Biosynthesis of Plant Triterpenoid Saponins in Microbial Cell Factories

Review Cite This: J. Agric. Food Chem. 2018, 66, 12155−12165

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Biosynthesis of Plant Triterpenoid Saponins in Microbial Cell Factories Yu-jia Zhao and Chun Li*

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Institute for Synthetic Biosystem, Department of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China ABSTRACT: Triterpenoid saponins are triterpenoid glycoside compounds which have been widely used in pharmaceutical, agricultural, and food industries. Traditionally, they are extracted from plants, which is time-consuming and environmentally unfriendly. Recently, de novo synthesis of triterpenoid saponins in microbial cell factories was realized, which provides a promising and green approach to alter the traditional supply way. However, the complex biosynthetic pathway and the poor suitability between the endogenous and heterogeneous pathways tremendously limit the yield of triterpenoid saponins. We introduce the biosynthetic pathways of triterpenoid saponins first, and we then summarize the microbial cell factories developed to produce these compounds. Further, we discuss the strategies applied to enhance the production. This paper systematically illustrates the biosynthesis of plant triterpenoid saponins in microbial cell factories. KEYWORDS: triterpenoid saponins, cytochrome P450 monooxygenase, glycosyltransferase, microbial cell factories, biomanufacturing

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oleanolic acid,17,18 betulinic acid,19 glycyrrhetinic acid,20 and β-carotene.21,22 The high production of oleanolic acid (606.9 ± 9.1 mg/L) and ginsenoside Rh2 (∼300 mg/L) have highlighted the commercial feasibility of this approach.12,17 Producing triterpenoid saponins by microbial manufacturing has been considered as a promising approach to substitute the traditional manner of supply. Although triterpenoid saponins have been successfully synthesized in microbial hosts, there are still tough challenges to boost the production. For example, most triterpenoid saponins biosynthetic pathways are unclear, and some key enzymes from plants are hard to express in the microbial host; metabolic flux through the heterogeneous pathway is generally low; and some triterpenoid saponins exhibit toxicity to the microbial cell. In order to solve these key issues, multiple metabolic engineering strategies and novel tools have been employed, such as protein engineering, morphology engineering, dynamic regulation, subcellular compartmentation, and so on. In this paper, we systematically illustrate the decoded biosynthetic pathway of triterpenoid saponins, especially the decoded cytochrome P450 monooxygenases (CYP450s) and uridine diphosphate glucuronosyltransferases (UGTs). We then briefly summarize the microbial cell factories which have been developed to produce triterpenoid saponins. Furthermore, novel strategies and tools used to boost triterpenoid saponins production are discussed.

INTRODUCTION Triterpenoid saponins are glycoside compounds which consist of a hydrophobic triterpenoid aglycone and one or more hydrophilic sugar moieties.1 As secondary metabolites of plants, triterpenoid saponins take part in the regulation of plant communication, defense, and sense.2 They act as herbicides and pesticides in the agricultural industry. For example, oleanane saponins isolated from Bellis sylvestris exhibit strong phytotoxic activity against Aegilops geniculate, and saponins from alfalfa can cause a decrease of food metabolized by Tenebrio molitor.3,4 Generally, these molecules are considered as the major bioactive constituents of some traditional herbal medicines, such as ginsenosides, saikosaponins, and glycyrrhizin, used as pharmaceuticals and nutraceuticals by human for thousands of years.5 Some triterpenoid saponins impart a strong sweetness; for instance, glycyrrhetinic acid monoglucuronide and mogroside V are approximately 941-fold and 300fold that of sucrose, respectively.6,7 These sweet-tasting compounds are used in the food industry. As a kind of amphiphilic compounds, triterpenoid saponins can form stable soap-like foams in aqueous solutions and be used in the detergent and cosmetic industry.8 Triterpenoid saponins are mainly extracted from plants and generally need a long cultivation period, but they are still produced with a low yield.9 Therefore, it is a time-consuming, labor-intensive, and environmentally unfriendly way to supply these compounds. Thus, there is a conflict between the huge market demand and the inefficient supply. Thus, it is of great significance and potential to develop novel approaches to replace the traditional supply way. Compared with plants, microbes exhibit many advantages, including fast growth, land saving, and large-scale fermentation.10 Recently, with the development of sequencing technology and synthetic biology, some triterpenoid saponins and aglycones have been successfully synthesized in microbial cell factories, including ginsenosides,11−13 β-amyrin,14−16 © 2018 American Chemical Society

Received: Revised: Accepted: Published: 12155

August 27, 2018 October 30, 2018 November 2, 2018 November 2, 2018 DOI: 10.1021/acs.jafc.8b04657 J. Agric. Food Chem. 2018, 66, 12155−12165

Review

Journal of Agricultural and Food Chemistry

Figure 1. Scheme for triterpenoid saponins biosynthetic pathways. Abbreviations: OSC, oxidosqualene cyclase; CYP450, cytochrome P450 monooxygenase; UGT, uridine diphosphate glycosyltransferase; IPP, 3-isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate.

