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Advances in the metabolic engineering of Yarrowia lipolytica for the production of terpenoids
Bioresource Technology 281 (2019) 449–456
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Review
Advances in the metabolic engineering of Yarrowia lipolytica for the production of terpenoids
T
Yi-Rong Maa, Kai-Feng Wanga, Wei-Jian Wanga, Ying Dinga, Tian-Qiong Shia, He Huangb,c,d, ⁎ Xiao-Jun Jia,d, a
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China College of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China c State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China d Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Terpenoids Yarrowia lipolytica Metabolic engineering Mevalonate pathway
Terpenoids are a large class of natural compounds based on the C5 isoprene unit, with many biological effects such activity against cancer and allergies, while some also have an agreeable aroma. Consequently, they have received extensive attention in the food, pharmaceutical and cosmetic fields. With the identification and analysis of the underlying natural product synthesis pathways, current microbial-based metabolic engineering approaches have yielded new strategies for the production of highly valuable terpenoids. Yarrowia lipolytica is a non-conventional oleaginous yeast that is rapidly emerging as a valuable host for the production of terpenoids due to its own endogenous mevalonate pathway and high oil production capacity. This review aims to summarize the status and strategies of metabolic engineering for the heterologous synthesis of terpenoids in Y. lipolytica in recent years and proposes new methods aiming towards further improvement of terpenoid production.
1. Introduction Terpenoids are a large class of mainly plant-derived natural compounds with extensive commercial applications (Maimone and Baran, 2007). Terpenoids are generally composed of isoprene (C5) units, and can be divided based on their number into hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), sesterterpenoids (C25), triterpenoids (C30), tetraterpenoids (C40) and polyterpenoids (C > 40) (Chandran et al., 2011). Due to their wide variety and complex structure, terpenoids are of great value in the fields of pharmaceuticals, flavors, fragrances, and high-quality liquid fuel alternatives (Zhou, 2018). Examples include the anti-malarial drug artemisinin (Ro et al., 2006), the anti-cancer drug paclitaxel (Ajikumar et al., 2010), the anti-oxidant lycopene (Alper et al., 2005), and the valuable natural perfume sandalwood oil (Diaz-Chavez et al., 2013). The large-scale production of terpenoids is mainly conducted via biological extraction or chemical synthesis. However, a majority of terpenoids are only present at extremely low concentrations in their natural sources, and it is difficult to obtain pure extracts because of
many impurities. Furthermore, many rare medicinal plants grow slowly and are subject to strict geographical and climate conditions, which makes them unsuitable for large-scale industrial production. Most terpenoids are structurally complex and their synthesis encompasses the generation of a number of chiral centers, making it difficult to produce them by chemical synthesis (Chen et al., 2015; Schempp et al., 2017). In order to meet the requirements of industrialization, we urgently need to find a sustainable way to produce terpenoids. Recently, biotechnological production has emerged as an alternative source of terpenoids for industrial applications (King et al., 2016). Escherichia coli and yeast are well-characterized hosts and have been used in the production of a variety of chemicals (Kong et al., 2019; Liu et al., 2017b,c; Rodriguez et al., 2017). However, E. coli is limited because its native 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway is less efficient in supplying isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) for terpenoid synthesis and it lacks post-transcriptional modification mechanisms that is difficult to express cytochrome P450 monooxygenases (P450s) (Chang et al., 2007; Wang et al., 2018). The nonconventional oleaginous yeast Yarrowia lipolytica
⁎ Corresponding author at: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China. E-mail address: [email protected] (X.-J. Ji).
https://doi.org/10.1016/j.biortech.2019.02.116 Received 24 January 2019; Received in revised form 24 February 2019; Accepted 26 February 2019 Available online 27 February 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Comparison of different terpenoid production in engineered Saccharomyces cerevisiae and Yarrowia lipolytica. Terpenoids
Limonene Linalool α-Farnesene Lyconene Ginsenoside compound K β-Carotene Astaxanthin
S. cerevisiae
Y. lipolytica
Yield/Titer
Refs.
Yield/Titer
Refs.
0.49 mg/L 240.00 μg/L 163.00 mg/L 115.64 mg/L 1.60 mg/L 6.29 mg/g DCW 29.00 μg/g DCW
Jongedijk et al.(2014) Deng et al. (2016) Tippmann et al. (2017) Li et al. (2019b) Yan et al. (2014) Yan et al. (2012) Ukibe et al.(2009)
23.56 mg/L 6.96 mg/L 259.98 mg/L 242.00 mg/L 161.80 mg/L 49.00 mg/g DCW 3.50 mg/g DCW
Cao et al. (2016) Cao et al. (2017) Yang et al. (2016) Nambou et al. (2015) Li et al. (2019a) Gao et al. (2017b) Kildegaard et al. (2017)
3. Regulation strategies for the synthesis of terpenoids in Y. Lipolytica
is rapidly emerging as a valuable host for the production terpenoids. On one hand, Y. lipolytica is able to synthesize a massive amount of acetylCoA as initial substrate of the mevalonate (MVA) pathway, which can provide more geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) for the accumulation of a variety of terpenoids. On the other hand, large amounts of oil are accumulated in lipid droplets that can store lipophilic terpenoids (Wang et al., 2018; Zeng et al., 2018; Zhu and Jackson, 2015). More importantly, Y. lipolytica has accumulated considerable knowledge about its genome, which makes its genetic manipulation more convenient and faster (Liu et al., 2017a; Shi et al., 2018). Thus, the reconstruction and optimization of terpenoid biosynthesis pathways in Y. lipolytica that are suitable for large-scale cultivation of cell factories holds the promise of providing a sustainable biological alternative to conventional production methods (Pateraki et al., 2015) (Table 1).
3.1. Overexpression of structural genes in the mevalonate pathway Generally, the engineering strategies of terpenoid biosynthesis pathways in Y. lipolytica can be divided into two categories. The upstream pathway is the MVA pathway that diverts acetyl-CoA towards the synthesis of the basic C5 substrate IPP and its isomer DMAPP, while the downstream pathway encompasses the condensation of IPP and DMAPP to yield a variety of terpenoids by terpene synthases. Since mevalonate synthesis is irreversible, HMG-CoA reductase (HMGR) is considered to be the first rate-limiting enzyme in the mevalonate pathway and is an important regulatory point for the metabolism of terpenoids (Polakowski et al., 1998). HMGR is typically an endoplasmic reticulum (ER)-resident integral membrane protein consisting of an Nterminal membrane anchor and a C-terminal catalytic domain that extends into the cytoplasm (Burg and Espenshade, 2011). Truncating the 500 N-terminal amino acids of HMGR can prevent the self-degradation mediated by this domain, and the shortened protein is thus stabilized in the cytoplasm in Y. lipolytica. Overexpression of truncated HMGR (tHMG/tHMGR) increased β-carotene production by 134% (Gao et al., 2017b). Another strategy to enhance the MVA pathway is overexpression of IPP isomerase (IDI) to balance the amount of IPP and DMAPP. IDI acts crucially in the distribution of GPP and FPP fluxes and is employed to improve the production of monoterpenoids and sesquiterpenoids such as linalool and α-farnesene. In addition, ERG8, ERG10, ERG12 and ERG19 are considered to enhance the precursor pool of terpenoids (Cao et al., 2017).
