Engineered Biosynthesis of Natural Products in Heterologous Hosts

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Engineered biosynthesis of natural products in heterologous hosts Cite this: DOI: 10.1039/c5cs00025d 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 Received 12th January 2015 low productivity and titers. Here we summarize the recent applications of heterologous biosynthesis for the DOI: 10.1039/c5cs00025d 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, www.rsc.org/csr pathway, genome and community levels for highly eﬃcient heterologous biosynthesis of natural products.

1. Introduction market share in pharmaceuticalindustryinthepast30years, comprising 61% of anticancer compounds and 49% of anti- Molecules from natural sources, especially bacteria, fungi and infectives.2 For example, erythromycin from Saccharopolyspora ery- plants, have proven to be a large reservoir for new pharmaceuticals, thraea is an antibiotic, which has an antimicrobial spectrum similar therapeutic agents and industrially useful compounds.1 To date, to that of penicillin and can be used to treat various diseases.3 natural products and their derivatives command a substantial Lovastatin (Merck’s Mevacor) isolated from Aspergillus terreus is often used to reduce risk of cardiovascular disease, while paclitaxel a Key Laboratory of Systems Bioengineering (Ministry of Education), extracted from the bark of the Pacific yew, Taxus brevifolia,isa Tianjin University, Tianjin, 300072, P. R. China. E-mail: [email protected]; mitotic inhibitor used in cancer chemotherapy. There are also Fax: +86-22-27403888 important natural products discovered from other diverse sources, b Department of Chemical and Biomolecular Engineering and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Ave, such as insects and marine sources. In the old days before 1940s, Urbana, IL 61801, USA natural products of interest are isolated directly from plant sources c SynBio Research Platform, Collaborative Innovation Center of Chemical Science (Fig. 1A). However, bioactive compounds from natural sources are

Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. and Engineering (Tianjin), School of Chemical Engineering and Technology, usually secondary metabolites of their native producers, thus in Tianjin University, Tianjin, 300072, P. R. China some cases, they exist at a relatively low level comparing with the † Current address: State Key Laboratory of Biotherapy/Collaborative Innovation production of primary metabolites. Therefore, in most cases, direct Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, P. R. China. Renmin South Road 3, No. 17, Chengdu, Sichuan, 610041, P. R. China. isolation and extraction is not an environmental friendly, sustainable ‡ Equal contribution. and economical solution. Combinatorial chemistry was also

Dr Yunzi Luo received her PhD in Dr Bing-Zhi Li is an associate Chemical and Biomolecular Engi- professor of Pharmaceutical Engi- neering from the University of neering in Tianjin University. He Illinois at Urbana and Champaign received his BS degree from in 2014. Her research has focused Nankai University in 2005 and his on natural products discovery and PhD from Tianjin University in synthetic biology. Dr Luo was a 2010. His research expertise is in postdoctoral research fellow at the synthetic biology and lignocellulose Institute for Genomic Biology of bioconversion. He has published University of Illinois at Urbana and more than 20 peer-reviewed inter- Champaign. She has published national papers. 11 papers in peer-reviewed inter- Yunzi Luo national journals and 2 book Bing-Zhi Li chapters.

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introduced to synthesize and screen small molecules of medicinal engineering of microbial biosynthetic pathways towards the design importance (Fig. 1B). Still, due to the structure complexity of and creation of novel metabolic circuits and biological systems. With natural products, chemical synthesis of these compounds is the newly developed biosystems, the yield of target natural products often impractical. could be increased noticeably in a heterologous host. For example, To solve these problems, researchers have engineered and opti- Keasling and his colleagues successfully redesigned the biosynthesis mized natural products biosynthetic pathways for decades. As process of the artemisinin precursor, artemisinic acid, in hetero- genetic manipulation is either difficult or yet-to-be established for logous hosts Escherichia coli and Saccharomyces cerevisiae,5–7 which the majority of organisms, heterologous expression of a single gene, produced the artemisinic acid with a much higher productivity, titer acassetteofgenes,oranentirebiosyntheticgeneclusterina and yield and now the process has been transferred to a pharma- genetically tractable host is a practical alternative route for identify- ceutical company for commercialization. In addition, with the ing and engineering the corresponding natural product. In recent versatility of synthetic biology approaches, new compounds could years, with the third revolution in biotechnology, which requires the be discovered or generated by pathway refactoring or advanced convergence of life science, physical science and engineering dis- combinatorial biosynthesis (Fig. 1C).8,9 ciplines, new solutions have beenproposedfordrugdiscovery, Microbes, especially E. coli and yeast, have proven to be energy and food crisis.4 The first revolution of biology was evidenced useful organisms for heterologous biosynthesis of secondary by the discovery of the double helix structure of DNA by James metabolites for decades. They have been engineered to balance Watson and Francis Crick, while the second revolution began with and optimize natural product biosynthetic pathways for cost- the human genome project. Now, with the dawn of the post- effective production of high value compounds, as well as to genomics era came the third revolutionary change, which shifts discover novel compounds.10 Recent advances in synthetic the paradigm of the biology field from the understanding and biology and genome engineering facilitate the development of new tools for construction, controlling and optimization of metabolic pathways in microbes for heterologous biosynthesis. Herein, we review the recent progresses made in heterologous Duo Liu is currently a PhD biosynthesis of several different classes of important natural candidate in the Department of products, as well as how the newly developed synthetic biology Biochemical Engineering in Tianjin tools could help future heterologous biosynthesis of valuable University. He has worked on the natural products. In addition, we will highlight some of the most modular strategies of synthetic recent advances in heterologous biosynthesis of natural product biology for combinatorial construc- in the past five years. tion of yeast metabolic system for natural product biosynthesis. 2. Heterologous biosynthesis of major classes of high value natural products

Natural products are a very important source for the develop- Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. Duo Liu ment of medicines. Many natural products and the derivatives

Dr Huimin Zhao is the Centennial Dr Ying-Jin Yuan is currently a Endowed Chair Professor of professor of Biochemical Engi- chemical and biomolecular engineering in Tianjin University. He neering, and professor of chemistry, received his PhD degree from biochemistry, biophysics, and Tianjin University in 1991. He is bioengineering at the University of now the director of Key Laboratory Illinois at Urbana-Champaign of Systems Bioengineering, Ministry (UIUC). He received his BS degree of Education, China. His research in Biology from the University of interests include synthetic biology, Science and Technology of China systems biotechnology, bioenergy, in 1992 and his PhD degree in biopharmaceutical engineering Chemistry from the California and plant cell culture. Dr Yuan is Huimin Zhao Institute of Technology in 1998. Ying-Jin Yuan the Fellow of IChemE. Dr Yuan has His primary research interests are published more than 180 inter- in the development and applications of synthetic biology tools to national peer-reviewed papers and address society’s most daunting challenges in human health and 30 patents. energy, and in the fundamental aspects of enzyme catalysis and gene regulation.

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

have been developed as antibacterial, anticancer, antifungal, the MVA pathway in yeast-like fungi. In yeast, some highly antiviral and antiparasitic drugs.2 Butler et al. systematically active IPP compounds can also be synthesized based on the summarized the development of drugs in clinical trials from basic C5 units. For example, geranyl pyrophosphate (GPP) is a natural products and the derivatives.11 In this section, we will C10 structure generated by condensing one molecule IPP and provide a comprehensive overview of the production of different one molecule DMAPP. Following this reaction, an additional classes of high-value compounds from microbes. In addition, we will IPP molecule can be combined to make a C15 structure, discuss the recently developed metabolic engineering and synthetic farnesyl pyrophosphate (FPP), and further with another IPP to biology approaches employed to engineer microorganisms for make a C20 structure, geranylgeranyl pyrophosphate (GGPP). In heterologous expression of natural product pathways, especially native yeast, ergosterol, as the final metabolite of the successive for higher production of practically important natural products. condensation reactions, is derived from C30 squalene which is Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. synthesized by condensation of two FPP molecules (Fig. 2). 2.1 Terpenoids These reactions in bacteria and fungi enable the introduction of Terpenoids, also called isoprenoids, are the largest class of genes of plant origins to construct the heterologous terpenoids plant metabolites, consisting of more than 40 000 molecules.12 biosynthetic pathways. Monoterpenoids are synthesized from The most famous plant terpenoids have been used as the major GPP; sesquiterpenoids from FPP; diterpenoids and carotenoids drugs to treat life-threatening diseases, such as the anticancer from GGPP; and triterpenoids from squalene. drug paclitaxel and the antimalarial drug artemisinin.13 Some 2.1.1 Monoterpenoids. The most well-known monoterpenoids other terpenoids are supplemented with daily nutritional diets, that have been heterologously synthesized in engineered yeast are such as lycopene and linalool. Terpenoids usually consist of linalool and geraniol. Although wild-type yeast does not synthesize different numbers of basic five-carbon isopentenyl diphosphate these monoterpenes, the mutants with blocked FPP synthetase that (IPP) units with a series of assembly, cyclization and group excretes geraniol and linalool have been characterized previously.15 modification reactions.14 Traditional methods for producing However, the enzyme involved in GPP dephosphorylation has not these compounds usually rely on multistep extraction from been identified yet. plants or organic chemical synthesis, both of which can result Linalool is a value-added fine chemical, and can be used in low yield, high cost and sometimes severe pollutions to the to synthesize linalyl acetate, geraniol, linalyl methyl sulfide, environment. vitamin A, vitamin K and other medical intermediates.16 Linalool is In microbes, such as E. coli and yeast, the terpenoids synthesized via the condensation of IPP and DMAPP by the endo- biosynthetic pathways typically work with very low fluxes. The genous mevalonate pathway. Several cis or trans linalool synthases important precursors for terpenoids, IPP and its isomer were identified from different origins.17–19 The engineered industrial dimethylallyl pyrophosphate (DMAPP), are synthesized from yeast strain with a linalool synthase from Clarkia breweri produced acetyl-CoA through the DXP pathway in E. coli-like bacteria and linalool with no by-products.20 In addition, over-expression of

