and MicrobialTechnology vol 51 pp 211ndash216 2012

[75] C G Lim Z L Fowler T Hueller S Schaffer and M AG Koffas ldquoHigh-yield resveratrol production in engineeredEscherichia colirdquo Applied and Environmental Microbiology vol77 no 10 pp 3451ndash3460 2011

[76] J Beekwilder R Wolswinkel H Jonker R Hall C H De RieVos and A Bovy ldquoProduction of resveratrol in recombinantmicroorganismsrdquoApplied and Environmental Microbiology vol72 no 8 pp 5670ndash5672 2006

[77] K TWatts P C Lee and C Schmidt-Dannert ldquoBiosynthesis ofplant-specific stilbene polyketides in metabolically engineeredEscherichia colirdquo BMC Biotechnology vol 6 article 22 2006

[78] L L Huang Z Xue andQ Q Zhu ldquoMethod for the productionof resveratrol in a recombinant bacterial host cellrdquo US Patent20070031951A1 2007

[79] Y Katsuyama N Funa I Miyahisa and S Horinouchi ldquoSyn-thesis of unnatural flavonoids and stilbenes by exploiting theplant biosynthetic pathway in Escherichia colirdquo Chemistry andBiology vol 14 no 6 pp 613ndash621 2007

[80] J V W Becker G O Armstrong M J Van Der Merwe MG Lambrechts M A Vivier and I S Pretorius ldquoMetabolicengineering of Saccharomyces cerevisiae for the synthesis of thewine-related antioxidant resveratrolrdquo FEMS Yeast Research vol4 no 1 pp 79ndash85 2003

[81] Y Zhang S Z Li J Li et al ldquoUsing unnatural protein fusionsto engineer resveratrol biosynthesis in yeast and mammaliancellsrdquo Journal of the American Chemical Society vol 128 no 40pp 13030ndash13031 2006

[82] S Y Shin N S Han Y C Park M D Kim and J H Seo ldquoPro-duction of resveratrol from p-coumaric acid in recombinantSaccharomyces cerevisiae expressing 4-coumaratecoenzyme Aligase and stilbene synthase genesrdquo Enzyme and MicrobialTechnology vol 48 no 1 pp 48ndash53 2011

[83] Y Wang and O Yu ldquoSynthetic scaffolds increased resveratrolbiosynthesis in engineered yeast cellsrdquo Journal of Biotechnologyvol 157 pp 258ndash260 2012

[84] Y Wang C Halls J Zhang M Matsuno Y Zhang andO Yu ldquoStepwise increase of resveratrol biosynthesis in yeastSaccharomyces cerevisiae by metabolic engineeringrdquo MetabolicEngineering vol 13 pp 455ndash463 2011

[85] T Sydor S Schaffer and E Boles ldquoConsiderable increase inresveratrol production by recombinant industrial yeast strainswith use of rich mediumrdquo Applied and Environmental Microbi-ology vol 76 no 10 pp 3361ndash3363 2010

[86] A V Shivange J Marienhagen H Mundhada A Schenk andU Schwaneberg ldquoAdvances in generating functional diversityfor directed protein evolutionrdquo Current Opinion in ChemicalBiology vol 13 no 1 pp 19ndash25 2009

[87] G Yang and S G Withers ldquoUltrahigh-throughput FACS-basedscreening for directed enzyme evolutionrdquo ChemBioChem vol10 no 17 pp 2704ndash2715 2009

[88] Z Shao H Zhao and H Zhao ldquoDNA assembler an invivo genetic method for rapid construction of biochemicalpathwaysrdquoNucleic Acids Research vol 37 no 2 article e16 2009


[89] M Blanusa A Schenk H Sadeghi J Marienhagen and USchwaneberg ldquoPhosphorothioate-based ligase-independentgene cloning (PLICing) an enzyme-free and sequence-independent cloning methodrdquo Analytical Biochemistry vol406 no 2 pp 141ndash146 2010

[90] J Liang J C Ning and H Zhao ldquoCoordinated induction ofmulti-gene pathways in Saccharomyces cerevisiaerdquoNucleic AcidsResearch vol 41 article e54 2013

[91] J Du Y Yuan T Si J Lian andH Zhao ldquoCustomized optimiza-tion of metabolic pathways by combinatorial transcriptionalengineeringrdquo Nucleic Acids Research vol 40 article e142 2012

[92] P Xu N Bhan and M A G Koffas ldquoPathway and proteinengineering approaches to produce novel and commodity smallmoleculesrdquo Current Opinion in Biotechnology In press