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DECODING SYNTHETIC PATHWAYS OF TRITERPENOID SAPONINS Triterpenoid saponins biosynthetic pathways are shown in Figure 1. First, the five-carbon building blocks, 3-isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), were synthesized through mevalonic acid (MVA) or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.23 Then, six building blocks were condensed to synthesize 2,3oxidosqualene. Subsequently, 2,3-oxidosqualene was cyclized to polycyclic triterpenoid skeletons by oxidosqualene cyclases (OSCs). These molecules were further oxidized to aglycones by CYP450s. Finally, the aglycones were glycosylated to triterpenoid saponins by UGTs. Cyclization by OSCs. Cyclization of 2,3-oxidosqualene was the first key step in biosynthesis of triterpenoid saponins. In this reaction, internal bonds were introduced into 2,3oxidosqualene backbone to form polycyclic molecules.24 More than 100 kinds of triterpenoid skeletons can be generated during cyclization due to the multiple possible combination of internal bonds. However, only a few cyclization products were further oxidized by CYP450s.25 In addition, OSCs in plants usually generate the products of different conformation. For example, amyrin synthase from Glycyrrhiza uralensis can produce not only α-amyrin but also β-amyrin.26 This was considered as a part of plant defense.27 Oxidization by CYP450s. The polycyclic triterpenoid skeletons were further oxidized by CYP450s, which introduced an oxygen atom into the specific site of their substrates to primarily form hydroxyl, carboxyl, or epoxy groups.28 The CYP450s decoding process was complicated. First, the specific CYP450 should be isolated from plant or expressed in heterogeneous hosts such as Escherichia coli and Saccharomyces cerevisiae. Then, the reaction to the target substrate and the purified CYP450 should be done in vitro. Finally, the products

of in vitro reaction need to be detected by chromatography, mass spectrum, and NMR. To date, only 48 CYP450s that are involved in the biosynthesis of triterpenoid saponins have been identified. They were summarized in Table 1. Glycosylation by UGTs. Glycosylation is the last step of triterpenoid saponins biosynthesis that links one or more hydrophilic sugar moieties to the hydrophobic aglycone by UGTs. The introduced hydroxy- and carboxy-moieties from sugar moieties can improve the bioactivities of triterpenoid saponins. Triterpenoid saponins glycosylation patterns usually link sugar chains at the positions C-3 and/or C-28. In some cases, it occurs at the positions C-4, C-16, C-20, C-21, C-22, and/or C-23.52,53 In addition, the introduced monosaccharide units were generally glucose, glucuronic acid, galactose, rhamnose, xylose, and arabinose. In view of the tremendous amounts of UGTs in plants, more than 120 genes encoding family 1 UGTs have been identified in Arabidopsis thaliana genome.51 Decoding the specific UGT involved in target triterpenoid saponins biosynthesis has been very difficult. So far, only 23 UGTs that are involved in triterpenoid saponins biosynthesishave been identified. They were summarized in Table 2.

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DE NOVO BIOSYNTHESIS OF TRITERPENOID SAPONINS IN MICROBIAL CELL FACTORIES With the decoding of plant biosynthetic pathways, some triterpenoid saponins have been successfully synthesized in microbial cell factories and the high production of ginsenoside Rh2 (∼300 mg/L) has highlighted the commercial feasibility of this approach. In the following, we systematically describe the biosynthetic pathways and the cell-factory construction methods for ginsenosides, saikosaponins, and mogrosides which are typical triterpenoid saponins produced in microorganisms. 12156

DOI: 10.1021/acs.jafc.8b04657 J. Agric. Food Chem. 2018, 66, 12155−12165

Review

Journal of Agricultural and Food Chemistry Table 1. Overview of Plant CYP450s in Biosynthesis of Triterpenoid Saponins no.

