CRITICAL REVIEWS IN BIOTECHNOLOGY, 2017
`VOL. 37, NO. 8, 974–989
`http://dx.doi.org/10.1080/07388551.2017.1299679
`
`REVIEW ARTICLE
`
`Progress in terpene synthesis strategies through engineering of
`Saccharomyces cerevisiae
`Kalaivani Paramasivan and Sarma Mutturi
`
`CSIR-Central Food Technological Research Institute, Mysore, India
`
`ABSTRACT
`Terpenes are natural products with a remarkable diversity in their chemical structures and they
`hold a significant market share commercially owing to their distinct applications. These potential
`molecules are usually derived from terrestrial plants, marine and microbial sources. In vitro pro-
`duction of terpenes using plant tissue culture and plant metabolic engineering, although receiv-
`ing some success, the complexity in downstream processing because of the interference of
`phenolics and product commercialization due to regulations that are significant concerns.
`Industrial workhorses’ viz., Escherichia coli and Saccharomyces cerevisiae have become microorgan-
`isms to produce non-native terpenes in order to address critical issues such as demand-supply
`imbalance, sustainability and commercial viability. S. cerevisiae enjoys several advantages for syn-
`thesizing non-native terpenes with the most significant being the compatibility for expressing
`cytochrome P450 enzymes from plant origin. Moreover, achievement of high titers such as 40 g/l
`of amorphadiene, a sesquiterpene, boosts commercial interest and encourages the researchers to
`envisage both molecular and process strategies for developing yeast cell factories to produce
`these compounds. This review contains a brief consideration of existing strategies to engineer S.
`cerevisiae toward the synthesis of terpene molecules. Some of the common targets for synthesis
`of terpenes in S. cerevisiae are as follows: overexpression of tHMG1, ERG20, upc2-1 in case of all
`classes of terpenes; repression of ERG9 by replacement of the native promoter with a repressive
`methionine promoter in case of mono-, di- and sesquiterpenes; overexpression of BTS1 in case of
`di- and tetraterpenes. Site-directed mutagenesis such as Upc2p (G888A) in case of all classes of
`terpenes, ERG20p (K197G) in case of monoterpenes, HMG2p (K6R) in case of mono-, di- and ses-
`quiterpenes could be some generic targets. Efforts are made to consolidate various studies
`(including patents) on this subject to understand the similarities, to identify novel strategies and
`to contemplate potential possibilities to build a robust yeast cell factory for terpene or terpenoid
`production. Emphasis is not restricted to metabolic engineering strategies pertaining to sterol
`and mevalonate pathway, but also other holistic approaches for elsewhere exploitation in the S.
`cerevisiae genome are discussed. This review also focuses on process considerations and chal-
`lenges during the mass production of these potential compounds from the engineered strain for
`commercial exploitation.
`
`ARTICLE HISTORY
`Received 5 May 2016
`Revised 20 August 2016
`Accepted 28 November 2016
`
`KEYWORDS
`Terpenes; terpenoids;
`Saccharomyces cerevisiae;
`metabolic engineering;
`sterol pathway
`
`Introduction
`
`Natural products have their origin from diverse living
`organisms including terrestrial plants, microbes and
`marine species. There are more than 200,000 plant nat-
`ural products identified so far [1]. Natural products
`could be either primary or secondary metabolites. The
`classes of secondary metabolites include polyketides
`(antibiotics), phenylpropanoids
`(flavonoids,
`stilbenes
`and lignin), alkaloids and terpenoids. Terpenes or terpe-
`noids (oxygenated forms of terpenes) otherwise called
`as isoprenoids (as they are built from isoprene, C5H8
`units) are the oldest known, structurally diverse and
`largest category of molecules with more than 55,000
`
`structures elucidated so far [2]. Terpenes include both
`primary as well as secondary metabolites and forms the
`main constituent of essential oils. Terpenes could be
`used as flavors, fragrances, colorants and medicine in
`the food, cosmetics and pharmaceutical industries and
`have a surging demand in forthcoming years [3]. The
`Asian flavor and fragrance industry is estimated to reach
`$9.6 billion and the global flavor and fragrance industry
`is estimated to reach a total market size of $33.5 billion
`in 2019 [4].
