Biotransformation of Oleanane and Ursane Triterpenic Acids

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Review Biotransformation of Oleanane and Ursane Triterpenic Acids

Natalia A. Luchnikova 1,2 , Victoria V. Grishko 3 and Irina B. Ivshina 1,2,*

1 Institute of Ecology and Genetics of Microorganisms, Perm Federal Research Center, Ural Branch of the Russian Academy of Sciences, 614081 Perm, Russia; [email protected] 2 Department of Microbiology and Immunology, Perm State National Research University, 614990 Perm, Russia 3 Institute of Technical Chemistry, Perm Federal Research Center, Ural Branch of the Russian Academy of Sciences, 614013 Perm, Russia; [email protected] * Correspondence: [email protected]; Tel.: +7-342-2808114

Academic Editor: Derek J. McPhee Received: 9 October 2020; Accepted: 23 November 2020; Published: 25 November 2020

Abstract: Oleanane and ursane pentacyclic triterpenoids are secondary metabolites of plants found in various climatic zones and regions. This group of compounds is highly attractive due to their diverse biological properties and possible use as intermediates in the synthesis of new pharmacologically promising substances. By now, their antiviral, anti-inﬂammatory, antimicrobial, antitumor, and other activities have been conﬁrmed. In the last decade, methods of microbial synthesis of these compounds and their further biotransformation using microorganisms are gaining much popularity. The present review provides clear evidence that industrial microbiology can be a promising way to obtain valuable pharmacologically active compounds in environmentally friendly conditions without processing huge amounts of plant biomass and using hazardous and expensive chemicals. This review summarizes data on distribution, microbial synthesis, and biological activities of native oleanane and ursane triterpenoids. Much emphasis is put on the processes of microbial transformation of selected oleanane and ursane pentacyclic triterpenoids and on the bioactivity assessment of the obtained derivatives.

Keywords: biological activity; biotransformation; glycyrrhetinic acid; oleanolic acid; ursolic acid

1. Introduction Drugs derived from secondary plant metabolites make up about 25% of the global pharmaceutical market [1]. Secondary metabolites of plants are several groups of compounds; the most numerous (about 25,000 representatives) and diverse group is terpenic hydrocarbons and their oxygen-containing derivatives (terpenoids). Depending on the number of isoprene units (C5H8) in their structure, they contain a certain number of carbon atoms and are classified into mono-(C10), sesqui-(C15), di-(C20), triterpenoids (C30), etc. Naturally occurring triterpenoids are represented by more than 100 various types of skeletons [2]. Native triterpenoids, in particular, oleanane and ursane representatives, are of interest for researchers due to their availability and multiple biological activities, including antimicrobial, anti-inflammatory, antitumor, cytotoxic, hepatoprotective, and other activities [3–7]. Triterpenic molecules, however, are highly hydrophobic which significantly limits their use as effective pharmacological agents. At present, one of the most common ways to increase the effectiveness and bioavailability of triterpenoids is by chemical modification. This usually requires high temperature and pH, use of expensive reagents, and introduction of protective groups of molecule reactive centers [8–11]. An alternative way to obtain valuable derivatives is by biotransformation under normal and environmentally friendly conditions employing the catalytic activity of microorganisms with high regio- and stereoselectivity in one

Molecules 2020, 25, 5526; doi:10.3390/molecules25235526 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 34 Molecules 2020, 25, 5526 2 of 31 [8–11]. An alternative way to obtain valuable derivatives is by biotransformation under normal and environmentally friendly conditions employing the catalytic activity of microorganisms with high technologicalregio- and stereoselectivity stage. Furthermore, in one microbial technological conversion stage. Furthermore, ensures specific microbial modifications conversion of triterpenic ensures moleculespecific modifications sites that are ofeither triterpenic not modified molecule orsites poorly that are modified either not by modified synthetic or transformations poorly modified [12 by]. Notesynthetic that, transformations among the known [12]. microbialNote that, biocatalysts,among the known members microbial of mycelial biocatalysts, fungi are members the most of studiedmycelial[ 13fungi–15] arewhereas the most bacterial studied catalysts [13–15] are whereas only represented bacterial catalysts by a few are gram-positive only represented species by [16 a– few20]. Thegram-positive first papers species on microbial [16–20]. The transformation first papers ofon triterpenoids microbial transformation were published of triterpenoids in the 1960s were [21]. Thepublished earliest in informationthe 1960s [21]. related The earliest to bioconversion information processes related to of bioconversion oleanane derivatives processes catalyzed of oleanane by fungi,derivatives such catalyzed as Curvularia by fungi, lunata suchATCC as 13432,CurvulariaTrichotecium lunata ATCC roseum 13432,ATCC Trichotecium 8685, Cunninghamella roseum ATCCsp. ATCC8685, Cunninghamella 3229, Mucor griseo-cyanus sp. ATCC 3229,ATCC Mucor 1207-a, griseo-cyanusHelicostylum ATCC piriforme 1207-a, HelicostylumATCC 8992, piriformeFusarium ATCC lini, and8992,Cunninghamella Fusarium lini, and blakesleana Cunninghamelladates back blakesleana to about dates the sameback to time about [22– the25]. same The time data [22–25]. on bacterial The transformationdata on bacterial of oleananetransformation triterpenoids of oleanane by Streptomyces triterpenoidssp. G-20by Streptomyces and Chainia sp. antibiotica G-20 andIFO Chainia 12,246 wereantibiotica reported IFO in12,246 the second were reported half of the in1980s the second [26,27]. half As forof microbialthe 1980s transformations[26,27]. As for ofmicrobial ursane pentacyclictransformations triterpenoids of ursane bypentacyclic both fungal triterpenoids (Mucor plumbeus by both ATCCfungal 4740(Mucor [28 plumbeus]) and bacterial ATCC 4740 (Nocardia [28]) sp.and NRRLbacterial 5646 (Nocardia [29]) strains, sp. NRRL those 5646 studies [29]) were strains, initiated those only studies in the were 2000s. initiated Henceforth, only in the the interest 2000s. inHenceforth, the topic discussedthe interest has in beenthe topic increasing discussed and thehas Activebeen increasing Triterpenoid and Biocatalysts the Active ListTriterpenoid has been expandedBiocatalysts every List has year, been as isexpanded the number every of year, various as is bioactive the number triterpenic of various derivatives bioactive formed triterpenic via biotransformationsderivatives formed [via30– biot32].ransformations [30–32]. Now, preparationpreparation of biologicallybiologically active compounds based on pentacyclic triterpenoids is an actual research discussed in plenty of experimental and review publications [8,12,32–34]. [8,12,32–34]. However, However, the reviews are are overwhelmingly overwhelmingly focused focused on on chemical chemical transformations transformations or or describe describe specific specific types types of ofbiological biological activities activities of triterpenoids. of triterpenoids. Less Less frequently, frequently, they they deal dealwith with biological biological transformations. transformations. The Thelatest latest of the of thefew few reviews reviews on onmicrobial microbial transformati transformationsons of of pentacyclic pentacyclic triterpenoids triterpenoids include include literature data upup to to 2016 2016 [12 ,[12,32,33,35].32,33,35]. Our Our review review summarizes summarizes the data the from data 2013 from to the 2013 present to the on distribution,present on microbialdistribution, biosynthesis, microbial biosynthesis, biological activity, biological and ac mainlytivity, biotransformationand mainly biotransformation of oleanane of and oleanane ursane pentacyclicand ursane triterpenoidspentacyclic totriterpenoids obtain promising to obtain biologically promising active biologically compounds active or intermediatescompounds or of theirintermediates synthesis. of their synthesis.