2. The terpene biosynthesis pathway Terpenoids are derived from the universal precursors IPP and its allylic isomer DMAPP, also called isoprene (C5) units (Roberts, 2007). IPP and DMAPP are synthesized in two different pathways: the mevalonate (MVA) pathway and the MEP pathway (Chappell, 1995; Tholl, 2006; Wang et al., 2017). In general, the choice of different pathways depends on the type of organism and the subcellular localization of the target product. Plants employ the MVA and MEP pathways to respectively produce IPP and DMAPP, whereby the MVA pathway operates in the cytosol and the MEP pathway is localized to the plastids (Bouvier et al., 2005; Lange and Ahkami, 2013). Prokaryotes such as E. coli convert glyceraldehyde 3-phosphate (G3P) and pyruvate to IPP and DMAPP using the MEP pathway, whereas eukaryotes, archaea and a few bacteria use the MVA pathway (Hemmerlin et al., 2012; Lombard and Moreira, 2011). The MVA pathway entails the condensation of two molecules of the initial substrate acetyl-CoA to mevalonate and further phosphorylation reactions (Liu et al., 2018). In the detailed process, two molecules acetyl-CoA condense to acetoacetyl-CoA under the action of acetyl-CoA thiolase (EC 2.3.1.9), after which acetoacetyl-CoA is converted to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) under the catalysis of hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) followed by the removal of one molecule of coenzyme A from HMG-CoA to form mevalonate by HMG-CoA reductase (EC 1.1.1.34) with electrons provided by NADPH. Finally, IPP and DMAPP are synthesized under the action of mevalonate kinase (EC 2.7.1.36), phosphomevalonate kinase (EC 2.7.4.2), mevalonate diphosphate decarboxylase (EC 4.1.1.33) and IPP isomerase (EC 5.3.3.2). IPP and DMAPP act as terpene synthesis precursors for a wide variety of terpenoids produced by different terpene synthases (Broker et al., 2018) (Fig. 1).
3.2. Increasing the fluxes of precursors towards terpenoids Terpene synthases (TPSs) comprise a large family of mechanistically related enzymes that are involved in both primary and secondary metabolism and play a crucial role in the synthesis of terpenoids (Keeling and Bohlmann, 2006). A large number of terpene synthases come from plants, where they produce high-value-added terpenoids such as (+)-nootkatone from grapefruit oil and valencene from orange peel (Schempp et al., 2017). With genetic engineering tools, codon-optimized terpene synthases weTare integrated into the genome of Y. lipolytica to heterologously produce valuable terpenoids (Cao et al., 2016, 2017; Yang et al., 2016). In order to further obtain the final bioactive structure, cytochrome P450 superfamily are often essential to modify the terpene skeleton (Chang et al., 2007). NADPH-dependent cytochrome P450 reductase (CPR) acts as a redox partner that provides electrons to P450s. However, unlike plant cell, there is no P450s-CPR system associated with terpenoids in Y. lipolytica. The co-expression of heterologous P450s and CPR using linkers or direct connection accelerated the effective electron transfer from NADPH to P450s. For example, a fusion between CYP706M1 and opt46AtCPR1 via a GSTSSG linker increased the efficiency of the conversion of (+)-valencene to (+)-nootkatone (Guo et al., 2018). In addition, many enzymes also 450
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Fig. 1. Two different synthetic routes for terpenoids: the MVA pathway and the MEP pathway. Blue arrows, endogenous metabolic pathway in Y. lipolytica; red arrows, exogenous genes introduced into Y. lipolytica; yellow frame, glycolysis pathway; pink frame, MVA pathway; gray frame, MEP pathway. Dashed lines represent multiple steps. Metabolites: G3P, D-glyceraldehyde 3-phosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-Derythritol 4-phosphate; HMB-PP, 4-hydroxy-3-methylbut-2-enyldiphosphate. Enzymes: ERG10, acetyl-CoA thiolase; ERG13, HMG-CoA synthase; ERG1, HMG-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; ERG19/MVD1, mevalonate diphosphate decarboxylase; IDI, IPP isomerase; DXS, 1-deoxyD-xylulose 5-phosphate (DXP) synthase; DXR, DXP reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-(cytidine 5-diphospho)-2-C-methyl-D-erythritol kinase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, 4-hydroxy-3-methylbut-2enyl diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; DNPS1, neryl diphosphate synthase 1; LS, limonene synthase; LIS, linalool synthase; OptFS, codon-optimized α-farnesene synthase; CnVS, (+)-valencene synthase; CYP706M1, (+)-nootkatone synthase; AtCPR1, NADPH-cytochrome P450 reductase; LUP, lupeol synthase; GGS1 or CrtE, geranylgeranyl diphosphate synthase; CrtB, phytoene synthase; CrtI, lycopene synthase; carRP, phytoene synthase/lycopene cyclase; carB, phytoene dehydrogenase; CrtYB, phytoene synthase and lycopene cyclase; crtW, β-carotene ketolase; crtZ, β-carotene hydrolase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
451
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16.00 mg/g DCW 242.00 mg/L 21.10 mg/g DCW 2.20 mg/g DCW 0.41 mg/g DCW 4.00 g/L 6.50 g/L 54.60 mg/L H222 Po1g Po1f ATCC 201,249 ATCC MYA 2613 ATCC MYA 2613 Po1d GB20 Lycopene Lycopene Lycopene β-Carotene β-Carotene β-Carotene β-Carotene Astaxanthin Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid
ATCC 201249 Ginsenoside compound K Triterpenoid
ATCC 201249 Betulinic acid Triterpenoid
For simplicity copy number information has been removed. Genomic knockouts are represented by Δ followed by the gene name.
fermenter 1.5L fermenter 1L fermenter Flask Flask 2L bioreactor 5L bioreactor 96-well plates
Li et al. (2019a, b) 161.08 mg/L 5L fermenter
Sun et al. (2018) 26.53 mg/L Flask
Cao et al. (2016) Cao et al. (2017) Yang et al. (2016) Guo et al. (2018) 23.56 mg/L 6.96 mg/L 259.98 mg/L 978.20 μg/L Flask Flask 1.5L fermenter Flask
HMGR, ERG12, tLS ERG20F88W-N119W, HMGR, IDI tHMGR, IDI, ERG20 (GSG) OptFS tHMGR, ERG20, opCYP706M1 (GSTSSG) opt46AtCPR1 tHMGR,ERG9 PEXP1-opMtCYP716A12-t46opAtCPR tHMGR, ERG9, ERG20, PPDS (GSTSSG) ATR1, UGT1 CrtB, CrtI, GGS1, HMGR, △POX1-6, △GUT2 CrtE, CrtB, CrtI HMGR, CrtI, CrtE ,CrtB, MVD1,ERG8, △MFE1, △PAH1, △GSY1 GGS1, carB, carRP △ku70, △ku80, PGPD-carS, GGS1 △ku70, tHMGR, carRP, carB, ERG10, GGS1, ERG13ERG19, ERG12ERG8, IDIERG20 DGA2, GPD1, △POX1-6, △TGL4 CrtYB, CrtI, CrtE, PSQS1-50bp::SQS1, HMGR, crtW, crtZ Po1f Po1f Po1h ATCC 201249
Strategy
Limonene Linalool α-Farnesene (+)-Nootkatone
Monoterpenoids can be derived from GPP, NPP, LPP, or CPP, but geranyl diphosphate (GPP) and neryl diphosphate (NPP) are the precursors for the biosynthesis of most monoterpenoids (Demissie et al., 2013; Ignea et al., 2014). Cao et al. (2017) were able to heterologously synthesize linalool by integrating a codon-optimized linalool synthase (LIS) gene from Actinidia arguta into the genome of Y. lipolytica, resulting in (S)-(+)-linalool production 0.09 mg/L, which was significantly higher than the S. cerevisiae strain harboring the same LIS gene (0.06 mg/L). To further improve linalool production, they cooverexpressed HMGR and the IDI, and linalool production of 1.44 mg/L was achieved, approximately 16-fold higher than that of the control strain. After optimizing the medium, linalool production was increased to 6.96 mg/L. Furthermore, Cao et al. (2016) also introduced codonoptimized neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) into Y. lipolytica and optimized the MVA pathway by overexpressing HMGR and ERG12, which, along with the addition of 4 g/L
Monoterpenoid Monoterpenoid Sesquiterpenoid Sesquiterpenoid
4.1. Monoterpenoids
Parental strain
Since Y. lipolytica is as a naturally oleaginous yeast, it only requires relatively simple metabolic engineering such as heterologous introduction of terpene synthase and optimizing the MVA pathway, to transform acetoacetyl-CoA into valuable terpenoids. In the past years, researchers have achieved heterologous synthesis of monoterpenes, sesquiterpenes, triterpenes, tetraterpenes and derivatives. These studies are of great significance for guiding the synthesis of further terpenoids in Y. lipolytica (Table 2).
