Overview of plant CYP450s in triterpenoid saponins biosynthesis.
Abstract
Plant natural products possess versatile biological activities including antiviral, anticancer and hepatoprotective activities, which are widely used in pharmaceutical and many other health-related fields. However, current production of such compounds relies on plant culture and extraction, which brings about severe concerns for environmental, ecological and amount of agricultural lands used. With the increasing awareness of environmental sustainability and shortage of lands, yeasts are engineered to produce natural products, for its inherent advantages such as the robustness, safety and sufficient supply of precursors. This chapter focused on the recent progress of yeast as a platform for the biosynthesis of plant natural products.
Keywords
- natural products
- flavonoids
- alkaloids
- terpenoids
- terpenoids saponins
- biomanufacturing
- heterogeneous synthesis
- yeast
1. Introduction
Plant natural products were a kind of active compounds including flavonoids, alkaloids, terpenoids and saponins etc. As the main composition of plants secondary metabolites, these compounds play an important role in plant communication and defensing, so these compounds have been widely used as herbicide and pesticide in the agricultural industry [1]. For example, oleanane saponins isolated from
Currently, the production of natural products are mainly based on extraction from plants, which is a low yield, time consuming, labor intensive and environment unfriendly supply way [4]. The inefficient approach could not match the huge demands of market, and further limit the application of these compounds in the pharmaceutical, agricultural, food, cosmetic and detergent industries. Thus, developing novel approaches are of great significance to replace the traditional method.
Producing natural products via microorganism cell factories turned out to be a promising solution. Compared with plants, microbes exhibit many advantages, including fast growing, land saving and controllable. Yeast especially
In this chapter, we systematically illustrate the decoded biosynthetic pathway of flavonoids, alkaloids, terpenoids and terpenoids saponins assisted by yeast. Then briefly summarize the progress of yeast to produce plant natural products. Furthermore, novel strategies and tools used to boost their production were discussed.
2. Synthetic pathways of natural products
Flavonoids and alkaloids are usually derived from the shikimate pathway, which exists in prokaryotic, eukaryotic, and archaeal microorganisms. The synthesis pathway start from the stereo-specific condensation catalyzed by 3-deoxy-Darabino-heptulosonate-7-phosphate synthase to generate 3-deoxy-Darabino-heptulosonate-7-phosphate (DAHP), which is further catalyzed to form chorismate a common precursor for various aromatic compounds, including aromatic amino acids, then these aromatic compound will be converted to flavonoids and alkaloids Figure 1(a).
Terpenes and saponins are usually synthesized from the common five-carbon building blocks, 3-isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are synthesized through mevalonic acid (MVA) or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway [5]. These five-carbon units are condensed to geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), and farnesyl pyrophosphate (FPP). These precursors are then diverted to specialized terpenes by terpene synthases. GPP is converted to monoterpene by monoterpene synthases, GGPP were used to synthesize diterpenes which can be further converted to tretaterpenes, and FPP is the precursor for triterpenes which can be converted to saponins by UGTs Figure 1(b).
In order to produce flavonoids, alkaloids, monoterpene, diterpene and tretaterpene heterogeneously,
During triterpenoids synthesis, FPP is condensed to 2,3-oxidosqualene, which is subsequently cyclized to polycyclic triterpenoid skeletons by oxidosqualene cyclases (OSCs). These molecules are oxidized by CYP450s forming aglycones, which are further glycosylated to triterpenoid saponins by UGTs.