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

the hmg1 gene in the engineered yeast strain enhanced the ( )-nootkatol or ( )-nootkatone by different kinds of P450 1 21 À À production of linalool to 133 25 mgLÀ . cytochrome oxidases (CYP71D51v2 from Nicotiana tabacum, Æ Geraniol, as a widely used floral fragrance, is a typical CYP71AV8 from Cichorium intybus and CnVO from C. nootka- monoterpene, and can be further transformed into ester flavors tensis).31,32 Patchoulol, the major constituent of patchouli oil, Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. for food flavors, fruit flavors, ginner flavors, and cinnamon was synthesized by PTS (patchoulol synthase from Pogostemon flavors.22 It is also used for clinical treatment of chronic bronchitis cablin).25,26 In addition, other valuable sesquiterpenes, includ- and improvement of pulmonary ventilation function.22 Geraniol is ing arteminisic acid, cubeol, germacrene D, b-caryophyllene, usually obtained from myrcene by esterification, saponification a-santalene, were recently produced in engineered microbes.30,33,34 and fractionation in current industrial production. However, Importantly, the biosynthesis of artemisinin, a valuable and geraniol can also be synthesized from linalool. The geraniol powerful antimalarial natural product extracted from Artemisia synthase (GES) was isolated from Ocimum basilicum and expressed annua L,wasidentifiedandengineered.Firstly,FPPwasconverted in S. cerevisiae to produce geraniol.23,24 Furthermore, blocking of to amorphadiene by ADS (amorphadiene synthase). Then amor- FPP synthase increased geraniol synthesis.24 phadiene was oxidized to artemisinic alcohol by CYP71AV1 (a P450 2.1.2 Sesquiterpenoids. Sesquiterpenoids are the most diverse cytochrome oxidase). It was then reduced by DBR2 (artemisinic class of terpenoids, including industrially valuable products for aldehyde reductase) to dihydroartemisinic aldehyde and oxidized fragrances, repellents, food colorants and high-value therapeutic to dihydroartemisinic acid by ALDH1 (aldehyde dehydrogenase molecules, such as epicedrol, artemisin, valencene, cubebol, from A. annua). Finally, dihydroartemisinic acid was converted to patchoulol and santalene.25–31 All these sesquiterpenoids are artemisinin via an allylic hydroperoxide intermediate, and light and derived from a 15-carbon precursor, FPP. Different sesquiter- oxygen were required for this conversion.5 pene synthases were used to catalyze the synthesis of different Most of these sesquiterpenes have been successfully produced sesquiterpenes from FPP (Fig. 3). b-Sesquiphellandrene, with in engineered microbes such as E. coli, S. cerevisiae and other efficient anti-microbial and anti-oxidant activity, was synthesized industrial microorganisms. To increase the production of these by PMSTS (Persicaria minor sesquiterpene synthase).28,29 Valencene, sesquiterpenes, the enzymes from plants or the flux of the whole an odorant molecule, was synthesized by CsPTS (Citrus sinensis pathway need to be modified. For example, in engineering patch- valencene synthase), and could be further oxidized to (+)-nootkatol, oulol producing yeast, PMSTS was fused with ERG20 (FPP synthase)

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Fig. 3 Schematic of sesquiterpenoids biosynthesis. Farnesyl diphosphate (FPP), the 15-carbon precursor, is converted to diﬀerent sesquiterpene by various sesquiterpenoid synthases (ADS, amorphadienesynthase;CsPTS,valencenesynthase;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 by various 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).

to optimize the local concentration and spatial organization of synthesis de novo.39 Therefore, heterologous expression of the path- these two enzymes and to redirect the metabolic flux towards the ways in microbes is very promising for diterpenoids production. product.26 Combined with the repression of ERG9, the final Tanshinones are the main lipophilic components of the 1 26 patchoulol titer reached 23 mg LÀ . In addition, higher FPP Chinese medicinal herb danshen (Salvia miltiorrhiza). Modular concentration and increased production of valencene was pathway engineering (MOPE) was successfully developed at Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. achieved by localizing the enzyme to the mitochondrion.35 assemble and optimize the biosynthetic pathway of miltiradiene, Another successful example of heterologous biosynthesis of the precursor to tanshinones, in S. cerevisiae.40 The fusion of sesquiterpene is the optimized biosynthesis of artemisinic acid.6 SmCPS and SmKSL and the fusion of GGPPS (GGPP synthase) The systematic optimization started with regulating the propor- and FPPS, together with overexpression of tHMGR led to significant 1 tion of CYP71AV1 and AaCPR1, followed by overexpressing improvement of miltiradiene production (365 mg LÀ )andreduced AaCYB5 (cytochrome b5 from A. annua) and native HEM1 to byproduct accumulation significantly.40 Based on the increased optimize the oxidation of amorphadiene which is the key step of production of miltiradiene, incorporation of genes coding for the synthesis of artemisinic acid. At the same time, the native CYP76AH1 and phyto-CYP reductase led to heterologous produc- CTT1 (catalase) is also overexpressed to reduce the oxidative tion of ferruginol, the precursor to danshinones.41 stress produced by CYP71AV1. AaADH1 (alcohol dehydrogenase Paclitaxel, as a diterpenoid, has been approved for treatment from A. annua) and AaALDH1 are overexpressed to promote the of breast, ovarian and lung cancers, as well as coronary artery conversion of artemisinic acid from artemisinic alcohol. The disease.42 Although some studies tried to figure out the biosynthetic 1 6 final production of artemisinic acid reached up to 25 g LÀ . pathway for paclitaxel in fungal endophytes and improve the 2.1.3 Diterpenoids. Diterpenoids are widely distributed natural paclitaxel production by fermentation,43 arecentstudyindicatedno products with important physiological eﬀects, such as paclitaxel paclitaxel pathway could be identified in fungal endophytes.44 Due against cancer, tanshinones against inflammation, and stevioside to the high clinical needs and high costs of production, researchers as sweetener.13,36,37 However, the amount of the diterpenoids in the have been mining the key genes for paclitaxel biosynthesis for host plants is usually very low, let alone the slow growth rate of decades. Most of the biosynthetic reactions have been elucidated, plants. For example, the paclitaxel content is only at 10 ppm level and several important genes or enzymes have been characterized in the most productive species, Taxus brevifolia.38 In addition, (Fig. 4). Based on the elucidated partial pathway for paclitaxel complex diterpenoids molecules are quite diﬃcult for chemical production, the heterologous synthesis of paclitaxel precursors

