Chem Soc Rev - University Of Illinoisscs.illinois.edu/~zhaogrp/publications/HZ202.pdfTerpenoids,...

This journal is©The Royal Society of Chemistry 2015 Chem. Soc. Rev.

Cite this:DOI: 10.1039/c5cs00025d

Engineered biosynthesis of natural products inheterologous hosts

Yunzi Luo,†‡ab Bing-Zhi Li,‡ac Duo Liu,‡ac Lu Zhang,ac Yan Chen,ac Bin Jia,ac

Bo-Xuan Zeng,ac Huimin Zhaob and Ying-Jin Yuan*ac

Natural products produced by microorganisms and plants are a major resource of antibacterial and

anticancer drugs as well as industrially useful compounds. However, the native producers often suffer from

low productivity and titers. Here we summarize the recent applications of heterologous biosynthesis for the

production of several important classes of natural products such as terpenoids, flavonoids, alkaloids, and

polyketides. In addition, we will discuss the new tools and strategies at multi-scale levels including gene,

pathway, genome and community levels for highly efficient heterologous biosynthesis of natural products.

1. Introduction

Molecules from natural sources, especially bacteria, fungi andplants, have proven to be a large reservoir for new pharmaceuticals,therapeutic agents and industrially useful compounds.1 To date,natural products and their derivatives command a substantial

market share in pharmaceutical industry in the past 30 years,comprising 61% of anticancer compounds and 49% of anti-infectives.2 For example, erythromycin from Saccharopolyspora ery-thraea is an antibiotic, which has an antimicrobial spectrum similarto that of penicillin and can be used to treat various diseases.3

Lovastatin (Merck’s Mevacor) isolated from Aspergillus terreus is oftenused to reduce risk of cardiovascular disease, while paclitaxelextracted from the bark of the Pacific yew, Taxus brevifolia, is amitotic inhibitor used in cancer chemotherapy. There are alsoimportant natural products discovered from other diverse sources,such as insects and marine sources. In the old days before 1940s,natural products of interest are isolated directly from plant sources(Fig. 1A). However, bioactive compounds from natural sources areusually secondary metabolites of their native producers, thus insome cases, they exist at a relatively low level comparing with theproduction of primary metabolites. Therefore, in most cases, directisolation and extraction is not an environmental friendly, sustainableand economical solution. Combinatorial chemistry was also

a Key Laboratory of Systems Bioengineering (Ministry of Education),

Tianjin University, Tianjin, 300072, P. R. China. E-mail: [email protected];

Fax: +86-22-27403888b Department of Chemical and Biomolecular Engineering and Institute for Genomic

Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Ave,

Urbana, IL 61801, USAc SynBio Research Platform, Collaborative Innovation Center of Chemical Science

and Engineering (Tianjin), School of Chemical Engineering and Technology,

Tianjin University, Tianjin, 300072, P. R. China

Yunzi Luo

Dr Yunzi Luo received her PhD inChemical and Biomolecular Engi-neering from the University ofIllinois at Urbana and Champaignin 2014. Her research has focusedon natural products discovery andsynthetic biology. Dr Luo was apostdoctoral research fellow at theInstitute for Genomic Biology ofUniversity of Illinois at Urbana andChampaign. She has published11 papers in peer-reviewed inter-national journals and 2 bookchapters.

Bing-Zhi Li

Dr Bing-Zhi Li is an associateprofessor of Pharmaceutical Engi-neering in Tianjin University. Hereceived his BS degree fromNankai University in 2005 and hisPhD from Tianjin University in2010. His research expertise is insynthetic biology and lignocellulosebioconversion. He has publishedmore than 20 peer-reviewed inter-national papers.

† Current address: State Key Laboratory of Biotherapy/Collaborative InnovationCenter of Biotherapy, West China Hospital, Sichuan University, Chengdu, P. R.China. Renmin South Road 3, No. 17, Chengdu, Sichuan, 610041, P. R. China.‡ Equal contribution.

Received 12th January 2015

DOI: 10.1039/c5cs00025d

www.rsc.org/csr

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introduced to synthesize and screen small molecules of medicinalimportance (Fig. 1B). Still, due to the structure complexity ofnatural products, chemical synthesis of these compounds isoften impractical.

To solve these problems, researchers have engineered and opti-mized natural products biosynthetic pathways for decades. Asgenetic manipulation is either difficult or yet-to-be established forthe majority of organisms, heterologous expression of a single gene,a cassette of genes, or an entire biosynthetic gene cluster in agenetically tractable host is a practical alternative route for identify-ing and engineering the corresponding natural product. In recentyears, with the third revolution in biotechnology, which requires theconvergence of life science, physical science and engineering dis-ciplines, new solutions have been proposed for drug discovery,energy and food crisis.4 The first revolution of biology was evidencedby the discovery of the double helix structure of DNA by JamesWatson and Francis Crick, while the second revolution began withthe human genome project. Now, with the dawn of the post-genomics era came the third revolutionary change, which shiftsthe paradigm of the biology field from the understanding and

engineering of microbial biosynthetic pathways towards the designand creation of novel metabolic circuits and biological systems. Withthe newly developed biosystems, the yield of target natural productscould be increased noticeably in a heterologous host. For example,Keasling and his colleagues successfully redesigned the biosynthesisprocess of the artemisinin precursor, artemisinic acid, in hetero-logous hosts Escherichia coli and Saccharomyces cerevisiae,5–7 whichproduced the artemisinic acid with a much higher productivity, titerand yield and now the process has been transferred to a pharma-ceutical company for commercialization. In addition, with theversatility of synthetic biology approaches, new compounds couldbe discovered or generated by pathway refactoring or advancedcombinatorial biosynthesis (Fig. 1C).8,9

Microbes, especially E. coli and yeast, have proven to beuseful organisms for heterologous biosynthesis of secondarymetabolites for decades. They have been engineered to balanceand optimize natural product biosynthetic pathways for cost-effective production of high value compounds, as well as todiscover novel compounds.10 Recent advances in syntheticbiology and genome engineering facilitate the development ofnew tools for construction, controlling and optimization ofmetabolic pathways in microbes for heterologous biosynthesis.

Herein, we review the recent progresses made in heterologousbiosynthesis of several different classes of important naturalproducts, as well as how the newly developed synthetic biologytools could help future heterologous biosynthesis of valuablenatural products. In addition, we will highlight some of the mostrecent advances in heterologous biosynthesis of natural productin the past five years.

2. Heterologous biosynthesis of majorclasses of high value natural products

Natural products are a very important source for the develop-ment of medicines. Many natural products and the derivatives

Huimin Zhao

Dr Huimin Zhao is the CentennialEndowed Chair Professor ofchemical and biomolecular engi-neering, and professor of chemistry,biochemistry, biophysics, andbioengineering at the University ofIllinois at Urbana-Champaign(UIUC). He received his BS degreein Biology from the University ofScience and Technology of Chinain 1992 and his PhD degree inChemistry from the CaliforniaInstitute of Technology in 1998.His primary research interests are

in the development and applications of synthetic biology tools toaddress society’s most daunting challenges in human health andenergy, and in the fundamental aspects of enzyme catalysis and generegulation.

Ying-Jin Yuan

Dr Ying-Jin Yuan is currently aprofessor of Biochemical Engi-neering in Tianjin University. Hereceived his PhD degree fromTianjin University in 1991. He isnow the director of Key Laboratoryof Systems Bioengineering, Ministryof Education, China. His researchinterests include synthetic biology,systems biotechnology, bioenergy,biopharmaceutical engineeringand plant cell culture. Dr Yuan isthe Fellow of IChemE. Dr Yuan haspublished more than 180 inter-national peer-reviewed papers and30 patents.

Duo Liu

Duo Liu is currently a PhDcandidate in the Department ofBiochemical Engineering in TianjinUniversity. He has worked on themodular strategies of syntheticbiology for combinatorial construc-tion of yeast metabolic system fornatural product biosynthesis.

Review Article Chem Soc Rev

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have been developed as antibacterial, anticancer, antifungal,antiviral and antiparasitic drugs.2 Butler et al. systematicallysummarized the development of drugs in clinical trials fromnatural products and the derivatives.11 In this section, we willprovide a comprehensive overview of the production of differentclasses of high-value compounds from microbes. In addition, we willdiscuss the recently developed metabolic engineering and syntheticbiology approaches employed to engineer microorganisms forheterologous expression of natural product pathways, especiallyfor higher production of practically important natural products.

2.1 Terpenoids

Terpenoids, also called isoprenoids, are the largest class ofplant metabolites, consisting of more than 40 000 molecules.12

The most famous plant terpenoids have been used as the majordrugs to treat life-threatening diseases, such as the anticancerdrug paclitaxel and the antimalarial drug artemisinin.13 Someother terpenoids are supplemented with daily nutritional diets,such as lycopene and linalool. Terpenoids usually consist ofdifferent numbers of basic five-carbon isopentenyl diphosphate(IPP) units with a series of assembly, cyclization and groupmodification reactions.14 Traditional methods for producingthese compounds usually rely on multistep extraction fromplants or organic chemical synthesis, both of which can resultin low yield, high cost and sometimes severe pollutions to theenvironment.