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


plants fungi bacteria and animals benzylisoquinolinealkaloids constitute a remarkable group of pharmaceuticalmolecules including for example the narcotic analgesicmorphine and the antibacterial agents berberine scoulerinepalmatine and magnoflorine Besides these alkaloids havebeen reported such as anti-human immunodeficiency virus(HIV) and antioxidant and anticancer agents (see [3 4]and the references therein) Because these compounds aretoo complex for cost-effective chemical total synthesismicrobial systems for benzylisoquinoline-type alkaloidproduction were used [9 10] but attempts to reconstructthe entire metabolic pathway leading to (S)-reticuline andbeyond this branch-point biochemical intermediate tothe synthesis of many other related alkaloids have beenachieved recently by the group of Minami and coworkers[11] They established a microbial system combining twodifferent microorganisms together with microbial andplant-derived genes Reticuline production was achievedfirst in engineered Escherichia coli and the biosynthesisof specific alkaloids was terminated using a coculture ofengineered E coli and Saccharomyces cerevisiae [11] Thisstudy resulted in (i) the biosynthesis of (S)-reticuline thekey intermediate in the formation of benzylisoquinolinealkaloids only when using a methyl group donor and(ii) the production of specific alkaloids magnoflorine andcorytuberine on one hand and on the other hand scoulerinedepending on the targeted genes Reticuline in engineeredE coli was obtained as follows (Figure 1) dopaminewas converted first to 34-dihydroxyphenylacetaldehydeby a monoamine oxidase from Micrococcus luteus (S)-norlaudanosoline was synthesized by the condensationof dopamine and 34-dihydroxyphenylacetaldehyde bya norcoclaurine synthase from Coptis japonica thenleading to (S)-31015840-hydroxycoclaurine via a norcoclaurine6-O-methyltransferase from C japonica (S)-31015840-hydroxy-N-methylcoclaurine was obtained through the action ofcoclaurine-N-methyltransferase from C japonica leadingto (S)-reticuline via the 31015840-hydroxy-N-methylcoclaurine-41015840-O-methyltransferase from C Japonica When S-adenosyl-L-methionine (SAM) was used as a methyl group donorthe engineered biosynthesis pathway leads predominantlyto the stereospecific (S)-reticuline with a yield of 55mgLwithin 1 h S cerevisiae expressing heterologous geneswere added to E coli cells engineered with the reticulinebiosynthetic genes with 5mM dopamine in the medium aftera certain period of culture S cerevisiae was engineered toharbor the genes encoding the P450 enzyme corytuberinesynthase (CYP80G2) and a CNMT from C japonica(possibly a CNMT-like methyltransferase) leading tomagnoflorine at a yield of 72mgL within 72 h without anyaddition of SAM Otherwise the second alkaloid produced(S)-scoulerine was obtained with a S cerevisiae strainexpressing the berberine bridge enzyme (BBE) from Cjaponica at a yield of 83mgL within 48 h The conversionefficiencies of reticuline to magnoflorine or scoulerine inS cerevisiae cells were 655 and 755 respectively Thisunderlines that such a working system results in the efficientproduction of benzylisoquinoline alkaloids in engineeredmicrobes The system could be generalized and applied to

MAODopamine 34-dihydroxyphenyl

acetaldehyde

NCS

(119878)-norlaudanosoline60MT

-hydroxycoclaurine

CNMT(119878)-3998400-hydroxy-119873-methylcoclaurine

4998400OMT

BBE(119878)-scoulerine

CYP80G2CorytuberineBerberine

Corytuberine-119873-methyltransferase

(CNMT)Magnoflorine

(119878)-reticuline (119878)-reticuline

(119878)-3998400

Saccharomyces cerevisiae

Escherichia coli

Figure 1 Benzylisoquinoline alkaloid biosynthetic pathway recon-structed in Escherichia coli and Saccharomyces cerevisiae For in vivoproduction of (S)-reticuline transgenicE coli expresses biosyntheticgenes MAO NCS 6OMT CNMT and 41015840OMT For in vivo produc-tion of (S)-scoulerine and magnoflorine from reticuline originatingfrom engineered E coli transgenic S cerevisiae expresses respec-tively the genes BBE CYP80G2 andCNMT All genes are fromCop-tis japonica except the geneMAOwhich originates fromMicrococcusluteus MAO monoamine oxidase NCS norcoclaurine synthase6OMT norcoclaurine 6-O-methyltransferase CNMT coclaurine-N-methyltransferase 41015840OMT 31015840-hydroxy-N-methylcoclaurine-41015840-O-methyltransferase BBE berberine bridge enzyme CYP80G2P450 corytuberine synthase

the production of many other alkaloids of medical interest[11]

Recently an important modification of the pathwayleading to (S)-reticuline was reported by the same group [12]An E coli strain that over produces L-tyrosine by amino-acidfermentation was first generated This was accomplished bythree steps of genetic engineering Production of dopaminewas then obtained via the introduction in E coli of a tyrosi-nase and its adaptor protein from Ralstonia solanacearumable to convert L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) along with a L-DOPA decarboxylase from Pseu-domonas putida leading to L-dopamine This short pathwayclosed the gap between L-tyrosine and the synthetic pathwayto reticuline via the monoamine oxidase from M luteusand leading to the 34-dihydroxyphenylacetaldehyde (see theprevious section) In the final step of the fermentative pro-duction of (S)-reticuline the dopamine-producing pathwaydescribed above was combined with the synthetic pathwayfrom dopamine to (S)-reticuline