name

accession number

plant species

substrate

loci

ref

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

CYP51H10 CYP71D353 CYP72A61v2 CYP72A63 CYP72A67 CYP72A68v2 CYP72A69 CYP72A154 CYP87D16 CYP88D6 CYP93E1 CYP93E2 CYP93E3 CYP93E4 CYP93E5 CYP93E6 CYP93E7 CYP93E8 CYP93E9 CYP716A12 CYP716A14v2 CYP716A15 CYP716A17 CYP716A44 CYP716A46 CYP716A47 CYP716A52v2 CYP716A53v2 CYP716A75 CYP716A78 CYP716A79 CYP716A80 CYP716A81 CYP716A83 CYP716A86 CYP716A140 CYP716A141 CYP716A180 CYP716A244 CYP716A254 CYP716AL1 CYP716C11 CYP716E41 CYP716E22 CYP716S5 CYP716Y1 CYP716A1 CYP716A2

ABG88965.1 AHB62239.1 BAL45199.1 BAL45200.1 ABC59075.1 BAL45204.1 BAW35014.1 BAL45207.1 AHF22090.1 AQQ13664.1 BAE94181.1 ABC59085.1 BAG68930.1 AIN25416.1 AIN25417.1 AIN25418.1 AIN25419.1 AIN25420.1 AIN25421.1 ABC59076.1 AHF22083.1 BAJ84106.1 BAJ84107.1 AEY75213.1 AFO63032.1 AFO63031.1 AHF22088.1 ANY30853.1 ANY30854.1 ALR73782.1 ALR73781.1 AOG74832.1 AOG74831.1 AOG74836.1 AOG74838.1 APZ88353.1 AEX07773.1 AOG74835.1 AOG74834.1 AOG74839.1 AHF45909.1 AED94045.1 BAU61505.1

Avena strigosa Lotus japonicus Medicago truncatula Medicago truncatula Medicago truncatula Medicago truncatula Glycine max Glycyrrhiza uralensis Maesa lanceolata Glycyrrhiza uralensis Glycine max Medicago truncatula Glycyrrhiza uralensis Arachis hypogaea Cicer arietinum Glycyrrhiza glabra Lens culinaris Pisum sativum Phaseolus vulgaris Medicago truncatula Artemisia annua Vitis vinifera Vitis vinifera Solanum lycopersicum Solanum lycopersicum Panax ginseng Panax ginseng Panax ginseng Maesa lanceolata Chenopodium quinoa Chenopodium quinoa Barbarea vulgaris Barbarea vulgaris Centella asiatica Centella asiatica Platycodon grandiflorus Platycodon grandiflorus Betula platyphylla Eleutherococcus senticosus Anemone flaccida Catharanthus roseus Centella asiatica Centella asiatica Solanum lycopersicum Platycodon grandiflorus Bupleurum falcatum Arabidopsis thaliana Arabidopsis thaliana

β-amyrin lupeol 24-OH-β-amyrin β-amyrin oleanolic acid oleanolic acid β-amyrin β-amyrin β-amyrin β-amyrin β-amyrin β-amyrin β-amyrin β-amyrin β-amyrin β-amyrin β-amyrin β-amyrin β-amyrin α-amyrin, β-amyrin, lupeol α-amyrin, β-amyrin α-amyrin, β-amyrin, lupeol α-amyrin, β-amyrin, lupeol α-amyrin, β-amyrin α-amyrin, β-amyrin dammarenediol-II β-amyrin dammarenediol-II β-amyrin α-amyrin, β-amyrin, lupeol α-amyrin, β-amyrin, lupeol α-amyrin, β-amyrin, lupeol α-amyrin, β-amyrin, lupeol β-amyrin β-amyrin β-amyrin, 24-OH-β-amyrin β-amyrin, 24-OH-β-amyrin lupeol β-amyrin β-amyrin α-amyrin, β-amyrin, lupeol oleanolic acid maslinic acid α-amyrin, β-amyrin β-amyrin, oleanolic acid α-amyrin, β-amyrin β-amyrin α-amyrin

C-12, 13, 16β C-20 C-23 C-30 C-2 C-25 C-21 C-30 C-16α C-11 C-24 C-24 C-24 C-24 C-24 C-24 C-24 C-24 C-24 C-28 C-3 C-28 C-28 C-28 C-28 C-12 C-28 C-20 C-28 C-28 C-28 C-28 C-28 C-28 C-28 C-28 C-28 C-28 C-28 C-28 C-28 C-2 C-6 C-6 C-12, 13 C-16α C-28 C-22

29 26 20 31 32 33 33 31 34 35 36 30 35 37 37 37 37 37 37 37 39 38 38 40 40 41 42 43,74