`Terpene biosynthesis involves two distinct pathways,
`produced by the mevalonate (MVA) pathway in
`yeast
`(Figure 1(A))
`[5], whereas in prokaryotes like
`
`CONTACT Sarma Mutturi, PhD
`sarma.mutturi@gmail.com; smutturi@cftri.res.in
`Supplemental data for this article can be accessed here.
` Both the authors contributed equally to this work
`ß 2017 Informa UK Limited, trading as Taylor & Francis Group
`
`CSIR-Central Food Technological Research Institute, Mysore, India
`
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`CRITICAL REVIEWS IN BIOTECHNOLOGY
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`Figure 1. (A) Compartmentalized depiction of sterol pathway in S. cerevisiae. The solid and dotted arrows indicate single and
`multiple steps, respectively. The genes involved in individual or multiple reactions are shown above the arrow in red font. The
`box around fecosterol and zymosterol indicates the reaction occurring inside mitochondrion via translocation reactions (SAM, S-
`adenosyl-L-methionine; SAH, S-Adenosyl-L-homocysteine) [114]. The energy (ATP) and electron ((NADPH) balances are stoichiomet-
`rically correct. (B) The depiction of MEP pathway in E. coli (adapted from ecocyc.org). The solid and dotted arrows indicate single
`and multiple steps, respectively. DXS, DOXP synthase; DXR, DOXP reductase; ispD, MEP cytidylyltransferase; ispE, 4-diphophocy-
`tidyl-2-C-methyl-D-erythritol kinase;
`ispF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase;
`ispG, HMB-PP synthase;
`ispH,
`HMB-PP reductase.
`
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`976
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`K. PARAMASIVAN AND S. MUTTURI
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`Escherichia coli, these compounds are produced via
`the methyl-D-erythritol-4-phosphate (MEP) pathway
`(Figure 1(B)) [6]. A study shows in some organisms pos-
`sess the both pathways. In such cases, MEP pathway is
`involved in primary metabolite formation whereas the
`MVA pathway in secondary metabolite formation [7].
`Plants employ both pathways, while the MEP pathway
`is localized in plastids. Pyruvate and glyceraldehyde-3-
`phosphate, G3P act as precursors for the MEP pathway
`while two molecules of acetyl-CoA form the basis for
`MVA pathway. The three main processes of terpenoid
`production are plant cell culture, microbial fermentation
`and chemical synthesis. However, microbial fermenta-
`tion is more desirable owing to its low cost of produc-
`tion and eco-friendly nature [8]. Since the publication of
`“Life with 6000 genes” [9] followed by release of the
`entire genome in the public domain, Saccharomyces cer-
`evisiae has become an indispensable eukaryotic system
`and considerable knowledge of its physiology, genetics,
`biochemistry, gene manipulation techniques [10], culti-
`vation strategies and computational methods has accu-
`mulated, thereby enhancing its scope for industrial
`exploitation. Synthetic biology efforts have led to the
`“cutting edge” development of the synthetic version of
`yeast cells with modification of the whole genome in a
`project called Sc.20 [11,12]. Though there are several
`advantages of using S. cerevisiae as a host organism, the
`pathways are compartmentalized in yeast which com-
`plicates the process as the intermediates have to trans-
`locate themselves from one compartment to another to
`carry out certain reactions in order to continue the
`pathway. Hence, the availability of precursors varies
`amongst
`the compartments. Unlike bacterial cells,
`S. cerevisiae cells do not have their genes organized as
`operons inside which make the genetic manipulation
`for strain improvement a more laborious process when
`compared to prokaryotes. Moreover, unlike plant cells,
`S. cerevisiae do not possess diverse class of Cytochrome
`P450 (CYP-CPR) enzymes which carry specific oxygen-
`ation reactions to produce structurally diverse terpenes.
`Hence, they needed to be heterologously expressed
`from plant origin. To some extent, these challenges
`have been addressed and are reported in subsequent
`sections in this review.
`Because of the exclusive nature so far published in
`terpenoid production studies, the need for a compre-
`hensive review article on S. cerevisiae considering the
`production of a diverse class of terpene molecules, the
`present review is relevant. Data “mining” was carried
`out with a special focus on the recent literature during
`in the last 5 years in order to consolidate information
`on this subject. This review covers the following topics:
`elucidation of the stoichiometric balance for terpene
`
`synthesis from the sterol pathway, description of differ-
`ent terpenes produced in yeast, genetic perturbations
`carried within and outside the sterol pathway to
`improve terpene production, application of
`in silico
`strategies, process considerations and finally, in conclu-
`sion, a consideration of
`future perspectives on this
`subject.