In microbes, such as E. coli and yeast, the terpenoidsbiosynthetic pathways typically work with very low fluxes. Theimportant precursors for terpenoids, IPP and its isomerdimethylallyl pyrophosphate (DMAPP), are synthesized fromacetyl-CoA through the DXP pathway in E. coli-like bacteria and

the MVA pathway in yeast-like fungi. In yeast, some highlyactive IPP compounds can also be synthesized based on thebasic C5 units. For example, geranyl pyrophosphate (GPP) is aC10 structure generated by condensing one molecule IPP andone molecule DMAPP. Following this reaction, an additionalIPP molecule can be combined to make a C15 structure,farnesyl pyrophosphate (FPP), and further with another IPP tomake a C20 structure, geranylgeranyl pyrophosphate (GGPP). Innative yeast, ergosterol, as the final metabolite of the successivecondensation reactions, is derived from C30 squalene which issynthesized by condensation of two FPP molecules (Fig. 2).These reactions in bacteria and fungi enable the introduction ofgenes of plant origins to construct the heterologous terpenoidsbiosynthetic pathways. Monoterpenoids are synthesized fromGPP; sesquiterpenoids from FPP; diterpenoids and carotenoidsfrom GGPP; and triterpenoids from squalene.

2.1.1 Monoterpenoids. The most well-known monoterpenoidsthat have been heterologously synthesized in engineered yeast arelinalool and geraniol. Although wild-type yeast does not synthesizethese monoterpenes, the mutants with blocked FPP synthetase thatexcretes geraniol and linalool have been characterized previously.15

However, the enzyme involved in GPP dephosphorylation has notbeen identified yet.

Linalool is a value-added fine chemical, and can be usedto synthesize linalyl acetate, geraniol, linalyl methyl sulfide,vitamin A, vitamin K and other medical intermediates.16 Linalool issynthesized via the condensation of IPP and DMAPP by the endo-genous mevalonate pathway. Several cis or trans linalool synthaseswere identified from different origins.17–19 The engineered industrialyeast strain with a linalool synthase from Clarkia breweri producedlinalool with no by-products.20 In addition, over-expression of

Fig. 1 Routes for heterologous biosynthesis of natural products. (A) Synthesis of natural products by traditional isolation from plants. (B) Synthesis andscreening of small molecules by combinatorial chemistry. (C) Production of natural products by heterologous biosynthesis in microbes.

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the hmg1 gene in the engineered yeast strain enhanced theproduction of linalool to 133 � 25 mg L�1.21

Geraniol, as a widely used floral fragrance, is a typicalmonoterpene, and can be further transformed into ester flavorsfor food flavors, fruit flavors, ginner flavors, and cinnamonflavors.22 It is also used for clinical treatment of chronic bronchitisand improvement of pulmonary ventilation function.22 Geraniol isusually obtained from myrcene by esterification, saponificationand fractionation in current industrial production. However,geraniol can also be synthesized from linalool. The geraniolsynthase (GES) was isolated from Ocimum basilicum and expressedin S. cerevisiae to produce geraniol.23,24 Furthermore, blocking ofFPP synthase increased geraniol synthesis.24

2.1.2 Sesquiterpenoids. Sesquiterpenoids are the most diverseclass of terpenoids, including industrially valuable products forfragrances, repellents, food colorants and high-value therapeuticmolecules, such as epicedrol, artemisin, valencene, cubebol,patchoulol and santalene.25–31 All these sesquiterpenoids arederived from a 15-carbon precursor, FPP. Different sesquiter-pene synthases were used to catalyze the synthesis of differentsesquiterpenes from FPP (Fig. 3). b-Sesquiphellandrene, withefficient anti-microbial and anti-oxidant activity, was synthesizedby PMSTS (Persicaria minor sesquiterpene synthase).28,29 Valencene,an odorant molecule, was synthesized by CsPTS (Citrus sinensisvalencene synthase), and could be further oxidized to (+)-nootkatol,

(�)-nootkatol or (�)-nootkatone by different kinds of P450cytochrome oxidases (CYP71D51v2 from Nicotiana tabacum,CYP71AV8 from Cichorium intybus and CnVO from C. nootka-tensis).31,32 Patchoulol, the major constituent of patchouli oil,was synthesized by PTS (patchoulol synthase from Pogostemoncablin).25,26 In addition, other valuable sesquiterpenes, includ-ing arteminisic acid, cubeol, germacrene D, b-caryophyllene,a-santalene, were recently produced in engineered microbes.30,33,34

Importantly, the biosynthesis of artemisinin, a valuable andpowerful antimalarial natural product extracted from Artemisiaannua L, was identified and engineered. Firstly, FPP was convertedto amorphadiene by ADS (amorphadiene synthase). Then amor-phadiene was oxidized to artemisinic alcohol by CYP71AV1 (a P450cytochrome oxidase). It was then reduced by DBR2 (artemisinicaldehyde reductase) to dihydroartemisinic aldehyde and oxidizedto dihydroartemisinic acid by ALDH1 (aldehyde dehydrogenasefrom A. annua). Finally, dihydroartemisinic acid was converted toartemisinin via an allylic hydroperoxide intermediate, and light andoxygen were required for this conversion.5

Most of these sesquiterpenes have been successfully producedin engineered microbes such as E. coli, S. cerevisiae and otherindustrial microorganisms. To increase the production of thesesesquiterpenes, the enzymes from plants or the flux of the wholepathway need to be modified. For example, in engineering patch-oulol producing yeast, PMSTS was fused with ERG20 (FPP synthase)

Fig. 2 Schematic of various biosynthetic pathways of terpenoids. Two pathways, the MVA pathway and the DXP pathway, provide the precursors for thebiosynthesis of terpenes, which are the backbones for the synthesis of terpenoids, including monoterpenoids, sesquiterpenoids diterpenoids,triterpenoids and carotenoids.


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to optimize the local concentration and spatial organization ofthese two enzymes and to redirect the metabolic flux towards theproduct.26 Combined with the repression of ERG9, the finalpatchoulol titer reached 23 mg L�1.26 In addition, higher FPPconcentration and increased production of valencene wasachieved by localizing the enzyme to the mitochondrion.35

Another successful example of heterologous biosynthesis ofsesquiterpene is the optimized biosynthesis of artemisinic acid.6

The systematic optimization started with regulating the propor-tion of CYP71AV1 and AaCPR1, followed by overexpressingAaCYB5 (cytochrome b5 from A. annua) and native HEM1 tooptimize the oxidation of amorphadiene which is the key step ofthe synthesis of artemisinic acid. At the same time, the nativeCTT1 (catalase) is also overexpressed to reduce the oxidativestress produced by CYP71AV1. AaADH1 (alcohol dehydrogenasefrom A. annua) and AaALDH1 are overexpressed to promote theconversion of artemisinic acid from artemisinic alcohol. Thefinal production of artemisinic acid reached up to 25 g L�1.6

2.1.3 Diterpenoids. Diterpenoids are widely distributed naturalproducts with important physiological effects, such as paclitaxelagainst cancer, tanshinones against inflammation, and steviosideas sweetener.13,36,37 However, the amount of the diterpenoids in thehost plants is usually very low, let alone the slow growth rate ofplants. For example, the paclitaxel content is only at 10 ppm levelin the most productive species, Taxus brevifolia.38 In addition,complex diterpenoids molecules are quite difficult for chemical

synthesis de novo.39 Therefore, heterologous expression of the path-ways in microbes is very promising for diterpenoids production.

Tanshinones are the main lipophilic components of theChinese medicinal herb danshen (Salvia miltiorrhiza). Modularpathway engineering (MOPE) was successfully developed atassemble and optimize the biosynthetic pathway of miltiradiene,the precursor to tanshinones, in S. cerevisiae.40 The fusion ofSmCPS and SmKSL and the fusion of GGPPS (GGPP synthase)and FPPS, together with overexpression of tHMGR led to significantimprovement of miltiradiene production (365 mg L�1) and reducedbyproduct accumulation significantly.40 Based on the increasedproduction of miltiradiene, incorporation of genes coding forCYP76AH1 and phyto-CYP reductase led to heterologous produc-tion of ferruginol, the precursor to danshinones.41

Paclitaxel, as a diterpenoid, has been approved for treatmentof breast, ovarian and lung cancers, as well as coronary arterydisease.42 Although some studies tried to figure out the biosyntheticpathway for paclitaxel in fungal endophytes and improve thepaclitaxel production by fermentation,43 a recent study indicated nopaclitaxel pathway could be identified in fungal endophytes.44 Dueto the high clinical needs and high costs of production, researchershave been mining the key genes for paclitaxel biosynthesis fordecades. Most of the biosynthetic reactions have been elucidated,and several important genes or enzymes have been characterized(Fig. 4). Based on the elucidated partial pathway for paclitaxelproduction, the heterologous synthesis of paclitaxel precursors

Fig. 3 Schematic of sesquiterpenoids biosynthesis. Farnesyl diphosphate (FPP), the 15-carbon precursor, is converted to different sesquiterpene by varioussesquiterpenoid synthases (ADS, amorphadiene synthase; CsPTS, valencene synthase; PMSTS, Persicaria minor sesquiterpene synthase; PTS, patchoulol synthase;VMPSTS, Vanda mimi palmer sesquiterpene synthase; SanSyn, santalene synthase). Some of these sesquiterpenes can be further oxidized to their derivatives byvarious cytochrome P450 oxidases (CYP71AV8, CnVO, CYP71D51V2 and CYP71AV1). In the biosynthesis pathway of artemisinin, the well-known sesquiterpenoid,the artemisinic alcohol can be further converted to artemisinin or artemisinic acid by several enzymes (AaALDH1, artemisinic aldehyde dehydrogenase; ADH1,artemisinic alcohol dehydrogenase; DBR2, artemisinic aldehyde reductase; CPR1, reductase of CYP71AV1; CYB5, cytochrome b5 from A. annua).