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were investigated in different microbes. E. coli was firstly used endogenous genes, the final engineered strain produced as the host cell for the heterologous biosynthesis of taxadiene.45 17.2 mg per L protopanaxadiol, 15.9 mg per L protopanaxatriol The heterologous biosynthesis of taxadiene in K-derived E. coli and 21.4 mg per L oleanolic acid. and B-derived E. coli indicated that the background of the The main tetracyclic triterpenoids successfully synthesized chassis affected taxadiene synthesis.46 A multivariate-modular by engineered microbes are amyrin-derivative compounds, 1 approach increased titers of taxadiene to approximately 1 g LÀ including a-amyrin type and b-amyrin type. Several key functional (B15 000-fold increase) in an engineered E. coli strain.47 genes were introduced into yeast cells to produce various amyrin Besides E. coli, S. cerevisiae was also used as the chassis to structures.53,56 The main product based on b-amyrin precursor is produce taxadiene.48 Five enzymes catalyzing sequential trans- oleanolic acid.55 Catharanthus roseus is an important medicinal formations from IPP and DMAPP were heterologously expressed in plant and commercial source of monoterpenoid indole alkaloids, 1 S. cerevisiae,andtaxadienetiterwasimprovedto8.7 0.85 mg LÀ ursolic acid and oleanolic acid. The EST collection from C. roseus Æ by overexpression of tHMGR and UPC2-1.49 Six different GGPPSs leaf epidermome was analyzed, and a cDNA (CrAS) encoding were analyzed using enzyme-substrate docking strategy, and GGPPS 2,3-oxidosqualenecyclase and a cDNA (CrAO) encoding amyrin from Taxus baccata cuspidate showed the best performance in C-28 oxidase were successfully discovered.57 The functions of docking with substrate molecule FPP and led to the highest CrAS and CrAO were analyzed in S. cerevisiae. The co-expression 1 50 taxadiene production (72.8 mg LÀ )everreportedinyeastsofar. of CrAO and CrAS in yeast led to ursolic- and oleanolic acids Although the paclitaxel precursors were produced with high production. In addition, the cells co-expressing CrAO and efficiency by heterologous biosynthesis in different chassis, the AtLUP1 from Arabidopsis thaliana produced betulinic acid. heterologous biosynthesis of paclitaxel is still impossible due to Another typical triterpenoid is ginsenosides compound K the lack of information for several key biosynthetic steps. (CK).58 Among the several structures of ginsenosides, CK is Therefore, some alternative bioconversion approaches were the main functional component presenting bioactivities of anti- developed to produce paclitaxel. An Enterobacter sp. was found inflammation, hepatoprotection, antidiabetes and anti-cancer. to convert 7-xylosyl-10-deacetyltaxol (7-XDT) to 10-deacetyltaxol The potential CK biosynthetic pathway was designed in yeast, (10-DT), which is the most abundant byproduct in Taxus. The including a cytochrome P450 (CYP716A47) from Panax ginseng, highest conversion rate and yield of 10-DT from 7-XDT reached a NADPH-cytochrome P450 reductase (ATR2-1) from Arabidopsis 1 51 92% and 764 mg LÀ , respectively. A xylosidase from Enter- thaliana, and a Dammarenediol-II synthase (PgDDS) from obacter sp. was discovered and heterologously expressed in P. ginseng, and the CK synthase was explored based on bio- yeast.52 The engineered yeast could robustly convert 7-XDT into informatics analysis. CK was synthesized heterologously for the 10-DT with over 85% conversion rate and the highest yield of first time in engineered microbes by two pathways, especially 1 52 8.42 mg mLÀ . considering active CK could not be detected in original Panax 1 2.1.4 Triterpenoids. Triterpenoids are synthesized by con- plants. The final production of CK reached 1.4 mg LÀ . densation of six C5 IPP basic units through end-to-end coupling 2.1.5 Tetraterpenoids. Tetraterpenoids consist of eight iso- reactions. Triterpenoids are widely distributed in nature and prene units and are mainly involved in metabolic precursors can be divided into two main categories, pentacyclic triterpe- and products in the biosynthesis of carotenoids with unique noids (such as ginsenoside) and tetracyclic triterpenoids (such UV-Vis absorption spectra.59 Carotenoids serve as precursors Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. as amyrin-derivative terpenoids), according to the number of for vitamin A biosynthesis, but more importantly, they demon- carbon rings contained in the compounds.53 strate coloring and antioxidant properties attributed to their The chemical compositions of the ginseng complex have structures. Therefore, their expanded production is appealing. extensive biological activities and unique pharmacological Many kinds of hosts can produce carotenoids, such as actions. Medical and pharmacological research proved that Agrobacterium aurantiacum, Blakeslea trispora, several Pantoea one of the main effective components of ginseng is a group bacteria, Xanthophyllomyces dendrorhous and Haematococcus of ginsenosides, which are considered to possess medical value pluvialis. Commonly in carotenogenic bacteria and algae, bio- as anti-tumor, antiviral and cholesterol-decreasing drugs and synthesis of carotenoids starts from GGPP, which is synthesized precursors. Genetic engineering of plants was tried to improve by GGPP synthase (CrtE) either through direct condensation of the ginsenoside production by overexpression of the key genes 5-carbon precursors, IPP and DMAPP, or from 15-carbon pre- coding dammarane synthase and amyrin synthase. However, cursor, FPP. A tail to tail condensation of two GGPP molecules limited success was achieved.54 catalysed by phytoene synthase (CrtB) leads to the formation of Three biosynthetic pathways for various ginsenosides were the primal carotenoid, phytoene, which is colorless. Then, successfully constructed in S. cerevisiae,55 and they are respon- through successive introduction of four double bonds in the sible for the alternative synthesis of protopanaxadiol, proto- phytoene skeleton by phytoene desaturase (CrtI), lycopene is panaxatriol and oleanolic acid, the three basic aglycons of synthesized. Using lycopene as substrate, lycopene b-cyclase ginsenosides. The engineered S. cerevisiae was transformed (CrtY) catalyzes the formation of b-ionone rings at both ends to with expression cassettes of b-amyrin synthase, oleanolic acid produce b-carotene. Different from some bacteria and algaes, the synthase, dammarenediol-II synthase, protopanaxadiol synthase, phytoene synthase and phytoene desaturase belong to a bifunctional protopanaxatriol synthase and NADPH-cytochrome P450 reductase enzyme in red yeast, X. dendrorhous, and other fungi, B. trispora. from different plants. Through additional overexpression of some 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. The core taxane structure is synthesized by the modification of the taxadiene, which is produced by the cyclization of GGPP. The solid arrows in the pink background mean the steps with known reactions and known enzymes. The solid arrows in the light blue background mean the steps with known reactions but unknown enzymes. The dotted arrows mean the steps with unknown reactions and unknown enzymes. FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate. TDC1, taxadiene synthase; CYP725A2: taxane 13 alpha-hydroxylase; CYP725A1, taxane 10 beta-hydroxylase; TBT, taxoid 2a-O-benzoyl transferase; DBAT, 10-deacetylbaccatin III 10-O-acetyltransferase; PAM, phenylalanine aminomutase.

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capable of synthesizing astaxanthin. In both prokaryotes and products are lycopene and b-carotene. By combining the native eukaryotes, biosynthesis of astaxanthin is the results of ketola- MEP pathway and the heterologous MVA pathway, 1.35 g per L 63 tion at the 4 and 40-position and hydroxylation at the 3 and 30 lycopene and 2.47 g per L b-carotene with a yield of 72 mg per positions of the b-ionone rings of b-carotene. In bacteria, keto- g DCW64 was produced by fed-batch fermentation in E. coli. lase (CrtW) and hydroxylase (CrtZ) are required for astaxanthin Most recently, 3.2 g per L b-carotene was achieved under flask biosynthesis. However, due to their weak selectivity toward keto- cultivation employing a similar strategy.65 With a targeted and hydroxyl-substrate, quite a few intermediates accumulated engineering strategy, a very high maximum productivity of 1 1 when combining bacteria crtW and crtZ to produce astax- 74.5 mg LÀ hÀ and up to 1.23 g per L of lycopene was anthin.60,61 In red yeast X. dendrorhous, astaxanthin synthase produced in 100 L fed-batch fermentation.66 ATP and NADPH (CrtS), a cytochrome P450 enzyme, in combination with its recycling was engineered in E. coli to enhance terpenoids cytochrome P450 reductase (CrtR), are responsible for astax- production.67 Five alternative functional modules were anthin biosynthesis.60 In plant Adonis aestivalis, the 4-position designed for combinatorial metabolic engineering, including of b-ring of b-carotene is activated by 4-dehydrogenase-b-ring the heterologous synthesis module, MEP module, ATP module, (CBFD), followed by further dehydrogenation to yield a carbonyl PPP module, and TCA module, which led to an optimized strain catalyzed by 4-hydroxy-b-ring 4-dehydrogenase (HBFD). And capable of producing 2.1 g per L b-carotene with a yield of then, the 3-position is added by CBFD with a hydroxyl group, 60 mg per g DCW. In addition, expression tuning the expression which leads to the synthesis of astaxanthin. This pathway was level of the genes encoding a-ketoglutarate dehydrogenase, proved to work eﬃciently in E. coli (Fig. 5).62 succinate dehydrogenase and transaldolase B increased NADPH Heterologous biosynthesis of carotenoids was mostly carried and ATP supplies, leading to 3.52 g lycopene per L (50.6 mg per g out in two hosts, E. coli and baker’s yeast. In E. coli, the main DCW) in fed-batch fermentation.68 Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08.