Cyclization of 2,3-oxidosqualene by OSCs, was the first step in triterpenoid biosynthesis. Because of the efficient 2,3-oxidosqualene supplement, most OSCs was verified by directly expressing in yeast [6]. Plants OSCs always possess promiscuous activities, and could cyclize 2,3-oxidosqualene to different conformations simultaneously. For example, amyrin synthase from
The triterpenoid skeletons could be further oxidized by CYP450s, introducing active groups such as hydroxyl, carboxyl or epoxy groups [9]. The CYP450s decoding process was complicated, for the identification of CYP450s need much chemical and bioinformatics information, and the membrane located plant CYP450s is hard to express in
No. | Name | Accession number | Plant species | Substrate | Loci |
---|---|---|---|---|---|
1 | CYP51H10 | ABG88965.1 | β-amyrin | C-12, 13, 16β | |
2 | CYP71D353 | AHB62239.1 | lupeol | C-20 | |
3 | CYP72A61v2 | BAL45199.1 | 24-OH-β-amyrin | C-23 | |
4 | CYP72A63 | BAL45200.1 | β-amyrin | C-30 | |
5 | CYP72A67 | ABC59075.1 | oleanolic acid | C-2 | |
6 | CYP72A68v2 | BAL45204.1 | oleanolic acid | C-25 | |
7 | CYP72A69 | BAW35014.1 | β-amyrin | C-21 | |
8 | CYP72A154 | BAL45207.1 | β-amyrin | C-30 | |
9 | CYP87D16 | AHF22090.1 | β-amyrin | C-16α | |
10 | CYP88D6 | AQQ13664.1 | β-amyrin | C-11 | |
11 | CYP93E1 | BAE94181.1 | β-amyrin | C-24 | |
12 | CYP93E2 | ABC59085.1 | β-amyrin | C-24 | |
13 | CYP93E3 | BAG68930.1 | β-amyrin | C-24 | |
14 | CYP93E4 | AIN25416.1 | β-amyrin | C-24 | |
15 | CYP93E5 | AIN25417.1 | β-amyrin | C-24 | |
16 | CYP93E6 | AIN25418.1 | β-amyrin | C-24 | |
17 | CYP93E7 | AIN25419.1 | β-amyrin | C-24 | |
18 | CYP93E8 | AIN25420.1 | β-amyrin | C-24 | |
19 | CYP93E9 | AIN25421.1 | β-amyrin | C-24 | |
20 | CYP716A12 | ABC59076.1 | α-amyrin, β-amyrin, lupeol | C-28 | |
21 | CYP716A14v2 | AHF22083.1 | α-amyrin, β-amyrin | C-3 | |
22 | CYP716A15 | BAJ84106.1 | α-amyrin, β-amyrin, lupeol | C-28 | |
23 | CYP716A17 | BAJ84107.1 | α-amyrin, β-amyrin, lupeol | C-28 | |
24 | CYP716A44 | – | α-amyrin, β-amyrin | C-28 | |
25 | CYP716A46 | – | α-amyrin, β-amyrin | C-28 | |
26 | CYP716A47 | AEY75213.1 | dammarenediol-II | C-12 | |
27 | CYP716A52v2 | AFO63032.1 | β-amyrin | C-28 | |
28 | CYP716A53v2 | AFO63031.1 | dammarenediol-II | C-20 | |
29 | CYP716A75 | AHF22088.1 | β-amyrin | C-28 | |
30 | CYP716A78 | ANY30853.1 | α-amyrin, β-amyrin, lupeol | C-28 | |
31 | CYP716A79 | ANY30854.1 | α-amyrin, β-amyrin, lupeol | C-28 | |
32 | CYP716A80 | ALR73782.1 | α-amyrin, β-amyrin, lupeol | C-28 | |
33 | CYP716A81 | ALR73781.1 | α-amyrin, β-amyrin, lupeol | C-28 | |
34 | CYP716A83 | AOG74832.1 | β-amyrin | C-28 | |
35 | CYP716A86 | AOG74831.1 | β-amyrin | C-28 | |
36 | CYP716A140 | AOG74836.1 | β-amyrin, 24-OH-β-amyrin | C-28 | |
37 | CYP716A141 | AOG74838.1 | β-amyrin, 24-OH-β-amyrin | C-28 | |
38 | CYP716A180 | – | lupeol | C-28 | |
39 | CYP716A244 | APZ88353.1 | β-amyrin | C-28 | |
40 | CYP716A254 | – | β-amyrin | C-28 | |
41 | CYP716AL1 | AEX07773.1 | α-amyrin, β-amyrin, lupeol | C-28 | |
42 | CYP716C11 | AOG74835.1 | oleanolic acid | C-2 | |
43 | CYP716E41 | AOG74834.1 | maslinic acid | C-6 | |
44 | CYP716E22 | – | α-amyrin, β-amyrin | C-6 | |
45 | CYP716S5 | AOG74839.1 | β-amyrin, oleanolic acid | C-12, 13 | |
46 | CYP716Y1 | AHF45909.1 | α-amyrin, β-amyrin | C-16α | |
47 | CYP716A1 | AED94045.1 | β-amyrin | C-28 | |