Product
4. Metabolic engineering of Y. Lipolytica for terpenoid production
Classification
Table 2 Summary of recently reported genetic strategies for the synthesis of terpenoids in Yarrowia lipolytica.
Scale
Titer/Yield
Refs.
accept a wide range of natural and non-natural substrates, and occasionally exhibit promiscuous catalytic functions (Khersonsky et al., 2006). For example, the enzyme ERG20 is both a geranyl diphosphate synthase (EC 2.5.1.1) and a farnesyl diphosphate synthase (EC 2.5.1.10), which makes it challenging to separate its functions during the synthesis of monoterpenoids. To improve the specificity of ERG20, Wu et al. (2018) used two point-mutations (F96W and N127W) that were introduced into ERG20 by overlap-extension PCR. Overexpressing the resulting ERG20 mutant diverted the carbon flux from FPP to GPP in Saccharomyces cerevisiae. To ensure adequate availability of GPP, a similar strategy was also employed to modify ERG20 in Y. lipolytica. Based on amino acid alignment analysis of Y. lipolytica ERG20 and S. cerevisiae ERG20, the residues F88 and N119 of Y. lipolytica ERG20 were identified as targets, and the two F88W and N119W were introduced. When the mutant ERG20F88W-N119W was expressed in Y. lipolytica to produce linalool, the resulting strain could yield 5.34 mg/L linalool, which was 3.7-fold higher than in the parent strain (Cao et al., 2017). FPP is a common precursor of various metabolites of Y. lipolytica, including ubiquinone, dolichol, squalene, cholesterol and other essential sterols (Kuranda et al., 2010). Squalene synthase SQS1 (EC 2.5.1.21) is a membrane-bound enzyme that catalyzes the condensation of two molecules of FPP into squalene and is considered as a potential candidate offering a competitive route for the synthesis of terpenoids. Since ergosterol is an essential component of the cell membrane of Y. lipolytica, completely inhibiting squalene synthase causes a strong growth defect (Tanaka and Tani, 2018). Downregulation of the SQS1 is the potential means of redirecting FPP away from sterol production towards the synthesis of interesting terpenoids after sufficient biomass has been accumulated. At present, there are two main ways to decrease the transcription of the native SQS1 – truncating the native promoter of SQS1 and replacing the native SQS1 promoter in the genome with a weak promoter or inducible promoter, such as PERG1 (squalene epoxidase promoter), PERG11 (lanosterol 14-α-demethylase promoter) or PALK1 (repressed by glucose and glycerol). This downregulation strategy has been applied to the production of astaxanthin and β-carotene (Gao et al., 2017b; Kildegaard et al., 2017).
Matthaus et al. (2014) Nambou et al. (2015) Schwartz et al. (2017) Gao et al. (2014) Gao et al. (2017a) Gao et al. (2017b) Larroude et al. (2018) Kildegaard et al. (2017)
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appeared orange in color, showing that lycopene can be synthesized in Y. lipolytica. The recombinant strain produced 142 µg of lycopene per g DCW after 96 h in yeast extract peptone dextrose (YPD) shake flask culture. The additional overexpression of GGS1 and HMGR led to a 10.8-fold increase of lycopene content to 1540 µg/g DCW. In order to improve the storage capacity of hydrophobic lycopene, researchers deleted POX1 to POX6 and GUT2 leading to increased lipid body content. The lycopene content eventually reached 16 mg/g DCW by controlled fed-batch fermentation. Schwartz et al. (2017) used linear DNA fragments encoding HMGR from the MVA pathway and CrtE/B/I from lycopene pathway to transform Y. lipolytica Po1f, and the resulting strain produced a lycopene titer of 23 mg/L when grown in shake flasks for 4 days in YPD medium. By alleviating the auxotrophies for leucine and uracil, the lycopene titer was increased 1.9-fold to 44 mg/L. They further overexpressed four enzymes in the pathway from acetyl-CoA to lycopene: HMGR, MVD1, ERG8, and CrtI, and the lycopene titer reached 213 mg/ L after continuous growth for 12 days in shake flasks and again in a 10day fed batch bioreactor. Similarly, Nambou et al. (2015) combined flux balance analysis (FBA) and Plackett-Burman design, to improve the lycopene concentration to 242 mg/L. Zhao et al. (2017) compared the metabolome data of Po1g-pINA1269-CrtEBI and Po1g-pINA1269, interpreted the effects of the Crt genes in a metabolic view and revealed remarkable effects on fructose and mannose metabolism, citrate cycle, glyoxylate and dicarboxylate metabolism. β-Carotene is a natural pigment that is used as a coloring additive or nutritional supplement for humans and domestic animals. It can be viewed as a downstream product of lycopene via cyclization by lycopene cyclase (carRP). Gao et al. (2014) reported a new multiple fragment assembly method they used to successfully construct the entire βcarotene pathway by overlap-extension PCR (OE-PCR), which they integrated into the rDNA locus of Y. lipolytica. They simultaneously expressed carB and carRP from Mucor circinelloides and GGS1, which led to a β-carotene production of 2.22 mg/g DCW. The product of the carRP genes has phytoene synthesis activity and lycopene cyclase activity in fungi and can catalyze multiple reactions. Gao et al. (2017a) used the BLASTP tool to find the multifunctional carotene synthase carS from Schizochytrium sp. They transformed Y. lipolytica with a carS expression cassette encoding three enzymatic functions under the control of the PGPD promoter, which resulted in a β-carotene production of 0.41 mg/g DCW. To further enhance β-carotene production, Gao et al. (2017b) expressed tHMGR and carRP to enhance the HMG-CoA and GGPP pools, which resulted in a 14-fold increase of β-carotene production. Production was further improved approximately 100-fold by optimizing the tHMGR and carRP gene dosage and thereby enhancing the activity of the MVA pathway. The recombinant strain was subjected to highcell-density fermentation in a nitrogen-limited medium, and the βcarotene content reached 4 g/L, most of which was stored in lipid droplets. In another study, Larroude et al. (2018) transformed a lipid overproducer strain with a tHMGR and car expression-cassette obtaining the three genes GGS1, carB and carRP, with the aim to maximize β-carotene production. The resulting strain produced 35.7 mg/L β-carotene. Subsequently, they used Golden Gate assembly to construct a new car-cassette as a second copy under the control of the TEF1 promoter, then transformed the previous recombinant strain. The second strain was able to produce 111.8 mg/L β-carotene. Finally, a fed-batch fermentation lead to a β-carotene production of 6.5 g/L after 122 h. Astaxanthin is a red-colored carotenoid that is often added to foods and cosmetics due to its powerful antioxidant activity (Guerin et al., 2003). Kildegaard et al. (2017) successfully produced astaxanthin in Y. lipolytica by genetic engineering. They screened a robust strain producing β-carotene from 14 strains with integrated β-carotene biosynthesis genes. As two important genes for β-carotene synthesis, HMGR and GGS1 were introduced into Y. lipolytica. Additional downregulation of the competing squalene synthase SQS1 by truncating the native