Fig. 5 Schematic of the various biosynthetic pathways of carotenoids. (A) The common pathway for lycopene and b-carotene biosynthesis in bacteria and 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 in carotenogenic bacteria and algae (B), red yeast X. dendrorhous (C), and plant Adonis aestivalis (D). CrtZ, b-carotene hydroxylase; CrtW, b-carotene ketolase; CrtS, P450 cytochrome monooxygenase; CrtR, cytochrome P450 reductase; CBFD, carotenoid b-ring 4-dehydrogenase; HBFD, carotenoid 4-hydroxy-b-ring 4-dehydrogenase.

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In yeast, carotenoids are the first class of terpenoids to be tyrosine ammonia lyase (TAL) activity and thus was able to bypass produced. In the early period, like the studies in E. coli, C4H to produce p-coumaric acid from tyrosine.79 engineering efforts sourced carotenoid biosynthetic genes from The synthesis of naringenin chalcone was first achieved in several microorganisms, in particular the red yeast X. dendrorhous an engineered yeast, through co-expression of three different and the soil bacterium Pantoea ananatis,andachievedlow-level enzymes, including R. toruloides PAL, Arabidopsis thaliana production of target carotenoidsfromendogenousprecursors.69 4-coumarate:CoA ligase (4CL), and Hypericum androsaeemum Since then, researchers began to engineer yeast strains to accumu- chalcone synthase (CHS).80 The following chalcone isomerase late more quantities of GGPP precursor for carotenoid production (CHI) could catalyze the cyclization of chalcones to flavanones by either introducing a heterologous GGPP synthase gene crtE69,70 (Fig. 6).77,81–83 or overexpressing the native gene bts1.60,71 By using this combina- Based on the biosynthesis of flavonones, the introduce of torial genetic engineering strategy, the expressions of several genes cytochrome P450 enzymes, such as isoflavone synthase (IFS), were optimized, leading to 5.9 mg per g DCW b-carotene and flavone synthase II (FSII) and flavone synthase I (FSI), can lead astaxanthin.60,69–71 Recently, a set of marker recyclable integrative to the production of isoflavone genistein and the flavone plasmids (pMRI) was employed for decentralized assembly of the apigenin.77,81,82 Co-expression of flavanone 3b-hydroxylase 72 b-carotene biosynthetic pathways. By using GAL inducible (F3H), flavonoid 3-hydroxylase (F30H), and flavonol synthase promoters, the pathway could be switched on at certain time, (FLS) achieved the synthesis of kaempferol and quercetin in leading to production of 11 mg per g DCW of total carotenoids yeast.77 The production of some stilbenoids, such as resvera- 1 77,84 (72.57 mg LÀ ) and 7.41 mg per g DCW of b-carotene. The trol, has also been achieved in yeast. engineered yeast strain exhibited high genetic stability after 20 generations of subculturing. After down-regulating the squalene 2.3 Alkaloids 1 synthase (ERG9), 1156 mg LÀ (20.79 mg per g DCW) of total Alkaloids are a large class of nitrogen-containing compounds in carotenoids was achieved by high-cell density fermentation.73 plant secondary metabolites with low molecular weight.2 Most Interestingly, by exploiting the antioxidant properties of carote- alkaloids are derived from amines produced by the decarboxylation noids, adaptive evolution was successfully applied to boost of amino acids, such as histidine, lysine, ornithine, tryptophan, and carotenoids production.74 In another study, carotenoid was used tyrosine. One of the largest groups of pharmaceutical alkaloids is as a phenotypic marker to screen the yeast deletion collection to benzylisoquinoline alkaloids (BIAs), including the antitussive reveal new genes with roles in enhancing terpenoid production,75 codeine, the analgesic morphine,andtheantibacterialdrugs which provides potential targets for subsequent metabolic berberine. BIAs are widely used in human health and nutrition, engineering. at present they are mainly obtained by extraction from plants, which limited the diversity of BIAs structures available for drug discovery due to their low abundance. Many of the native plant 2.2 Flavonoids hosts accumulate only a selected few BIA products. BIAs are Flavonoids are a large group of plant secondary metabolites produced through (S)-reticuline, which begins with the condensa- containing at least one hydroxylated aromatic ring. Flavonoids, tion of two L-tyrosine derivatives, 3,4-dihydroxyphenylacetaldehyde when consumed in human diet, could help to promote human (3,4-DHPAA) and dopamine (Fig. 7). (S)-Reticuline is a main branch- Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. health and prevent certain diseases, as it can prevent cancer point metabolite in the biosynthesis of many kinds of BIAs, and it by preventing cellular oxidation processes and stopping cell plays an impartment role in the development of novel antimalarial degradation and aging.76 The fact that only a few of these and anticancer drugs. A number of recent reports have successfully compounds can be produced as pure products from limited transplanted portions of (S)-reticuline biosynthesis pathways into plant species makes flavonoids as attractive candidates to be microbial hosts, such as E. coli and S. cerevisiae.85–89 synthesized in engineered microbes, such as E. coli and (S)-Reticuline and some other intermediates in the berber- S. cerevisiae.76–78 ine and morphinan branches could be synthesized in yeast by S. cerevisiae is a suitable host to synthesize flavonoid com- overexpressing enzymes from three plant sources and humans. pounds as its natural metabolism provides necessary amino A glucocorticoid inducible promoter and in situ promoter acid precursors tyrosine and phenylalanine for downstream titration were applied to tune enzyme expression levels and exogenous pathways. To date, more than 20 pathways from optimize the productivity.85 Reticuline could also be produced fungus and plant have been implanted into S. cerevisiae to from dopamine in E. coli by heterologous expression of biosyn- produce different kinds of flavonoid products, including flava- thetic enzymes (i.e., MAO, NCS, 6-OMT, CNMT, and 4-OMT).86 nones, flavones, isoflavones, and flavonols.77,78 About 7.2 mg per L magnoflorine and 8.3 mg per L scoulerine p-Coumaric acid is a common intermediate for almost all were produced from dopamine via reticuline by using different flavonoids biosynthesis. Phenylalanine ammonia lyase (PAL) combination cultures of engineered E. coli and S. cerevisiae.86 first catalyzes the deamination of phenylalanine to cinnamic acid, The productivity of reticuline could be improved by fine-tuning and cytochrome P450 cinnamate-4-hydroxylase (C4H) acting with the possible rate-limiting step.88 Notably, S. cerevisiae expressing partner reductase (CPR) subsequently catalyzes the hydroxylation of CYP80G2 and CNMT converted reticuline to magnoflorine.86 An cinnamic acid to p-coumaric acid. The enzyme from the fungus alkaloid biosynthetic pathway from L-tyrosine cells was constructed Rhodospridium toruloides was introduced into yeast to exhibit a in E. coli,whichproduced46.0mgperLof(S)-reticuline from

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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, flavone synthase I; FSII, flavone synthase II; IFS, isoflavone synthase; TAL, tyrosine ammonia lyase.

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

2.4 Polyketides Polyketides are another diverse group of natural products with important applications in the pharmaceutical industry. They have been isolated and characterized from a variety of natural Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. sources, including plants, fungi, and bacteria. They are of high interests as they often exhibit a broad spectrum of biological activities, which are attributed to their structural complexity and diversity. Those diverse and complex structures are produced by polyketide 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 by the Gram-positive bacterium Saccharopolyspora erythraea.90 It can be used to treat various diseases, such as respiratory infections, whooping cough, syphilis, Legionnaires’ disease, gastrointestinal infections, acne, as well as used as a substitution for penicillin. After the identification and characterization of the Fig. 7 Schematic of the various biosynthetic pathways of alkaloids. CNMT, DEBS PKSs gene cluster responsible for biosynthesis of erythro- coclaurine-N-methyltransferase; DODC, DOPA decarboxylase; MAO, mono- mycin, functional heterologous expression of these proteins and amine; NCS, norcoclaurine; TYR, tyrosinase; 3,4-DHPAA, 3,4-dihydroxyphenyl- their mutants in S. coelicolor and E. coli were employed.91,92 This acetaldehyde; 6-OMT, norcoclaurine 6-O-methyltransferase; 40-OMT, not only helped to reveal the biochemical basis for these highly 3 -hydroxy-N-methylcoclaurine 4 -O-methyltransferase. 0 0 controlled megasynthases, but also allowed the rational design of its biosynthetic products, and led to the generation of diverse glycerol.87 Recently, a biosynthesis pathway for the production of polyketide libraries. Researchers have engineered the microbes dihydrosanguinarine and sanguinarine from (R,S)-norlaudano- for heterologous production of various erythromycin derivatives soline was constructed with 10 genes in S. cerevisiae.89 It represents for further studies in the hope of improving their pharmacological