34 44 44 45 45 46 47 47 47 21 47 48 49 46 46 41 46 50 51 51

Protopanaxadiol (PPD) was the first ginsenoside aglycone produced by microbial manufacturing. It was synthesized through the coexpression of P. ginseng dammarenediol-II synthase (PgDS), P. ginseng CYP716A47, and A. thaliana CYP450 reductase 1 (AtCPR1) in S. cerevisiae.41 Subsequently, protopanaxatriol (PPT), another dammarane-type ginsenosides aglycone, was produced by feeding PPD to the engineered yeast, which coexpresses P. ginseng CYP716A53v2 and AtCPR1.74 Soon after that, oleanolic acid (OA), the oleanane-type ginsenosides aglycone, was synthesized by coexpressing of P. ginseng β-amyrin synthase (PNY1),

Ginsenosides. Ginsenosides are one of the most famous triterpenoid saponins which are the major bioactive ingredients of Panax plants. Ginsenosides exhibit great pharmaceutical value.71−73 De novo synthesis of ginsenosides in microbial cell factories has been a hot spot in recent years. To date, three aglycones (protopanaxadiol, protopanaxatriol, and oleanolic acid), two unnatural ginsenosides (compound K and 3β, 12βDi-O-Glc-PPD), and four natural ginsenosides (Rh2, Rg3, RF1, and Rh1) have been successfully biosynthesized in S. cerevisiae (Figure 2). 12157


Review

Journal of Agricultural and Food Chemistry Table 2. Overview of Plant UGTs in Biosynthesis of Triterpenoid Saponins no.

name

accession number

plant species

substrate

loci

ref

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

UGT71G1 UGT73AD1 UGT73AE1 UGT73AH1 UGT73C10 UGT73C11 UGT73C12 UGT73C13 UGT73F2 UGT73F3 UGT73F4 UGT73F17 UGT73K1 UGT74AE2 UGT74M1 UGT94Q2 UGTPg1 UGTPg100 UGTPg101 Pg3-O-UGT1 GmSGT2 GmSGT3 UDPG

AAW56092.1 ALD84259.1 AJT58578.1 AUR26623.1 AFN26666.1 AFN26667.1 AFN26668.1 AFN26669.1 BAM29362.1 ACT34898.1 BAM29363.1 AXS75258. AAW56091.1 ABK76266.1 BAI99584.1 BAI99585.1 -

Medicago truncatula Centella asiatica Carthamus tinctorius Centella asiatica Barbarea vulgaris Barbarea vulgaris Barbarea vulgaris Barbarea vulgaris Glycine max Medicago truncatula Glycine max Glycyrrhiza uralensis Medicago truncatula Panax quinquefolium Vaccaria hispanica Panax quinquefolium Panax ginseng Panax ginseng Panax ginseng Panax quinquefolius Glycine max Glycine max Panax ginseng

medicagenic acid, UDP-glucose asiatic acid, madecassic acid, UDP-glucose glycyrrhetinic acid, UDP-glucose asiatic acid, UDP-glucose hederagenin, oleanolic acid, UDP-glucose glycyrrhetinic acid, oleanolic acid, UDP-glucose hederagenin, oleanolic acid, UDP-glucose hederagenin, oleanolic acid, UDP-glucose saponin A0-gα, UDP-xylose hederagenin, UDP-glucose saponin A0-gα, UDP-xylose glycyrrhizin, UDP-glucose hederagenin, soyasapogenols B and E, UDP-glucose protopanaxadiol, UDP-glucose gypsogenic acid, UDP-glucose ginsenoside Rh2, UDP-glucose protopanaxadiol, UDP-glucose ginsenoside RF1, protopanaxatriol, UDP-glucose ginsenoside RF1, protopanaxatriol, UDP-glucose protopanaxadiol, UDP-glucose soyasapogenol B monoglucuronide, UDP-galactose soyasaponin III, UDP-rhamnose ginsenoside Rd, UDP-glucose

C-3, 28 C-28 C-3 C-28 C-3 C-3 C-3 C-3 C-22 C-28 C-22 C-30 C-3, 28 C-3 C-28 C-3 C-3 C-6 C-6, 20 C-3 C-3 C-3

54 55 56 57 58 58,59

58 58 60 61 60 62 63 64 65 66 63 67 67 68 69 69 70

Figure 2. Scheme for ginsenosides biosynthesized in S. cerevisiae. The green arrows represent OSCs, the yellow arrows represent CYP450s, and the blue arrows represent UGTs.