`
`Sterol pathway and terpene theoretical yield
`calculations
`
`The MVA pathway extends into ergosterol (ERG) biosyn-
`thesis in S. cerevisiae, and the synthesis of ERG from ace-
`tyl-CoA is referred as the sterol pathway (Figure 1(A)).
`Figure 1(A) depicts the compartmentalized sterol path-
`way spanning the cytoplasm and endoplasmic reticu-
`lum of S. cerevisiae. The synthesis of ERG from acetyl-
`CoA involves 20 distinct metabolic steps with special-
`ized structural genes which fuel these reactions as
`shown in Table S1. Following assumptions have been
`considered for framing the stoichiometric equations:
` Glucose to pyruvate formation does not include
`pentose-phosphate pathway.
` Pyruvate to acetyl-CoA is via pyruvate dehydrogen-
`ase (PDH)-bypass where NAD is selected as cofactor
`for reaction involving ALD5.
`
`The overall stoichiometry for squalene production
`from acetyl-CoA summarized below indicates that 18
`moles both acetyl-CoA and ATP are required in this
`energy-intensive pathway to produce a single mole of
`squalene:
`18AcetylCoA þ 13NADPH þ 18ATP þ 6H2O
`¼ Squalene þ 18CoA þ 13NADP þ 18ADP
`þ 6CO2 þ 6PPi þ 6Pi:
`From glucose, the overall stoichiometry for squalene
`production is as follows:
`9GlucoseðC6H12O6Þ þ 36NAD þ 13NADPH
`þ 12Pi þ 18ATP þ 6H2O ¼ SqualeneðC30H50Þ
`þ 24CO2 þ 13NADP þ 36NADH
`þ 18AMP þ 24PPi:
`Further downstream reactions of squalene leading to
`ERG require oxygen and the stoichiometric balance is as
`follows:
`Squalene þ 12O2 þ 14NADPH þ SAdenosyl
`Lmethionine ¼ Ergosterol þ 14NADP
`þ 2CO2 þ Formate þ SAdenosylL
`homocysteine þ 17H2O:
`
`(3)
`
`(1)
`
`(2)
`
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`Also, both Equations (1) and (3) demonstrate the
`high requirement of NADPH as a source of electrons. In
`Equation (1), the reactions carried by HMG-CoA reduc-
`tases (Hmg1p, Hmg2p) and squalene synthase (Erg9p)
`are NADPH-dependent (Figure 1(A)). Whereas,
`in the
`lower half of sterol pathway represented by Equation
`(3), NADPH is utilized in most of the reactions including
`Erg11p and Erg5p, which belong to the cytochrome
`P450 family.
`The stoichiometric closure for overproduction or
`heterologous production of a compound facilitates and
`sometimes becomes a priori for pathway engineering as
`it dictates and demonstrates the energy requirement
`that governs the efficiency of producing the required
`compound. Based on stoichiometric calculations [13], it
`was observed that the pathways where reducing equiv-
`alents are generated (NADH/NADPH) along with prod-
`uct formation are usually inefficient in terms of yield in
`comparison to those pathways which require supply of
`reducing equivalents for product formation. The individ-
`ual stoichiometric balance for each of the terpene class
`precursors from glucose as the substrate is provided in
`separate equations below:
`3C6H12O6 þ 4Pi þ 4NADPH þ 6ATP þ 2H2O
`þ 12NAD ¼ GPPðC10H20O7P2Þ þ 7PPi
`þ 4NADP þ 6AMP þ 8CO2 þ 12NADH:
`4:5C6H12O6 þ 18NAD þ 6Pi þ 3H2O þ 6NADPH
`þ 9ATP ¼ FPPðC15H28O7P2Þ þ 18NADH
`þ 11PPi þ 12CO2 þ 6NADP þ 9AMP:
`6C6H12O6 þ 24NAD þ 8Pi þ 4H2O þ 8NADPH
`þ 12ATP ¼ GGPPðC20H36O7P2Þ þ 15PPi
`þ 24NADH þ 16CO2 þ 8NADP þ 12AMP:
`9C6H12O6 þ 36NAD þ 12Pi þ 5H2O þ 14NADPH
`ð
`Þ
`þ 18ATP þ O2 ¼ oxidosqualene C30H50O
`þ 36NADH þ 24PPi þ 14NADP
`þ 24CO2 þ 18AMP:
`These Equations (4–7) indicate the requirement of
`NADPH and ATP for the synthesis of the precursor mol-
`ecules with concomitant release of NADH. This NADH
`can be either utilized during other biosynthetic path-
`ways or end up in a futile pathway as a sink. The yield
`for the different classes of terpene molecules were cal-
`culated according to the framework developed by
`Dugar and Stephanopoulos [13] and is tabulated in
`Table 1.