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were investigated in different microbes. E. coli was firstly usedas the host cell for the heterologous biosynthesis of taxadiene.45

The heterologous biosynthesis of taxadiene in K-derived E. coliand B-derived E. coli indicated that the background of thechassis affected taxadiene synthesis.46 A multivariate-modularapproach increased titers of taxadiene to approximately 1 g L�1

(B15 000-fold increase) in an engineered E. coli strain.47

Besides E. coli, S. cerevisiae was also used as the chassis toproduce taxadiene.48 Five enzymes catalyzing sequential trans-formations from IPP and DMAPP were heterologously expressed inS. cerevisiae, and taxadiene titer was improved to 8.7� 0.85 mg L�1

by overexpression of tHMGR and UPC2-1.49 Six different GGPPSswere analyzed using enzyme-substrate docking strategy, and GGPPSfrom Taxus baccata cuspidate showed the best performance indocking with substrate molecule FPP and led to the highesttaxadiene production (72.8 mg L�1) ever reported in yeast so far.50

Although the paclitaxel precursors were produced with highefficiency by heterologous biosynthesis in different chassis, theheterologous biosynthesis of paclitaxel is still impossible due tothe lack of information for several key biosynthetic steps.Therefore, some alternative bioconversion approaches weredeveloped to produce paclitaxel. An Enterobacter sp. was foundto convert 7-xylosyl-10-deacetyltaxol (7-XDT) to 10-deacetyltaxol(10-DT), which is the most abundant byproduct in Taxus. Thehighest conversion rate and yield of 10-DT from 7-XDT reached92% and 764 mg L�1, respectively.51 A xylosidase from Enter-obacter sp. was discovered and heterologously expressed inyeast.52 The engineered yeast could robustly convert 7-XDT into10-DT with over 85% conversion rate and the highest yield of8.42 mg mL�1.52

2.1.4 Triterpenoids. Triterpenoids are synthesized by con-densation of six C5 IPP basic units through end-to-end couplingreactions. Triterpenoids are widely distributed in nature andcan be divided into two main categories, pentacyclic triterpe-noids (such as ginsenoside) and tetracyclic triterpenoids (suchas amyrin-derivative terpenoids), according to the number ofcarbon rings contained in the compounds.53

The chemical compositions of the ginseng complex haveextensive biological activities and unique pharmacologicalactions. Medical and pharmacological research proved thatone of the main effective components of ginseng is a groupof ginsenosides, which are considered to possess medical valueas anti-tumor, antiviral and cholesterol-decreasing drugs andprecursors. Genetic engineering of plants was tried to improvethe ginsenoside production by overexpression of the key genescoding dammarane synthase and amyrin synthase. However,limited success was achieved.54

Three biosynthetic pathways for various ginsenosides weresuccessfully constructed in S. cerevisiae,55 and they are respon-sible for the alternative synthesis of protopanaxadiol, proto-panaxatriol and oleanolic acid, the three basic aglycons ofginsenosides. The engineered S. cerevisiae was transformedwith expression cassettes of b-amyrin synthase, oleanolic acidsynthase, dammarenediol-II synthase, protopanaxadiol synthase,protopanaxatriol synthase and NADPH-cytochrome P450 reductasefrom different plants. Through additional overexpression of some

endogenous genes, the final engineered strain produced17.2 mg per L protopanaxadiol, 15.9 mg per L protopanaxatrioland 21.4 mg per L oleanolic acid.

The main tetracyclic triterpenoids successfully synthesizedby engineered microbes are amyrin-derivative compounds,including a-amyrin type and b-amyrin type. Several key functionalgenes were introduced into yeast cells to produce various amyrinstructures.53,56 The main product based on b-amyrin precursor isoleanolic acid.55 Catharanthus roseus is an important medicinalplant and commercial source of monoterpenoid indole alkaloids,ursolic acid and oleanolic acid. The EST collection from C. roseusleaf epidermome was analyzed, and a cDNA (CrAS) encoding2,3-oxidosqualenecyclase and a cDNA (CrAO) encoding amyrinC-28 oxidase were successfully discovered.57 The functions ofCrAS and CrAO were analyzed in S. cerevisiae. The co-expressionof CrAO and CrAS in yeast led to ursolic- and oleanolic acidsproduction. In addition, the cells co-expressing CrAO andAtLUP1 from Arabidopsis thaliana produced betulinic acid.Another typical triterpenoid is ginsenosides compound K(CK).58 Among the several structures of ginsenosides, CK isthe main functional component presenting bioactivities of anti-inflammation, hepatoprotection, antidiabetes and anti-cancer.The potential CK biosynthetic pathway was designed in yeast,including a cytochrome P450 (CYP716A47) from Panax ginseng,a NADPH-cytochrome P450 reductase (ATR2-1) from Arabidopsisthaliana, and a Dammarenediol-II synthase (PgDDS) fromP. ginseng, and the CK synthase was explored based on bio-informatics analysis. CK was synthesized heterologously for thefirst time in engineered microbes by two pathways, especiallyconsidering active CK could not be detected in original Panaxplants. The final production of CK reached 1.4 mg L�1.

2.1.5 Tetraterpenoids. Tetraterpenoids consist of eight iso-prene units and are mainly involved in metabolic precursorsand products in the biosynthesis of carotenoids with uniqueUV-Vis absorption spectra.59 Carotenoids serve as precursorsfor vitamin A biosynthesis, but more importantly, they demon-strate coloring and antioxidant properties attributed to theirstructures. Therefore, their expanded production is appealing.

Many kinds of hosts can produce carotenoids, such asAgrobacterium aurantiacum, Blakeslea trispora, several Pantoeabacteria, Xanthophyllomyces dendrorhous and Haematococcuspluvialis. Commonly in carotenogenic bacteria and algae, bio-synthesis of carotenoids starts from GGPP, which is synthesizedby GGPP synthase (CrtE) either through direct condensation of5-carbon precursors, IPP and DMAPP, or from 15-carbon pre-cursor, FPP. A tail to tail condensation of two GGPP moleculescatalysed by phytoene synthase (CrtB) leads to the formation ofthe primal carotenoid, phytoene, which is colorless. Then,through successive introduction of four double bonds in thephytoene skeleton by phytoene desaturase (CrtI), lycopene issynthesized. Using lycopene as substrate, lycopene b-cyclase(CrtY) catalyzes the formation of b-ionone rings at both ends toproduce b-carotene. Different from some bacteria and algaes, thephytoene synthase and phytoene desaturase belong to a bifunctionalenzyme in red yeast, X. dendrorhous, and other fungi, B. trispora.X. dendrorhous and H. pluvialis are well-known microorganisms


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Fig. 4 Schematic of the paclitaxel biosynthetic pathway. Paclitaxel is synthesized by the combination of the core taxane and the side chain. Thecore taxane structure is synthesized by the modification of the taxadiene, which is produced by the cyclization of GGPP. The solid arrows in thepink background mean the steps with known reactions and known enzymes. The solid arrows in the light blue background mean the steps withknown reactions but unknown enzymes. The dotted arrows mean the steps with unknown reactions and unknown enzymes. FPP, farnesylpyrophosphate; GGPP, geranylgeranyl pyrophosphate. TDC1, taxadiene synthase; CYP725A2: taxane 13 alpha-hydroxylase; CYP725A1, taxane10 beta-hydroxylase; TBT, taxoid 2a-O-benzoyl transferase; DBAT, 10-deacetylbaccatin III 10-O-acetyltransferase; PAM, phenylalanineaminomutase.


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capable of synthesizing astaxanthin. In both prokaryotes andeukaryotes, biosynthesis of astaxanthin is the results of ketola-tion at the 4 and 40-position and hydroxylation at the 3 and 30

positions of the b-ionone rings of b-carotene. In bacteria, keto-lase (CrtW) and hydroxylase (CrtZ) are required for astaxanthinbiosynthesis. However, due to their weak selectivity toward keto-and hydroxyl-substrate, quite a few intermediates accumulatedwhen combining bacteria crtW and crtZ to produce astax-anthin.60,61 In red yeast X. dendrorhous, astaxanthin synthase(CrtS), a cytochrome P450 enzyme, in combination with itscytochrome P450 reductase (CrtR), are responsible for astax-anthin biosynthesis.60 In plant Adonis aestivalis, the 4-positionof b-ring of b-carotene is activated by 4-dehydrogenase-b-ring(CBFD), followed by further dehydrogenation to yield a carbonylcatalyzed by 4-hydroxy-b-ring 4-dehydrogenase (HBFD). Andthen, the 3-position is added by CBFD with a hydroxyl group,which leads to the synthesis of astaxanthin. This pathway wasproved to work efficiently in E. coli (Fig. 5).62

Heterologous biosynthesis of carotenoids was mostly carriedout in two hosts, E. coli and baker’s yeast. In E. coli, the main

products are lycopene and b-carotene. By combining the nativeMEP pathway and the heterologous MVA pathway, 1.35 g per Llycopene63 and 2.47 g per L b-carotene with a yield of 72 mg perg DCW64 was produced by fed-batch fermentation in E. coli.Most recently, 3.2 g per L b-carotene was achieved under flaskcultivation employing a similar strategy.65 With a targetedengineering strategy, a very high maximum productivity of74.5 mg L�1 h�1 and up to 1.23 g per L of lycopene wasproduced in 100 L fed-batch fermentation.66 ATP and NADPHrecycling was engineered in E. coli to enhance terpenoidsproduction.67 Five alternative functional modules weredesigned for combinatorial metabolic engineering, includingthe heterologous synthesis module, MEP module, ATP module,PPP module, and TCA module, which led to an optimized straincapable of producing 2.1 g per L b-carotene with a yield of60 mg per g DCW. In addition, expression tuning the expressionlevel of the genes encoding a-ketoglutarate dehydrogenase,succinate dehydrogenase and transaldolase B increased NADPHand ATP supplies, leading to 3.52 g lycopene per L (50.6 mg per gDCW) in fed-batch fermentation.68

Fig. 5 Schematic of the various biosynthetic pathways of carotenoids. (A) The common pathway for lycopene and b-carotene biosynthesis in bacteriaand fungi. IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; CrtE,GGPP synthase; CrtB, phytoene synthase; CrtI, phytoene desaturase; CrtY, lycopene b-cyclase; (B–D) The astaxanthin biosynthetic pathway incarotenogenic bacteria and algae (B), red yeast X. dendrorhous (C), and plant Adonis aestivalis (D). CrtZ, b-carotene hydroxylase; CrtW, b-caroteneketolase; CrtS, P450 cytochrome monooxygenase; CrtR, cytochrome P450 reductase; CBFD, carotenoid b-ring 4-dehydrogenase; HBFD, carotenoid4-hydroxy-b-ring 4-dehydrogenase.