CYP716A52v2 and AtCPR1 in S. cerevisiae.42 Although these three ginsenoside aglycones have been successfully synthesized in yeast, the yield was still not satisfactory. To increase PPD production, the truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMG1), farnesyl diphosphate synthase (ERG20), squalene synthase (ERG9), and 2,3-oxidosqualene synthase (ERG1) from S. cerevisiae have been overexpressed. In addition, the gene encoding CYP716A53v2 from P. ginseng has been codon optimized. On the basis of these strategies, the production of PPD was increased by 262-fold and up to nearly 1.2 g/L, which provided an efficient platform for further biosynthesis of ginsenosides.75 Similar strategies were used to

improve PPT production by overexpressing endogenous genes encoding tHMG1, ERG9, and ERG1 combined with optimization of the codons of heterogeneous genes. As a result, 17.2 mg/L PPD, 15.9 mg/L PPT, and 21.4 mg/L OA were achieved in engineered S. cerevisiae.18 Compound K (CK) is an unnatural ginsenoside which is considered as the metabolite of glycosidases.76 CK is the main functional compound of oral administration of ginseng.77 It was synthesized through coexpressing PgDS, CYP716A47, AtCPR2, and UGTPg1 in S. cerevisiae. Further optimization was conducted by (1) overexpressing tHMG1 and UPC2.1 and (2) inducing heterogeneous genes expression via adding 12158


Review


Figure 3. Scheme for strategies used to advance biosynthesis of triterpenoid saponins in microbial hosts. (a) Strategies to design triterpenoid saponins biosynthetic pathways; (b) Strategies to improve plant enzyme activity in microbial hosts; (c) Strategies to enhance metabolic flux in microbial hosts; (d) Strategies to reduce toxicity to the hosts.

galactose. On the basis of these strategies, the production of CK was up to 1.4 mg/L.63 Another unnatural ginsenoside

3β,12β-Di-O-Glc-PPD was produced by coexpression of PgDS, CYP716A47, AtCPR1 and UGT109A1 from B. subtilis in S. 12159


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Journal of Agricultural and Food Chemistry cerevisiae. Further optimization was carried out by (1) replacing the endogenous gene encoding lanosterol synthase to an antisense one, (2) overexpressing tHMG1, and (3) fusion expression PgDS in S. cerevisiae. Thus, the production of 3β,12β-Di-O-Glc-PPD was increased up to 9.05 mg/L.13 In addition to the unnatural ginsenosides, Rh2 and Rg3 are PPD-derived natural ginsenosides which have been successfully biosynthesized in S. cerevisiae. By coexpressing PgDS, CYP716A47, AtCPR2, PgUGT74AE2, and PgUGT94Q2, and by replacing the native promoter of lanosterol synthase gene to a methionine-repressible promoter (MET3), the production of Rg3 reached 1.3 mg/L.66 PPT-derived ginsenosides, RF1 and Rh1, were synthesized with the coexpression of PgDS, CYP716A47, CYP716A53v2, PgCPR1, and UGTPg1 (or UGTPg100) in S. cerevisiae. The production of RF1 and Rh1 reached 42.1 and 92.8 mg/L, respectively.67 Saikosaponins. Bupleurum falcatum is an important perennial herb widely used in traditional Chinese medicine to treat cold, fever and inflammatory disorders.78 Saikosaponins are the major pharmaceutical constituents of B. falcatum which exert multiple bioactivities.79,80 Currently, two saikosaponins aglycones, 16α-hydroxy α-amyrin and 16α-hydroxy βamyrin, have been synthesized in engineered S. cerevisiae. These compounds were synthesized by coexpressing CYP716Y1, AtCTR1, CaDDS (dammarenediol synthase from Centella asiatica) or GgbAS (β-amyrin synthase from Glycyrrhiza glabra) in S. cerevisiae, respectively.50 Mogrosides. Siraitia grosvenorii, also called “luo han guo”, is a perennial plant which grows in southern China natively.81 Mogrosides are cucurbitane-type triterpenoid saponins extracted from S. grosvenorii, which are the major bioactive constituents of S. grosvenorii. Among them, mogroside V is nearly 300 times sweeter than sucrose, and it acts as a food additive in low-calorie sweet beverages.7 Based on the transcriptome analysis of S. grosvenorii, genes encoding key enzymes involved in the biosynthesis pathway of mogroside V have been identified, including squalene synthase (SQS), squalene epoxidase (SQE), cycloartenol synthase (CAS), epoxide hydrolases (EPH), CYP102801, UGT94-289-3, and UGT720-269-1.7 By introducing AtCPR1 with these enzymes in S. cerevisiae, mogroside V has been successfully synthesized.7 These advances highlight the possibility to decrease the cost of producing natural, noncaloric sweetener mogrosides.