`It can be observed from this table, that the
`pathway yields (YP, YP,G and YCI
`P,G) are lower than the
`maximum yields for all the terpenes due to the gener-
`ation of excess reducing equivalents (NADH). This data
`also indicated that the actual yields will be either lower
`
`(6)
`
`(7)
`
`(4)
`
`(5)
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`CRITICAL REVIEWS IN BIOTECHNOLOGY
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`Table 1. Estimation of possible yields for different terpenes
`produced in S. cerevisiae [13].
`
`Terpene
`type
`Mono-
`
`Sesqui-
`
`Formula
`C10H18O
`C10H16
`C15H24
`
`P,G
`
`YCI
`0.102
`0.101
`0.076
`
`Yields (g terpene/g
`glucose)a
`YP
`YP,G
`0.317
`0.239
`0.324
`0.236
`0.236
`0.101
`
`YE
`0.317
`0.324
`0.324
`
`Compound/s
`Cineole, Geraniol
`Limonene, Sabinene
`Amorphadiene,
`Bisabolene,
`Caryophyllene,
`Copaene, Cubebene,
`Epiaristolochene,
`Humulene, Cadinene,
`Premnaspirodiene,
`Valencene,
`a-santalene
`Epicedrol, Farnesol
`Texadiene,
`Abietadiene,Casbene
`0.081
`0.108
`0.251
`0.345
`C20H34O
`Geranylgeraniol
`0.086
`0.114
`0.267
`0.367
`C20H36O2
`Scareol
`b-amyrin
`0.079
`0.111
`0.246
`0.338
`C30H50O
`Tri-
`b-carotene, Lycopene
`0.075
`0.099
`0.232
`0.331
`C40H56
`Tetra-
`aYE is maximum yield (cS/cP), YP is pathway yield (without cofactor imbal-
`ance), YP,G is terpene yield with concomitant production of glycerol,
`which is assumed to be a sink for excess NADH (without cofactor imbal-
`P,G is terpene yield with concomitant production of glycerol,
`ance), YCI
`a sink for NADH, with cofactor imbalance.
`
`Di-
`
`C15H26O
`C20H32
`
`0.352
`0.324
`
`0.256
`0.236
`
`0.110
`0.101
`
`0.083
`0.076
`
`or close to the estimated yields. Varman et al. [14] built
`an empirical equation from various experimental stud-
`ies for yield determination based on the type of gene
`manipulations carried out in S. cerevisiae for producing
`these compounds. Their methodology predicts the yield
`of a compound for a given set of genetic manipulations.
`Among these calculations, the yields of terpenes were
`in agreement with the yields compiled in Table 1. Thus,
`this kind of stoichiometric analysis would not only pro-
`vide insight into the yield but also indicates pointers for
`genetic manipulation.
`
`Genetic perturbations to improve terpene
`synthesis
`
`The essence of metabolic engineering is to systematic-
`ally improve the properties of cells by using analytical
`and computational methods to quantify fluxes and their
`control employing molecular
`tools and techniques
`[15–17]. Major strategies for global and pathway spe-
`cific metabolic engineering, as described in [18] are:
`
`Increasing precursor and cofactor supply.
`a.
`b. Heterologous expression of non-native genes.
`c.
`Blocking or down regulating competing pathways.
`d. Overexpression of transcriptional regulators.
`e.
`Improving enzyme specificity.
`
`The objective of this section is to present different
`specific strategies carried by several researchers in order
`to produce native and primarily non-native terpenes in
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`978
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`K. PARAMASIVAN AND S. MUTTURI
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`S. cerevisiae. The section is focused and classified based
`on the metabolic strategy rather than a class of ter-
`penes. Furthermore, it is divided into perturbations car-
`ried inside the sterol pathway and more generic
`manipulations outside the sterol pathway to produce
`the target terpene. The consolidated information on the
`list of terpenes produced in S. cerevisiae along with
`details of metabolic strategies, titers generated and fold
`increment are provided in Table S2. This table is referred
`to in subsequent sections to discuss specific strategies
`implemented leading to robust strain development and
`the increase of the specific terpene upon perturbation.