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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) Domain engineering; (C) domain inactivation; (D) engineering of tailoring reactions.

properties. One notable example is the incorporation of unnatural combined and applied to a S. coelicolor strain expressing the starter units to change the chain initiation process by replacing heterologous DEBS.101 Fermentation achieved the production the loading domain AT with otherATdomains,orincorporating of 1.3 g per L of 15-methyl-6-dEB, and the productivity was synthetic oligoketide precursors into the downstream DEBS path- increased by over 100% in a fermentation process. Furthermore, way.93–95 Besides the precursors, the extender unit can also be the introduction of an engineered mutase-epimerase pathway in Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. replaced in the erythromycin DEBS system.96–98 Since the DEBS E. coli enabled a 5-fold higher production of 6-dEB.102,103 In AT domains of each module are theprimarygatekeepersforthe addition, after the carbon flux of the biosynthetic pathway was incorporation of an extender unit into the polyketide chain, redirected and an extra copy of a key deoxysugar glycosyltrans- replacing AT domains with other ones that accept other extender ferase gene was introduced, a 7-fold increase in erythromycin A units could result in the incorporation of other functional groups titer was achieved.104 In those studies, E. coli-derived production into the final polyketide products. Later on, combinatorial poly- of erythromycin was enabled by the introduction of the entire ketide biosynthesis by de novo design and rearrangement of erythromycin pathway (20 genes in total) using separate expression modular DEBS genes with other eight PKS genes created 154 plasmids. However, this may cause metabolic burden and plasmid bimodular combinations and allowed the production of novel instability. Recently, the E. coli erythromycin A production system derivatives as well.99 Recently, a new E. coli platform for erythro- was upgraded by altering the design of the expression plasmids mycin analogue production was established by the production of needed for biosynthetic pathway introduction, and a 5-fold increased alternative final erythromycin compounds exhibiting bioactivity erythromycin A production was achieved.105 On the other hand, in against multiple antibiotic-resistant Bacillus subtilis strains.100 the native host, by the chromosome gene inactivation technique Both the native deoxysugartailoringreactions,D-desosamine based on homologous recombination with linearized DNA and L-mycarose deoxysugar pathways, were replaced with alter- fragments, one TetR family regulator SACE_7301 was identified 17 native D-mycaminose and D-olivose pathways to produce new to enhance erythromycin production in S. erythraea. erythromycin analogues in E. coli. Another recent example is for the biosynthesis of cholesterol- Besides understanding the basic biochemistry and production of lowering drug pravastatin106 derived from the natural product new derivatives, newly developed synthetic biology platforms oﬀer compactin.107 Pravastatin inhibit 3b-hydroxy-3-methylglutaryl new strategies for productivity enhancement. Random mutagenesis, CoA (HMG-CoA) reductase, which catalyzes the rate-limiting recombinant DNA techniques, and process development were step in cholesterol biosynthesis. Pravastatin is obtained after

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stereoselective hydroxylation of compactin at the C-6 position dogma process, including gene replication, transcription, and this is often achieved by a two-step production process in translation, and post-translation. The development of central industry.108 Novel expression vector has been used to carry a gene for dogma based engineering toolkits has attracted attention for cytochrome P450 monooxygenase, and3.3-foldincreasedproduction decades since the discovery of the double helix structure of DNA of pravastatin has been achieved. Optimized batch culture yielded and the lac operon.112,113 More recently, the rapid development of 14.3 g per L of pravastatin after 100 h.109 Metabolic reprogramming synthetic biology calls for much more powerful central dogma of this antibiotics has been achieved by introducing both the based engineering toolkits. These toolkits possess some common compactin pathway and a new cytochrome P450 from Amycolatopsis characteristics, such as standardization, universality, real-time orientalis (CYP105AS1) into the b-lactam–negative P. chrysogenum controllability, reusability, and modularity. Several strategies for DS50662. More than 6 g per L pravastatin was obtained via further standardized gene parts storage and ligation have been established engineering of the CYP105AS1 enzyme at a pilot production scale.110 recently, such as Biobrick, Bglbrick and SEVA.114–116 Gene parts have standard identification sites that can be digested by type I or type IIS restriction enzymes and be ligated to another part 3. New tools for heterologous containing the same couple of sites. However, there is a paradox biosynthesis of natural products about standardization and universality of the gene parts. Several excellent tools to tune gene expression were developed by Prior to synthetic biology, several genetic engineering and combination of standardized rules and universal manipulations metabolic engineering strategies were developed for the optimiza- (Fig. 9). At the gene level, variant copies of genes on different tion of heterologous pathway expression. For example, expression of plasmids showed obvious effects on the synthesis of target single genes can be optimized by codon-optimization, promoter products.117 At the transcriptional level, promoter strength, engineering, gene copy number, translation regulating sequence mutation of promoter functional sites, addition of the upstream mutation, post-translational modification and protein engineering. activation sites, and inducible artificial promoters have been While for multiple gene expressions, metabolic modelling is widely successfully used in optimization of multiple genes expres- used to guide the coordination of multiple gene expressions in the sion.118–124 At the translational level, special sequence repeats, metabolic network, such as identifying the targets of limiting steps. synthetic riboswitches, rational ribosome binding sites design However, the lack of the parameters during the heterologous gene tools, random translation starting sites, tuneable intergenic expression, such as the physiological changes of the host and the region options, and artificial codons have been used for fine- property parameters of the heterologous proteins in the host tuning of gene expression.125–130 Most of these methods showed 111 environment, limited the applications of metabolic modelling. great potentials in tuning of multiple genes and optimization of In the past few years, several powerful synthetic biology tools multiple genetic modules. In addition, novel 4-base codons and have been developed for the optimization of heterologous unnatural amino acids were introduced in the translation pro- pathways in engineered microbial hosts. As engineered hetero- cess for novel and orthogonal polypeptides elongation and logous microbes offer an optimized and standardized platform protein synthesis.130 Most recently, two synthetic bases X and or cell factory for natural product production, advances in their Y were added to genetic codons in addition to natural bases A, T, metabolism engineering would benefit the reconstruction of C and G.131 Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. the whole cellular network and heterologous natural product Based on these ideas of engineering the central dogma biosynthesis pathways. process, next generation of DNA synthesis technologies can be used to construct large libraries of genes with modified 3.1 Engineering at the parts Level codons and varying expression levels. By taking advantage of Besides the basic physical construction of a heterologous path- computational design and metagenomic information, de novo way, the achievement of maximal yield of target products relies DNA synthesis enables us to obtain artificial or natural genes on many other factors, including optimization of metabolic for the required enzymes in an expanding range (Fig. 9). For flux, reduction of intermediate toxicity, and tuning proper example, 89 methyl halide transferases found in metagenomic stress on the host cell. Some general strategies for the design sequences from diverse organisms were synthesized and of gene or protein parts are needed for optimal expression and screened for higher enzymatic activities.132 154 and 1468 organization of individual pathway enzymes. The expression of reporter genes were constructed and characterized as libraries gene parts can be tuned by modifying gene copy numbers, to study codon usage of genes.133 Oligo pools were combined transcriptional activity, or post-transcriptional processing. The with multiplexed reporter assays for construction of more than individual proteins can be organized as fusion proteins or in 14 000 reporter constructs. The transcriptional and transla- artificial scaffolds for coupled activities in metabolic pathways. tional rates of these constructs were used to analyze the effects The natural and heterologous enzymes can also be organized of N-terminal codon bias on protein expression levels.134 inside and outside of certain organelles to enhance the product 3.1.2 Making artificial protein fusions. Natural metabolism yields. is under tight regulations, such as positive or negative feedbacks. 3.1.1 Tuning the expression of gene parts. In engineered The heterologously expressed enzymes often suffer from flux microbes, expressions of gene parts, especially heterologous imbalances because of the lacking of well-established regulatory gene parts, can be fine-tuned throughout the whole central mechanisms. In addition, the enzymes in the same metabolic

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Fig. 9 Strategies to tune the gene expression levels in the natural product biosynthetic pathways. The expression of heterologous genes are regulated at the gene level (variation on copy numbers of genes, selection of plasmids), at the transcriptional level (promoter strength, mutation of promoter functional sites, addition of upstream activation sites, and inducible artificial promoters), and at the translational level (special sequence repeats, synthetic Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. riboswitches, rational ribosome binding sites, random translation starting sites, tuneable intergenic region options, and artificial codons). The codon optimization of the heterologous genes is another eﬃcient strategy to tune gene expression.