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In yeast, carotenoids are the first class of terpenoids to beproduced. In the early period, like the studies in E. coli,engineering efforts sourced carotenoid biosynthetic genes fromseveral microorganisms, in particular the red yeast X. dendrorhousand the soil bacterium Pantoea ananatis, and achieved low-levelproduction of target carotenoids from endogenous precursors.69

Since then, researchers began to engineer yeast strains to accumu-late more quantities of GGPP precursor for carotenoid productionby either introducing a heterologous GGPP synthase gene crtE69,70

or overexpressing the native gene bts1.60,71 By using this combina-torial genetic engineering strategy, the expressions of several geneswere optimized, leading to 5.9 mg per g DCW b-carotene andastaxanthin.60,69–71 Recently, a set of marker recyclable integrativeplasmids (pMRI) was employed for decentralized assembly of theb-carotene biosynthetic pathways.72 By using GAL induciblepromoters, the pathway could be switched on at certain time,leading to production of 11 mg per g DCW of total carotenoids(72.57 mg L�1) and 7.41 mg per g DCW of b-carotene. Theengineered yeast strain exhibited high genetic stability after 20generations of subculturing. After down-regulating the squalenesynthase (ERG9), 1156 mg L�1 (20.79 mg per g DCW) of totalcarotenoids was achieved by high-cell density fermentation.73

Interestingly, by exploiting the antioxidant properties of carote-noids, adaptive evolution was successfully applied to boostcarotenoids production.74 In another study, carotenoid was usedas a phenotypic marker to screen the yeast deletion collection toreveal new genes with roles in enhancing terpenoid production,75

which provides potential targets for subsequent metabolicengineering.

2.2 Flavonoids

Flavonoids are a large group of plant secondary metabolitescontaining at least one hydroxylated aromatic ring. Flavonoids,when consumed in human diet, could help to promote humanhealth and prevent certain diseases, as it can prevent cancerby preventing cellular oxidation processes and stopping celldegradation and aging.76 The fact that only a few of thesecompounds can be produced as pure products from limitedplant species makes flavonoids as attractive candidates to besynthesized in engineered microbes, such as E. coli andS. cerevisiae.76–78

S. cerevisiae is a suitable host to synthesize flavonoid com-pounds as its natural metabolism provides necessary aminoacid precursors tyrosine and phenylalanine for downstreamexogenous pathways. To date, more than 20 pathways fromfungus and plant have been implanted into S. cerevisiae toproduce different kinds of flavonoid products, including flava-nones, flavones, isoflavones, and flavonols.77,78

p-Coumaric acid is a common intermediate for almost allflavonoids biosynthesis. Phenylalanine ammonia lyase (PAL)first catalyzes the deamination of phenylalanine to cinnamic acid,and cytochrome P450 cinnamate-4-hydroxylase (C4H) acting withpartner reductase (CPR) subsequently catalyzes the hydroxylation ofcinnamic acid to p-coumaric acid. The enzyme from the fungusRhodospridium toruloides was introduced into yeast to exhibit a

tyrosine ammonia lyase (TAL) activity and thus was able to bypassC4H to produce p-coumaric acid from tyrosine.79

The synthesis of naringenin chalcone was first achieved inan engineered yeast, through co-expression of three differentenzymes, including R. toruloides PAL, Arabidopsis thaliana4-coumarate:CoA ligase (4CL), and Hypericum androsaeemumchalcone synthase (CHS).80 The following chalcone isomerase(CHI) could catalyze the cyclization of chalcones to flavanones(Fig. 6).77,81–83

Based on the biosynthesis of flavonones, the introduce ofcytochrome P450 enzymes, such as isoflavone synthase (IFS),flavone synthase II (FSII) and flavone synthase I (FSI), can leadto the production of isoflavone genistein and the flavoneapigenin.77,81,82 Co-expression of flavanone 3b-hydroxylase(F3H), flavonoid 3-hydroxylase (F30H), and flavonol synthase(FLS) achieved the synthesis of kaempferol and quercetin inyeast.77 The production of some stilbenoids, such as resvera-trol, has also been achieved in yeast.77,84

2.3 Alkaloids

Alkaloids are a large class of nitrogen-containing compounds inplant secondary metabolites with low molecular weight.2 Mostalkaloids are derived from amines produced by the decarboxylationof amino acids, such as histidine, lysine, ornithine, tryptophan, andtyrosine. One of the largest groups of pharmaceutical alkaloids isbenzylisoquinoline alkaloids (BIAs), including the antitussivecodeine, the analgesic morphine, and the antibacterial drugsberberine. BIAs are widely used in human health and nutrition,at present they are mainly obtained by extraction from plants,which limited the diversity of BIAs structures available for drugdiscovery due to their low abundance. Many of the native planthosts accumulate only a selected few BIA products. BIAs areproduced through (S)-reticuline, which begins with the condensa-tion of two L-tyrosine derivatives, 3,4-dihydroxyphenylacetaldehyde(3,4-DHPAA) and dopamine (Fig. 7). (S)-Reticuline is a main branch-point metabolite in the biosynthesis of many kinds of BIAs, and itplays an impartment role in the development of novel antimalarialand anticancer drugs. A number of recent reports have successfullytransplanted portions of (S)-reticuline biosynthesis pathways intomicrobial hosts, such as E. coli and S. cerevisiae.85–89

(S)-Reticuline and some other intermediates in the berber-ine and morphinan branches could be synthesized in yeast byoverexpressing enzymes from three plant sources and humans.A glucocorticoid inducible promoter and in situ promotertitration were applied to tune enzyme expression levels andoptimize the productivity.85 Reticuline could also be producedfrom dopamine in E. coli by heterologous expression of biosyn-thetic enzymes (i.e., MAO, NCS, 6-OMT, CNMT, and 4-OMT).86

About 7.2 mg per L magnoflorine and 8.3 mg per L scoulerinewere produced from dopamine via reticuline by using differentcombination cultures of engineered E. coli and S. cerevisiae.86

The productivity of reticuline could be improved by fine-tuningthe possible rate-limiting step.88 Notably, S. cerevisiae expressingCYP80G2 and CNMT converted reticuline to magnoflorine.86 Analkaloid biosynthetic pathway from L-tyrosine cells was constructedin E. coli, which produced 46.0 mg per L of (S)-reticuline from


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glycerol.87 Recently, a biosynthesis pathway for the production ofdihydrosanguinarine and sanguinarine from (R,S)-norlaudano-soline was constructed with 10 genes in S. cerevisiae.89 It represents

the most complex plant alkaloid biosynthetic pathway ever recon-structed in yeast and proves the potential of the synthesis of evermore complex plant natural products in engineered microbes.

2.4 Polyketides

Polyketides are another diverse group of natural products withimportant applications in the pharmaceutical industry. Theyhave been isolated and characterized from a variety of naturalsources, including plants, fungi, and bacteria. They are of highinterests as they often exhibit a broad spectrum of biologicalactivities, which are attributed to their structural complexity anddiversity. Those diverse and complex structures are produced bypolyketide synthases (PKSs), which polymerize acyl-Coenzyme As(CoAs) in a fashion similar to fatty acid synthesis (Fig. 8).

Erythromycin A is a potent polyketide antibiotic produced bythe Gram-positive bacterium Saccharopolyspora erythraea.90 Itcan be used to treat various diseases, such as respiratoryinfections, whooping cough, syphilis, Legionnaires’ disease,gastrointestinal infections, acne, as well as used as a substitutionfor penicillin. After the identification and characterization of theDEBS PKSs gene cluster responsible for biosynthesis of erythro-mycin, functional heterologous expression of these proteins andtheir mutants in S. coelicolor and E. coli were employed.91,92 Thisnot only helped to reveal the biochemical basis for these highlycontrolled megasynthases, but also allowed the rational design ofits biosynthetic products, and led to the generation of diversepolyketide libraries. Researchers have engineered the microbesfor heterologous production of various erythromycin derivativesfor further studies in the hope of improving their pharmacological

Fig. 6 Schematic of the various biosynthetic pathways of flavonoids. PAL, phenylalanine ammonia lyase; TAL, tyrosine ammonia lyase; 4CL,4-coumarate:Co1-ligase; C4H, cinnamate-4-hydroxylase; CHI, chalcone isomerase; CHR, chalcone reductase; CHS, chalcone synthase; FSI, flavonesynthase I; FSII, flavone synthase II; IFS, isoflavone synthase; TAL, tyrosine ammonia lyase.

Fig. 7 Schematic of the various biosynthetic pathways of alkaloids. CNMT,coclaurine-N-methyltransferase; DODC, DOPA decarboxylase; MAO, mono-amine; NCS, norcoclaurine; TYR, tyrosinase; 3,4-DHPAA, 3,4-dihydroxyphenyl-acetaldehyde; 6-OMT, norcoclaurine 6-O-methyltransferase; 40-OMT,30-hydroxy-N-methylcoclaurine 40-O-methyltransferase.