48 | CYP716A2 | BAU61505.1 | α-amyrin | C-22 |
Glycosylation is the last step of triterpenoid saponins biosynthesis that links hydrophilic sugar moieties to the hydrophobic aglycone by UGTs. By glycosylation, various monosaccharide units (including glucose, glucuronic acid, galactose, rhamnose, xylose and arabinose, etc.) could be linked to aglycone at the positions C-3, C-28, C-4, C-16, C-20, C-21, C-22 and/or C-23. The introduced of sugar moieties could improve triterpenoid saponins bioactivities. In view of the tremendous amounts of UGTs in plants, more than 120 genes encoding family 1 UGTs have been identified in
No. | Name | Accession number | Plant species | Substrate | Loci |
---|---|---|---|---|---|
1 | UGT71G1 | AAW56092.1 | Medicagenic acid UDP- glucose | C-3, 28 | |
2 | UGT73AD1 | ALD84259.1 | Asiatic acid, Madecassic acid UDP- glucose | C-28 | |
3 | UGT73AE1 | AJT58578.1 | Glycyrrhetinic acid UDP- glucose | C-3 | |
4 | UGT73AH1 | AUR26623.1 | Asiatic Acid UDP- glucose | C-28 | |
5 | UGT73C10 | AFN26666.1 | Hederagenin, Oleanolic acid UDP- glucose | C-3 | |
6 | UGT73C11 | AFN26667.1 | Glycyrrhetinic acid, Oleanolic acid UDP- glucose | C-3 | |
7 | UGT73C12 | AFN26668.1 | Hederagenin, Oleanolic acid UDP- glucose | C-3 | |
8 | UGT73C13 | AFN26669.1 | Hederagenin, Oleanolic acid UDP-glucose | C-3 | |
9 | UGT73F2 | BAM29362.1 | Saponin A0-gα UDP-xylose | C-22 | |
10 | UGT73F3 | ACT34898.1 | Hederagenin UDP- glucose | C-28 | |
11 | UGT73F4 | BAM29363.1 | Saponin A0-gα UDP-xylose | C-22 | |
12 | UGT73F17 | AXS75258. | Glycyrrhizin UDP-glucose | C-30 | |
13 | UGT73K1 | AAW56091.1 | Hederagenin, Soyasapogenols B and E UDP-glucose | C-3, 28 | |
14 | UGT74AE2 | – | Protopanaxadiol UDP-glucose | C-3 | |
15 | UGT74M1 | ABK76266.1 | Gypsogenic acid UDP-glucose | C-28 | |
16 | UGT94Q2 | – | Ginsenoside Rh2 UDP-glucose | C-3 | |
17 | UGTPg1 | – | Protopanaxadiol UDP-glucose | C-3 | |
18 | UGTPg100 | – | Ginsenoside RF1, Protopanaxatriol UDP-glucose | C-6 | |
19 | UGTPg101 | – | Ginsenoside RF1, Protopanaxatriol UDP-glucose | C-6, 20 | |
20 | Pg3-O-UGT1 | – | Protopanaxadiol UDP-glucose | C-3 | |
21 | GmSGT2 | BAI99584.1 | Soyasapogenol B monoglucuronide UDP-galactose | C-3 | |
22 | GmSGT3 | BAI99585.1 | Soyasaponin III UDP-rhamnose | C-3 | |
23 | UDPG | – | Ginsenoside Rd. UDP-glucose |
3. Biosynthesis of natural products in yeast
3.1 Biosynthesis of flavonoids in yeast
Flavonoids are among the most extensively investigated natural products, which could be divided into several subgroups, including common flavonoids (e.g., galangin, eriodictyol, catechin, quercetin, luteolin, myricetin and cyanidin), isoflavonoids (e.g., genistein) and neoflavonoids (e.g., calophyllolide, isodispar B). Due to their physiological activity and decoded synthesis pathways, the heterologous biosynthesis of flavonoids and their derivatives, have been extensively studied in microbial hosts mostly in
Based on yeast platform, 531 mg/L resveratrol production was achieved via the tyrosine pathway directly using glucose and ethanol as substrate in fed-batch fermentation. Through the subsequent pull-push-block strain engineering strategy, more resveratrol production formed via the phenylalanine pathway increased up to 800 mg/L directly from glucose which was the highest titer of resveratrol up to now.