Engineering microbes also offers great opportunitiesfor the biosynthesis of plant polyketides of medical interestincluding antibiotics (erythromycin rifamycin) anticancerdrugs (tetracyclines anthracyclines and epothilones)antiparasitic agents (avermectin) cholesterol-lowering


SCoA SCoA

O OHCO2H





Erythromycin A




SSSSSSSO

O

OH

OH

OH

OH

O

O

O

OO

O

O

O

O

OOH

OH

OHOH

OHOH

HOHO

N(CH3)2

OCH3

O

O

OH

OH

O

O

OH

OH

O

O

OH

OH

OH

O

OH

OH

OOOH

6x









600to 2008mgLOD

600




4 Anticancer Drugs


5units respec-


5units and larger


Sugars

PyruvateG3P


FPPsynthase

GPPsynthase


synthase

Taxadienesynthase

BaccatinIII

Taxol

Taxadiene5120572-

hydroxylase

Taxadiene5120572-OL

Taxa-4(5)11(12)

-diene

Geranyl-geranyl PP















Acetoacetyl-CoA

Mevalonate pathway


synthaseERG20

FPPsynthaseERG20

Squalene GPP


Semisynthesis



Artemisinicacid


411-diene




























Shikimatepathway

Phenylalanine

PALTyrosine

Cinnamic acidTAL

4CH

4CL

+

Sugarmetabolism

Malonyl-coenzyme A

STS CHS













9 Conclusions









References








































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


SCoA SCoA

O OHCO2H





Erythromycin A




SSSSSSSO

O

OH

OH

OH

OH

O

O

O

OO

O

O

O

O

OOH

OH

OHOH

OHOH

HOHO

N(CH3)2

OCH3

O

O

OH

OH

O

O

OH

OH

O

O

OH

OH

OH

O

OH

OH

OOOH

6x









600to 2008mgLOD

600




4 Anticancer Drugs


5units respec-


5units and larger


Sugars

PyruvateG3P


FPPsynthase

GPPsynthase


synthase

Taxadienesynthase

BaccatinIII

Taxol

Taxadiene5120572-

hydroxylase

Taxadiene5120572-OL

Taxa-4(5)11(12)

-diene

Geranyl-geranyl PP















Acetoacetyl-CoA

Mevalonate pathway


synthaseERG20

FPPsynthaseERG20

Squalene GPP


Semisynthesis



Artemisinicacid


411-diene




























Shikimatepathway

Phenylalanine

PALTyrosine

Cinnamic acidTAL

4CH

4CL

+

Sugarmetabolism

Malonyl-coenzyme A

STS CHS













9 Conclusions









References








































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






600to 2008mgLOD

600




4 Anticancer Drugs


5units respec-


5units and larger


Sugars

PyruvateG3P


FPPsynthase

GPPsynthase


synthase

Taxadienesynthase

BaccatinIII

Taxol

Taxadiene5120572-

hydroxylase

Taxadiene5120572-OL

Taxa-4(5)11(12)

-diene

Geranyl-geranyl PP















Acetoacetyl-CoA

Mevalonate pathway


synthaseERG20

FPPsynthaseERG20

Squalene GPP


Semisynthesis



Artemisinicacid


411-diene




























Shikimatepathway

Phenylalanine

PALTyrosine

Cinnamic acidTAL

4CH

4CL

+

Sugarmetabolism

Malonyl-coenzyme A

STS CHS













9 Conclusions









References








































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Sugars

PyruvateG3P


FPPsynthase

GPPsynthase


synthase

Taxadienesynthase

BaccatinIII

Taxol

Taxadiene5120572-

hydroxylase

Taxadiene5120572-OL

Taxa-4(5)11(12)

-diene

Geranyl-geranyl PP















Acetoacetyl-CoA

Mevalonate pathway


synthaseERG20

FPPsynthaseERG20

Squalene GPP


Semisynthesis



Artemisinicacid


411-diene




























Shikimatepathway

Phenylalanine

PALTyrosine

Cinnamic acidTAL

4CH

4CL

+

Sugarmetabolism

Malonyl-coenzyme A

STS CHS













9 Conclusions









References








































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Acetoacetyl-CoA

Mevalonate pathway


synthaseERG20

FPPsynthaseERG20

Squalene GPP


Semisynthesis



Artemisinicacid


411-diene




























Shikimatepathway

Phenylalanine

PALTyrosine

Cinnamic acidTAL

4CH

4CL

+

Sugarmetabolism

Malonyl-coenzyme A

STS CHS













9 Conclusions









References








































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Shikimatepathway

Phenylalanine

PALTyrosine

Cinnamic acidTAL

4CH

4CL

+

Sugarmetabolism

Malonyl-coenzyme A

STS CHS













9 Conclusions









References








































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








9 Conclusions









References








































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





References








































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Review Article Engineering Microbial Cells for the...

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