`
`Manipulations inside sterol pathway
`
`Heterologous expression of terpene synthases
`
`As discussed earlier, the sterol pathway leading to ERG
`formation involves intermittent compounds such farne-
`syl pyrophosphate (FPP), geranyl pyrophosphate (GPP),
`geranylgeranyl pyrophosphate (GGPP) and squalene
`epoxide which can be diverted to specialized terpenes
`(Figure 2). This diversion of
`flux involves a class of
`enzymes known as terpene synthases that are not pre-
`sent in S. cerevisiae in contrast to plants and other
`organisms. The striking feature among all these syn-
`thases contains aspartate-rich motifs (DDXXDD) as con-
`served portions. Most of the heterologous genes for the
`functional
`expression of
`terpenes
`synthases
`are
`obtained from plant sources, or they are synthetically
`generated (from NCBI gene accession number)
`for
`expression in S. cerevisiae. S-Linalool synthase (LIS) was
`one among the earlier identified monoterpene synthase
`(MTS) and was isolated from stigmata of Clarkia breweri,
`a flowering plant [19]. Rico et al. [20] reported the suc-
`cessful expression of LIS cDNA in S. cerevisiae to produce
`132 lg/l of linalool, a monoterpene used as a perfume
`ingredient. The studies on C. breweri was followed by
`isolation of terpene synthases from selected plant spe-
`cies and a list was compiled for both MTS and sesquiter-
`pene synthases [21], which also includes gene bank
`accession numbers. Both monoterpenes and sesquiter-
`penes production in S. cerevisiae have been widely
`studied in comparison to others (Figure 2(A) and (B)).
`The diterpene synthases (DTS) list has also been com-
`piled which provides details on its sources covering bac-
`teria, fungi and plants [22]. Taxadiene synthase, a DTS
`from Taxus brevifolia, was successfully expressed in S.
`cerevisiae to produce taxadiene, a precursor for anti-
`cancer drug taxol [23]. Kirby et al. [24] investigated DTS
`produced in plants belonging to the Euphorbiaceae fam-
`ily for functional expression in S. cerevisiae to produce
`potential diterpenes such as neocembrene and casbene
`
`(Figure 2(C)). The details on triterpene synthases (TTS),
`often referred to as oxido squalene cyclases, are com-
`prehensively covered by [25]. Several TTS have been
`expressed in S. cerevisiae, the prominent being for the
`production of b-amyrin (Figure 2(D)]. In the case of tetra-
`terpenoids, the synthesis begins by condensation of two
`GGPP molecules
`to form phytoene (Figure 2(E)].
`Tetraterpenoids derived from phytoene, often referred
`to as carotenoids, includes lycopene, b-carotene, c-caro-
`tene, torulene and astaxanthin. S. cerevisiae does not
`have the natural ability to produce these carotenoids.
`A red yeast species, Xanthophyllomyces dendrorhous has
`the capacity to produce carotenoids and is the source
`for isolation of genes for heterologous expression of car-
`otenoids in S. cerevisiae [26] (Figure 2(E)].
`
`Overexpression of structural genes in sterol pathway
`
`One of the easiest and most widely applied strategy in
`metabolic engineering is to identify a rate-limiting reac-
`tion and overexpress the gene or genes that affect this
`reaction. However, it must be understood that the con-
`cept of a single rate-limiting step in isolation does not
`justify flux distribution for the entire pathway [27,28].
`Hence,
`the term flux control coefficient has been
`adopted to suggest that some reactions possess a higher
`flux control coefficient relative to other reactions in a
`pathway. One such flux control reaction in the S. cerevi-
`siae sterol pathway has been identified as HMG-CoA con-
`version to MVA which is catalyzed by Hmg1p or Hmg2p,
`products of HMG1 and HMG2, respectively (Table S1) [29].