pyruvic acid and 8% dodecane, that led to an initial limonene production of 23.56 mg/L. 4.2. Sesquiterpenoids Sesquiterpenoids are a large group of terpenoids with FPP as direct precursor, which in turn is synthesized by two sequential condensation reaction of IPP with DMAPP into GPP, followed by its condensation with another IPP molecule into FPP. Researchers have attempted to increase the yield of sesquiterpenoids by increasing the supply of FPP. In order to heterologously produce α-farnesene in Y. lipolytica, Yang et al. (2016) constructed a vector with a codon-optimized α-farnesene synthase gene expression cassette and integrated it into the genome of Y. lipolytica Po1h. The resulting engineered Y. lipolytica successfully synthesized α-farnesene at a low initial level. They further overexpressed tHMGR, IDI and ERG20 aiming to increase the FPP content and produced 57.08 mg/L of α-farnesene in shake flasks, corresponding to a 20.8-fold increase over the initial production of 2.75 mg/L. Recently, Guo et al. (2018) succeeded in the heterologous production of (+)-nootkatone in Y. lipolytica by co-expressing (+)-valencene synthase (CnVS), a codon-optimized version of the (+)-nootkatone synthase opCYP706M1 from Callitropsis nootkatensis and a codon-optimized version of the NADPH-dependent cytochrome P450 reductase opAtCPR1 from Arabidopsis thaliana. They fused opCYP706M1 and opt46AtCPR1 (truncated N-terminal 46 amino acids) and overexpressed tHMGR and ERG20, improving the (+)-nootkatone production. The final engineered strain achieved a (+)-nootkatone titer of 978.2 µg/L, which was a 20.5-fold increase compared to the simple co-expression of CnVS, opCYP706M1 and opAtCPR1. 4.3. Triterpenoids Over 23,000 triterpenoid structures have been found in nature, which makes them the largest group of compounds in the terpenoid family (Pateraki et al., 2015). Generally, the triterpene intermediate squalene is synthesized by coupling two molecules of FPP head to head in a reaction catalyzed by squalene synthase, followed by oxidation into 2,3-squalene epoxide by squalene epoxidase at the expense of NADPH. The resulting 2,3-squalene epoxide is cyclized by various cyclases to form mono- to pentacyclic backbone structures (Dong et al., 2018; Zhang et al., 2017). Sun et al. (2018) integrated the lupeol synthase AtLUP1 and cytochrome P450 reductase AtCPR1 from Arabidopsis thaliana, cytochrome P450 monooxygenase MtCYP716A12 from Medicago truncatula into Y. lipolytica genome after codon optimization. The resulting strain successfully synthesized the pentacyclic triterpene betulinic acid with a titer of 0.32 mg/L. This titer was increased to 9.41 mg/L following the fusion of cytochrome P450 enzymes and overexpression of key genes from the upstream MVA pathway. 4.4. Tetraterpenoids Carotenoids are typical tetraterpenoids. They are derived from phytoene, which is formed from two molecules of GGPP. Carotenoids are a general term for an important class of natural products that are ubiquitous as yellow, orange-red or red pigments of higher plants, fungi and algae (Mata-Gómez et al., 2014). Furthermore, carotenoids are the main source of vitamin A in the body, and also have properties related to antioxidation, immune regulation, cancer prevention, anti-aging, and so on (Milani et al., 2017; Ribeiro et al., 2018). The most studied major carotenoids are lycopene, β-carotene, astaxanthin and lutein. Lycopene is an important intermediate in the synthetic pathway of carotenoids and is produced by the consecutive action of three enzymes: geranylgeranyl diphosphate synthase (GGS1/CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI). Matthaus et al. (2014) expressed codon-optimized CrtB and CrtI genes from Pantoea ananatis under the control of the TEF1 promoter in Y. lipolytica. The transformants 453
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promoter of SQS1 to 50 bp resulted in a β-carotene titer of 797.1 mg/L. They next expressed β-carotene ketolase (crtW) from the marine bacterium Paracoccus sp. N81106 and β-carotene hydroxylase (crtZ) from the enterobacterium P. ananatis to divert the flux from β-carotene to astaxanthin. To further improve astaxanthin production, they optimized the copy numbers of crtZ and crtW to obtain 54.6 mg/L of astaxanthin in a microtiter plate cultivation.
precursor (+)-epi-α-bisabolol from FPP, the P450s enzyme that catalyzes the next reaction to form hernandulcin remains unknown (Attia et al., 2012). It is therefore necessary to more broadly investigate the functions of cytochrome P450 enzymes and provide a basis for research on the synthesis of more valuable terpenoids, especially triterpenoids.
5. The search for new strategies and their application
Y. lipolytica can assimilate different carbon sources, including hydrophilic materials (glucose, glycerol, alcohols and acetate) and hydrophobic substrates (fatty acids, triacylglycerols and alkanes) (Liu et al., 2015; Sun et al., 2017). In order to realize the industrial production of terpenoids using Y. lipolytica, reducing the cost of fermentation is a problem that must be considered. Glycerol is the main byproduct of the biodiesel production chain and has been used extensively as a substrate in the production of single cell oil and organic acids (André et al., 2009). Replacing glucose with inexpensive carbon sources can reduce the cost of materials for the production of terpenoids. The titer of betulinic acid in Y. lipolytica using glycerol as the sole carbon source and using glucose as carbon source in S. cerevisiae in shake flask cultivation were almost the same (Sun et al., 2018). This confirms that cheap carbon sources are also available for the synthesis of terpenoids in Y. lipolytica. In addition, extensive use of carbon sources has been achieved through metabolic engineering. Y. lipolytica has achieved the use of xylose as the sole carbon source, produced over 15 g/L of lipid by introducing a heterologous oxidoreductase pathway (Li and Alper, 2016). Y. lipolytica can utilize various carbon sources, which contributes to the development of carbon sources for economical production of terpenoids.
5.3. Optimization of culture media
Y. lipolytica is a promising biorefinery platform strain for the production of bulk and value-added terpenoids owing to its endogenous MVA pathway and native high-level oil production capacity. It is currently possible to quickly and easily achieve the heterologous synthesis of plant-derived terpenoids in Y. lipolytica by synthetic pathway rewiring and genome editing. In order to further explore ways to improve terpenoid production, more genetic modification strategies can be adopted from the eukaryotic model strain S. cerevisiae. 5.1. Modification of enzymes Protein degradation is an efficient mechanism to control carbon allocation to competing pathways and avoid an insufficiency of target precursors. Peng et al. (2017) added a PEST sequence responsible for ER-associated protein degradation to the C-terminus of squalene synthase to shorten its half-life in S. cerevisiae, and the nerolidol titer improved by 86% to about 100 mg/L. Recently, Peng et al. (2018) explored the effect of degradation of ERG20 on the carbon flux balance between monoterpene synthesis and cell growth by fusing an N-degron to the N-terminus of ERG20. To avoid a severe lack of sterols causing cell lethal, they replaced the promoter of ERG20 with a sterol-responsive promoter. This regulatory strategy increased monoterpene production in S. cerevisiae with a heterologous monoterpene synthesis pathway. In addition, affibody scaffolds can be used to co-localize multiple enzymes via noncovalent interactions to reduce substrate loss and accumulation of toxic intermediates. Compared to enzymes fusions, affibody scaffolds can generate a close spatial concentration of multiple enzymes and thereby guarantee efficient substrate channeling. More importantly, formed subcellular compartments avoid the negative impact of the intracellular environment on synthetic target products. Affibody scaffolds based on anti-idiotypic partners have been employed for the in vivo co-localization of farnesyl diphosphate synthase and farnesene synthase in S. cerevisiae (Tippmann et al., 2017). Han et al. (2018) described the use of Tya protein as a scaffold in combination with three key enzymes (including tHMGR, ispA, AFS1or DPP1) involved in sesquiterpene biosynthesis, which improved farnesene and farnesol production in S. cerevisiae. The application of these new strategies has undoubtedly provided new references and guidance for the synthesis of terpenoids in Y. lipolytica, which developed a convenient means for us to further use this platform to achieve the synthesis of more terpenoids.