pathway may be distributed at different regions inside the cell, conditions, most of the FPP is used for production of ergosterol, thus diffusion or degradation of the intermediates occurs and which is essential for the cellular maintenance. Therefore, the FPP the efficiency of the whole pathway would be decreased. Simply is a main branching point for the synthesis of sesquiterpenoids and blocking the competitive pathways, in many cases, is not sufficient triterpenes (Fig. 2). Fusing FPPS together with patchoulol synthase since it might be lethal or hard to implement. In fact, the high (PTS, encoded by Pogostemon cablin gene PatTps177)andGGPPS expression level of engineered pathways may compete with biomass redirected the flux to the production of the sesquiterpene patch- production or other primary metabolite pathways for nucleotides oulol and GGPPS respectively, and enhanced the production by and amino acids, which results in opposite to what is expected. 2-fold and 8-fold, respectively.26,135 Besides fusion of these two Fusion of proteins could bring active sites of enzymes into a endogenous proteins, heterologous enzymes copalyl diphosphate closer proximity, which facilitates substrate channelling synthase (SmCPS from Salvia miltiorrhiza) and kaurene synthase- (Fig. 10) and relieves the side-pathway competition. Protein like (SmKSL from Salvia miltiorrhiza) were fused for biosynthesis fusion strategies have been used in the endogenous MVA of tanshionones precursor miltiradiene in yeast.40 Simply fusing 26 1 pathway. FPPS, encoded by ERG20, catalyses the condensa- FPPS with Bst1p made the production from trace to 1.0 mg LÀ . tion of 5-carbon units IPP and DMAPP to produce FPP, which is According to the coimmunoprecipitation experiments, they the general precursor for biosynthesis of sesquiterpenes and predicted SmKSL and SmCPS may form a complex in vivo, geranylgeranyl pyrophosphate (GGPP), and then GGPP can be which inspired them to fuse these two enzymes for more converted into diterpenes and tetraterpenes. In natural growth efficient production. By comparison of fused SmCPS-SmKSL

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Fig. 10 Strategies to enhance the pathway flux by modifying the distances of proteins and protein re-localization. In order to enhance heterologous metabolic flux, fusion of proteins with different linkers is efficient. DNA-, RNA- and protein-based scaffolds are used to assemble the heterologous enzymes to improve the sequential reactions. Re-localization of enzymes switches the subcellular position of heterologous proteins to enhance the Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. production by either enhancing enzyme concentration or taking advantage of environmental differences. Colourful solid arrows represent the heterologous enzymes.

and SmKSL-SmCPS, the latter pattern was more effective by of bacterial metabolism. The synthetic RNA modules were 1 40 offering a 2.9-fold increase of miltiradiene to 3.1 mg LÀ . functionally discrete and formed 1D and 2D scaffolds in which Construction of scaffolds is another commonly used strategy the palindromic regions were disfavoured and the assembly to couple several enzymes catalysing sequential reactions in the order of RNA strands were controlled by insuring tile formation same pathway. This type of constructs will not affect other before polymerization. This RNA-based scaffolding system was metabolic pathways significantly due to its controllability. applied to increase the production of hydrogen, and an increase There are different types of scaffolds, such as plasmid scaffolds, of 48-fold over the unscaffolded, tagged enzymes in E. coli was RNA scaffolds and protein scaffolds (Fig. 10). Plasmid scaffolds observed.138 Besides plasmid scaffolds and RNA scaffolds, protein possess the advantage of accommodating many interaction scaffolds were constructed by co-localization of pathway enzymes to motifs and linkers of variable lengths without solubility issues, synthetic complexes using well-characterized protein–protein inter- such as the linking of the glucose oxidase and horse radish action domains and their specific ligands. This strategy is able to peroxidase via a lysine residue to short DNA oligo nucleo- increase the effective concentrations of the metabolic intermediates tides.136 However, the disadvantage is that enzymes must be around the enzymes. significantly modified with multiple zinc finger domains The mevalonate producing pathway is consisted of three (usually 3–4 domains with a total addition of 90–120 amino enzymes (acetoacetyl-CoA thiolase (AtoB) from E. coli, hydroxy- acids).137 RNA scaffolds were designed and constructed using methylglutaryl-CoA synthase (HMGS) and HMGR from S. cere- multidimensional RNA structures for the spatial organization visiae) in the engineered E. coli.139 Difference in the expression

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levels of these 3 enzymes led to a ‘‘bottle-neck’’ effect and the a standard, flexible set of vectors to target identical pathways accumulation of toxic intermediate HMG-CoA. Thus the synthetic to different subcellular parts. Targeting Ll-kivd (a-ketoacid protein scaffolds were applied to spatially recruit metabolic decarboxylase from Lactococcus lactis) and Sc-adh7 (alcohol enzymes, and the 1 : 2 : 2 ratio produced 77-fold of mevalonate dehydrogenase from S. cerevisiae) to mitochondria increased 139 1 143 compared with the wild strain. No harm to the growth of the the titer of isobutanol approximately 220% to 486 mg LÀ . strain was observed, no matter how the inducer concentration The results showed that the compartmentalization the pathway varied.139 At the same time, increasing the copy number of the into mitochondria achieved higher local enzyme concentra- protein scaffolds led to enhanced productivity. tions and increased the availability of intermediates and 3.1.3 Re-localizing proteins. Primarily for eukaryotic microbial released the restriction on transportation. hosts, many enzymes, such as cytochrome P450s, are thought to bind to the endoplasmic reticulum or mitochondrial membrane via 3.2 Engineering at the pathway level the hydrophobic membrane binding domain at the N-terminus in Similar to organic synthesis of a target chemical, the heterologous their native hosts. Therefore, this membrane binding domain may production of a target product in engineered microbe hosts is also be required for function, especially when these cytochrome P450s affected by common factors such as substrate supply, reaction are expressed in heterologous hosts. Mature forms of these efficiency, reaction thermodynamics and kinetics, yield, productivity, enzymes without the signal peptides are often used in heterologous toxicity, and cost-efficiency. Although some operation units are expression. However, the cytochrome P450 with both N-terminal different for alternative products and can be optimized case by case, membrane insertion and C-terminal HA epitope-tag deletion exhib- some universal pathway engineering strategies would facilitate the ited the highest expression level than other forms with partial improvement of these biochemical reactions. The successful produc- deletion.140 Relatively upstream pathways are also effective targets tion of artemisinic acid synthesis in a heterologous host offered us a for compartmentalization. S. cerevisiae was engineered to produce good example of how to optimize cellular metabolic pathways for 2,3-butanediol (BDO) by the introduction of a cytosolic acetolactate terpenoid production, and these strategies could be applied to other synthase (cytoILV2).141 Acetyl-CoA level was improved in the cytosol biochemical molecules as well. by the combined disruption of competing pathways and introduc- 3.2.1 Mining of heterologous pathways. A typical synthetic tion of heterologous biosynthetic pathways to supply more pre- biology approach for heterologous biosynthesis of target products cursors for desired product biosynthesis in yeast.142 n-Butanol begins with the exploration of genes and pathways encoding production was improved to 3-fold through acetyl-CoA enhance- functional enzymes. However, there are still many pathways remain- ment by introduction of heterologous acetyl-CoA biosynthetic path- ing to be explored, and the typical strategy the ones studied is to ways including pyruvate dehydrogenase (PDH), ATP-dependent express them heterologously, either to make more use of the known citratelyase (ACL), and PDH-bypass.142 pathway or decipher the cryptic pathway (Fig. 11). The first trial to In contrast with expressing mitochondrial enzymes in cyto- introduce a whole pathway heterologously was done for terpenoid sol, some enzymes and metabolic pathways were also expressed production.144 Targeted amorpha-4,11-diene, the sesquiterpene in mitochondria instead of in cytoplasm, because mitochondria olefin precursor to artemisinin, as the final product, they introduced have many potential advantages for metabolic engineering, the mevalonate terpenoid pathway from S. cerevisiae into E. coli and such as the sequestration of diverse metabolites, containing the concentration reached 24 mgcaryophylleneequivalentpermL, Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. intermediates of many central metabolic pathways, and the which approved the functionalization of fungi pathway in the environment in the mitochondrial matrix is highly different cytosol of bacteria. For cryptic pathway exploration, besides the from that in the cytoplasm.143 Mitochondrion was also used as analysis of systems biology, synthetic biology also takes in practice an alternative location for heterologous expression of metabolic the construction of transcription units of any possible ORFs based engineering instead of cytosol to enhance the flux to FPP.35 on bioinformatics analysis, as intheexampleoftheminingofthe CsTPS1 (valencene synthase from Citrus sinensis) was located gene encoding the CK synthase (Fig. 11).58 CK, as the main into mitochondria by the fusion with mitochondrial targeting functional component of ginsenoside, possesses bioactivities of signal peptide of COX4, which brought a 3-fold rise in valen- anti-inflammation, hepatoprotection, antidiabetes and anti- cene titers compared with cytosol CsTPS1. The expression of cancer.145 The potential CK biosynthetic pathway was designed in mitochondrion-targeted FPPS (mtFPPS) further increased the yeast, including a cytochrome P450 (CYP716A47) from Panax gin- production by 40%. Addition of another copy of cytosolic seng,aNADPH-cytochromeP450reductase(ATR2-1)fromArabidop- 1 CsTPS1 further increased the production to 1.5 mg LÀ , which sis thaliana,andaDammarenediol-IIsynthase(PgDDS)fromPanax was an 8-fold enhancement. This strategy was also performed ginseng,withotherinvolvedenzymesoriginatedfromS. cerevisiae.In on amorph-4,11-dienesynthase (ADS), and mtADS was con- this study, a proprietary cDNA database including 479 689 structed, which strongly enhanced the amorphadiene produc- assembled cDNA contigs was established based on 9 Panax EST 1 tion to 20 mg LÀ . Accordingly, for upstream pathways, Avalos datasets available from the NCBI GenBank. 16 ORFs were amplified et al. engineered yeast mitochondria to produce isobutanol, and expressed in S. cerevisiae,throughwhichway,theenzyme isopentanol and 2-methyl-1-butanol through Ehrlich degrada- coding CK synthase was obtainedfromnaturalresourcesand tion pathway, which originally occurs in cytoplasm, while the primarily verified and the strain produced 1.4 mg per L of CK.58 upstream pathway is confined to mitochondria, which creates a Sometimes a novel pathway for product synthesis can also substantial bottleneck in the transportation.143 They developed be designed. For example, an unprecedented artificial salvianic