conditions. These increase the difficulty to elucidate biosynthetic pathways for triterpenoid saponins. Thanks to sequencing technology, the genomic and transcriptome information on many medicinal plants were decoded and published online (http:// medicinalplantgenomics.msu.edu). The sequencing information helps to predict genes involved in triterpenoid saponins biosynthesis. Key genes can be identified by comparing the transcriptome information between high- and low-producing plants or tissues. For example, genes involved in quinoa saponins biosynthesis were discovered through comparative transcriptome profiling of high- and low-producing varieties. Subsequently, the function of bAS1, CYP716A78, and CYP716A79 from Chenopodium quinoa were identified by expressing these genes in yeast.44 Another case, genes take part in maesasaponins biosynthesis were discovered by comparing the transcriptome profiling between methyl jasmonate and ethyl alcohol elicited conditions. Then, the function of bAS, CYP716A75 and CYP87D16 from Maesa lanceolata were identified by expressing these genes in yeast.34 On the basis of comparing the transcriptome information between different varieties or elicited conditions, key genes involved in triterpenoid saponins biosynthesis can be predicted and further identified by heterologous expressed in microbes. Generally, the expression patterns of genes involved in one biological process are strongly correlated. Thus, gene coexpression analysis was applied to predict genes function. On the basis of this approach, CYP716A12, CYP93E2, CYP72A61v2, and CYP72A68v2 from M. truncatula have been identified.30 In addition to the substrate-specificity enzymes, some substrate-promiscuous enzymes were used to reconstruct the triterpenoid saponins biosynthetic pathways. For example, ginsenoside Rh1 and four unnatural ginsenosides were synthesized by Bs-YjiC, a substrate-promiscuous glycosyltransferase from Bacillus subtilis 168.82,83 UDP-glycosyltransferase UGT109A1 from B. subtilis is another substrate-promiscuous enzyme, which was used to produce the unnatural ginsenosides 3β-O-Glc-DM, 3β,20S-Di-O-Glc-DM, 3β,12β-Di-O-Glc-PPD, and 3β,12β-Di-O-Glc-PPT.13 The poor enzyme activity on the unnatural substrate led to low production of the synthesized compounds. Protein engineering improves the “substrate specificity” of the substrate-promiscuous enzymes migrates from natural to unnatural substrates. UGT51, a UDP-glucose: sterol glucosyltransferase from S. cerevisiae, is a substrate-promiscuous enzyme which can glycosylate not only ergosterol but also a series of analogues, including cholesterol, sitosterol, estradiol, pregnenolone, diosgenin, PPD, and PPT. On the basis of this characteristic, UGT51 has been used to produce ginsenosides Rh2 with coexpression of PgDS, CYP716A47 and AtCPR1 in S. cerevisiae, unfortunately only a trace amount of Rh2 was synthesized. On the basis of the crystal structure of UGT51 (PDB code: 5gl5), a semirational design strategy was introduced to improve enzyme activity. The best UGT51 mutant presented an 1800-fold enhanced catalytic efficiency (kcat/Km) in converting PPD to ginsenoside Rh2 in vitro, and the Rh2 production reached up to 300 mg/L in vivo.12 Using substrate-promiscuous enzymes to biosynthesize the triterpenoid saponins is an efficient approach to construct triterpenoid saponins biosynthetic pathways in microbes. Strategies To Improve Plant Enzyme Activity in Microbial Hosts. Plant enzymes that are heterogeneously expressed in microbial hosts usually exhibited incorrect folding

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STRATEGIES FOR BOOSTING BIOSYNTHESIS OF TRITERPENOID SAPONINS Currently, main challenges about microbial manufacturing are (1) the biosynthetic pathways of most triterpenoid saponins are not fully elucidated; (2) some key enzymes from plants often exhibit no activity or poor activity in microbial hosts; (3) metabolic flux throughout the heterogeneous pathways is generally low in microbial hosts; (4) most of triterpenoid saponins exhibit cytotoxicity to the microbial hosts. Strategies and tools contribute to settle these issues and facilitate the microbial biosynthesis of triterpenoid saponins have been developed based on omics, protein engineering, metabolic flow dynamic regulation, and compartmentalization (Figure 3). Strategies To Design Triterpenoid Saponins Biosynthetic Pathways. In plants, genes encoding enzymes which take part in triterpenoid saponins biosynthesis are distributed among the whole genome rather than in a gene cluster. In addition, they are generally expressed under intricate inducible 12160