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properties. One notable example is the incorporation of unnaturalstarter units to change the chain initiation process by replacingthe loading domain AT with other AT domains, or incorporatingsynthetic oligoketide precursors into the downstream DEBS path-way.93–95 Besides the precursors, the extender unit can also bereplaced in the erythromycin DEBS system.96–98 Since the DEBSAT domains of each module are the primary gatekeepers for theincorporation of an extender unit into the polyketide chain,replacing AT domains with other ones that accept other extenderunits could result in the incorporation of other functional groupsinto the final polyketide products. Later on, combinatorial poly-ketide biosynthesis by de novo design and rearrangement ofmodular DEBS genes with other eight PKS genes created 154bimodular combinations and allowed the production of novelderivatives as well.99 Recently, a new E. coli platform for erythro-mycin analogue production was established by the production ofalternative final erythromycin compounds exhibiting bioactivityagainst multiple antibiotic-resistant Bacillus subtilis strains.100

Both the native deoxysugar tailoring reactions, D-desosamineand L-mycarose deoxysugar pathways, were replaced with alter-native D-mycaminose and D-olivose pathways to produce newerythromycin analogues in E. coli.

Besides understanding the basic biochemistry and production ofnew derivatives, newly developed synthetic biology platforms offernew strategies for productivity enhancement. Random mutagenesis,recombinant DNA techniques, and process development were

combined and applied to a S. coelicolor strain expressing theheterologous DEBS.101 Fermentation achieved the productionof 1.3 g per L of 15-methyl-6-dEB, and the productivity wasincreased by over 100% in a fermentation process. Furthermore,the introduction of an engineered mutase-epimerase pathway inE. coli enabled a 5-fold higher production of 6-dEB.102,103 Inaddition, after the carbon flux of the biosynthetic pathway wasredirected and an extra copy of a key deoxysugar glycosyltrans-ferase gene was introduced, a 7-fold increase in erythromycin Atiter was achieved.104 In those studies, E. coli-derived productionof erythromycin was enabled by the introduction of the entireerythromycin pathway (20 genes in total) using separate expressionplasmids. However, this may cause metabolic burden and plasmidinstability. Recently, the E. coli erythromycin A production systemwas upgraded by altering the design of the expression plasmidsneeded for biosynthetic pathway introduction, and a 5-fold increasederythromycin A production was achieved.105 On the other hand, inthe native host, by the chromosome gene inactivation techniquebased on homologous recombination with linearized DNAfragments, one TetR family regulator SACE_7301 was identifiedto enhance erythromycin production in S. erythraea.17

Another recent example is for the biosynthesis of cholesterol-lowering drug pravastatin106 derived from the natural productcompactin.107 Pravastatin inhibit 3b-hydroxy-3-methylglutarylCoA (HMG-CoA) reductase, which catalyzes the rate-limitingstep in cholesterol biosynthesis. Pravastatin is obtained after

Fig. 8 Schematic of the biosynthetic pathway of erythromycin. (A) The biosynthetic assembly line for the polyketide antibiotic erythromycin. AT:acyltransferase; ACP: acyl carrier protein; KS: ketosynthase; KR: ketoreductase; DH: dehydratase; ER: enoylreductase; TE: thioesterase. (B) Domainengineering; (C) domain inactivation; (D) engineering of tailoring reactions.


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stereoselective hydroxylation of compactin at the C-6 positionand this is often achieved by a two-step production process inindustry.108 Novel expression vector has been used to carry a gene forcytochrome P450 monooxygenase, and 3.3-fold increased productionof pravastatin has been achieved. Optimized batch culture yielded14.3 g per L of pravastatin after 100 h.109 Metabolic reprogrammingof this antibiotics has been achieved by introducing both thecompactin pathway and a new cytochrome P450 from Amycolatopsisorientalis (CYP105AS1) into the b-lactam–negative P. chrysogenumDS50662. More than 6 g per L pravastatin was obtained via furtherengineering of the CYP105AS1 enzyme at a pilot production scale.110

3. New tools for heterologousbiosynthesis of natural products

Prior to synthetic biology, several genetic engineering andmetabolic engineering strategies were developed for the optimiza-tion of heterologous pathway expression. For example, expression ofsingle genes can be optimized by codon-optimization, promoterengineering, gene copy number, translation regulating sequencemutation, post-translational modification and protein engineering.While for multiple gene expressions, metabolic modelling is widelyused to guide the coordination of multiple gene expressions in themetabolic network, such as identifying the targets of limiting steps.However, the lack of the parameters during the heterologous geneexpression, such as the physiological changes of the host and theproperty parameters of the heterologous proteins in the hostenvironment,111 limited the applications of metabolic modelling.

In the past few years, several powerful synthetic biology toolshave been developed for the optimization of heterologouspathways in engineered microbial hosts. As engineered hetero-logous microbes offer an optimized and standardized platformor cell factory for natural product production, advances in theirmetabolism engineering would benefit the reconstruction ofthe whole cellular network and heterologous natural productbiosynthesis pathways.

3.1 Engineering at the parts Level

Besides the basic physical construction of a heterologous path-way, the achievement of maximal yield of target products relieson many other factors, including optimization of metabolicflux, reduction of intermediate toxicity, and tuning properstress on the host cell. Some general strategies for the designof gene or protein parts are needed for optimal expression andorganization of individual pathway enzymes. The expression ofgene parts can be tuned by modifying gene copy numbers,transcriptional activity, or post-transcriptional processing. Theindividual proteins can be organized as fusion proteins or inartificial scaffolds for coupled activities in metabolic pathways.The natural and heterologous enzymes can also be organizedinside and outside of certain organelles to enhance the productyields.

3.1.1 Tuning the expression of gene parts. In engineeredmicrobes, expressions of gene parts, especially heterologousgene parts, can be fine-tuned throughout the whole central

dogma process, including gene replication, transcription,translation, and post-translation. The development of centraldogma based engineering toolkits has attracted attention fordecades since the discovery of the double helix structure of DNAand the lac operon.112,113 More recently, the rapid development ofsynthetic biology calls for much more powerful central dogmabased engineering toolkits. These toolkits possess some commoncharacteristics, such as standardization, universality, real-timecontrollability, reusability, and modularity. Several strategies forstandardized gene parts storage and ligation have been establishedrecently, such as Biobrick, Bglbrick and SEVA.114–116 Gene partshave standard identification sites that can be digested by type Ior type IIS restriction enzymes and be ligated to another partcontaining the same couple of sites. However, there is a paradoxabout standardization and universality of the gene parts. Severalexcellent tools to tune gene expression were developed bycombination of standardized rules and universal manipulations(Fig. 9). At the gene level, variant copies of genes on differentplasmids showed obvious effects on the synthesis of targetproducts.117 At the transcriptional level, promoter strength,mutation of promoter functional sites, addition of the upstreamactivation sites, and inducible artificial promoters have beensuccessfully used in optimization of multiple genes expres-sion.118–124 At the translational level, special sequence repeats,synthetic riboswitches, rational ribosome binding sites designtools, random translation starting sites, tuneable intergenicregion options, and artificial codons have been used for fine-tuning of gene expression.125–130 Most of these methods showedgreat potentials in tuning of multiple genes and optimization ofmultiple genetic modules. In addition, novel 4-base codons andunnatural amino acids were introduced in the translation pro-cess for novel and orthogonal polypeptides elongation andprotein synthesis.130 Most recently, two synthetic bases X andY were added to genetic codons in addition to natural bases A, T,C and G.131

Based on these ideas of engineering the central dogmaprocess, next generation of DNA synthesis technologies canbe used to construct large libraries of genes with modifiedcodons and varying expression levels. By taking advantage ofcomputational design and metagenomic information, de novoDNA synthesis enables us to obtain artificial or natural genesfor the required enzymes in an expanding range (Fig. 9). Forexample, 89 methyl halide transferases found in metagenomicsequences from diverse organisms were synthesized andscreened for higher enzymatic activities.132 154 and 1468reporter genes were constructed and characterized as librariesto study codon usage of genes.133 Oligo pools were combinedwith multiplexed reporter assays for construction of more than14 000 reporter constructs. The transcriptional and transla-tional rates of these constructs were used to analyze the effectsof N-terminal codon bias on protein expression levels.134

3.1.2 Making artificial protein fusions. Natural metabolismis under tight regulations, such as positive or negative feedbacks.The heterologously expressed enzymes often suffer from fluximbalances because of the lacking of well-established regulatorymechanisms. In addition, the enzymes in the same metabolic


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pathway may be distributed at different regions inside the cell,thus diffusion or degradation of the intermediates occurs andthe efficiency of the whole pathway would be decreased. Simplyblocking the competitive pathways, in many cases, is not sufficientsince it might be lethal or hard to implement. In fact, the highexpression level of engineered pathways may compete with biomassproduction or other primary metabolite pathways for nucleotidesand amino acids, which results in opposite to what is expected.

Fusion of proteins could bring active sites of enzymes into acloser proximity, which facilitates substrate channelling(Fig. 10) and relieves the side-pathway competition. Proteinfusion strategies have been used in the endogenous MVApathway.26 FPPS, encoded by ERG20, catalyses the condensa-tion of 5-carbon units IPP and DMAPP to produce FPP, which isthe general precursor for biosynthesis of sesquiterpenes andgeranylgeranyl pyrophosphate (GGPP), and then GGPP can beconverted into diterpenes and tetraterpenes. In natural growth

conditions, most of the FPP is used for production of ergosterol,which is essential for the cellular maintenance. Therefore, the FPPis a main branching point for the synthesis of sesquiterpenoids andtriterpenes (Fig. 2). Fusing FPPS together with patchoulol synthase(PTS, encoded by Pogostemon cablin gene PatTps177) and GGPPSredirected the flux to the production of the sesquiterpene patch-oulol and GGPPS respectively, and enhanced the production by2-fold and 8-fold, respectively.26,135 Besides fusion of these twoendogenous proteins, heterologous enzymes copalyl diphosphatesynthase (SmCPS from Salvia miltiorrhiza) and kaurene synthase-like (SmKSL from Salvia miltiorrhiza) were fused for biosynthesisof tanshionones precursor miltiradiene in yeast.40 Simply fusingFPPS with Bst1p made the production from trace to 1.0 mg L�1.According to the coimmunoprecipitation experiments, theypredicted SmKSL and SmCPS may form a complex in vivo,which inspired them to fuse these two enzymes for moreefficient production. By comparison of fused SmCPS-SmKSL

Fig. 9 Strategies to tune the gene expression levels in the natural product biosynthetic pathways. The expression of heterologous genes are regulated atthe gene level (variation on copy numbers of genes, selection of plasmids), at the transcriptional level (promoter strength, mutation of promoterfunctional sites, addition of upstream activation sites, and inducible artificial promoters), and at the translational level (special sequence repeats, syntheticriboswitches, rational ribosome binding sites, random translation starting sites, tuneable intergenic region options, and artificial codons). The codonoptimization of the heterologous genes is another efficient strategy to tune gene expression.