2. Distribution in Nature Plant pentacyclic triterpenoids are represented by more than two dozen structural types; the most common are oleanane and ursane ones (Figure1 1).). InIn freefree forms,forms, triterpenoidstriterpenoids areare nonvolatilenonvolatile lipophilic substances that are soluble in organic solventssolvents andand insolubleinsoluble inin water.water. The mostmost availableavailable oleanane triterpenoids are oleanolic acid 1 (OA, 3β-hydroxy-olean-12-en-28-oic acid, C H O , CAS oleanane triterpenoids are oleanolic acid 1 (OA, 3β-hydroxy-olean-12-en-28-oic acid, C3030H4848O33, CAS 508-02-1) and glycyrrhetinic acid 2 (GA, 3β-hydroxy-11-oxo-18β-olean-12-en-30-oic acid, C H O , 508-02-1) and glycyrrhetinic acid 2 (GA, 3β-hydroxy-11-oxo-18β-olean-12-en-30-oic acid, C3030H4646O4, CAS 471-53-4), and ursolic acid 3 (UA, 3β-hydroxy-urs-12-en-28-oic acid, C H O , CAS 77-52-1) is CAS 471-53-4), and ursolic acid 3 (UA, 3β-hydroxy-urs-12-en-28-oic acid, C3030H4848O3, CAS 77-52-1) is the most available ursane triterpenoid. Biosynthesis of oleanane and ursane pentacyclic triterpenoids in plants occurs occurs by by conversion conversion of of the the acyclic acyclic triterpene triterpene squalene squalene (4) ( 4to) to2,3-oxidosqualene 2,3-oxidosqualene (5) (and5) and its β itsfurther further cyclization cyclization by byspecific specific enzyme enzyme complexes complexes (oxidosqualene (oxidosqualene cyclase) cyclase) via viaβ-amyrin-amyrin (6) (or6) α or- α amyrin-amyrin (7), (7 respectively), respectively [36,37]. [36,37 ].The The carbon carbon skeletons skeletons of of these these triterpenoids triterpenoids consist consist of of five five condensed condensed cyclohexane rings. What differentiates them is one methyl group (CH -29) located in oleanane and cyclohexane rings. What differentiates them is one methyl group (СН33-29) located in oleanane and ursane derivatives at C20 and C19 of the E ring, respectively.respectively.

30 29 30

20 29 19 21 20 E 19 21 22 22 12 18 11 13 17 12 18 25 26 17 D 28 25 11 26 13 C 14 16 28 15 14 16 1 9 15 2 10 8 1 9 A B 2 10 8 27 3 5 7 27 4 6 3 5 7 4 6 23 24 23 24 A B

Figure 1. Representative skeletons of oleanane (A) and ursane (B) pentacyclic triterpenoids.

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30 29 30 O 29 30 OH 29 20 20 19 21 20 19 21 H 19 21 H 22 H 22 12 18 22 28 O 12 18 25 11 13 17 OH 12 18 11 17 28 25 11 13 17 25 13 OH 14 16 28 15 14 16 14 16 1 9 O 1 9 15 2 10 8 1 9 15 O 8 2 10 8 7 27 2 10 3 5 3 5 7 27 4 6 1 3 5 7 27 2 6 3 HO 4 6 4 HO HO 23 24 23 24 23 24

4 5 6 7

HO O HO

RepresentativesRepresentatives of variousvarious higher higher plant plant families families are are active active producers producers of both of oleananeboth oleanane and ursane and ursanetriterpenoids triterpenoids (Table1 (Table). Frequently, 1). Frequently, OA and OA UA and are simultaneouslyUA are simultaneously detected detected in the same in the plant same sources. plant sources.OA and OA UA and contents UA contents in Meconopsis in Meconopsis henrici, Dracocephalum henrici, Dracocephalum tanguticum tanguticum, Comastoma, Comastoma pulmonaria pulmonaria, Corydalis, impatiens, and Swertia racemosa—traditionally used in Chinese medicine—can reach 0.96 0.01 mg/g Corydalis impatiens, and Swertia racemosa—traditionally used in Chinese medicine—can reach± 0.96 ± and 0.64 0.01 mg/g dry weight, respectively [38]. Flowers and leaves of the shrubs Ocimum tenuiflorum 0.01 mg/g± and 0.64 ± 0.01 mg/g dry weight, respectively [38]. Flowers and leaves of the shrubs Ocimum tenuiflorumand Syzygium and aromaticum Syzygium andaromaticum the herbs andOriganum the herbs vulgare Origanum, Rosmarus vulgare offi, cinalisRosmarus, and officinalisSalvia offi, cinalisand Salviaused officinalisas condiments used as contain condiments up to 15.3 contain mg/g up OA to and 15.3 up mg/g to 26.2 OA mg and/g up UA to (wet 26.2 weight) mg/g UA [39 (wet]. The weight) main source [39]. Theof OA main is consideredsource of OA to beis considered the fruits and to be leaves the fruits of Olea and europaea leaves. of The Olea acid europaea content. The in oliveacid content leaves can in reach 27.16 mg/g wet weight and 25.09 0.72 mg/g dry weight [39,40]. GA is commonly extracted olive leaves can reach 27.16 mg/g wet ±weight and 25.09 ± 0.72 mg/g dry weight [39,40]. GA is commonlyfrom herbaceous extracted plants from ofherbaceous the genus plantsGlycyrrhiza of the genus[41–43 Glycyrrhiza]; the acid content[41–43]; the in their acid content roots can in reachtheir 10.2 1.7 mg/g wet weight [44]. roots± can reach 10.2 ± 1.7 mg/g wet weight [44]. TheThe amount amount of of pentacyclic pentacyclic triterpenoids triterpenoids in in plan plantsts is is not not constant constant and and can can significantly significantly vary vary dependingdepending on on the the activity activity of of enzyme enzyme systems systems and and exte externalrnal factors factors [45]. [45]. Thus, Thus, the the fruits fruits and and leaves leaves of of olive (Olea europaea) of various varieties contained OA from 0.4 0.1 mg/g to 0.81 0.16 mg/g dry olive (Olea europaea) of various varieties contained OA from 0.4 ±± 0.1 mg/g to 0.81 ±± 0.16 mg/g dry weight and from 29.2 1.8 mg/g to 34.5 3.1 mg/g dry weight, respectively [46,47]. The OA content weight and from 29.2 ±± 1.8 mg/g to 34.5 ±± 3.1 mg/g dry weight, respectively [46,47]. The OA content decreaseddecreased by by 70 70-80%‒80% during during olive olive fruit fruit ripening ripening [40]. [40]. The The same same tendency tendency was was observed observed when when grapes grapes ((VitisVitis vinifera vinifera)) ripen ripen [48]. [48]. Changes Changes in in pentacyclic pentacyclic triterpeno triterpenoidid concentrations concentrations in in plant plant sources sources may may be be relatedrelated to to specific specific climate, climate, season, season, land landscape,scape, and and cultivation cultivation strategies strategies [47]. [47].