6. Conclusions The metabolic engineering studies of the production of terpenoids reviewed here demonstrate that Y. lipolytica is a potential platform strain for the synthesis of plant-derived terpenoids. In the future, we should further develop more genetic tools that are able to achieve more efficient and rapid genetic operations for Y. lipolytica, such as CRISPR/ Cas9 genome-editing technology and protein engineering. With the identification of specific terpene synthases and the application of emerging tools, researchers have found a way to achieve heterologous synthesis of terpenoids in Y. lipolytica. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21776131), the Six Talent Peaks Project in Jiangsu Province of China (No. 2018-SWYY-047), and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (No. XTD1814). References
5.2. Decoration of terpenoids
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Review
Advances in the metabolic engineering of Yarrowia lipolytica for the production of terpenoids
T
Yi-Rong Maa, Kai-Feng Wanga, Wei-Jian Wanga, Ying Dinga, Tian-Qiong Shia, He Huangb,c,d, ⁎ Xiao-Jun Jia,d, a
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China College of Pharmaceutical Sciences, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China c State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China d Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Terpenoids Yarrowia lipolytica Metabolic engineering Mevalonate pathway
Terpenoids are a large class of natural compounds based on the C5 isoprene unit, with many biological effects such activity against cancer and allergies, while some also have an agreeable aroma. Consequently, they have received extensive attention in the food, pharmaceutical and cosmetic fields. With the identification and analysis of the underlying natural product synthesis pathways, current microbial-based metabolic engineering approaches have yielded new strategies for the production of highly valuable terpenoids. Yarrowia lipolytica is a non-conventional oleaginous yeast that is rapidly emerging as a valuable host for the production of terpenoids due to its own endogenous mevalonate pathway and high oil production capacity. This review aims to summarize the status and strategies of metabolic engineering for the heterologous synthesis of terpenoids in Y. lipolytica in recent years and proposes new methods aiming towards further improvement of terpenoid production.
1. Introduction Terpenoids are a large class of mainly plant-derived natural compounds with extensive commercial applications (Maimone and Baran, 2007). Terpenoids are generally composed of isoprene (C5) units, and can be divided based on their number into hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), sesterterpenoids (C25), triterpenoids (C30), tetraterpenoids (C40) and polyterpenoids (C > 40) (Chandran et al., 2011). Due to their wide variety and complex structure, terpenoids are of great value in the fields of pharmaceuticals, flavors, fragrances, and high-quality liquid fuel alternatives (Zhou, 2018). Examples include the anti-malarial drug artemisinin (Ro et al., 2006), the anti-cancer drug paclitaxel (Ajikumar et al., 2010), the anti-oxidant lycopene (Alper et al., 2005), and the valuable natural perfume sandalwood oil (Diaz-Chavez et al., 2013). The large-scale production of terpenoids is mainly conducted via biological extraction or chemical synthesis. However, a majority of terpenoids are only present at extremely low concentrations in their natural sources, and it is difficult to obtain pure extracts because of
many impurities. Furthermore, many rare medicinal plants grow slowly and are subject to strict geographical and climate conditions, which makes them unsuitable for large-scale industrial production. Most terpenoids are structurally complex and their synthesis encompasses the generation of a number of chiral centers, making it difficult to produce them by chemical synthesis (Chen et al., 2015; Schempp et al., 2017). In order to meet the requirements of industrialization, we urgently need to find a sustainable way to produce terpenoids. Recently, biotechnological production has emerged as an alternative source of terpenoids for industrial applications (King et al., 2016). Escherichia coli and yeast are well-characterized hosts and have been used in the production of a variety of chemicals (Kong et al., 2019; Liu et al., 2017b,c; Rodriguez et al., 2017). However, E. coli is limited because its native 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway is less efficient in supplying isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) for terpenoid synthesis and it lacks post-transcriptional modification mechanisms that is difficult to express cytochrome P450 monooxygenases (P450s) (Chang et al., 2007; Wang et al., 2018). The nonconventional oleaginous yeast Yarrowia lipolytica
⁎ Corresponding author at: State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China. E-mail address: [email protected] (X.-J. Ji).
https://doi.org/10.1016/j.biortech.2019.02.116 Received 24 January 2019; Received in revised form 24 February 2019; Accepted 26 February 2019 Available online 27 February 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Comparison of different terpenoid production in engineered Saccharomyces cerevisiae and Yarrowia lipolytica. Terpenoids
Limonene Linalool α-Farnesene Lyconene Ginsenoside compound K β-Carotene Astaxanthin
S. cerevisiae
Y. lipolytica
Yield/Titer
Refs.
Yield/Titer
Refs.
0.49 mg/L 240.00 μg/L 163.00 mg/L 115.64 mg/L 1.60 mg/L 6.29 mg/g DCW 29.00 μg/g DCW
Jongedijk et al.(2014) Deng et al. (2016) Tippmann et al. (2017) Li et al. (2019b) Yan et al. (2014) Yan et al. (2012) Ukibe et al.(2009)
23.56 mg/L 6.96 mg/L 259.98 mg/L 242.00 mg/L 161.80 mg/L 49.00 mg/g DCW 3.50 mg/g DCW
Cao et al. (2016) Cao et al. (2017) Yang et al. (2016) Nambou et al. (2015) Li et al. (2019a) Gao et al. (2017b) Kildegaard et al. (2017)
3. Regulation strategies for the synthesis of terpenoids in Y. Lipolytica
is rapidly emerging as a valuable host for the production terpenoids. On one hand, Y. lipolytica is able to synthesize a massive amount of acetylCoA as initial substrate of the mevalonate (MVA) pathway, which can provide more geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) for the accumulation of a variety of terpenoids. On the other hand, large amounts of oil are accumulated in lipid droplets that can store lipophilic terpenoids (Wang et al., 2018; Zeng et al., 2018; Zhu and Jackson, 2015). More importantly, Y. lipolytica has accumulated considerable knowledge about its genome, which makes its genetic manipulation more convenient and faster (Liu et al., 2017a; Shi et al., 2018). Thus, the reconstruction and optimization of terpenoid biosynthesis pathways in Y. lipolytica that are suitable for large-scale cultivation of cell factories holds the promise of providing a sustainable biological alternative to conventional production methods (Pateraki et al., 2015) (Table 1).
3.1. Overexpression of structural genes in the mevalonate pathway Generally, the engineering strategies of terpenoid biosynthesis pathways in Y. lipolytica can be divided into two categories. The upstream pathway is the MVA pathway that diverts acetyl-CoA towards the synthesis of the basic C5 substrate IPP and its isomer DMAPP, while the downstream pathway encompasses the condensation of IPP and DMAPP to yield a variety of terpenoids by terpene synthases. Since mevalonate synthesis is irreversible, HMG-CoA reductase (HMGR) is considered to be the first rate-limiting enzyme in the mevalonate pathway and is an important regulatory point for the metabolism of terpenoids (Polakowski et al., 1998). HMGR is typically an endoplasmic reticulum (ER)-resident integral membrane protein consisting of an Nterminal membrane anchor and a C-terminal catalytic domain that extends into the cytoplasm (Burg and Espenshade, 2011). Truncating the 500 N-terminal amino acids of HMGR can prevent the self-degradation mediated by this domain, and the shortened protein is thus stabilized in the cytoplasm in Y. lipolytica. Overexpression of truncated HMGR (tHMG/tHMGR) increased β-carotene production by 134% (Gao et al., 2017b). Another strategy to enhance the MVA pathway is overexpression of IPP isomerase (IDI) to balance the amount of IPP and DMAPP. IDI acts crucially in the distribution of GPP and FPP fluxes and is employed to improve the production of monoterpenoids and sesquiterpenoids such as linalool and α-farnesene. In addition, ERG8, ERG10, ERG12 and ERG19 are considered to enhance the precursor pool of terpenoids (Cao et al., 2017).