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Fig. 11 Exploration and construction of novel natural product biosynthetic pathways. (A) Heterologous expression of uncharacterized genes or proteins with potential functions to catalyze the specific reaction is applied to explore new pathways for natural product biosynthesis. (B) Heterologous expression of uncharacterized genes to explore the biosynthetic pathway for ginsenoside CK. (C) Heterologous expression of protein with similar activity to

Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. construct a novel biosynthetic pathway for salvianic acid A. Colourful solid arrows represent the heterologous enzymes. Adapted by permission from Macmillan Publishers Ltd: [Cell Research] (ref. 58), copyright (2014). Reprinted from Y. F. Yao, C. S. Wang, J. Qiao and G. R. Zhao, Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway, Metab. Eng., 19, 79–87, copyright (2013), with permission from Elsevier.

acid A (SAA) biosynthesis pathway was developed in E. coli.146 strategies of engineering central carbon metabolisms, which Two enzymes D-lactate dehydrogenase (D-LDH) from Lactobacillus can be summarized as combinatorial engineering (Fig. 12). pentosus and hydroxylase complex encoded by hpaBC from E. coli, Most of these strategies were designed rationally; however, were introduced into E. coli to convert 4-hydroxyphenylpyruvate some could also be utilized to build up a randomized library in (4HPP) into SAA. D-LDH and its variants were selected to catalyze the pre-design frame. In some strategies, variation in expression the chiral reduction of ketone group in 4HPP according to their levels of individual genes were introduced, and those alternative performance in transformation of phenylpyruvate into phenyllactic expressions were combined during DNA assembly process (Fig. 12). acid (Fig. 11). Co-expression of d-ldh and its mutants and hpaBC One prominent example is the Customized Optimization of Meta- together with optimizations on the upstream pathway resulted in bolic Pathways by Combinatorial Transcriptional Engineering 1 146 7.1 g per L of SAA with a yield of 0.47 mol molÀ glucose. (COMPACTER) method in which assembly of promoter mutants 3.2.2 DNA assembly-assisted pathway construction. To and pathway genes resulted in the combination of diﬀerent gene synthesize a desired product in heterologous organisms, the expression levels in a target pathway.147 Amorerecentworkutilized introduced biosynthetic pathways and upstream metabolic Gibson assembly method to introduce coding sequences after the pathways are required to be considered together as the supply initial codon ATG for expanding expression range of the genes of precursors aﬀects the ultimate yield of the final product. In coding violacein synthesizing enzymes, causing variant combina- recent years, the studies on synthetic biology also expand to tions of multi-gene transcriptional levels.128

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3.2.3 Modular pathway engineering for metabolic precursor to increase the production of the target compounds. Chen et al. synthesis. To produce high-level products, simple modification enhanced the supply of acetyl-CoA in the cytoplasm through a of the expression levels of genes in the target pathway is the most combined push–pull-block strategy. The push part included the rational method for the pathway optimization. These methods over-expression of the endogenous alcohol dehydrogenase could be classified into 3 categories (Fig. 13): (1) increasing the (ADH2) and NADP-dependent aldehyde dehydrogenase (ALD6), supplement of the substrate, (2) increasing the whole flux of the and a codon-optimized acetyl-CoA synthase variant from Salmo- L641P desired biosynthetic pathway, and (3) decreasing or eliminating nella enterica (ACSSE ). The pulling of acetyl-CoA towards the the flux of the branch pathway. Here, we take the optimization of products of interest was enhanced by the over-expression of the MVA pathway as an example to demonstrate how to optimize ERG10, encoding acetyl-CoA C-acetyltransferase, that catalyzes a biosynthesis process at the pathway level. the conversion of acetyl-CoA to acetoacetyl-CoA. The block part Acetyl coenzyme A (acetyl-CoA) is a central metabolite in of the strategy decreased reduction of acetyl-CoA and was carbon and energy metabolism. The mevalonate pathway in achieved through removing two key enzymes involved in the S. cerevisiae is initiated from acetyl-CoA, a key precursor to a consumption of acetyl-CoA. One is the peroxisomal citrate wide range of valuable secondary metabolites.148 Moreover, synthase and the other is cytosolic malate synthase. This strategy acetyl-CoA supply in the mitochondrial matrix is essential for improved the production of a-santalene by 4-fold.150 Lian et al. cell growth because it is used to replenish the mitochondrial redirected the glycolytic flux towards acetyl-CoA by inactivating tricarboxylic acid (TCA) cycle with C2 units and produce ATP.149 ADH1 and ADH4 for ethanol formation and GPD1 and GPD2 Optimizing the supply of acetyl-CoA becomes a logical approach for glycerol production, resulting in 4-fold improvement in Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08.

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

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n-butanol production. Subsequent introduction of heterologous For production of terpenes, the native biosynthesis pathway acetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase of ergosterol always needs to be down-regulated. The most (PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, further useful method is to reduce the expression level of essential increased n-butanol production. Among them recombinant PDHs gene ERG9 by diﬀerent weaker promoters such as pMET, localized in the cytosol (cytoPDHs) was most efficient and increased pCTR3, and pHXT1.6,7 Knocking out the lppA and STC genes n-butanol production by additional 3-fold.142 reduced the production of fansesol while the important precursor To increase the overall flux of the MVA pathway, enhancing FPP was accumulated.33 the reaction rate of the limiting step (HMG-CoA to mevalonate) Optimization of the MVA pathway needs the combination of was considered. tHMG1,thetruncatedHMG1 which only contains diﬀerent strategies. A very successful example is the biosynthesis the catalytic domain of HMG1, is commonly used in engineering of of amorphadiene, which can be synthesized from FPP by amor- the MVA pathway in yeast. tHMG1 was often overexpressed by phadiene synthase encode by ADS. Westfall et al. overexpressed strong promoter and has many copies in the cell.7,33,151 On the all genes responsible for the MVA pathway from ERG10 to ERG20 other hand, UPC2-1,amodifiedglobaltranscriptionalregulatorof under the GAL promoter and integrated all these genes into ergosterol biosynthesis was oftenusedtoup-regulatetheexpres- genome. Among them, the key enzyme tHMG1 had three copies sion level of the entire MVA pathway.7,33,151 to enhance the limiting-step of the MVA pathway. ERG9 was Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08.