TableTable 1. 1. SomeSome plant plant sources sources of of oleanolic oleanolic acid acid (OA), (OA), ursolic ursolic acid acid (UA), (UA), and and glycyrrhetinic glycyrrhetinic acid acid (GA). (GA).

CompoundCompound Plant Plant Source Source Part of Part Plant of Plant Reference Reference ApocynaceaeApocynaceae Juss.,Juss., nom. cons.cons. OA, OA,UA UA Alstonia scholaris scholaris(L.) (L.) R.Br. R.Br. Leaves Leaves [49 [49]] OA, OA,UA UA Plumeria obtusa obtusaL.L. var. var.sericifolia sericifolia LeavesLeaves [50[50]] AquifoliaceaeAquifoliaceae DC.DC. ex. A.Rich. OA, OA,UA UA IlexIlex guayusa guayusaLoes. Loes. LeavesLeaves [39[39]] AraliaceaeAraliaceae Juss.,Juss., nom. nom. cons. OA Panax stipuleanatus H.T.Tsai & K.M.Feng Roots [51] OA Panax stipuleanatus H.T.Tsai & K.M.Feng Roots [51] Asteraceae Bercht. & J.Presl, nom. cons. Asteraceae Bercht. & J.Presl, nom. cons. OA Baccharis uncinella DC. Leaves [52] OA Baccharis uncinellaBetulaceaeDC. Gray Leaves [52] OA Betula pendulaBetulaceae Roth. Gray Bark [3,53] OA CornaceaeBetula pendula Bercht.Roth. & J.Presl, nom. cons. Bark [3,53] UA Cornus officinalis Torr. ex Dur. Seeds [54] Ebenaceae Gürke, nom. cons. OA, UA Diospyros L. Fruits [55]

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Table 1. Cont.

Compound Plant Source Part of Plant Reference Cornaceae Bercht. & J.Presl, nom. cons. UA Cornus officinalis Torr. ex Dur. Seeds [54] Ebenaceae Gürke, nom. cons. OA, UA Diospyros L. Fruits [55] Ericaceae Juss., nom. cons. OA, UA Arctostaphylos uva-ursi (L.) Spreng. Fruits [39] OA, UA Vaccinium vitis-idaea L. Fruits [53] Fabaceae Lindl. Glycyrrhiza glabra L. Roots [41] GA Glycyrrhiza uralensis Fisch. Roots [42] Glycyrrhiza inflate Batalin Roots [43] Gentianaceae Juss., nom. cons. Comastoma pulmonaria Turcz. Sprouts, roots OA, UA [38] Swertia racemosa Wall. ex Griseb. Sprouts, roots Lamiaceae Martinov Ocimum tenuiflorum L. Leaves Lavandula angustifolia Mill. Flowers Origanum vulgare L. Leaves [39] OA, UA Rosmarinus officinalis L. Leaves Salvia officinalis L. Leaves Dracocephalum tanguticum Maxim Sprouts, roots [38] Lythraceae J.St.-Hil. Punica granatum L. Fruits [38] OA, UA Punica granatum L.cv. Daqingpi Flowers [56] Malvaceae Juss., nom. cons. OA Durio zibethinus Murr. Fruits [7] Myrtaceae Juss., nom. cons. Syzygium aromaticum (L.) Merr. & L.M.Perry Flowers OA, UA [39] Eucalyptus globules Labill. Leaves Oleaceae Hoffmanns. & Link, nom. cons. Olea europaea L.cv. Picual OA Fruits, leaves [40] Olea europaea L. cv. Cornezuelo OA, UA Olea europaea L. Leaves [39] Papaveraceae Juss. Meconopsis henrici Bureau & Franch. Sprouts, roots OA, UA [38] Corydalis impatiens (Pall.) Fisch. ex DC. Sprouts, roots Pinaceae Lindl. UA Picea abies (L.) H.Karst. Resin, cons, sprouts [57] Rosaceae Juss. Malus P. Mill. Fruits OA, UA [55] Pyrus L. Fruits Fragaria ananassa (Duchesne ex Weston) UA Perianth [4] Duchesne ex Rozier UA Potentilla fulgens Wall. ex Hook. Roots [58] OA, UA Eriobotrya japonica (Thunb.) Lindl Leaves [59] Molecules 2020, 25, 5526 5 of 31

Table 1. Cont.

Compound Plant Source Part of Plant Reference Rubiaceae Juss., nom. cons. OA Uncaria laevigata Wall. ex G.Don Stem bark [60] UA Emmenopterys henryi Oliv. Leaves, sprouts [61] Vitaceae Juss., nom. cons. OA Vitis vinifera L. Fruits [55] Note: GA, glycyrrhetinic acid; OA, oleanolic acid; UA, ursolic acid. Molecules 2020, 25, x FOR PEER REVIEW 5 of 34