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and SmKSL-SmCPS, the latter pattern was more effective byoffering a 2.9-fold increase of miltiradiene to 3.1 mg L�1.40

Construction of scaffolds is another commonly used strategyto couple several enzymes catalysing sequential reactions in thesame pathway. This type of constructs will not affect othermetabolic pathways significantly due to its controllability.There are different types of scaffolds, such as plasmid scaffolds,RNA scaffolds and protein scaffolds (Fig. 10). Plasmid scaffoldspossess the advantage of accommodating many interactionmotifs and linkers of variable lengths without solubility issues,such as the linking of the glucose oxidase and horse radishperoxidase via a lysine residue to short DNA oligo nucleo-tides.136 However, the disadvantage is that enzymes must besignificantly modified with multiple zinc finger domains(usually 3–4 domains with a total addition of 90–120 aminoacids).137 RNA scaffolds were designed and constructed usingmultidimensional RNA structures for the spatial organization

of bacterial metabolism. The synthetic RNA modules werefunctionally discrete and formed 1D and 2D scaffolds in whichthe palindromic regions were disfavoured and the assemblyorder of RNA strands were controlled by insuring tile formationbefore polymerization. This RNA-based scaffolding system wasapplied to increase the production of hydrogen, and an increaseof 48-fold over the unscaffolded, tagged enzymes in E. coli wasobserved.138 Besides plasmid scaffolds and RNA scaffolds, proteinscaffolds were constructed by co-localization of pathway enzymes tosynthetic complexes using well-characterized protein–protein inter-action domains and their specific ligands. This strategy is able toincrease the effective concentrations of the metabolic intermediatesaround the enzymes.

The mevalonate producing pathway is consisted of threeenzymes (acetoacetyl-CoA thiolase (AtoB) from E. coli, hydroxy-methylglutaryl-CoA synthase (HMGS) and HMGR from S. cere-visiae) in the engineered E. coli.139 Difference in the expression

Fig. 10 Strategies to enhance the pathway flux by modifying the distances of proteins and protein re-localization. In order to enhance heterologousmetabolic flux, fusion of proteins with different linkers is efficient. DNA-, RNA- and protein-based scaffolds are used to assemble the heterologousenzymes to improve the sequential reactions. Re-localization of enzymes switches the subcellular position of heterologous proteins to enhance theproduction by either enhancing enzyme concentration or taking advantage of environmental differences. Colourful solid arrows represent theheterologous enzymes.


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levels of these 3 enzymes led to a ‘‘bottle-neck’’ effect and theaccumulation of toxic intermediate HMG-CoA. Thus the syntheticprotein scaffolds were applied to spatially recruit metabolicenzymes, and the 1 : 2 : 2 ratio produced 77-fold of mevalonatecompared with the wild strain.139 No harm to the growth of thestrain was observed, no matter how the inducer concentrationvaried.139 At the same time, increasing the copy number of theprotein scaffolds led to enhanced productivity.

3.1.3 Re-localizing proteins. Primarily for eukaryotic microbialhosts, many enzymes, such as cytochrome P450s, are thought tobind to the endoplasmic reticulum or mitochondrial membrane viathe hydrophobic membrane binding domain at the N-terminus intheir native hosts. Therefore, this membrane binding domain maybe required for function, especially when these cytochrome P450sare expressed in heterologous hosts. Mature forms of theseenzymes without the signal peptides are often used in heterologousexpression. However, the cytochrome P450 with both N-terminalmembrane insertion and C-terminal HA epitope-tag deletion exhib-ited the highest expression level than other forms with partialdeletion.140 Relatively upstream pathways are also effective targetsfor compartmentalization. S. cerevisiae was engineered to produce2,3-butanediol (BDO) by the introduction of a cytosolic acetolactatesynthase (cytoILV2).141 Acetyl-CoA level was improved in the cytosolby the combined disruption of competing pathways and introduc-tion of heterologous biosynthetic pathways to supply more pre-cursors for desired product biosynthesis in yeast.142 n-Butanolproduction was improved to 3-fold through acetyl-CoA enhance-ment by introduction of heterologous acetyl-CoA biosynthetic path-ways including pyruvate dehydrogenase (PDH), ATP-dependentcitratelyase (ACL), and PDH-bypass.142

In contrast with expressing mitochondrial enzymes in cyto-sol, some enzymes and metabolic pathways were also expressedin mitochondria instead of in cytoplasm, because mitochondriahave many potential advantages for metabolic engineering,such as the sequestration of diverse metabolites, containingintermediates of many central metabolic pathways, and theenvironment in the mitochondrial matrix is highly differentfrom that in the cytoplasm.143 Mitochondrion was also used asan alternative location for heterologous expression of metabolicengineering instead of cytosol to enhance the flux to FPP.35

CsTPS1 (valencene synthase from Citrus sinensis) was locatedinto mitochondria by the fusion with mitochondrial targetingsignal peptide of COX4, which brought a 3-fold rise in valen-cene titers compared with cytosol CsTPS1. The expression ofmitochondrion-targeted FPPS (mtFPPS) further increased theproduction by 40%. Addition of another copy of cytosolicCsTPS1 further increased the production to 1.5 mg L�1, whichwas an 8-fold enhancement. This strategy was also performedon amorph-4,11-dienesynthase (ADS), and mtADS was con-structed, which strongly enhanced the amorphadiene produc-tion to 20 mg L�1. Accordingly, for upstream pathways, Avaloset al. engineered yeast mitochondria to produce isobutanol,isopentanol and 2-methyl-1-butanol through Ehrlich degrada-tion pathway, which originally occurs in cytoplasm, while theupstream pathway is confined to mitochondria, which creates asubstantial bottleneck in the transportation.143 They developed

a standard, flexible set of vectors to target identical pathwaysto different subcellular parts. Targeting Ll-kivd (a-ketoaciddecarboxylase from Lactococcus lactis) and Sc-adh7 (alcoholdehydrogenase from S. cerevisiae) to mitochondria increasedthe titer of isobutanol approximately 220% to 486 mg L�1.143

The results showed that the compartmentalization the pathwayinto mitochondria achieved higher local enzyme concentra-tions and increased the availability of intermediates andreleased the restriction on transportation.

3.2 Engineering at the pathway level

Similar to organic synthesis of a target chemical, the heterologousproduction of a target product in engineered microbe hosts is alsoaffected by common factors such as substrate supply, reactionefficiency, reaction thermodynamics and kinetics, yield, productivity,toxicity, and cost-efficiency. Although some operation units aredifferent for alternative products and can be optimized case by case,some universal pathway engineering strategies would facilitate theimprovement of these biochemical reactions. The successful produc-tion of artemisinic acid synthesis in a heterologous host offered us agood example of how to optimize cellular metabolic pathways forterpenoid production, and these strategies could be applied to otherbiochemical molecules as well.

3.2.1 Mining of heterologous pathways. A typical syntheticbiology approach for heterologous biosynthesis of target productsbegins with the exploration of genes and pathways encodingfunctional enzymes. However, there are still many pathways remain-ing to be explored, and the typical strategy the ones studied is toexpress them heterologously, either to make more use of the knownpathway or decipher the cryptic pathway (Fig. 11). The first trial tointroduce a whole pathway heterologously was done for terpenoidproduction.144 Targeted amorpha-4,11-diene, the sesquiterpeneolefin precursor to artemisinin, as the final product, they introducedthe mevalonate terpenoid pathway from S. cerevisiae into E. coli andthe concentration reached 24 mg caryophyllene equivalent per mL,which approved the functionalization of fungi pathway in thecytosol of bacteria. For cryptic pathway exploration, besides theanalysis of systems biology, synthetic biology also takes in practicethe construction of transcription units of any possible ORFs basedon bioinformatics analysis, as in the example of the mining of thegene encoding the CK synthase (Fig. 11).58 CK, as the mainfunctional component of ginsenoside, possesses bioactivities ofanti-inflammation, hepatoprotection, antidiabetes and anti-cancer.145 The potential CK biosynthetic pathway was designed inyeast, including a cytochrome P450 (CYP716A47) from Panax gin-seng, a NADPH-cytochrome P450 reductase (ATR2-1) from Arabidop-sis thaliana, and a Dammarenediol-II synthase (PgDDS) from Panaxginseng, with other involved enzymes originated from S. cerevisiae. Inthis study, a proprietary cDNA database including 479 689assembled cDNA contigs was established based on 9 Panax ESTdatasets available from the NCBI GenBank. 16 ORFs were amplifiedand expressed in S. cerevisiae, through which way, the enzymecoding CK synthase was obtained from natural resources andprimarily verified and the strain produced 1.4 mg per L of CK.58

Sometimes a novel pathway for product synthesis can alsobe designed. For example, an unprecedented artificial salvianic