2. The terpene biosynthesis pathway Terpenoids are derived from the universal precursors IPP and its allylic isomer DMAPP, also called isoprene (C5) units (Roberts, 2007). IPP and DMAPP are synthesized in two different pathways: the mevalonate (MVA) pathway and the MEP pathway (Chappell, 1995; Tholl, 2006; Wang et al., 2017). In general, the choice of different pathways depends on the type of organism and the subcellular localization of the target product. Plants employ the MVA and MEP pathways to respectively produce IPP and DMAPP, whereby the MVA pathway operates in the cytosol and the MEP pathway is localized to the plastids (Bouvier et al., 2005; Lange and Ahkami, 2013). Prokaryotes such as E. coli convert glyceraldehyde 3-phosphate (G3P) and pyruvate to IPP and DMAPP using the MEP pathway, whereas eukaryotes, archaea and a few bacteria use the MVA pathway (Hemmerlin et al., 2012; Lombard and Moreira, 2011). The MVA pathway entails the condensation of two molecules of the initial substrate acetyl-CoA to mevalonate and further phosphorylation reactions (Liu et al., 2018). In the detailed process, two molecules acetyl-CoA condense to acetoacetyl-CoA under the action of acetyl-CoA thiolase (EC 2.3.1.9), after which acetoacetyl-CoA is converted to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) under the catalysis of hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) followed by the removal of one molecule of coenzyme A from HMG-CoA to form mevalonate by HMG-CoA reductase (EC 1.1.1.34) with electrons provided by NADPH. Finally, IPP and DMAPP are synthesized under the action of mevalonate kinase (EC 2.7.1.36), phosphomevalonate kinase (EC 2.7.4.2), mevalonate diphosphate decarboxylase (EC 4.1.1.33) and IPP isomerase (EC 5.3.3.2). IPP and DMAPP act as terpene synthesis precursors for a wide variety of terpenoids produced by different terpene synthases (Broker et al., 2018) (Fig. 1).
3.2. Increasing the fluxes of precursors towards terpenoids Terpene synthases (TPSs) comprise a large family of mechanistically related enzymes that are involved in both primary and secondary metabolism and play a crucial role in the synthesis of terpenoids (Keeling and Bohlmann, 2006). A large number of terpene synthases come from plants, where they produce high-value-added terpenoids such as (+)-nootkatone from grapefruit oil and valencene from orange peel (Schempp et al., 2017). With genetic engineering tools, codon-optimized terpene synthases weTare integrated into the genome of Y. lipolytica to heterologously produce valuable terpenoids (Cao et al., 2016, 2017; Yang et al., 2016). In order to further obtain the final bioactive structure, cytochrome P450 superfamily are often essential to modify the terpene skeleton (Chang et al., 2007). NADPH-dependent cytochrome P450 reductase (CPR) acts as a redox partner that provides electrons to P450s. However, unlike plant cell, there is no P450s-CPR system associated with terpenoids in Y. lipolytica. The co-expression of heterologous P450s and CPR using linkers or direct connection accelerated the effective electron transfer from NADPH to P450s. For example, a fusion between CYP706M1 and opt46AtCPR1 via a GSTSSG linker increased the efficiency of the conversion of (+)-valencene to (+)-nootkatone (Guo et al., 2018). In addition, many enzymes also 450
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Fig. 1. Two different synthetic routes for terpenoids: the MVA pathway and the MEP pathway. Blue arrows, endogenous metabolic pathway in Y. lipolytica; red arrows, exogenous genes introduced into Y. lipolytica; yellow frame, glycolysis pathway; pink frame, MVA pathway; gray frame, MEP pathway. Dashed lines represent multiple steps. Metabolites: G3P, D-glyceraldehyde 3-phosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-Derythritol 4-phosphate; HMB-PP, 4-hydroxy-3-methylbut-2-enyldiphosphate. Enzymes: ERG10, acetyl-CoA thiolase; ERG13, HMG-CoA synthase; ERG1, HMG-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; ERG19/MVD1, mevalonate diphosphate decarboxylase; IDI, IPP isomerase; DXS, 1-deoxyD-xylulose 5-phosphate (DXP) synthase; DXR, DXP reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-(cytidine 5-diphospho)-2-C-methyl-D-erythritol kinase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, 4-hydroxy-3-methylbut-2enyl diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; DNPS1, neryl diphosphate synthase 1; LS, limonene synthase; LIS, linalool synthase; OptFS, codon-optimized α-farnesene synthase; CnVS, (+)-valencene synthase; CYP706M1, (+)-nootkatone synthase; AtCPR1, NADPH-cytochrome P450 reductase; LUP, lupeol synthase; GGS1 or CrtE, geranylgeranyl diphosphate synthase; CrtB, phytoene synthase; CrtI, lycopene synthase; carRP, phytoene synthase/lycopene cyclase; carB, phytoene dehydrogenase; CrtYB, phytoene synthase and lycopene cyclase; crtW, β-carotene ketolase; crtZ, β-carotene hydrolase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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16.00 mg/g DCW 242.00 mg/L 21.10 mg/g DCW 2.20 mg/g DCW 0.41 mg/g DCW 4.00 g/L 6.50 g/L 54.60 mg/L H222 Po1g Po1f ATCC 201,249 ATCC MYA 2613 ATCC MYA 2613 Po1d GB20 Lycopene Lycopene Lycopene β-Carotene β-Carotene β-Carotene β-Carotene Astaxanthin Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid Tetraterpenoid
ATCC 201249 Ginsenoside compound K Triterpenoid
ATCC 201249 Betulinic acid Triterpenoid
For simplicity copy number information has been removed. Genomic knockouts are represented by Δ followed by the gene name.
fermenter 1.5L fermenter 1L fermenter Flask Flask 2L bioreactor 5L bioreactor 96-well plates
Li et al. (2019a, b) 161.08 mg/L 5L fermenter
Sun et al. (2018) 26.53 mg/L Flask
Cao et al. (2016) Cao et al. (2017) Yang et al. (2016) Guo et al. (2018) 23.56 mg/L 6.96 mg/L 259.98 mg/L 978.20 μg/L Flask Flask 1.5L fermenter Flask
HMGR, ERG12, tLS ERG20F88W-N119W, HMGR, IDI tHMGR, IDI, ERG20 (GSG) OptFS tHMGR, ERG20, opCYP706M1 (GSTSSG) opt46AtCPR1 tHMGR,ERG9 PEXP1-opMtCYP716A12-t46opAtCPR tHMGR, ERG9, ERG20, PPDS (GSTSSG) ATR1, UGT1 CrtB, CrtI, GGS1, HMGR, △POX1-6, △GUT2 CrtE, CrtB, CrtI HMGR, CrtI, CrtE ,CrtB, MVD1,ERG8, △MFE1, △PAH1, △GSY1 GGS1, carB, carRP △ku70, △ku80, PGPD-carS, GGS1 △ku70, tHMGR, carRP, carB, ERG10, GGS1, ERG13ERG19, ERG12ERG8, IDIERG20 DGA2, GPD1, △POX1-6, △TGL4 CrtYB, CrtI, CrtE, PSQS1-50bp::SQS1, HMGR, crtW, crtZ Po1f Po1f Po1h ATCC 201249
Strategy
Limonene Linalool α-Farnesene (+)-Nootkatone
Monoterpenoids can be derived from GPP, NPP, LPP, or CPP, but geranyl diphosphate (GPP) and neryl diphosphate (NPP) are the precursors for the biosynthesis of most monoterpenoids (Demissie et al., 2013; Ignea et al., 2014). Cao et al. (2017) were able to heterologously synthesize linalool by integrating a codon-optimized linalool synthase (LIS) gene from Actinidia arguta into the genome of Y. lipolytica, resulting in (S)-(+)-linalool production 0.09 mg/L, which was significantly higher than the S. cerevisiae strain harboring the same LIS gene (0.06 mg/L). To further improve linalool production, they cooverexpressed HMGR and the IDI, and linalool production of 1.44 mg/L was achieved, approximately 16-fold higher than that of the control strain. After optimizing the medium, linalool production was increased to 6.96 mg/L. Furthermore, Cao et al. (2016) also introduced codonoptimized neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) into Y. lipolytica and optimized the MVA pathway by overexpressing HMGR and ERG12, which, along with the addition of 4 g/L
Monoterpenoid Monoterpenoid Sesquiterpenoid Sesquiterpenoid
4.1. Monoterpenoids
Parental strain
Since Y. lipolytica is as a naturally oleaginous yeast, it only requires relatively simple metabolic engineering such as heterologous introduction of terpene synthase and optimizing the MVA pathway, to transform acetoacetyl-CoA into valuable terpenoids. In the past years, researchers have achieved heterologous synthesis of monoterpenes, sesquiterpenes, triterpenes, tetraterpenes and derivatives. These studies are of great significance for guiding the synthesis of further terpenoids in Y. lipolytica (Table 2).