3.3. Biosynthesis of Pentacyclic TriterpenicTriterpenic AcidsAcids UsingUsing MicroorganismsMicroorganisms Today,Today, pentacyclic pentacyclic triterpenoids triterpenoids andand theirtheir naturalnatural derivativesderivatives areare mainlymainly obtainedobtained byby extractionextraction fromfrom plantplant sources.sources. However,However, thethe extractionextraction andand separationseparation ofof thesethese compoundscompounds (often(often withwith organicorganic solvents)solvents) areare extremely extremely labor-intensive, labor-intensive, and energy-and energy- and time-consuming. and time-consuming. Besides, mostBesides, of pentacyclic most of triterpenoidspentacyclic triterpenoids are found in are relatively found lowin relatively concentrations low concentrations in plants, entailing in plants, the use entailing of huge the amounts use of ofhuge plant amounts raw materials of plant andraw thematerials formation and the of wasteformation biomass of waste in large biomass volumes in large [62 ].volumes An alternative [62]. An sourcealternative of pentacyclic source of pentacyclic triterpenoids triterpenoids seems to be seems highly to ebeﬃ highlycient cell efficient factories, cell factories, increasingly increasingly popular inpopular the last in decade.the last decade. They allow They to allow obtain to valuableobtain valuable biologically biologically active compoundsactive compounds of plant of origin plant inorigin environmentally in environmentally friendly friendly conditions conditions using using available available compounds compounds as as the the sole sole source source of of carboncarbon (glucose,(glucose, galactose,galactose, andand ethanol)ethanol) [[63].63]. CellCell factoriesfactories areare usuallyusually yeastyeast cells—naturalcells—natural catalystscatalysts ofof thethe mevalonatemevalonate (MVA)(MVA) pathway—withpathway—with plantplant genesgenes responsibleresponsible forfor pentacyclicpentacyclic triterpenoidtriterpenoid synthesissynthesis introducedintroduced intointo their genome. The The MVA MVA pathway pathway includes includes formation formation of of mevalonate mevalonate involving involving 3- 3-hydroxy-3-methylglutaryl-CoAhydroxy-3-methylglutaryl-CoA reductases reductases (HMG1). (HMG1). The The mevalonate mevalonate formed formed is further transformedtransformed intointo isopentenylisopentenyl diphosphate diphosphate and dimethylallyland dimethylallyl diphosphate, diphosphate, being converted being converted to farnesyl diphosphateto farnesyl bydiphosphate a farnesyl phosphateby a farnesyl synthase phosphate (ERG20). synthase This pathway(ERG20). providesThis pathway natural provi synthesisdes natural of squalene synthesis (4)—a of commonsqualene precursor(4)—a common of triterpenoids—by precursor of triterpenoids—by the squalene synthase the squalene (ERG9) synthase based on(ERG9) two based molecules on two of farnesylmolecules diphosphate of farnesyl and diphosphate its further transformationand its further into transformation 2,3-oxidosqualene into (2,3-oxidosqualene5) by squalene epoxidase (5) by (ERG1)squalene [64 epoxidase]. Subsequent (ERG1) synthesis [64]. Subsequent of pentacyclic synthesis triterpenoids of pentacyclic involves planttriterpenoids genes encoding involves amyrin plant synthase,genes encoding CYP450, amyrin and CYP450 synthase, reductase CYP450, (Scheme and CYP4501). reductase (Scheme 1).

Scheme 1. Biosynthesis of oleanane and ursane triterpenoids in engineered yeasts. Enzymes (in frames): Scheme 1. Biosynthesis of oleanane and ursane triterpenoids in engineered yeasts. Enzymes (in HMG1, 3-hydroxy-3-methylglutaryl-CoA reductase; ERG20, farnesyl phosphate synthase; ERG9, frames): HMG1, 3-hydroxy-3-methylglutaryl-CoA reductase; ERG20, farnesyl phosphate synthase; squalene synthase; ERG1, squalene epoxidase; βAS, β-amyrin synthase; mix-AS, mixed amyrin ERG9, squalene synthase; ERG1, squalene epoxidase; βAS, β-amyrin synthase; mix-AS, mixed amyrin synthase; CYP450, cytochrome P450. Metabolites: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl synthase; CYP450, cytochrome P450. Metabolites: IPP, isopentenyl diphosphate; DMAPP, diphosphate; and FPP, farnesyl diphosphate. Modiﬁed from Lu et al., 2018 and Zhao et al., 2018 [64,65]. dimethylallyl diphosphate; and FPP, farnesyl diphosphate. Modified from Lu et al., 2018 and Zhao et al., 2018 [64,65].

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Genes encoding β-amyrin synthase (βAS) that catalyze the formation of β-amyrin (6)—a precursor of oleanane pentacyclic triterpenoids—from 2,3-oxidosqualene (5) were isolated from the genomes of Glycyrrhiza glabra, Panax ginseng, Catharanthus roseus, Lotus japonicus, Artemisia annua, Chenopodium quinoa, and others [65–69]. Because no enzymes were found to synthesize exclusively α-amyrin (7), a precursor of ursane pentacyclic triterpenoids, this reaction involves mixed amyrin synthases (mix-AS) that catalyze the formation of both α- and β-amyrin from Eriobotrya japonica and C. roseus [64,70]. Medicago truncatula is most often used to search for genes encoding CYP450 enzymes that catalyze subsequent conversion of α- and β-amyrin [64–67,69,71]. Less frequently, Phaseolus vulgaris [72], Bupleurum falcatum [71], G. uralensis [62], C. roseus [64], Crataegus pinnatifida [70], Solanum lycoperum, P. ginseng [68], and others are used to search for these genes. Native microbial cytochrome P450 reductases are often unable to transfer electrons to foreign CYP450s, as required for catalysis, and the source of additional CYP450 reductases (CPR and ATR) is usually Arabidopsis thaliana [62,64–66,69,71]. In a few studies, CYP450 reductases were obtained from M. truncatula [65], L. japonicus [67], G. uralensis [62], and V. vinifera [70]. Various approaches, including modification, overexpression, or inactivation of microorganisms’ own genes; insertion of plant genes in the yeast genome by various techniques; as well as combinatorial biosynthesis, are used to enhance microbial biosynthesis and to achieve an increased yield of pentacyclic triterpenoids (Table2). Overexpression of genes ERG1, ERG9, ERG20, and, more frequently, tHMG1 (HMG1 with truncated N-terminal 511 amino acids) involved in the natural microbial synthesis of 2,3-oxidosqualene (Scheme1) can enhance the biosynthesis of triterpenoids [64–66,69]. Along with overexpression, the enhancement can also be facilitated by inactivation of TRP1 (phosphoribosylanthranilate isomerase), GAL1 (galactokinase), and GAL80 (galactose/lactose metabolism regulatory protein) involved in metabolic processes that “distract” cells from biosynthesis of triterpenoids [65,66]. Thus, overexpression of tHMG1, ERG1, and ERG9 and inactivation of GAL1 and GAL80 in the chromosome of Saccharomyces cerevisiae JDY52 and its use in a 5-L fermenter with 40 g/L glucose resulted in 606.9 9.1 mg/L OA after 144 ± h. To date, it is the highest yield reported [65]. In another study, the use of Yarrowia lipolytica ATCC 201249 with overexpressed ERG1, ERG9, ERG20, and tHMG1 and the inserted expression modules βAS and CYP716A2-linker(GSTSSG)-t46ATR1 (ATR1 with truncated N-terminal 46 amino acids) provided the yield of 540.7 mg/L OA after 82 h in a 5-L fermenter with 100 g/L glucose [69]. Despite the fact that S. cerevisiae JDY52 produced relatively higher OA amounts (606.9 9.1 mg/L) [65], the productivity of ± Y. lipolytica was 6.59 mg/L/h and exceeded that of S. cerevisiae (4.214 mg/L/h). Various techniques of yeast genome modification were also applied to intensify the biosynthesis of triterpenoids. It was shown that the expression of inserted plant genes from low-copy and single-copy plasmids was more effective than that from integrated, high-copy, and multicopy plasmids [71,73]. In Reference [71], the activities of S. cerevisiae TM30 and S. cerevisiae TM44 obtained from the same parent strain by including different plasmid variations were evaluated. In the first case, the strain expressing CYP716Y1 and CYP716A12 to obtain a self-processing polyprotein with two enzymes bound via oligopeptide 2A catalyzed the formation of β-amyrin (6), erythrodiol (8), OA, oleanolic aldehyde (9), and 16α-hydroxy-oleanolic aldehyde (10), while the second strain produced two self-processing polyproteins, one consisting of CYP716Y1 and CYP716A12 and the other consisting of AtATR1 and UDP-dependent glycosyl transferase UGT73C11, and catalyzed the formation of 3-O-Glc-echinocystic acid (11) and 3-O-Glc-OA (12). Molecules 2020, 25, 5526 7 of 31