Review


Pushing more precursors to the heterogeneous pathway is an effective strategy to enhance terpenoids production. IPP and DMAPP, the five-carbon building blocks of terpenoids, are naturally synthesized through either eukaryotic MVA or prokaryotic MEP pathway. In MVA pathway, IPP and DMAPP are synthesized through acetyl-coenzyme A (acetylCoA), whereas in the MEP pathway, they are produced via glyceraldehyde-3-phosphate and pyruvate. To combine MVA and MEP pathways into one host can effectively supply precursors of terpenoids. By expressing the heterogeneous MVA pathway, Keasling has produced amorphadiene in E. coli, with a yield of 27.0 g/L.88 The synergy of the MEP pathway and the MVA pathway also successfully increased the isoprene (24.0 g/L),89 valerenadiene (62.0 mg/L)84 and lycopene (47.0 mg/L)89 in E. coli. In addition to synergy of the two pathways in E. coli, Yu has established dual MVA pathways simultaneously act in cytoplasmic and mitochondrial of yeast to improve the utilization of acetyl-CoA and achieved 2.5 mg/ L isoprene.90 Furthermore, efforts have been exerted toward the combination of MVA and MEP pathway in yeast.91,92 Besides, catalyzes the conversion of HMG-CoA to mevalonate by HMG-CoA reductase is a rate-limiting step in the MVA pathway. Overexpressing this rate-limiting enzyme (tHMG1) is one of the most commonly used methods for bioengineering of terpenoids in yeast.84 Another commonly used method is overexpressing UPC2−1, which is a sterol regulatory element binding protein to induce sterol biosynthetic genes expression.93 Combined MVA and MEP pathways, overexpressed key enzymes and transcriptional elements can push more precursors to the heterogeneous pathway. Pulling more precursors from the competing endogenous pathways is another effective strategy to enhance terpenoids production. However, most enzymes of the competing endogenous pathways are essential to the hosts. Thus, decreasing the concentration of key enzyme in the critical process to partially redirect the metabolic flux is an effective method to enhance the terpenoids production. The cellular protein concentration is regulated by transcription, RNA degradation, translation and protein degradation. Promoter replacement is commonly used to decrease the transcriptional level of key genes. The methionine repressible promoter (PMET3) was used to replace the native promoter of lanosterol synthase gene (ERG7) to redirect the metabolic flux from ergosterol pathway to the heterogeneous β-amyrin pathway.47 Additionally, the copper repressible promoter (PCRT3) and low concentration glucose repressible promoter (PHXT1) were used to replace the native promoter of squalene synthase gene (ERG9) to redirect metabolic flux from native ergosterol pathway to the heterogeneous α-santalene pathway.94 Recently, protein destabilization was developed to reduce functional enzyme concentration. The cytosolic proteins can be destabilized by attaching a PEST sequence, an N-terminal degron or a C-terminal degron. Based on ER-associated protein degradation system, the G1 cyclin PEST sequence was used to label the cytosolic end of squalene synthase to degrade the squalene synthase, which enhanced sesquiterpene transnerolidol production.95 Furthermore, based on N-end rule pathway, a designed N-terminal degron was fused to the Nterminus of the farnesyl pyrophosphate synthetase to degrade the farnesyl pyrophosphate synthetase, which enhanced monoterpene linalool titer.96 Strategies To Reduce Toxicity to the Hosts. Most terpenoids exhibit toxic to the microbial hosts, which can