`Further studies showed that these two isoforms contain
`a membrane binding domain (1–552 amino acids) which
`spans ER and a catalytic domain (553–1046 amino acids)
`[30]. Later,
`it was observed that overexpression of the
`only catalytic domain of HMG1, termed tHMG1 (truncated
`HMG1), has improved squalene concentrations in S. cere-
`visiae [31]. This laid the foundation for overexpression of
`tHMG1 in a large number of studies involving the pro-
`duction of terpenes in S. cerevisiae (Table S2). Hmg2p, an
`isoform of this reductase, is usually dominant during low
`oxygen conditions and hence has become the target for
`overexpression during anaerobic cultivation to produce
`squalene [32]. Products of
`IDI1 and ERG20 are key
`enzymes which lead to GPP and FPP formation that in
`turn could be diverted to monoterpene and sesquiter-
`pene synthesis, respectively. In various studies, shown in
`Table S2, both these enzymes are overexpressed to pro-
`duce the terpenes of interest in S. cerevisiae. A branch of
`the reaction from the main sterol pathway, catalyzed by
`Bts1p brings FPP and isopentenyl-PP (IPP), to synthesize
`GGPP,
`forms the precursor
`for diterpenes synthesis
`(Figure 2(B)]. Hence, diterpene synthesis in S. cerevisiae is
`
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`evisiae.(A)Monoterpenes.(B)Sesquiterpenes.(C)Diterpenes.(D)Triterpenes.(E)Tetraterpenes.
`Figure2.BiosyntheticroutestoproducedifferentclassofterpenesfromtheircorrespondingprecursorinvolvingtheheterologousexpressionofspecificterpenesynthasesinS.cer-
`
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`K. PARAMASIVAN AND S. MUTTURI
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`sometimes facilitated by overexpression of BTS1 [23,33]
`(Table S2). It is interesting to see in the studies by [34],
`they have overexpressed all the structural genes in the
`MVA pathway (ERG10, tHMG1, ERG13, ERG12, ERG8) and
`ERG20 during synthesis of amorphadiene (Table S2) and,
`as a result of this study, artemisinic acid production has
`been doubled whereas amorphadiene production has
`been increased 10-fold to a titer value of 40 g/l. During
`synthesis of acchileol A, a triterpene [35] overexpressed
`lanosterol synthase coded by the ERG7 and different var-
`iants of the enzyme by introducing mutations in the
`ERG7 gene. Essentially, overexpression of
`structural
`genes, prominently the ones upstream of squalene in the
`sterol pathway, has been targetted for producing ter-
`penes in S. cerevisiae.
`
`Knockout and knockdown
`
`Deletion or down-regulation of a competing pathway has
`been proven as a successful strategy for developing an
`efficient cell factory [18]. Usually, this strategy is imple-
`mented by completely eliminating a gene involved in a
`competing pathway (referred as knockout) or lowering its
`expression levels by incorporating a weak promoter
`(referred as knockdown) and also sometimes enabling a
`mechanism to control the expression using a regulated
`or tunable promoter (referred as repression). The knock-
`out strategy is preferred only if the target is a non-essen-
`tial gene. ERG6 is one of the non-essential genes that has
`been used as a target during squalene synthesis in S. cere-
`visiae [32]. Squalene synthase, coded by ERG9, is one of
`the key enzymes involved in sterol formation. Hence,
`ERG9 has become the key target. Moreover, it is an essen-
`tial gene. Knockdown and repression strategies for this
`gene have been widely studied and have proven success-
`ful. The ERG9 gene was down-regulated by replacing its
`endogenous promoter with the tunable methionine pro-
`moter, PMET3 [24,34,36,37]. ERG9 downregulation using
`the same strategy when combined with overexpression
`of tHMG1 expression lead to a two-fold increase in amor-
`phadiene production [37]. Glucose-regulated promoters,
`PHXT1 and PHXT2 have also been used to exert tight control
`over the ERG9 gene and has been successful in downre-
`gulation [38–40]. Comprehensive information on the level
`of expression using various promoters in S. cerevisiae can
`be obtained [41]. Single allele deletion of ERG9 in a dip-
`loid strain was used as a strategy [42]. Homologous dele-
`tion of a gene in a diploid strain leads to a 50% reduction
`in its protein level in most of the cases [38].
`
`Site-directed mutagenesis
`
`Site-directed mutagenesis is an indispensable molecular
`tool
`for
`the
`direct
`evolution
`of
`enzymes.