Table 2. Biosynthesis of pentacyclic triterpenoids using engineered yeasts.

Parent Strain Modiﬁcation Product (Yield) Reference Insertion of βAS, CPR (Lotus japonicus), CYP93E2, and CYP72A61 β-Amyrin (6 **, 1.07 mg/L), 24-hydroxy-β-amyrin (13, 0.27 mg/L), (Medicago truncatula) and soyasapogenol B (21, 1.35 mg/L) β-Amyrin (6, 0.55 mg/L), erythrodiol (8, 0.09 mg/L), and gypsogenic acid Saccharomyces cerevisiae Insertion of βAS, CPR (L. japonicus), CYP716A12, and CYP72A68 (M. truncatula) (22, 0.96 mg/L) [67] INVSc1 Insertion of βAS, CPR (L. japonicus), CYP716A12, and CYP93E2 (M. truncatula) Erythrodiol (8), OA, 4-epi-hederagenin (24), and others (trace amount) Insertion of βAS, CPR (L. japonicus), CYP72A63, and CYP716A12 (M. truncatula) Erythrodiol (8), OA, queretaroic acid (25), and others (trace amount) Insertion of βAS, CPR (L. japonicus), CYP93E2, and CYP72A63 (M. truncatula) Probable 11-deoxo-GA (23) and others (trace amount) Insertion of βAS (Glycyrrhiza glabra) β-Amyrin (6, 36.2 3.9 mg/L) S. cerevisiae BY4742 ± Insertion of βAS (M. truncatula) β-Amyrin (6, 19.0 1.0 mg/L) ± Insertion of AtATR1 (Arabidopsis thaliana), CYP716Y1 (Bupleurum falcatum), β-Amyrin (6), erythrodiol (8), OA (1), oleanolic aldehyde (9), [71] and CYP716A12 (M. truncatula) and 16α-hydroxy-oleanolic aldehyde (10) Insertion of CYP716Y1 (B. falcatum), CYP716A12 (M. truncatula), AtATR1 3-O-Glc-Echinocystic acid (11), 3-O-Glc-OA (12) (A. thaliana), and UGT73C11 (Barbarea vulgaris) Insertion of AtATR1 (A. thaliana) and CYP93E2 (M. truncatula) 24-Hydroxy-β-amyrin (13, 1.3%) Insertion of AtATR1 (A. thaliana) and CYP93E7 (Lens culinaris) 24-Hydroxy-β-amyrin (13, 16.2%) 24-Hydroxy-β-amyrin (13, 37.6%), probable Insertion of AtATR1 (A. thaliana) and CYP93E8 (Pisum sativum) 3β-hydroxy-olean-12-en-24-oic acid (14, 3.2%) S. cerevisiae TM3 24-Hydroxy-β-amyrin (13, 51.3%), probable Insertion of AtATR1 (A. thaliana) and CYP93E5 (Cicer arietinum) 3β-hydroxy-olean-12-en-24-oic acid (14, 7.6%) 24-Hydroxy-β-amyrin (13, 50.2%), probable Insertion of AtATR1 (A. thaliana) and CYP93E6 (G. glabra) [72] 3β-hydroxy-olean-12-en-24-oic acid (14, 6.3%) 24-Hydroxy-β-amyrin (13, 47.0%), probable Insertion of AtATR1 (A. thaliana) and CYP93E4 (Arachis hypogaea) 3β-hydroxy-olean-12-en-24-oic acid (14, 5.5%) 24-Hydroxy-β-amyrin (13, 58.8%), probable Insertion of AtATR1 (A. thaliana) and CYP93E1 (Glycine max) 3β-hydroxy-olean-12-en-24-oic acid (14, 13.5%) 24-Hydroxy-β-amyrin (13, 67.6%), probable Insertion of AtATR1 (A. thaliana) and CYP93E9 (Phaseolus vulgaris) 3β-hydroxy-olean-12-en-24-oic acid (14, 11.8%) S. cerevisiae BY4742 Deletion of TRP1 Squalene (4, 9.6 mg/L) S. cerevisiae BY4742-TRP Overexpression of tHMG1 and LYS2 Squalene (4, 150.9 mg/L) Insertion of βAS (G. glabra) β-Amyrin (6, 107.0 mg/L), squalene (4, 183.4 mg/L) Overexpression of ERG1 and ERG9 S. cerevisiae BY-T1 [66] Insertion of βAS (Panax ginseng) β-Amyrin (6, 1.9 mg/L) Overexpression of ERG1 and ERG9 S. cerevisiae BY-βA-G Insertion of βAS (G. glabra), OAS (M. truncatula), and AtCPR1 (A. thaliana) OA (1, 71.0 mg/L), β-amyrin (6, 88.6 mg/L), and squalene (4, 141.2 mg/L) Molecules 2020, 25, 5526 8 of 31

Table 2. Cont.