structure, low expression, and poor catalytic efficiency, especially CYP450s. Plant CYP450s are key enzymes involved in biosynthesis of triterpenoid saponin . As membrane-bound oxidoreductase enzymes, CYP450s are anchored in the endoplasmic reticulum (ER) inner-membrane of plants and require pairing with CPRs, which transfers an electron from NADPH to CYP450s. Improving the enzyme activities of heterogeneous expressed plant CYP450s is a huge challenge to establish high-yield microbial cell factories for triterpenoid saponins. Currently, massive efforts have been done to correct folding, improve expression level, and regulate the pairing ratio to CPRs of plant CYP450s in microbial hosts. Codon optimization is one of the most common strategies for improving enzyme expression level in heterologous hosts, and it is also effectively used to improve plant CYP450s expression level in microbial hosts.84 Improved transcription efficiency of CYP450s by applying a strong promoter is another common strategy.14 Protein fusion expression has been used to correct folding of plant CYP450s in microbial hosts.80 Protein-directed evolution was also applied to enhance the activity of plant enzymes.85 As plant CYP450s are anchored in the ER membrane, the activity can be improved by replacing the native N-terminal sequence with ERmembrane-bound proteins of yeast to facilitate correct folding and anchoring.86 Besides the traditional regulatory strategies, morphology engineering is also applicable in this field.87 Because plant CYP450s and CPRs are both anchored in the ER membrane, enlarged intracellular ER membranes can supply more accommodation for ER-localized CYP450s and CPRs. Gene PAH1 encoding phosphatidic acid phosphatase has been knocked out to tremendously expand the ER membranes of S. cerevisiae, which stimulated the expression of CYP716A12, CYP72A67, and CYP72A68, and ultimately resulted in the increased accumulation of triterpenoid and triterpenoid saponins.87 Approaches like codon optimization, protein fusion expression, protein-directed evolution, and ER enlargement have been used to improve CYP450s expression level in heterologous hosts. To regulate the target CYP450 with different CPRs is an efficient strategy to improve the pairing efficiency. For example, the combination between CYP716A12 and M. truncatula CPR have been proved to be the most efficient among CPRs from A. thaliana, Lotus japonicus, and G. uralensis that boost OA production in yeast.17 In addition, the combination between CYP88D6/CYP72A63 and G. uralensis CPR has been proved to be the most efficient among CPRs from A. thaliana, M. truncatula, L. japonicus, and G. uralensis that boost GA production in yeast.20 Besides, to regulate the ratios between one combination of CYP450 and CPR is another strategy to improve the pairing efficiency. Experimental results have verified that the best ratio between CYP716Y1 and CPR1 from A. thaliana was 5−9, which increases 16-α-hydroxy amyrin production.50 To improve the pairing efficiency and pairing ratio between CYP450 and CPR can improve triterpenoids production in microbes. Strategies To Enhance Metabolic Flux in Microbial Hosts. The biosynthetic pathways of plant triterpenoid saponins in the microbial host includes multiple steps and interacts with the host’s metabolic network in many ways, including cosubstrates, cofactors, and metabolites that reverse influence. Thus, balancing metabolic flux between heterologous and endogenous metabolic pathways plays a key role in enhancing the production. 12161



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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).

influence cell growth and further cause low production. Strategies like two-stage fermentation, subcellular compartmentalization, and membrane transporter engineering proved to effectively reduce the toxic effects to the microbial hosts. Two-stage fermentation is a traditional strategy: in the first fermentation stage, cells grow fast and the precursor accumulates, whereas target compounds are induced to produce in the second stage.97 Organelles like mitochondria, peroxisome, and vacuole possess intracellular membrane structure and are relatively independent from the cytoplasm. The intracellular membrane structure can reduce or prevent toxic intermediates and products from invading into the cytoplasm to disrupt cell growth. Furthermore, construction of terpenoids biosynthesis pathways in organelles allows higher concentrations of substrates, intermediates, and enzymes, which can improve the production of terpenoids. For example, the biosynthetic pathway of amorpha-4,11-diene, including eight endogenous enzymes and the heterogeneous amorpha4,11-diene synthase, has been constructed in the mitochondria of yeast in which amorpha-4,11-diene production increased by 63% compared with constructed in the cytosol. Besides, the mitochondrial compartment has proved to be a barrier for the translocation of FPP from mitochondria into the cytosol, which reduced loss of FPP.98 For another example, production of valencene increased 8-fold by constructing its biosynthetic pathway into the mitochondria of yeast.99 Besides, lycopene pathway genes have been expressed into the peroxisome of Pichia pastoris that significantly improved lycopene production up to 73.9 mg/L.100 Thus, the peroxisome was considered as another potential subcellular factory to produce terpenoids. Therefore, subcellular compartmentalization was considered as a pioneering strategy to reduce products cytotoxicity to the microbial hosts. Membrane transporter engineering has been considered to reduce the toxic effects as efflux membrane transporters contribute significantly to transport the product to the extracellular space. AcrAB-TolC efflux pump system has been engineered to improve the efflux of α-pinene in E. coli. By protein engineering of AcrB, one variant can effectively improve the α-pinene efflux.101 Although efflux membrane transporter engineering was proved to reduce the toxic effects of the products, the transport mechanisms and crystal structures of most membrane transporters have not been fully elucidated, which limit its application. In this Review, we systematically summarized the biosynthetic pathways of triterpenoid saponins and introduced microbial cell factories that are constructed to produce these valuable compounds. Although triterpenoid saponins have been successfully synthesized by biomanufacturing, there are still tough challenges in boosting the production. Strategies such as protein engineering, metabolic network dynamic regulation, subcellular localization, and fermentation control were used to increase triterpenoid saponins productivity in microbial cell factories.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Chun Li: 0000-0003-4262-6848 Notes

The authors declare no competing financial interest. 12162


Review

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