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acid A (SAA) biosynthesis pathway was developed in E. coli.146

Two enzymes D-lactate dehydrogenase (D-LDH) from Lactobacilluspentosus and hydroxylase complex encoded by hpaBC from E. coli,were introduced into E. coli to convert 4-hydroxyphenylpyruvate(4HPP) into SAA. D-LDH and its variants were selected to catalyzethe chiral reduction of ketone group in 4HPP according to theirperformance in transformation of phenylpyruvate into phenyllacticacid (Fig. 11). Co-expression of d-ldh and its mutants and hpaBCtogether with optimizations on the upstream pathway resulted in7.1 g per L of SAA with a yield of 0.47 mol mol�1 glucose.146

3.2.2 DNA assembly-assisted pathway construction. Tosynthesize a desired product in heterologous organisms, theintroduced biosynthetic pathways and upstream metabolicpathways are required to be considered together as the supplyof precursors affects the ultimate yield of the final product. Inrecent years, the studies on synthetic biology also expand to

strategies of engineering central carbon metabolisms, whichcan be summarized as combinatorial engineering (Fig. 12).Most of these strategies were designed rationally; however,some could also be utilized to build up a randomized library inthe pre-design frame. In some strategies, variation in expressionlevels of individual genes were introduced, and those alternativeexpressions were combined during DNA assembly process (Fig. 12).One prominent example is the Customized Optimization of Meta-bolic Pathways by Combinatorial Transcriptional Engineering(COMPACTER) method in which assembly of promoter mutantsand pathway genes resulted in the combination of different geneexpression levels in a target pathway.147 A more recent work utilizedGibson assembly method to introduce coding sequences after theinitial codon ATG for expanding expression range of the genescoding violacein synthesizing enzymes, causing variant combina-tions of multi-gene transcriptional levels.128

Fig. 11 Exploration and construction of novel natural product biosynthetic pathways. (A) Heterologous expression of uncharacterized genes or proteinswith potential functions to catalyze the specific reaction is applied to explore new pathways for natural product biosynthesis. (B) Heterologous expressionof uncharacterized genes to explore the biosynthetic pathway for ginsenoside CK. (C) Heterologous expression of protein with similar activity toconstruct a novel biosynthetic pathway for salvianic acid A. Colourful solid arrows represent the heterologous enzymes. Adapted by permission fromMacmillan Publishers Ltd: [Cell Research] (ref. 58), copyright (2014). Reprinted from Y. F. Yao, C. S. Wang, J. Qiao and G. R. Zhao, Metabolic engineering ofEscherichia coli for production of salvianic acid A via an artificial biosynthetic pathway, Metab. Eng., 19, 79–87, copyright (2013), with permission fromElsevier.


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3.2.3 Modular pathway engineering for metabolic precursorsynthesis. To produce high-level products, simple modificationof the expression levels of genes in the target pathway is the mostrational method for the pathway optimization. These methodscould be classified into 3 categories (Fig. 13): (1) increasing thesupplement of the substrate, (2) increasing the whole flux of thedesired biosynthetic pathway, and (3) decreasing or eliminatingthe flux of the branch pathway. Here, we take the optimization ofthe MVA pathway as an example to demonstrate how to optimizea biosynthesis process at the pathway level.

Acetyl coenzyme A (acetyl-CoA) is a central metabolite incarbon and energy metabolism. The mevalonate pathway inS. cerevisiae is initiated from acetyl-CoA, a key precursor to awide range of valuable secondary metabolites.148 Moreover,acetyl-CoA supply in the mitochondrial matrix is essential forcell growth because it is used to replenish the mitochondrialtricarboxylic acid (TCA) cycle with C2 units and produce ATP.149

Optimizing the supply of acetyl-CoA becomes a logical approach

to increase the production of the target compounds. Chen et al.enhanced the supply of acetyl-CoA in the cytoplasm through acombined push–pull-block strategy. The push part included theover-expression of the endogenous alcohol dehydrogenase(ADH2) and NADP-dependent aldehyde dehydrogenase (ALD6),and a codon-optimized acetyl-CoA synthase variant from Salmo-nella enterica (ACSSE

L641P). The pulling of acetyl-CoA towards theproducts of interest was enhanced by the over-expression ofERG10, encoding acetyl-CoA C-acetyltransferase, that catalyzesthe conversion of acetyl-CoA to acetoacetyl-CoA. The block partof the strategy decreased reduction of acetyl-CoA and wasachieved through removing two key enzymes involved in theconsumption of acetyl-CoA. One is the peroxisomal citratesynthase and the other is cytosolic malate synthase. This strategyimproved the production of a-santalene by 4-fold.150 Lian et al.redirected the glycolytic flux towards acetyl-CoA by inactivatingADH1 and ADH4 for ethanol formation and GPD1 and GPD2for glycerol production, resulting in 4-fold improvement in

Fig. 12 Strategies to optimize natural product biosynthetic pathways assisted by DNA assembly methods. A heterologous pathway can be optimized bychanging different promoters to regulate the expression of different genes. DNA assembly in vivo and in vitro can be used to introduce variations eitherinside the genes or in regulation parts (promoters or regulators) to build a library, which can be used to select the desired strains with the heterologouspathway. Colourful solid arrows represent the heterologous enzymes.


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n-butanol production. Subsequent introduction of heterologousacetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase(PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, furtherincreased n-butanol production. Among them recombinant PDHslocalized in the cytosol (cytoPDHs) was most efficient and increasedn-butanol production by additional 3-fold.142

To increase the overall flux of the MVA pathway, enhancingthe reaction rate of the limiting step (HMG-CoA to mevalonate)was considered. tHMG1, the truncated HMG1 which only containsthe catalytic domain of HMG1, is commonly used in engineering ofthe MVA pathway in yeast. tHMG1 was often overexpressed bystrong promoter and has many copies in the cell.7,33,151 On theother hand, UPC2-1, a modified global transcriptional regulator ofergosterol biosynthesis was often used to up-regulate the expres-sion level of the entire MVA pathway.7,33,151

For production of terpenes, the native biosynthesis pathwayof ergosterol always needs to be down-regulated. The mostuseful method is to reduce the expression level of essentialgene ERG9 by different weaker promoters such as pMET,pCTR3, and pHXT1.6,7 Knocking out the lppA and STC genesreduced the production of fansesol while the important precursorFPP was accumulated.33

Optimization of the MVA pathway needs the combination ofdifferent strategies. A very successful example is the biosynthesisof amorphadiene, which can be synthesized from FPP by amor-phadiene synthase encode by ADS. Westfall et al. overexpressedall genes responsible for the MVA pathway from ERG10 to ERG20under the GAL promoter and integrated all these genes intogenome. Among them, the key enzyme tHMG1 had three copiesto enhance the limiting-step of the MVA pathway. ERG9 was

Fig. 13 Strategies of modular pathway engineering for metabolic precursor synthesis. Strategies to increase metabolic precursors include: (1) increasingthe supply of the substrate, such as adding new biosynthetic pathway of precursor; (2) increasing the whole flux of the desired biosynthetic pathway, suchas improving the gene expression for precursor synthesis; (3) decreasing or eliminating the flux of the branch pathways, such as deleting the branchpathways. These strategies can be achieved by manipulating the copy number of genes, changing promoters and introducing either heterologousupstream or heterologous downstream pathways. Colourful solid arrows represent the heterologous enzymes. Red arrow with two tailors means thedown regulation.


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down-regulated by substitution of the native promoter of ERG9by pMET. During fermentation, ethanol was the sole carbonsource for production, which was helpful for supplying moreacetyl-CoA for biosynthesis. The accumulated precursor FPP,could be converted into amorphadiene efficiently by highlyexpressed ADS. Subsequent fermentation optimization led to40 g per L products, which was the highest production comparedto previous reports.7

3.2.4 Biosensor-directed real-time control and evolution.In recent years, development of intracellular small molecule screen-ing methods led to generation of cellular self-modification systemswith the small molecules functioning as signal molecules (Fig. 14).Some of the small molecules are produced by the cell, such asN-acylhomoserine lactones (AHL) and N-butyrylhomoserine lactone(BHL), which could be synthesized by special enzymes in cells anddiffused to coherent cells to regulate multi-cell activities throughgene circuits. While some other similar molecules could only be

added from outside to tune genes’ transcription and translationprocesses, such as isopropyl thiogalactoside, arabinose, anhydrote-tracycline, and theophylline. Light is also used as a signal to regulategene expression and change cellular activity.152–154 However, most ofthese earlier systems could only be operated in a pre-determined wayor via serendipity. Recent advances offered us some biosensor-directed real-time metabolic control methods (Fig. 14). It is oftendifficult to accumulate the products in engineered cells due to thetoxicity of target intermediates. Metabolite response promoters wereexplored and utilized in metabolic self-regulation.155 The whole-genome transcript arrays were employed to identify promoters thatresponded to these intermediates. The promoters could controlexpression of certain genes in a real-time manner, causing enhancedamorphadiene production by 2-fold over traditional constructions inE. coli. This approach could also be extended to other toxic inter-mediates for dynamic regulation.155 A similar metabolite biosensorsystem was constructed to achieve the feedback regulated evolution

Fig. 14 Biosensor-directed real-time control and evolution. The promoters inhibited by the target products are used as the biosensors to control theexpression of DNA mutator proteins, such as mutD5. This design can be used to generate a mutant library. Meanwhile, the same promoter also controlthe expression of a fluorescent protein, which is used as the reporter for phenotype selection. Colourful solid arrows represent the heterologousenzymes. Adapted by permission from Macmillan Publishers Ltd: [Nature Communication] (ref. 156), copyright (2013).