Product
4. Metabolic engineering of Y. Lipolytica for terpenoid production
Classification
Table 2 Summary of recently reported genetic strategies for the synthesis of terpenoids in Yarrowia lipolytica.
Scale
Titer/Yield
Refs.
accept a wide range of natural and non-natural substrates, and occasionally exhibit promiscuous catalytic functions (Khersonsky et al., 2006). For example, the enzyme ERG20 is both a geranyl diphosphate synthase (EC 2.5.1.1) and a farnesyl diphosphate synthase (EC 2.5.1.10), which makes it challenging to separate its functions during the synthesis of monoterpenoids. To improve the specificity of ERG20, Wu et al. (2018) used two point-mutations (F96W and N127W) that were introduced into ERG20 by overlap-extension PCR. Overexpressing the resulting ERG20 mutant diverted the carbon flux from FPP to GPP in Saccharomyces cerevisiae. To ensure adequate availability of GPP, a similar strategy was also employed to modify ERG20 in Y. lipolytica. Based on amino acid alignment analysis of Y. lipolytica ERG20 and S. cerevisiae ERG20, the residues F88 and N119 of Y. lipolytica ERG20 were identified as targets, and the two F88W and N119W were introduced. When the mutant ERG20F88W-N119W was expressed in Y. lipolytica to produce linalool, the resulting strain could yield 5.34 mg/L linalool, which was 3.7-fold higher than in the parent strain (Cao et al., 2017). FPP is a common precursor of various metabolites of Y. lipolytica, including ubiquinone, dolichol, squalene, cholesterol and other essential sterols (Kuranda et al., 2010). Squalene synthase SQS1 (EC 2.5.1.21) is a membrane-bound enzyme that catalyzes the condensation of two molecules of FPP into squalene and is considered as a potential candidate offering a competitive route for the synthesis of terpenoids. Since ergosterol is an essential component of the cell membrane of Y. lipolytica, completely inhibiting squalene synthase causes a strong growth defect (Tanaka and Tani, 2018). Downregulation of the SQS1 is the potential means of redirecting FPP away from sterol production towards the synthesis of interesting terpenoids after sufficient biomass has been accumulated. At present, there are two main ways to decrease the transcription of the native SQS1 – truncating the native promoter of SQS1 and replacing the native SQS1 promoter in the genome with a weak promoter or inducible promoter, such as PERG1 (squalene epoxidase promoter), PERG11 (lanosterol 14-α-demethylase promoter) or PALK1 (repressed by glucose and glycerol). This downregulation strategy has been applied to the production of astaxanthin and β-carotene (Gao et al., 2017b; Kildegaard et al., 2017).
Matthaus et al. (2014) Nambou et al. (2015) Schwartz et al. (2017) Gao et al. (2014) Gao et al. (2017a) Gao et al. (2017b) Larroude et al. (2018) Kildegaard et al. (2017)
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appeared orange in color, showing that lycopene can be synthesized in Y. lipolytica. The recombinant strain produced 142 µg of lycopene per g DCW after 96 h in yeast extract peptone dextrose (YPD) shake flask culture. The additional overexpression of GGS1 and HMGR led to a 10.8-fold increase of lycopene content to 1540 µg/g DCW. In order to improve the storage capacity of hydrophobic lycopene, researchers deleted POX1 to POX6 and GUT2 leading to increased lipid body content. The lycopene content eventually reached 16 mg/g DCW by controlled fed-batch fermentation. Schwartz et al. (2017) used linear DNA fragments encoding HMGR from the MVA pathway and CrtE/B/I from lycopene pathway to transform Y. lipolytica Po1f, and the resulting strain produced a lycopene titer of 23 mg/L when grown in shake flasks for 4 days in YPD medium. By alleviating the auxotrophies for leucine and uracil, the lycopene titer was increased 1.9-fold to 44 mg/L. They further overexpressed four enzymes in the pathway from acetyl-CoA to lycopene: HMGR, MVD1, ERG8, and CrtI, and the lycopene titer reached 213 mg/ L after continuous growth for 12 days in shake flasks and again in a 10day fed batch bioreactor. Similarly, Nambou et al. (2015) combined flux balance analysis (FBA) and Plackett-Burman design, to improve the lycopene concentration to 242 mg/L. Zhao et al. (2017) compared the metabolome data of Po1g-pINA1269-CrtEBI and Po1g-pINA1269, interpreted the effects of the Crt genes in a metabolic view and revealed remarkable effects on fructose and mannose metabolism, citrate cycle, glyoxylate and dicarboxylate metabolism. β-Carotene is a natural pigment that is used as a coloring additive or nutritional supplement for humans and domestic animals. It can be viewed as a downstream product of lycopene via cyclization by lycopene cyclase (carRP). Gao et al. (2014) reported a new multiple fragment assembly method they used to successfully construct the entire βcarotene pathway by overlap-extension PCR (OE-PCR), which they integrated into the rDNA locus of Y. lipolytica. They simultaneously expressed carB and carRP from Mucor circinelloides and GGS1, which led to a β-carotene production of 2.22 mg/g DCW. The product of the carRP genes has phytoene synthesis activity and lycopene cyclase activity in fungi and can catalyze multiple reactions. Gao et al. (2017a) used the BLASTP tool to find the multifunctional carotene synthase carS from Schizochytrium sp. They transformed Y. lipolytica with a carS expression cassette encoding three enzymatic functions under the control of the PGPD promoter, which resulted in a β-carotene production of 0.41 mg/g DCW. To further enhance β-carotene production, Gao et al. (2017b) expressed tHMGR and carRP to enhance the HMG-CoA and GGPP pools, which resulted in a 14-fold increase of β-carotene production. Production was further improved approximately 100-fold by optimizing the tHMGR and carRP gene dosage and thereby enhancing the activity of the MVA pathway. The recombinant strain was subjected to highcell-density fermentation in a nitrogen-limited medium, and the βcarotene content reached 4 g/L, most of which was stored in lipid droplets. In another study, Larroude et al. (2018) transformed a lipid overproducer strain with a tHMGR and car expression-cassette obtaining the three genes GGS1, carB and carRP, with the aim to maximize β-carotene production. The resulting strain produced 35.7 mg/L β-carotene. Subsequently, they used Golden Gate assembly to construct a new car-cassette as a second copy under the control of the TEF1 promoter, then transformed the previous recombinant strain. The second strain was able to produce 111.8 mg/L β-carotene. Finally, a fed-batch fermentation lead to a β-carotene production of 6.5 g/L after 122 h. Astaxanthin is a red-colored carotenoid that is often added to foods and cosmetics due to its powerful antioxidant activity (Guerin et al., 2003). Kildegaard et al. (2017) successfully produced astaxanthin in Y. lipolytica by genetic engineering. They screened a robust strain producing β-carotene from 14 strains with integrated β-carotene biosynthesis genes. As two important genes for β-carotene synthesis, HMGR and GGS1 were introduced into Y. lipolytica. Additional downregulation of the competing squalene synthase SQS1 by truncating the native