The co-culture system was also developed for flavonoids production. In the collaboration system of
3.2 Biosynthesis of alkaloids in yeast
Alkaloid compounds, especially plant-derived benzylisoquinoline alkaloids and monoterpene indole alkaloids are considered as a valuable source of pharmaceuticals for its anticancer, antiviral and antimalarial activities, et al. In order to replace plant-extracting method, the reconstruction of plant-derived alkaloid biosynthetic pathways in microbes are extensively studied. Though always achieve much lower titers than in
By overexpressing 14 known monoterpene indole alkaloid pathway genes and an enhancement of secondary metabolism through overexpression of additional seven genes and deletion of three genes, strictosidine was produced in yeast with a production of 0.53 mg/L. (S)-reticuline (around 80 μg/L), baine (6.4 μg/L) and hydrocodone (0.3 μg/L) have been produced in yeast from simple sugars. Indeed, the titers was low; However, very recently, the total assembly and optimization of the noscapine biosynthetic pathway involving over 30 enzymes in yeast was realized, which was very hard to reconstruct in
3.3 Biosynthesis of terpenes and saponins in yeast
Similar with flavonoids and alkaloids, some simple terpenoids skeletons such as monoterpenoid and diterpenoid, which were produced directly by terpenoid synthase, were mostly studied in
By expressing geraniol synthase from
Moreover, the heterologous biosynthesis of diterpenoid by yeast also became a new trend and gained more and more attention. Through introducing diterpenoids synthase and metabolic optimization, several plant diterpenoids including taxadiene and miltiradiene were produced in yeast. With the introducing of the taxadiene biosynthetic pathways, and the optimization of precursors supplement by strengthen the MVA pathway through overexpressing tHMG1 and UPC2-1 (a global transcription factor of MVA), the taxadiene was successfully produce in the engineered yeast and achieved a production of 8.7 mg/L, though the production was lower than that produced in
As an exception, the heterogeneous biosynthesis of triterpenoid and triterpenoid saponins were mostly studied in yeast, for the membrane located plant OSC and CYP450 are challengeable to correctly express in
Benefit from the yeast hosts, the more complex saponins were also heterologously synthesized. By expressing
Protopanaxadiol (PPD) is an important starting material for the biosynthesis of ginsenoside, which is synthesized from 2,3-oxidosqualene by dammarenediol-II synthase and CYP450s. Through expressing
The widely studied ginsenosides Rh2 and Rg3 which are synthesized from PPD have been successfully biosynthesized in
It is demonstrated that compound K (CK), generally considered as the metabolite of glycosidases [24], is the main functional form of oral administration of ginsenosides [25]. By the co-expression of PgDS, AtCPR2, CYP716A47 and UGTPg1, CK has already been synthesized in
Besides ginsenosides, the heterogeneous biosynthesis of other triterpenoids saponins with markedly physiological function also attracted much attention. The natural sweeter mogroside V from S. grosvenorii, which is nearly 300 times sweeter than sucrose, is widely used as a food additive in low-calorie sweet beverages [28]. Through analysis of S. grosvenorii transcriptome data and gene mining, the key genes involved in mogroside V synthesis including cycloartenol synthase (CAS) gene, epoxide hydrolases (EPH) gene, CYP102801, UGT94-289-3 and UGT720-269-1 have been identified. By introducing these enzymes together with squalene synthase (SQS), squalene epoxidase (SQE) and AtCPR1 in
Saikosaponins are the major pharmaceutical constituents of
Moreover, by overexpressing lycopene synthetic genes from
All these progresses indicate the great potential of yeast for heterologous synthesis of natural products, especially for these with complex molecular structure and synthetic pathways such as terpenoids and their saponins. Recently, various kinds of terpenoids and saponins were heterologously produced in yeast, but most of them had a low final concentration far from to instead of plant extracting methods. To boost the production efficiency of engineered yeast strains, various strategies need intensively study.
4. Strategies for boosting biosynthesis of terpenoids in yeast
Although various kinds of natural products could be produced by yeast, many challenges still remain for this approach. The main bottleneck of building an efficient yeast cell factory was that the biosynthetic pathways are not totally elucidated and the poor or disappeared activity of plant enzymes when expressed in yeast. Moreover, the destabilization on the native metabolic flux caused by the heterogeneous pathways could lead to low cell growth and low final products concentration, and the cytotoxicity most of natural products also restrict the use of microbial hosts for producing natural products. Strategies and biotools focused on settling such issues to accelerate the microbial natural products biosynthesis in yeast host have been developed based on omics, metabolic engineering and protein engineering (Figure 3).
4.1 Strategies to redesign natural biosynthetic pathways
Unlike some prokaryotic biosynthetic pathway genes, which always locate on a gene cluster, the genes involved in triterpenoid saponins biosynthesis always distributes among the whole genome in plants. Moreover, the expression of these genes generally needs intricate inducible conditions, which increase the difficulty to elucidate triterpenoid saponins biosynthetic pathways.