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of phenotype (FREP) based on production of target products.156 Inthe designed adaptive control system, the genomic mutation ratewould be increased by expression of mutD5 to generate diversity inthe population, and be decreased only when the concentration oftarget metabolites came to required amounts. The synthetic tran-scription factors were utilized to construct artificial biosensors torespond for metabolites without natural sensors. The FREP wasverified by evolving increased tyrosine and terpenoid production.

3.3 Engineering at the genome level

Genome engineering approaches can be grouped into threecategories: genome editing, transcriptome engineering, andgenome synthesis.157 Genome editing precisely or combinato-rially modifies the target genome at multiple loci while tran-scriptome engineering essentially targets regulatory elementsby mutating endogenous regulators or introducing artificialones. Genome synthesis involves hierarchical assembly of short

chemically synthesized DNA fragments into microbial gen-omes. Because of the large size of a genome, genome engineer-ing is typically coupled with high throughput screeningtechnologies. In the past few years, genome engineering hasbeen increasingly used for heterologous biosynthesis of naturalproducts.

Based on the allelic replacement on the lagging strand bythe oligos during replication, multiplex automated genomeengineering (MAGE) was developed in E. coli, and applied toimprove the biosynthesis of lycopene by simultaneously mod-ifying 24 genes in the corresponding biosynthetic pathway.158

This method was recently extended to S. cerevisiae.159 Byapplying the same principle, multiplex iterative plasmid engi-neering (MIPE) was developed to engineer the heterologousmetabolic pathway in the plasmid, and a clone with 2.67-foldimproved production of riboflavin was achieved in less than aweek.160 Another related approach, RNA interference (RNAi)-

Fig. 15 The SCRaMbLE system based on genome synthesis. Based on the Cre-LoxP system, the SCRaMbLE (synthetic chromosome rearrangement andmodification by loxP-mediated evolution) is able to generate a mutant library by gene deletion, gene reversion, gene insertion, and gene translocation ata genome scale. Colourful solid arrows represent the heterologous enzymes. Reproduced with permission from ref. 175, copyright 2014 John Wiley andSons.


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assisted genome evolution (RAGE) could continuously improvetarget trait(s) by accumulating multiplex beneficial geneticmodifications in an evolving yeast genome.161

Modular artificial nucleases have been increasingly usedfor genome editing. The zinc-finger domain, which typicallyrecognizes 3-nucleotide DNA motifs, was the first to be exploited.Sequence-specificity was further increased by engineering zinc-fingernucleases (ZFNs) in a way that requires their heterodimerizationthrough the FokI domain for efficient cleavage.162 The modularapproach was taken a step further with the discovery of the TALeffector (TALE) DNA-binding modules of Xanthomonas bacteria, andtheir simple DNA recognition code.163 However, to target uniquesequences in a eukaryotic genome, long TAL arrays need to beassembled in order, because each TAL repeat targets a singlenucleotide. More recently, clustered regularly interspaced shortpalindromic repeats (CRISPR) have been developed as a generalmethod for genome engineering including activating transcription/translation, silencing transcription/translation, up/down regulatinggene expression, and modifying multiple sites simultaneously,each accompanied with permanent changes of genome sequence.Compared to ZFNs and TALENs, the CRISPR technology hasradically improved the accessibility of gene targeting due to itsstraightforward approach for customizing sequence specificity viatarget-specific guide RNAs. Its targeting efficiency is comparablewith the best efficiency achieved using TALENs in a wide rangeof animals and plants.164 Recently, it was shown that theendonuclease domains of the Cas9 protein can be mutatedand co-expressed with a guide RNA to specifically interfere withtranscriptional elongation and work like RNAi. This system wasnamed CRISPR interference (CRISPRi), which can efficientlyrepress expression of a target gene with no detectable off-targeteffects.165 In addition, dCas9 was used with existing optogeneticor chemical-induced proximity systems to dynamically analyzerecruitment of a broad range of chromatin modifiers to a specificDNA sequence, both for customized activation and repression.166

CRISPR/Cas transcription factors (CRISPR-TFs) have the potentialto be integrated for the tunable modulation of gene networks.167 Arapid, efficient, and potentially scalable strategy based on CRISPR/Cas system was recently developed to generate multiple genedisruptions simultaneously in S. cerevisiae.168

Genome synthesis with novel design is the extreme ofgenome editing for natural product synthesis. So far, genomesynthesis based on the natural genome sequence has succeededin several virus and bacteria, such as Mycoplasma genitalium,Mycoplasma mycoides and Phaeodactylum tricornutum.169–171

However, due to the lack of understanding of the biology,the de novo design of a genome has not been attempted.On the other hand, an initiative to redesigning the genome ofS. cerevisiae named SC 2.0 was launched recently. NumerousloxP sites were introduced into the redesigned genome and theSCRaMbLE (synthetic chromosome rearrangement and modi-fication by loxP-mediated evolution) was developed by combin-ing loxP sites with the inducible Cre recombinase (Fig. 15).172

The SCRaMbLE system in Sc2.0 is a very useful tool for mini-mization of the yeast genome and generation of a library ofmutant genomes at a large scale.173–175 Such mutant genomes

may offer the potential chassis for heterologous biosynthesis ofdifferent natural products.

3.4 Engineering at the community level

Many complex natural products are synthesized by very longpathways such as paclitaxel. It is often a challenge for one cell toexpress such long heterologous pathways due to the heavy metabolicburden associated with expression of so many heterologous enzymesand production burden.176,177 Most of the microbial consortiumengineering focus on the lignocelluloses bioconversion to producechemicals and fuels.132,178 The progress on the fundamental studiesand the applications of artificial consortium was reviewedrecently.179 More recently, engineering of the microbial consortiumhas been applied to biosynthesis of complex natural products.177

Taxadiene, as the scaffold molecule of paclitaxel, can be currentlyproduced at much higher titer in the engineered E. coli comparedto engineered yeast.47,50 However, heterologous expression of cyto-chrome P450s, the downstream enzymes for taxadiene transforma-tion, is more suitable in yeast than in E. coli,47 because yeastpossesses the advanced protein expression machinery and abun-dant intracellular membranes. Therefore, metabolic pathway fortaxane biosynthesis was distributed into the consortium of E. coliand S. cerevisiae, and this synthetic consortium produced 33 mg perL oxygenated taxanes (Fig. 16).177 Using the similar strategy,biosynthesis of ferruginol were also achieved in the artificialconsortium of E. coli with the heterologous pathway of miltiradienesynthesis and S. cerevisiae with an cytochrome P450.177 The conceptof distributing the long metabolic pathways into consortium would

Fig. 16 Engineering of the microbial consortium to improve heterolo-gous biosynthesis of complex natural products. (A) Schematic of thedistribution of the long synthetic pathway for complex natural productsinto different strains of the consortium. (B) The artificial consortium withengineering E. coli and S. cerevisiae produce oxygenated taxanes usingxylose and ethanol. Colourful solid arrows represent the heterologousenzymes. Adapted by permission from Macmillan Publishers Ltd: [NatureBiotechnology] (ref. 177), copyright (2015).


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significantly enhance the heterologous biosynthesis of naturalproducts (Fig. 16).

4. Conclusion and future prospects

Natural products have been and continue to be the source andinspiration for a substantial fraction of human therapeutics.The successfully commercialized production of several drugs ordrug candidates shed lights on the future heterologous bio-synthesis of important natural products. With the emergence ofmore recently genomics- and bioinformatics-guided syntheticbiology approaches, alternative strategies have been developedfor better heterologous expression of natural products bio-synthesis pathways. In the near future, better designed parts,modules or biosystems could be created to facilitate the hetero-logous biosynthesis of natural products. Also, the new toolsdeveloped in the synthetic biology field, such as CRISPR/Cas system or RNAi system would be engineered for manipula-tion of biosynthetic pathways via gene activation/inactivation.Moreover, these techniques not only can help improving theproduction of value-added compounds, but also can lead toflourish in natural product discovery field, including genomics-driven discovery combined with genetic manipulations inheterologous hosts. In addition, the separation technologycoupled with specific artificial systems will be developed tofurther enhance the heterologous biosynthesis of naturalproducts.

E. coli, Streptomyces species, yeast and Aspergillus species areoften used as heterologous hosts for expression of a singlegene, a cassette of genes, or an entire biosynthetic gene clusterfor identifying the corresponding natural product. Genes orpathways from bacteria were often heterologously expressed inE. coli or Streptomyces species, resulting in the synthesis ofnovel compounds.52,62–74 To facilitate the discovery of novelcompounds in a bacteria heterologous host, several strategieshave already been tested, such as activation of cryptic pathwaysvia promoter replacement, generating genome-minimized het-erologous hosts with clean background and combinatorialbiosynthesis for new derivatives. Meanwhile, S. cerevisiae andAspergillus species have often been used as heterologous hostsexpressing genes from fungi.180–190 Several strategies for acti-vating silent gene clusters in fungal genomes have previouslybeen developed and were recently reviewed elsewhere.183,190 Aspowerful analytical techniques (MS, NMR, and software forcomparative data analysis) continue to evolve, we will continueto explore novel natural products from ‘silent’ natural productbiosynthesis pathways by enabling detection and characteriza-tion of compounds present in minute quantities.

With the advent of new technologies for manipulation bothof biosynthetic gene clusters and heterologous hosts, it isforeseeable that improved production of known compounds wouldbe achieved; novel bioactive compounds with pharmaceutical orindustrial importance could be discovered or generated and agreater understanding of the molecular complexities of naturalproducts biosynthesis would be guaranteed.

Acknowledgements

This work was financially supported by the Ministry of Scienceand Technology of China (863 program: 2012AA02A701, 973program: 2013CB733600), and the National Natural ScienceFoundation of China (major project: 21390203). HZ would liketo acknowledge the financial support by National Institutes ofHealth (GM077596).

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