pyruvic acid and 8% dodecane, that led to an initial limonene production of 23.56 mg/L. 4.2. Sesquiterpenoids Sesquiterpenoids are a large group of terpenoids with FPP as direct precursor, which in turn is synthesized by two sequential condensation reaction of IPP with DMAPP into GPP, followed by its condensation with another IPP molecule into FPP. Researchers have attempted to increase the yield of sesquiterpenoids by increasing the supply of FPP. In order to heterologously produce α-farnesene in Y. lipolytica, Yang et al. (2016) constructed a vector with a codon-optimized α-farnesene synthase gene expression cassette and integrated it into the genome of Y. lipolytica Po1h. The resulting engineered Y. lipolytica successfully synthesized α-farnesene at a low initial level. They further overexpressed tHMGR, IDI and ERG20 aiming to increase the FPP content and produced 57.08 mg/L of α-farnesene in shake flasks, corresponding to a 20.8-fold increase over the initial production of 2.75 mg/L. Recently, Guo et al. (2018) succeeded in the heterologous production of (+)-nootkatone in Y. lipolytica by co-expressing (+)-valencene synthase (CnVS), a codon-optimized version of the (+)-nootkatone synthase opCYP706M1 from Callitropsis nootkatensis and a codon-optimized version of the NADPH-dependent cytochrome P450 reductase opAtCPR1 from Arabidopsis thaliana. They fused opCYP706M1 and opt46AtCPR1 (truncated N-terminal 46 amino acids) and overexpressed tHMGR and ERG20, improving the (+)-nootkatone production. The final engineered strain achieved a (+)-nootkatone titer of 978.2 µg/L, which was a 20.5-fold increase compared to the simple co-expression of CnVS, opCYP706M1 and opAtCPR1. 4.3. Triterpenoids Over 23,000 triterpenoid structures have been found in nature, which makes them the largest group of compounds in the terpenoid family (Pateraki et al., 2015). Generally, the triterpene intermediate squalene is synthesized by coupling two molecules of FPP head to head in a reaction catalyzed by squalene synthase, followed by oxidation into 2,3-squalene epoxide by squalene epoxidase at the expense of NADPH. The resulting 2,3-squalene epoxide is cyclized by various cyclases to form mono- to pentacyclic backbone structures (Dong et al., 2018; Zhang et al., 2017). Sun et al. (2018) integrated the lupeol synthase AtLUP1 and cytochrome P450 reductase AtCPR1 from Arabidopsis thaliana, cytochrome P450 monooxygenase MtCYP716A12 from Medicago truncatula into Y. lipolytica genome after codon optimization. The resulting strain successfully synthesized the pentacyclic triterpene betulinic acid with a titer of 0.32 mg/L. This titer was increased to 9.41 mg/L following the fusion of cytochrome P450 enzymes and overexpression of key genes from the upstream MVA pathway. 4.4. Tetraterpenoids Carotenoids are typical tetraterpenoids. They are derived from phytoene, which is formed from two molecules of GGPP. Carotenoids are a general term for an important class of natural products that are ubiquitous as yellow, orange-red or red pigments of higher plants, fungi and algae (Mata-Gómez et al., 2014). Furthermore, carotenoids are the main source of vitamin A in the body, and also have properties related to antioxidation, immune regulation, cancer prevention, anti-aging, and so on (Milani et al., 2017; Ribeiro et al., 2018). The most studied major carotenoids are lycopene, β-carotene, astaxanthin and lutein. Lycopene is an important intermediate in the synthetic pathway of carotenoids and is produced by the consecutive action of three enzymes: geranylgeranyl diphosphate synthase (GGS1/CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI). Matthaus et al. (2014) expressed codon-optimized CrtB and CrtI genes from Pantoea ananatis under the control of the TEF1 promoter in Y. lipolytica. The transformants 453
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promoter of SQS1 to 50 bp resulted in a β-carotene titer of 797.1 mg/L. They next expressed β-carotene ketolase (crtW) from the marine bacterium Paracoccus sp. N81106 and β-carotene hydroxylase (crtZ) from the enterobacterium P. ananatis to divert the flux from β-carotene to astaxanthin. To further improve astaxanthin production, they optimized the copy numbers of crtZ and crtW to obtain 54.6 mg/L of astaxanthin in a microtiter plate cultivation.
precursor (+)-epi-α-bisabolol from FPP, the P450s enzyme that catalyzes the next reaction to form hernandulcin remains unknown (Attia et al., 2012). It is therefore necessary to more broadly investigate the functions of cytochrome P450 enzymes and provide a basis for research on the synthesis of more valuable terpenoids, especially triterpenoids.
5. The search for new strategies and their application
Y. lipolytica can assimilate different carbon sources, including hydrophilic materials (glucose, glycerol, alcohols and acetate) and hydrophobic substrates (fatty acids, triacylglycerols and alkanes) (Liu et al., 2015; Sun et al., 2017). In order to realize the industrial production of terpenoids using Y. lipolytica, reducing the cost of fermentation is a problem that must be considered. Glycerol is the main byproduct of the biodiesel production chain and has been used extensively as a substrate in the production of single cell oil and organic acids (André et al., 2009). Replacing glucose with inexpensive carbon sources can reduce the cost of materials for the production of terpenoids. The titer of betulinic acid in Y. lipolytica using glycerol as the sole carbon source and using glucose as carbon source in S. cerevisiae in shake flask cultivation were almost the same (Sun et al., 2018). This confirms that cheap carbon sources are also available for the synthesis of terpenoids in Y. lipolytica. In addition, extensive use of carbon sources has been achieved through metabolic engineering. Y. lipolytica has achieved the use of xylose as the sole carbon source, produced over 15 g/L of lipid by introducing a heterologous oxidoreductase pathway (Li and Alper, 2016). Y. lipolytica can utilize various carbon sources, which contributes to the development of carbon sources for economical production of terpenoids.
5.3. Optimization of culture media
Y. lipolytica is a promising biorefinery platform strain for the production of bulk and value-added terpenoids owing to its endogenous MVA pathway and native high-level oil production capacity. It is currently possible to quickly and easily achieve the heterologous synthesis of plant-derived terpenoids in Y. lipolytica by synthetic pathway rewiring and genome editing. In order to further explore ways to improve terpenoid production, more genetic modification strategies can be adopted from the eukaryotic model strain S. cerevisiae. 5.1. Modification of enzymes Protein degradation is an efficient mechanism to control carbon allocation to competing pathways and avoid an insufficiency of target precursors. Peng et al. (2017) added a PEST sequence responsible for ER-associated protein degradation to the C-terminus of squalene synthase to shorten its half-life in S. cerevisiae, and the nerolidol titer improved by 86% to about 100 mg/L. Recently, Peng et al. (2018) explored the effect of degradation of ERG20 on the carbon flux balance between monoterpene synthesis and cell growth by fusing an N-degron to the N-terminus of ERG20. To avoid a severe lack of sterols causing cell lethal, they replaced the promoter of ERG20 with a sterol-responsive promoter. This regulatory strategy increased monoterpene production in S. cerevisiae with a heterologous monoterpene synthesis pathway. In addition, affibody scaffolds can be used to co-localize multiple enzymes via noncovalent interactions to reduce substrate loss and accumulation of toxic intermediates. Compared to enzymes fusions, affibody scaffolds can generate a close spatial concentration of multiple enzymes and thereby guarantee efficient substrate channeling. More importantly, formed subcellular compartments avoid the negative impact of the intracellular environment on synthetic target products. Affibody scaffolds based on anti-idiotypic partners have been employed for the in vivo co-localization of farnesyl diphosphate synthase and farnesene synthase in S. cerevisiae (Tippmann et al., 2017). Han et al. (2018) described the use of Tya protein as a scaffold in combination with three key enzymes (including tHMGR, ispA, AFS1or DPP1) involved in sesquiterpene biosynthesis, which improved farnesene and farnesol production in S. cerevisiae. The application of these new strategies has undoubtedly provided new references and guidance for the synthesis of terpenoids in Y. lipolytica, which developed a convenient means for us to further use this platform to achieve the synthesis of more terpenoids.
6. Conclusions The metabolic engineering studies of the production of terpenoids reviewed here demonstrate that Y. lipolytica is a potential platform strain for the synthesis of plant-derived terpenoids. In the future, we should further develop more genetic tools that are able to achieve more efficient and rapid genetic operations for Y. lipolytica, such as CRISPR/ Cas9 genome-editing technology and protein engineering. With the identification of specific terpene synthases and the application of emerging tools, researchers have found a way to achieve heterologous synthesis of terpenoids in Y. lipolytica. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Nos. 21776131), the Six Talent Peaks Project in Jiangsu Province of China (No. 2018-SWYY-047), and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (No. XTD1814). References
5.2. Decoration of terpenoids
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