Benefit from the rapid progress of sequencing technology, genome and transcriptome of many medicinal plants have been sequenced, and the information has been publicly available online (http://medicinalplantgenomics.msu.edu). The analysis of the genome and transcriptome date facilitates the prediction of genes involved in the targeted compound biosynthesis. Through the comparison of the transcriptome information between plants or tissues with high- and low-production of the target compounds, several key genes could be predicted. For example, through comparing the transcriptome data between high- and low-producing varieties, the genes including bAS1, CYP716A79 and CYP716A78 that involved in quinoa saponins biosynthesis were targeted from
Besides mining enzymes from native host, the application of the substrate-promiscuous enzymes turned out to be an alternative approach to reconstruct the target compounds biosynthetic pathways. For instance, the nonnative substrate-promiscuous glycosyltransferase Bs-YjiC from
The low final concentration of the synthesized compounds is always caused by the poor enzyme activity on the unnatural substrate. Protein evaluation, which could improve the catalytic characteristic involving “substrate specificity” of the substrate-promiscuous enzymes has been developed for the specific decoration of natural and unnatural substrates. One example is the engineering of the substrate-promiscuous UDP-glucose sterol glucosyltransferase UGT51 from
4.2 Strategies to improve plant enzyme activity
Plant CYP450s are indispensable enzymes for the C–H bounds oxidation of triterpenoid skeletons. However, heterogeneous expression of plant CYP450s in yeasts hosts usually exists problems as low expression level, poor catalytic efficiency or even incorrect folding structure. Plant CYP450s involved in triterpenoids synthesis are membrane-bound oxidase enzymes, which anchor in the endoplasmic reticulum (ER) of plants cells and requires electrons transferred by CYP450 reductase. Although CYP450 is essential for the hydroxylation of C–H bounds and can further oxidize the alcohol products to aldehyde and acid, it always shows poor activity on such substrates. As a result, it is challenging to establish high-yield triterpenoid saponins in yeast cell factories, and improving the plant CYP450s is essential to improve the situation. Currently, many strategies have been developed to improve the expression level and regulate the pairing efficiency of CPRs to plant CYP450s in microbial hosts.
Codon optimization and application of a strong promoter are the most common strategies to improve enzyme expression level in heterologous hosts and it is also effectively used to improve plant CYP450s expression level in microbial hosts [36]. Chimeric protein has been used to correct the folding of plant CYP450s in microbial hosts. As plant CYP450s are anchored in ER membrane, the activity can be improved by replacing the native N-terminal sequence with ER-membrane bound proteins of yeast to facilitate correctly folding and anchoring. Protein directed evolution was also applied to enhance the activity of plant enzymes. In consideration of that the enlargement of ER would provide more room for the ER-located CYP450s and CPRs leading to higher protein abundance, a novel ER morphology engineering strategy is developed. Through the deletion of PAH1 gene encoding phosphatidic acid phosphatase, the ER membranes of
The pairing efficiency of CYP450s and CPRs plays an important role for the catalytic activity of CYP450s. Mining novel CPRs is a straightforward way to improve the CYP450 activities as different CPRs has different pairing efficiency with CYP450s. For example, the CPR from
4.3 Strategies to enhance metabolic flux
The plant natural products biosynthetic pathways always include multiple steps. When introduced in yeast, the heterogeneous pathways would intensively interact with the native metabolic network, by means of competing substrates and co-factors as well as metabolites reverse influence. The disturbance will restrict the targeted compound production. Therefore, balancing metabolic flux distribution between heterologous pathways and native metabolic networks plays an important role in promoting the production of targeted compound.