Parent Strain Modiﬁcation Product (Yield) Reference Insertion of βAS (G. glabra) β-Amyrin (6, 4.16 mg/L) Insertion of βAS (G. glabra) and ERG1 (Candida albicans) β-Amyrin (6, 24.50 mg/L) Insertion of βAS (G. glabra), ERG1 (C. albicans), IDI (Escherichia coli) β-Amyrin (6, 36.50 mg/L, 75.50 mg/L *) S. cerevisiae INVSc1 Overexpression of ERG9 and ERG20 [73] Insertion of βAS (G. glabra), ERG1 (C. albicans), IDI (Escherichia coli) Overexpression of ERG9 and ERG20 β-Amyrin (6, 85.78 mg/L, 108.60 mg/L *) Reconstruction of SRE into promoter S. cerevisiae W303-1a Overexpression of tHMG1 and ERG20 Squalene (4, 165.28 mg/L) Insertion of mix-AS (Catharanthus roseus) β-Amyrin (6, 1.64 mg/L), α-amyrin (7, 5.64 mg/L) Overexpression of tHMG1 and ERG20 S. cerevisiae WTE Insertion of mix-AS (Catharanthus roseus) and ERG1 (C. albicans) β-Amyrin (6, 24.95 mg/L, 44.92 mg/L *), α-amyrin (7, 97.31 mg/L, Overexpression of tHMG1 175.15 mg/L *) [64] Insertion of CrOAS (C. roseus) and AtCPR1 (A. thaliana) OA (1, 29.49 mg/L), UA (3, 24.58 mg/L) Insertion of MtOAS (M. truncatula) and LjCPR1 (L. japonicus) OA (1, 24.34 mg/L), UA (3, 23.37 mg/L) S. cerevisiae ScLCZ08 Insertion of CrOAS (C. roseus) and LjCPR1 (L. japonicus) OA (1, 23.91 mg/L), UA (3, 22.96 mg/L) Insertion of MtOAS (M. truncatula) and AtCPR1 (A. thaliana) OA (1, 31.41 mg/L, 155.58 mg/L *), UA (3, 25.85 mg/L, 123.37 mg/L *) Insertion of GgβAS (G. glabra), MtCYP716A12 (M. truncatula), and AtCPR1 OA (1, 2.5 0.2 mg/L) (A. thaliana) ± Insertion of GgβAS (G. glabra), MtCYP716A12 (M. truncatula), and LjCPR OA (1, 6.3 0.3 mg/L) (L. japonicus) ± Insertion of GgβAS (G. glabra), MtCYP716A12 (M. truncatula), and GuCPR OA (1, 7.1 0.5 mg/L) (G. uralensis) ± S. cerevisiae JDY52 Insertion of GgβAS (G. glabra), MtCYP716A12, and MtCPR (M. truncatula) OA (1, 9.0 0.7 mg/L) [65] ± Insertion of GgβAS (G. glabra), MtCYP716A12, and MtCPR (M. truncatula) OA (1, 41.3 3.4 mg/L) Knocking out of GAL1 ± Insertion of GgβAS (G. glabra), MtCYP716A12, and MtCPR (M. truncatula) OA (1, 70.3 7.0 mg/L) Knocking out of GAL1 and GAL80 ± Insertion of GgβAS (G. glabra), MtCYP716A12, and MtCPR (M. truncatula) Knocking out of GAL1 and GAL80 OA (1, 186.1 12.4 mg/L, 606.9 9.1 mg/L *) ± ± Overexpression of tHMG1, ERG1, and ERG9 Molecules 2020, 25, 5526 9 of 31

Table 2. Cont.

Parent Strain Modiﬁcation Product (Yield) Reference GA (2, 20.4 7.7 µg/L), 11-oxo-β-amyrin (15, 0.5 0.1 mg/L), β-amyrin ± ± (6, 21.23 1.64 mg/L), 11α-hydroxy-β-amyrin (16), S. cerevisiae INVSc1 Insertion of CYP88D6, CYP72A154 (G. uralensis), and ATR1 (A. thaliana) ± 30-hydroxy-11-oxo-β-amyrin (17), and glycyrrhetaldehyde (18) (trace amount) S. cerevisiae INVSc1 Insertion of CYP88D6, CYP72A154 (G. uralensis), and ATR1 (A. thaliana) GA (2, 31.8 6.6 µg/L), 11-oxo-β-amyrin (15, 0.5 0.06 mg/L) (diploid) ± ± Insertion of CYP88D6, CYP72A154 (G. uralensis), and ATR1 (A. thaliana) GA (2, 33.7 7.4 µg/L), 11-oxo-β-amyrin (15, 7.5 0.5 mg/L) [62] ± ± Insertion of CYP88D6 and ATR1 (A. thaliana) 11-oxo-β-amyrin (15, 3.1 0.05 mg/L) ± Insertion of Unigene25647 and ATR1 (A. thaliana) 11-oxo-β-amyrin (15, 6.0 0.1 mg/L) ± Insertion of CYP72A63, Unigene25647 (G. uralensis), and ATR1 (A. thaliana) GA (2, 40.9 6.4 µg/L), 11-oxo-β-amyrin (15, 9.16 1.20 mg/L) S. cerevisiae SGib ± ± Insertion of CYP72A154, Unigene25647 (G. uralensis), and ATR1 (A. thaliana) GA (2, 42.3 5.8 µg/L), 11-oxo-β-amyrin (15, 7.22 1.58 mg/L) ± ± Insertion of CYP72A154, Unigene25647, and GuCPR1 (G. uralensis) GA (2, 517.4 35.5 µg/L), 11-oxo-β-amyrin (15, 15.3 1.6 mg/L) ± ± GA (2, 7.4 1.0 mg/L, 18.9 2.0 mg/L *), 11-oxo-β-amyrin (15, Insertion of CYP72A63, Unigene25647, and GuCPR1 (G. uralensis) ± ± 22.6 0.9 mg/L, ~80 mg/L *) ± Insertion of CYP716C49 (Crataegus pinnatiﬁda) Maslinic acid (19, 0.06 mg/L/OD600) Insertion of CaCYP716C49 (Centella asiatica) Maslinic acid (19, 0.2 mg/L/OD600) Insertion of codon-optimized CaCYP716C49 (C. asiatica) Maslinic acid (19, 0.45 mg/L/OD , 384.3 mg/L *) S. cerevisiae BY-OA 600 [70] Insertion of mix-AS (Eriobotrya japonica), VvCYP716A15, and CPR (Vitis vinifera) UA (3, 1.76 mg/L/OD600), OA (2, 0.61 mg/L/OD600) Insertion of mix-AS (E. japonica), codon-optimized CaCYP716C49 (C. asiatica), Corosolic acid (20, 0.39 mg/L/OD , 141.0 mg/L*) VvCYP716A15, and CPR (V. vinifera) 600 Insertion of AaβAS (Artemisia annua) β-Amyrin (6, 10.8 1.0 mg/L) ± Insertion of CqβAS (Chenopodium quinoa) β-Amyrin (6, 10.8 1.0 mg/L) ± Insertion of PtβAS (Polygala tenuifolia) β-Amyrin (6, 9.0 0.7 mg/L) ± Insertion of LjβAS (L. japonicus) β-Amyrin (6, 8.2 1.0 mg/L) ± Insertion of EtβAS (Euphorbium tirucalli) β-Amyrin (6, 8.0 0.2 mg/L) ± Insertion of SlβAS (Solanum lycopersicum) β-Amyrin (6, 2.9 0.3 mg/L) ± OA (1, 14.3 1.6 mg/L), erythrodiol (8), and oleanolic aldehyde (9) (trace Insertion of CYP716AL1 (C. roseus) and ATR2 (A. thaliana) ± S. cerevisiae BY4742 amount) [68] OA (1, 3.0 0.0 mg/L), erythrodiol (8), and oleanolic aldehyde (9) (trace Insertion of CYP716A52v2 (P. ginseng) and ATR2 (A. thaliana) ± amount) Insertion of SlβAS (S. lycopersicum), CYP716AL1 (C. roseus), and ATR2 OA (1, 3.9 0.2 mg/L) (A. thaliana) ± Insertion of SlβAS (S. lycopersicum), CYP716A52v2 (P. ginseng), and ATR2 OA (1, 2.8 0.0 mg/L) (A. thaliana) ± Insertion of AaβAS (A. annua), CYP716AL1 (C. roseus), and ATR2 (A. thaliana) OA (1, 8.5 0.2 mg/L) ± Insertion of βAS (C. roseus) β-Amyrin (6, 16.0 mg/L) Insertion of βAS (C. roseus), CYP716A12 (M. truncatula), and ATR1 (A. thaliana) OA (1, 16.3 mg/L) Insertion of βAS (C. roseus), CYP716A12 (M. truncatula), and ATR1(A. thaliana) Yarrowia lipolytica ATCC OA (1, 92.1 mg/L) Overexpression of ERG1, ERG9, ERG20, tHMG1 [69] 201249 Insertion of βAS (C. roseus) and CYP716A2-linker (GSTSSG)-t46ATR1 OA (1, 129.9 mg/L, 540.7 mg/L*) Overexpression of ERG9, ERG20, and tHMG1 Note: * Yield of the compound in a 5-L fermenter. ** The number in bold is the number of the compound structural formula. GA, glycyrrhetinic acid; OA, oleanolic acid; UA, ursolic acid. Molecules 2020, 25, x FOR PEER REVIEW 6 of 34