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

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down-regulated by substitution of the native promoter of ERG9 added from outside to tune genes’ transcription and translation by pMET. During fermentation, ethanol was the sole carbon processes, such as isopropyl thiogalactoside, arabinose, anhydrote- source for production, which was helpful for supplying more tracycline, and theophylline. Light is also used as a signal to regulate acetyl-CoA for biosynthesis. The accumulated precursor FPP, gene expression and change cellular activity.152–154 However, most of could be converted into amorphadiene eﬃciently by highly these earlier systems could only be operated in a pre-determined way expressed ADS. Subsequent fermentation optimization led to or via serendipity. Recent advances offered us some biosensor- 40 g per L products, which was the highest production compared directed real-time metabolic control methods (Fig. 14). It is often to previous reports.7 difficult to accumulate the products in engineered cells due to the 3.2.4 Biosensor-directed real-time control and evolution. toxicity of target intermediates. Metabolite response promoters were In recent years, development of intracellular small molecule screen- explored and utilized in metabolic self-regulation.155 The whole- ing methods led to generation of cellular self-modification systems genome transcript arrays were employed to identify promoters that with the small moleculesfunctioningassignalmolecules(Fig.14). responded to these intermediates. The promoters could control Some of the small molecules are produced by the cell, such as expression of certain genes in a real-time manner, causing enhanced N-acylhomoserine lactones (AHL) and N-butyrylhomoserine lactone amorphadiene production by 2-fold over traditional constructions in (BHL), which could be synthesized by special enzymes in cells and E. coli.Thisapproachcouldalsobeextendedtoothertoxicinter- diﬀused to coherent cells to regulate multi-cell activities through mediates for dynamic regulation.155 Asimilarmetabolitebiosensor gene circuits. While some other similar molecules could only be system was constructed to achieve the feedback regulated evolution Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08.

Fig. 14 Biosensor-directed real-time control and evolution. The promoters inhibited by the target products are used as the biosensors to control the expression of DNA mutator proteins, such as mutD5. This design can be used to generate a mutant library. Meanwhile, the same promoter also control the expression of a fluorescent protein, which is used as the reporter for phenotype selection. Colourful solid arrows represent the heterologous enzymes. 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 In chemically synthesized DNA fragments into microbial gen- the designed adaptive control system, the genomic mutation rate omes. Because of the large size of a genome, genome engineer- would be increased by expression of mutD5togeneratediversityin ing is typically coupled with high throughput screening the population, and be decreased only when the concentration of technologies. In the past few years, genome engineering has target metabolites came to required amounts. The synthetic tran- been increasingly used for heterologous biosynthesis of natural scription factors were utilized to construct artificial biosensors to products. respond for metabolites without natural sensors. The FREP was Based on the allelic replacement on the lagging strand by verified by evolving increased tyrosine and terpenoid production. the oligos during replication, multiplex automated genome engineering (MAGE) was developed in E. coli, and applied to improve the biosynthesis of lycopene by simultaneously mod- 3.3 Engineering at the genome level ifying 24 genes in the corresponding biosynthetic pathway.158 Genome engineering approaches can be grouped into three This method was recently extended to S. cerevisiae.159 By categories: genome editing, transcriptome engineering, and applying the same principle, multiplex iterative plasmid engi- 157 genome synthesis. Genome editing precisely or combinato- neering (MIPE) was developed to engineer the heterologous rially modifies the target genome at multiple loci while tran- metabolic pathway in the plasmid, and a clone with 2.67-fold scriptome engineering essentially targets regulatory elements improved production of riboflavin was achieved in less than a by mutating endogenous regulators or introducing artificial week.160 Another related approach, RNA interference (RNAi)- ones. Genome synthesis involves hierarchical assembly of short Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08.

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

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assisted genome evolution (RAGE) could continuously improve may offer the potential chassis for heterologous biosynthesis of target trait(s) by accumulating multiplex beneficial genetic different natural products. modifications in an evolving yeast genome.161 Modular artificial nucleases have been increasingly used 3.4 Engineering at the community level for genome editing. The zinc-finger domain, which typically recognizes 3-nucleotide DNA motifs, was the first to be exploited. Many complex natural products are synthesized by very long Sequence-specificity was further increased by engineering zinc-finger pathways such as paclitaxel. It is often a challenge for one cell to nucleases (ZFNs) in a way that requires their heterodimerization express such long heterologous pathways due to the heavy metabolic through the FokI domain for efficient cleavage.162 The modular burden associated with expression of so many heterologous enzymes 176,177 approach was taken a step further with the discovery of the TAL and production burden. Most of the microbial consortium effector (TALE) DNA-binding modules of Xanthomonas bacteria, and engineering focus on the lignocelluloses bioconversion to produce 132,178 their simple DNA recognition code.163 However, to target unique chemicals and fuels. The progress on the fundamental studies sequences in a eukaryotic genome, long TAL arrays need to be and the applications of artificial consortium was reviewed 179 assembled in order, because each TAL repeat targets a single recently. More recently, engineering of the microbial consortium 177 nucleotide. More recently, clustered regularly interspaced short has been applied to biosynthesis of complex natural products. palindromic repeats (CRISPR) have been developed as a general Taxadiene, as the scaffold molecule of paclitaxel, can be currently method for genome engineering including activating transcription/ produced at much higher titer in the engineered E. coli compared 47,50 translation, silencing transcription/translation, up/down regulating to engineered yeast. However, heterologous expression of cyto- gene expression, and modifying multiple sites simultaneously, chrome P450s, the downstream enzymes for taxadiene transforma- 47 each accompanied with permanentchangesofgenomesequence. tion, is more suitable in yeast than in E. coli, because yeast Compared to ZFNs and TALENs, the CRISPR technology has possesses the advanced protein expression machinery and abun- radically improved the accessibility of gene targeting due to its dant intracellular membranes. Therefore, metabolic pathway for straightforward approach for customizing sequence specificity via taxane biosynthesis was distributed into the consortium of E. coli target-specific guide RNAs. Its targeting efficiency is comparable and S. cerevisiae,andthissyntheticconsortiumproduced33mgper 177 with the best efficiency achieved using TALENs in a wide range Loxygenatedtaxanes(Fig.16). Using the similar strategy, of animals and plants.164 Recently, it was shown that the biosynthesis of ferruginol were also achieved in the artificial endonuclease domains of the Cas9 protein can be mutated consortium of E. coli with the heterologous pathway of miltiradiene 177 and co-expressed with a guide RNA to specifically interfere with synthesis and S. cerevisiae with an cytochrome P450. The concept transcriptional elongation and work like RNAi. This system was of distributing the long metabolic pathways into consortium would named CRISPR interference (CRISPRi), which can efficiently repress expression of a target gene with no detectable off-target effects.165 In addition, dCas9 was used with existing optogenetic or chemical-induced proximity systems to dynamically analyze recruitment of a broad range of chromatin modifiers to a specific DNA sequence, both for customized activation and repression.166 Published on 11 May 2015. Downloaded by University of Illinois - Urbana 26/05/2015 23:14:08. CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to be integrated for the tunable modulation of gene networks.167 A rapid, efficient, and potentially scalable strategy based on CRISPR/ Cas system was recently developed to generate multiple gene disruptions simultaneously in S. cerevisiae.168 Genome synthesis with novel design is the extreme of genome editing for natural product synthesis. So far, genome synthesis based on the natural genome sequence has succeeded in 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 of S. cerevisiae named SC 2.0 was launched recently. Numerous loxP sites were introduced into the redesigned genome and the Fig. 16 Engineering of the microbial consortium to improve heterolo- SCRaMbLE (synthetic chromosome rearrangement and modi- gous biosynthesis of complex natural products. (A) Schematic of the fication by loxP-mediated evolution) was developed by combin- distribution of the long synthetic pathway for complex natural products ing loxP sites with the inducible Cre recombinase (Fig. 15).172 into different strains of the consortium. (B) The artificial consortium with engineering E. coli and S. cerevisiae produce oxygenated taxanes using The SCRaMbLE system in Sc2.0 is a very useful tool for mini- xylose and ethanol. Colourful solid arrows represent the heterologous mization of the yeast genome and generation of a library of enzymes. Adapted by permission from Macmillan Publishers Ltd: [Nature mutant genomes at a large scale.173–175 Such mutant genomes Biotechnology] (ref. 177), copyright (2015).

Review Article Chem Soc Rev

significantly enhance the heterologous biosynthesis of natural Acknowledgements products (Fig. 16). This work was financially supported by the Ministry of Science and Technology of China (863 program: 2012AA02A701, 973 program: 2013CB733600), and the National Natural Science 4. Conclusion and future prospects Foundation of China (major project: 21390203). HZ would like Natural products have been and continue to be the source and to acknowledge the financial support by National Institutes of inspiration for a substantial fraction of human therapeutics. Health (GM077596). The successfully commercialized production of several drugs or drug candidates shed lights on the future heterologous bio- References synthesis of important natural products. With the emergence of more recently genomics- and bioinformatics-guided synthetic 1 Y. Luo, R. E. Cobb and H. Zhao, Curr. Opin. 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