Enhancing the precursors supplement to the targeted pathway is a straightforward strategy to enhance natural products production. As demonstrated, the five-carbon building blocks IPP and DMAPP are naturally synthesized through either eukaryotic MVA or prokaryotic MEP pathway in microorganisms. The combination of MVA and MEP pathways in one host could take advantages of both pathways and lead to more efficient precursor supplement for terpenoids. Through introducing a heterogeneous MVA pathway, 27.0 g/L amorphadiene was achieved in
Decreasing the metabolic flux of competing pathways is efficient to strengthen the flux to targeted pathway. However, in most cases, directly deletion of the enzymes involved in the competing could lead to lethality, for many of the genes are essential to the hosts. Therefore, decrease the metabolic flux to the competing pathways by down-regulation of key enzymes is a proper approach to strengthen the final production. Generally, the cellular protein concentration is regulated by transcription, RNA degradation, translation and protein degradation. The application of weaker promoters is the most commonly used strategy to down-regulate the transcriptional level of key genes. To decrease the sterol synthesis and redirect the metabolic flux to the β-amyrin synthesis pathway in yeast. ERG7 (lanosterol synthase gene) promoter was replaced by a methionine repressible promoter (PMET3). Moreover, in order to improve the α-santalene accumulation in yeast, the native promoter of ERG9 (squalene synthase gene) was replaced by PCRT3, the copper repressible promoter and PHXT1, a low concentration glucose repressible promoter which resulted in decreased metabolic flux to ergosterol synthesis and increased α-santalene production [39]. Recently, dynamic protein degradation was developed to weaken the competing pathways. Depending on the ER-associated protein degradation system, the cytosolic proteins can be degraded when attached by a PEST sequence. As a result, the strategy of using the G1 cyclin PEST sequence as a degradation degron to label the cytosolic term of squalene synthase was developed for the production of trans-nerolidol. Once labeled by degron, the squalene synthase will be degraded dynamically, resulting in enhanced sesquiterpene trans-nerolidol production. By the similar strategy, farnesyl pyrophosphate synthetase was labeled by a designed N-terminal degron on the N-terminus, which increased the titer of monoterpene linalool [40].
4.4 Strategies to reduce toxicity to the hosts
Natural products always exhibit cytotoxicity to the microbial hosts, leading to decreased cell growth and finally impair the production. In order to solve these problems, various strategies were developed including two-stage fermentation, pathway compartmentalization and transporters mediated compound secretion. In order to alleviate the negative influences on cell growth, the fermentation course is divided into two stages. In first fermentation stage, heterogeneous pathway keep silence and cells grow fast with precursor accumulated, while in the second stage, target pathway would be induced to produce the target compounds [41]. In addition to the traditional two-stage fermentation, the organelles including mitochondria, peroxisome and vacuole were also used to compartmentalize the heterogeneous pathways. Because the integrated membrane structure, these organelles are relatively independent from the cytoplasm, which could prevent the toxic precursors and products from distributing in cytoplasm to disrupt cell growth. Furthermore, subcellular compartmentalization of target biosynthetic pathways can concentrate the substrates, intermediates and enzymes in a more narrow space, which can improve the reaction efficiency of enzymes. Through locating the amorpha-4,11-diene biosynthetic pathways in the mitochondria of yeast, the amorpha-4,11-diene production increased by 63% compared with locating in the cytosol. By this strategy, the precursor FPP was restricted in mitochondria by the membrane structure, which reduced the loss of FPP. Using similar strategy, the valencene biosynthetic pathway was reconstructed into the mitochondria of yeast resulting in eight-fold increase of valencene production. Besides the mitochondria, peroxisome was also used to compartmentalize the heterogeneous pathways, by introducing the lycopene synthesis pathway in peroxisome, lycopene production was improved up to 73.9 mg/L in Pichia pastoris [42]. Therefore, subcellular compartmentalization was a pioneering strategy to reduce products cytotoxicity to the microbial hosts.
Another strategy to reduce the inner cytotoxicity of natural products is to secrete these compounds outside the cell automatically. To achieve this goal, transporters were taken into account, for their significant contribution of transporting the products to the extracellular space. Due to the rarity of transporters that possess the ability to transport the complex natural products, transporter engineering has been developed to improve the situation. For example, through protein engineering, one variant of AcrB from AcrAB-TolC efflux pump can effectively improve the α-pinene efflux out of the
In this chapter, the biosynthetic pathways of natural products and their reconstruction in yeast cell factories were systematically summarized. The strategies developed to increase natural products productivity in yeast were also discussed including protein engineering, metabolic engineering, subcellular localization and fermentation control. With these endeavors, the engineered strains can produce these compounds in different levels. These achievements indicate yeast a promising chassis for the heterogeneous biosynthesis of natural products.
Acknowledgments
The authors kindly acknowledge financial support from the National Science Fund for Distinguished Young Scholars (NO. 21425624) and the National Natural Science Foundation of China (NO. 21506011, NO. 21476026).
References
- 1.
Thimmappa R, Geisler K, Louveau T, et al. Triterpene biosynthesis in plants. Annual Review of Plant Biology. 2014; 65 :225-257. DOI: 10.1146/annurev-arplant-050312-120229 - 2.