`
`Direct evolution leads to the enzyme characteristic
`enhancement such as their stability, solubility, substrate
`and cofactor specificity thereby leading to the develop-
`ment of tailor-made enzymes [43]. The crux of in vitro
`mutagenesis is to create a willful mutation at a specific
`position in the structural gene thereby generating a
`modified codon for translation. This approach has been
`an indispensable tool in protein engineering to enhance
`its functional properties and has been exploited to
`facilitate terpene production in S. cerevisiae. As ERG20
`fuels both FPP and GPP synthesis reactions, efforts have
`been made to manipulate this gene in order to change
`the ratios at which it catalyzes each of these condensa-
`tion reactions.
`In one of the early studies, the AAG
`codon of ERG20 gene was point mutagenized to GAG,
`thereby causing conversion of lysine (K) at the 197 pos-
`ition to glutamic acid (E) in the amino acid sequence of
`Erg20p [44]. This mutation has triggered production of
`prenyl alcohols (geraniol and linalool) which are other-
`wise not produced in S. cerevisiae. This study has
`inspired to probe into Erg20p conserved region, K197,
`in order
`to generate several point mutations and
`observe the response of monoterpene yields [45]. They
`have observed that K197G, A, L, C, S, T, R, D, E, N
`showed improved geraniol, linalool and citranellol pro-
`duction and the levels were improved by 10–20-folds
`when geraniol synthases obtained from Ocimum basili-
`cum was heterologously expressed in conjunction. The
`K197G strategy was also adopted in other studies to
`improve monoterpene in S. cerevisiae [46,47] (Table S2).
`According to patent US008481286B2 [48], several point
`mutations were generated in squalene synthase gene
`(ERG9) to improve FPP levels and also screened for via-
`bility so as to avoid the external addition of ERG for cul-
`tivation (Table S3). Amongst the isozymes, Hmg1p is
`quite stable contributing to 83% of reductase activity
`whereas its counterpart Hmg2p undergoes regulated
`degradation [29,49,50]. Essentially,
`it was lysine (K)
`at position 6 and 357 on Hmgp2 were found to be
`critical
`for degradation and these
`lysines were
`replaced to observe the stability of Hmgp2 [49]. Thus,
`the K6R (lysine to arginine replacement) mutant of
`Hmgp2 was utilized for improved reductase activity as a
`strategy for improved terpene production [32,42,51,52]
`(Table S2).
`
`Expression of cytochrome P450 (CYP-CPR) enzymes
`
`Cytochrome P450 (CYP) catalyze diverse metabolic reac-
`tions ranging from region- and specific- oxygenations
`to structural alterations such as ring modifications,
`deamination, dealkylation, decarboxylation and C-C
`cleavage.
`In the case of terpene synthesis, they carry
`
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`out several changes in intermediary terpene backbones
`and P450 monooxygenase is one of the major enzymes
`involved in such activities. The transfer of electrons
`from NADPH is via NADPH- dependent cytochrome
`P450 oxidoreductases (CPR) which acts as electron
`donors that colocalize along with monooxygenase [53].
`CPR is membrane bound and is localized in the ER-
`membrane in the case of S. cerevisiae. The CYPs are clas-
`sified into different clans, among which CYP71 com-
`prises more than half of CYPs in higher plants [54].
`Prominent clans involved during terpene synthesis in
`plants belong to CYP51, CYP71, CYP72, CYP76, CYP85
`and CYP97 [55]. Ro et al. [37], Shiba et al. [56], and
`Paddon et
`al.
`[57] have
`successfully
`expressed
`CYP71AV1 and CPR from Artemisia annua in S. cerevisiae
`for the production of artemisinic acid, a sesquiterpene
`(Table S2). CYP71AV1 catalyzes three successive oxida-
`tions that take place from amorphadiene to artemisinic
`acid [58]. Co-expression of CYP71AV1, along with
`NADPH-dependent CPR and cytochrome b5, has
`improved artemisinic acid production to 25 g/l after
`process optimization in fed-batch cultivation [57].
`Overexpression of CPR causes electron transfer coupling
`thereby leading to toxicity and also leads to reactive
`oxygen species (ROS) formation, whereas cytochrome
`b5 prevents reactive oxygen formation and toxic inter-
`mediates accumulation. Hence, cytochrome b5 is co-
`expressed along with CPR in yeast host cells. A similar
`strategy has been adopted where CYB5 and CPR from
`Catharanthus roseus were expressed in S. cerevisiae dur-
`ing the production of strictosidine [59]
`(Table S2).