66,69,71]. In a few studies, CYP450 reductases were obtained from M. truncatula [65], L. japonicus [67], G. uralensis [62], and V. vinifera [70]. Various approaches, including modification, overexpression, or inactivation of microorganisms’ own genes; insertion of plant genes in the yeast genome by various techniques; as well as combinatorial biosynthesis, are used to enhance microbial biosynthesis and to achieve an increased yield of pentacyclic triterpenoids (Table 2). Overexpression of genes ERG1, ERG9, ERG20, and, more frequently, tHMG1 (HMG1 with truncated N-terminal 511 amino acids) involved in the natural microbial synthesis of 2,3- oxidosqualene (Scheme 1) can enhance the biosynthesis of triterpenoids [64–66,69]. Along with overexpression, the enhancement can also be facilitated by inactivation of TRP1 (phosphoribosylanthranilate isomerase), GAL1 (galactokinase), and GAL80 (galactose/lactose metabolism regulatory protein) involved in metabolic processes that “distract” cells from biosynthesis of triterpenoids [65,66]. Thus, overexpression of tHMG1, ERG1, and ERG9 and inactivation of GAL1 and GAL80 in the chromosome of Saccharomyces cerevisiae JDY52 and its use in a 5-L fermenter with 40 g/L glucose resulted in 606.9 ± 9.1 mg/L OA after 144 h. To date, it is the highest yield reported [65]. In another study, the use of Yarrowia lipolytica ATCC 201249 with overexpressed ERG1, ERG9, ERG20, and tHMG1 and the inserted expression modules βAS and CYP716A2- linker(GSTSSG)-t46ATR1 (ATR1 with truncated N-terminal 46 amino acids) provided the yield of 540.7 mg/L OA after 82 h in a 5-L fermenter with 100 g/L glucose [69]. Despite the fact that S. cerevisiae JDY52 produced relatively higher OA amounts (606.9 ± 9.1 mg/L) [65], the productivity of Y. lipolytica was 6.59 mg/L/h and exceeded that of S. cerevisiae (4.214 mg/L/h). Various techniques of yeast genome modification were also applied to intensify the biosynthesis of triterpenoids. It was shown that the expression of inserted plant genes from low-copy and single- copy plasmids was more effective than that from integrated, high-copy, and multicopy plasmids [71,73]. In Reference [71], the activities of S. cerevisiae TM30 and S. cerevisiae TM44 obtained from the same parent strain by including different plasmid variations were evaluated. In the first case, the strain expressing CYP716Y1 and CYP716A12 to obtain a self-processing polyprotein with two enzymes bound via oligopeptide 2A catalyzed the formation of β-amyrin (6), erythrodiol (8), OA, oleanolic aldehyde (9), and 16α-hydroxy-oleanolic aldehyde (10), while the second strain produced two self-processing polyproteins, one consisting of CYP716Y1 and CYP716A12 and the other consistingMolecules 2020of ,AtATR125, 5526 and UDP-dependent glycosyl transferase UGT73C11, and catalyzed10 of the 31 formation of 3-O-Glc-echinocystic acid (11) and 3-O-Glc-OA (12).