Scognamiglio M, D’Abrosca B, Fiumano V, et al. Oleanane saponins from Bellis sylvestris Cyr. and evaluation of their phytotoxicity on Aegilops geniculata Roth. Phytochemistry. 2012;84 (12):125-134 - 3.
Zhao Y, Lv B, Feng X, et al. Perspective on biotransformation and de novo biosynthesis of licorice constituents. Journal of Agricultural and Food Chemistry. 2017; 65 (51):11147-11156. DOI: 10.1021/acs.jafc.7b04470 - 4.
Wang L, Weller CL. Recent advances in extraction of nutraceuticals from plants. Trends in Food Science & Technology. 2006; 17 (6):300-312 - 5.
Pablo P, Catalina P, Manuel RC. New insights into plant isoprenoid metabolism. Molecular Plant. 2012; 5 (5):964-967 - 6.
Zheyong X, Lixin D, Dan L, et al. Divergent evolution of oxidosqualene cyclases in plants. New Phytologist. 2012; 193 (4):1022-1038 - 7.
Aragão GF, Carneiro LMV, Júnior APF, et al. Antiplatelet activity of α.- and β.-Amyrin, isomeric mixture from Protium heptaphyllum. Pharmaceutical Biology. 2007; 45 (5):343-349 - 8.
Augustin JM, Kuzina V, Andersen SB, et al. Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry. 2011; 72 (6):435-457. DOI: 10.1016/j.phytochem.2011.01.015 - 9.
Loew GH, Harris DL. Role of the heme active site and protein environment in structure, spectra, and function of the cytochrome p450s. Chemical Reviews. 2010; 31 (16):407-420 - 10.
Yasumoto S, Fukushima EO, Seki H, et al. Novel triterpene oxidizing activity of Arabidopsis thaliana CYP716A subfamily enzymes. FEBS Letters. 2016;590 (4):533-540. DOI: 10.1002/1873-3468.12074 - 11.
Zhang R, Li C, Wang J, et al. Microbial production of small medicinal molecules and biologics: From nature to synthetic pathways. Biotechnology Advances. 2018; 36 (8):2219-2231. DOI: 10.1016/j.biotechadv.2018.10.009 - 12.
Li Y, Li S, Thodey K, et al. Complete biosynthesis of noscapine and halogenated alkaloids in yeast. Proceedings of the National Academy of Sciences of the United States of America. 2018: 115 (17):3922-3931 - 13.
Zhang G, Cao Q , Liu J, et al. Refactoring β-amyrin synthesis in Saccharomyces cerevisiae . AICHE Journal. 2015;61 (10):3172-3179. DOI: 10.1002/aic.14950 - 14.
Zhu M, Wang C, Sun W, et al. Boosting 11-oxo-beta-amyrin and glycyrrhetinic acid synthesis in Saccharomyces cerevisiae via pairing novel oxidation and reduction system from legume plants. Metabolic Engineering. 2018;45 :43-50. DOI: 10.1016/j.ymben.2017.11.009 - 15.
Zhao Y, Fan J, Wang C, et al. Enhancing oleanolic acid production in engineered Saccharomyces cerevisiae . Bioresource Technology. 2018;257 :339-343. DOI: 10.1016/j.biortech.2018.02.096 - 16.
Yu Y, Chang P, Yu H, et al. Productive Amyrin Synthases for efficient alpha-amyrin synthesis in engineered Saccharomyces cerevisiae . ACS Synthetic Biology. 2018;7 (10):2391-2402. DOI: 10.1021/acssynbio.8b00176 - 17.
Liu X, Zhang L, Feng X, et al. Biosynthesis of glycyrrhetinic acid-3-O-monoglucose using glycosyltransferase UGT73C11 from Barbarea vulgaris . Industrial & Engineering Chemistry Research. 2017;56 (51):14949-14958. DOI: 10.1021/acs.iecr.7b03391 - 18.
Endale M, Lee WM, Kamruzzaman SM, et al. Ginsenoside-Rp1 inhibits platelet activation and thrombus formation via impaired glycoprotein VI signalling pathway, tyrosine phosphorylation and MAPK activation. British Journal of Pharmacology. 2012; 167 (1):109-127 - 19.
Han JY, Kim HJ, Kwon YS, et al. The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng . Plant & Cell Physiology. 2011;52 (12):2062-2073. DOI: 10.1093/pcp/pcr150 - 20.
Han JY, Kim MJ, Ban YW, et al. The involvement of β-amyrin 28-oxidase (CYP716A52v2) in oleanane-type ginsenoside biosynthesis in Panax ginseng . Plant & Cell Physiology. 2013;54 (12):2034-2046 - 21.