`Selecting the right CYP-CPR system either from plants
`or of microbial origin to successfully express in S. cer-
`is a challenge [60]. For oxygenation of
`evisiae
`(þ)-valencene at
`the second position to produce
`(þ)-nootkatone, Gavira et al. [60] expressed CYP71D4
`from Solanum tuberosum, CYP71D51v2 from Nicotiana
`tabacum, CYP71D1 from C.
`roseus and CYP71D326
`from Ricinus communis separately in S. cerevisiae to
`observe the efficacy of this CYP clan in a gene min-
`ing technique. They have also observed that the syn-
`thesis of
`the alcohol derivative (nootkatol
`in their
`case) causes inhibition of CYP. Fukushima et al. [61]
`used a combinatorial approach based on [62] to het-
`erologously express CPR, CYP93E2 and CYP72A61v2
`from Medicago truncatula to produce soyasapogenol
`B and CPR, CYP716A12 and CYP72A68v2 from the
`same source to produce gypsogenic acid in S. cerevi-
`siae (Table S2). They have also observed that such
`combinations of CYP-CPR produce native as well as
`rare triterpenes (that are not produced in M. trunca-
`tula) in S. cerevisiae. Thus, heterologous expression of
`P450 enzymes in S. cerevisiae might be a critical step
`
`CRITICAL REVIEWS IN BIOTECHNOLOGY
`
`981
`
`in metabolic engineering to produce non-native
`terpenes.
`
`Miscellaneous metabolic strategies
`
`Some of the distinct metabolic engineering strategies
`that have been established to improve terpene produc-
`tion in S. cerevisiae are described in this section and
`apparently does not fall
`into generic categories as
`described in the above sub-sections. The native mito-
`chondrial signal peptide of COX4 of S. cerevisiae was
`fused to A. annua amorphadiene synthase,
`farnesyl
`diphosphate synthase from Arabidopsis thaliana and
`valencene synthase (TPS) from Camellia sinensis to be
`successfully expressed in S. cerevisiae [63]. This strategy
`has substantially increased the valacene and amorpha-
`diene production (Table S2) indicating the presence of
`the viable precursor pool
`in mitochondria. The same
`strategy was also later patented [64]. Heterologous
`expression of structural genes in the sterol pathway of
`S. cerevisiae could also be a potential strategy and
`Evolva Inc. adopted this in their patent where all the
`enzymes upstream of the DMAPP node starting from
`acetoacetyl transferase were heterologously expressed
`from a diverse class of sources to improve the target
`terpene, b-carotene [65] (Table S3). This strategy has
`essentially enabled them to improve the inherent avail-
`ability of prenyl phosphate precursor pools. In another
`patent by KRIBB, South Korea [66], ispA (geranyl diphos-
`phate synthase/FDS) from E. coli was heterologously
`expressed as a substitute ERG20 to achieve a squalene
`titer of 1.24 g/l (Table S3).
`One study recently reported that mutagenesis was
`selectively carried out to produce premnaspirodiene
`(sesquiterpene), farnesol (sesquiterpene), botryococcene
`(triterpene) and squalene (triterpene)
`[67].
`In their
`study, the BY4741 haploid laboratory strain was sub-
`jected to ethyl methanesulfonate mutagenesis to select
`colonies grown under a selective pressure of nystatin
`(selection for mutants which do not produce ERG),
`squalestatin (inhibition of squalene synthase, Erg9p)
`and cholesterol (for the restoration of growth). Here,
`the surviving colonies indicate that the cells are not
`producing ERG (due to squalestatin impairment of
`Erg9p) and ERG producing colonies do not survive as
`nystatin binds to ERG on the cell membrane eventually
`leading to their death. However, for the survivability of
`yeast, ERG is replaced with cholesterol (to which nysta-
`tin cannot bind). The viable colonies were further
`screened for growth in the presence of ERG and
`ERGþ squalestatin. A final selection was based on farne-
`sol response and the colonies accumulated more than
`50 mg/l farnesol. Thus, generated mutant strains were
`
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`982
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`K. PARAMASIVAN AND S. MUTTURI
`
`used as chassis for further rational design procedures to
`produce premnaspirodiene, botryococcene and squa