Dai Z, Liu Y, Zhang X, et al. Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metabolic Engineering. 2013;20 (5):146-156 - 22.
Suk-Chae J, Woohyun K, Sung Chul P, et al. Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg3 and Rd. Plant & Cell Physiology. 2014; 55 (12):2177 - 23.
Wei W, Wang P, Wei Y, et al. Characterizations of Panax ginseng UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive ginsenosides F1 and Rh1 in metabolically engineered yeasts. Molecular Plant. 2015;8 (9):1412-1424 - 24.
Quan LH, Min JW, Yang DU, et al. Enzymatic biotransformation of ginsenoside Rb1 to 20()-Rg3 by recombinant β-glucosidase from Microbacterium esteraromaticum. Applied Microbiology and Biotechnology. 2012; 94 (2):377-384 - 25.
Chen J, Wu H, Wang Q , et al. Ginsenoside metabolite compound K alleviates adjuvant-induced arthritis by suppressing T cell activation. Inflammation. 2014; 37 (5):1608-1615 - 26.
Xing Y, Yun F, Wei W, et al. Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Research. 2014; 24 (6):770-773 - 27.
Liang H, Hu Z, Zhang T, et al. Production of a bioactive unnatural ginsenoside by metabolically engineered yeasts based on a new UDP-glycosyltransferase from Bacillus subtilis . Metabolic Engineering. 2017;44 :60 - 28.
Itkin M, Davidovich-Rikanati R, Cohen S, et al. The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii. Proceedings of the National Academy of Sciences of the United States of America. 2016; 113 (47):E7619 - 29.
Aoyagi H, Kobayashi Y, Yamada K, et al. Efficient production of saikosaponins in Bupleurum falcatum root fragments combined with signal transducers. Applied Microbiology and Biotechnology. 2001;57 (4):482-488 - 30.
Tessa M, Jacob P, Lorena A, et al. Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase fromBupleurum falcatum . Proceedings of the National Academy of Sciences of the United States of America. 2014;111 (4):1634-1639 - 31.
Ma T, Shi B, Ye Z, et al. Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene. Metabolic Engineering. 2019;52 :134-142. DOI: 10.1016/j.ymben.2018.11.009 - 32.
Fiallos-Jurado J, Pollier J, Moses T, et al. Saponin determination, expression analysis and functional characterization of saponin biosynthetic genes in Chenopodium quinoa leaves. Plant Science. 2016;250 :188-197 - 33.
Moses T, Pollier J, Faizal A, et al. Unraveling the triterpenoid saponin biosynthesis of the African shrub Maesa lanceolata . Molecular Plant. 2015;8 (1):122-135. DOI: 10.1016/j.molp.2014.11.004 - 34.
Fukushima EO, Seki H, Sawai S, et al. Combinatorial biosynthesis of legume natural and rare triterpenoids in engineered yeast. Plant & Cell Physiology. 2013; 54 (5):740-749. DOI: 10.1093/pcp/pct015 - 35.
Dai L, Li J, Yang J, et al. Use of a promiscuous glycosyltransferase from Bacillus subtilis 168 for the enzymatic synthesis of novel protopanaxatriol-type ginsenosides. Journal of Agricultural and Food Chemistry. 2018;66 (4):943-949 - 36.
Nybo SE, Saunders J, Mccormick SP. Metabolic engineering of Escherichia coli for production of valerenadiene. Journal of Biotechnology. 2017;262 :60-66 - 37.
Lv X, Wang F, Zhou P, et al. Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae . Nature Communications. 2016;7 :12851. DOI: 10.1038/ncomms12851 - 38.
Zhubo D, Yi L, Luqi H, et al. Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae . Biotechnology and Bioengineering. 2012;109 (11):2845-2853 - 39.
Koch B, Schacher G, Inc HLR. Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode. Metabolic Engineering. 2012;14 (2):91-103 - 40.
Peng B, Nielsen LK, Kampranis SC, et al. Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae . Metabolic Engineering. 2018;47 :83-93 - 41.
Pingping Z, Lidan Y, Wenping X, et al. Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt. Applied Microbiology and Biotechnology. 2015;99 (20):8419-8428 - 42.
Bhataya A, Schmidt-Dannert C, Lee PC. Metabolic engineering of Pichia pastoris X-33 for lycopene production. Process Biochemistry. 2009; 44 (10):1095-1102