CHO COOH OH R R Glc HO 8 HO 9: R = H O 11: R = αOH

10: R = αOH 12: R = H

TheThe most most promising promising research research areaarea in in this this field fiel isd combinatorial is combinatorial biosynthesis. biosynthesis. Change Change in the natural in the naturalbiosynthesis biosynthesis pathway pathway or combination or combination of synthesis of synthesis genes from genes diff erentfrom sourcesdifferent leads sources to enhanced leads to enhancedefficiency efficiency of microbial of microbial biosynthesis biosynthesis or to the formation or to the offormation new compounds. of new compounds. The Moses group The (2014)Moses groupstudied (2014) the studied C24-oxidizing the C24-oxidizing activity of CYP93E2 activityorthologs of CYP93E2 from orthologs different speciesfrom different of the family speciesFabaceae of the. familyThe Fabaceae obtained. strainsThe obtained catalyzed strains the formation catalyzed of the 24-hydroxy- formationβ-amyrin of 24-hydroxy- (13). Almostβ-amyrin all strains (13). (except Almost all strainsone containing (except CYP93E7one containing) simultaneously CYP93E7) catalyzed simultaneously the formation catalyzed of 3 βthe-hydroxy-olean-12-en-24-oic formation of 3β-hydroxy- olean-12-en-24-oicacid (14). The highest acid ( conversion14). The highest (79.4%) conversion of β-amyrin (79.4%) (6) to 24-hydroxy- of β-amyrinβ -amyrin(6) to 24-hydroxy- was observedβ-amyrin using wassaccharomycetes observed using containing saccharomycetesCYP93E9 containing(P. vulgaris)[ CYP93E972]. Dale et(P. al. vulgaris (2020)) conducted [72]. Dale a comparativeet al. (2020) β β conductedstudy on a catalyticcomparative activities study of 12on catalyticASs and activities 16 enzymes of of12 theβASs CYP716A and 16 subfamily. enzymes of Of the all theCYP716AASs Artemisia annua Chenopodium quinoa subfamily.studied, Of synthases all the derivedβASs studied, from synthases derivedand from Artemisia annuaexhibited and theChenopodium highest catalytic quinoa activity (10.8 1.0 mg/L β-amyrin). Comparatively, CYP716AL1 (C. roseus) showed the highest exhibited the highest± catalytic activity (10.8 ± 1.0 mg/L β-amyrin). Comparatively, CYP716AL1 (C. activity (14.3 1.6 mg/L OA) of the 16 CYP716As studied. When a combination of genes encoding roseus) showed ±the highest activity (14.3 ± 1.6 mg/L OA) of the 16 CYP716As studied. When a these enzymes was used, the OA yield made up 8.5 0.2 mg/L. At the same time, saccharomycetes combination of genes encoding these enzymes was used,± the OA yield made up 8.5 ± 0.2 mg/L. At containing CYP716A75 (Maesa lanceolata), CYP716A79 (C. quinoa), CYP716A110 (Aquilegia coerulea), and CYP716A1 (A. thaliana) did not catalyze the formation of OA; erythrodiol (8) but not OA was detected in all cases [68]. Substitutions of CYP88D6 (C11 oxidase) for Unigene25647 (97% similarity) and ATR1 (A. thaliana) for GuCPR1 (G. uralensis) were performed, and a combination of GuCPR1 and two Unigene25647 cassettes was inserted into the genome of S. cerevisiae SGib. These manipulations resulted in the formation of 18.9 2.0 mg/L GA and about 80.0 mg/L 11-oxo-β-amyrin (15) after 144 h ± of the fed-batch fermentation with ethanol (30 mL every 24 h) compared with the previously obtained 20.4 7.7 µg/L GA and 0.5 0.1 mg/L 11-oxo-β-amyrin (15) and β-amyrin (6) and trace amounts of ± ± 11α-hydroxy-β-amyrin (16), 30-hydroxy-11-oxo-β-amyrin (17), and glycyrrhetaldehyde (18)[62]. In strain S. cerevisiae BY-OA, substitution of CYP716C49 (C. pinnatifida) for the homologue (47.9% similarity) CaCYP716C49 (C. asiatica) allowed for obtaining 384.3 mg/L maslinic acid (19, 2α-hydroxy-OA) after 96-h incubation in a 5-L fermenter with glucose (5 g/L). When S. cerevisiae BY-T3 containing mix-AS, VvCYP716A15, CPR, and CaCYP716C49 from different plant sources were used in the same conditions; corosolic acid (20, 2α-hydroxy-UA) was formed after 144 h [70]. The activity of various combinations of CYP450 from M. truncatula was studied in Reference [67]. The main products of synthesis in different cases were soyasapogenol B (21), gypsogenic acid (22), or 11-deoxo-GA (23). Importantly, saccharomycetes expressing CYP716A12 and CYP93E2 catalyzed the formation of 4-epi-hederagenin (24), and the yeasts expressing CYP716A12 and CYP72A63 catalyzed the formation of queretaroic acid (25). These compounds were not previously detected in M. truncatula tissues, indicating the great potential of combinatorial biosynthesis using microorganisms [67]. Molecules 2020, 25, x FOR PEER REVIEW 7 of 34 the same time, saccharomycetes containing CYP716A75 (Maesa lanceolata), CYP716A79 (C. quinoa), CYP716A110 (Aquilegia coerulea), and CYP716A1 (A. thaliana) did not catalyze the formation of OA; erythrodiol (8) but not OA was detected in all cases [68]. Substitutions of CYP88D6 (C11 oxidase) for Unigene25647 (97% similarity) and ATR1 (A. thaliana) for GuCPR1 (G. uralensis) were performed, and a combination of GuCPR1 and two Unigene25647 cassettes was inserted into the genome of S. cerevisiae SGib. These manipulations resulted in the formation of 18.9 ± 2.0 mg/L GA and about 80.0 mg/L 11- oxo-β-amyrin (15) after 144 h of the fed-batch fermentation with ethanol (30 mL every 24 h) compared with the previously obtained 20.4 ± 7.7 µg/L GA and 0.5 ± 0.1 mg/L 11-oxo-β-amyrin (15) and β- amyrin (6) and trace amounts of 11α-hydroxy-β-amyrin (16), 30-hydroxy-11-oxo-β-amyrin (17), and glycyrrhetaldehyde (18) [62]. In strain S. cerevisiae BY-OA, substitution of CYP716C49 (C. pinnatifida) for the homologue (47.9% similarity) CaCYP716C49 (C. asiatica) allowed for obtaining 384.3 mg/L maslinic acid (19, 2α-hydroxy-OA) after 96-h incubation in a 5-L fermenter with glucose (5 g/L). When S. cerevisiae BY-T3 containing mix-AS, VvCYP716A15, CPR, and CaCYP716C49 from different plant sources were used in the same conditions; corosolic acid (20, 2α-hydroxy-UA) was formed after 144 h [70]. The activity of various combinations of CYP450 from M. truncatula was studied in Reference [67]. The main products of synthesis in different cases were soyasapogenol B (21), gypsogenic acid (22), or 11-deoxo-GA (23). Importantly, saccharomycetes expressing CYP716A12 and CYP93E2 catalyzed the formation of 4-epi-hederagenin (24), and the yeasts expressing CYP716A12 and CYP72A63 catalyzed the formation of queretaroic acid (25). These compounds were not previously detectedMolecules 2020in M., 25 ,truncatula 5526 tissues, indicating the great potential of combinatorial biosynthesis using11 of 31 microorganisms [67].

R O

15: R = O 17 HO HO HO 13: R = OH 16: R = αOH R 14: R = COOH CHO

O COOH HO COOH HO

HO 18 HO 19 20 HO

COOH

OH COOH

HO 21 22 23 HO HO HOOC OH OH

COOH OH

HO 24 HO 25

4. Biological Activities of Triterpenic Acids and Their Native Derivatives Extracts obtained from plant sources using various solvents and containing the pentacyclic triterpenoids reviewed in this paper usually exhibit a wide range of biological properties. Methanolic OA-containing extracts from various parts of Betula pendula exhibited antibacterial activity against test cultures Staphylococcus aureus and Bacillus subtilis [3]. Methanolic extracts from the aerial part of the tropical plant Baccharis uncinella containing OA and UA showed antiparasitic activity by limiting the growth of promastigote and amastigote forms of Leishmania amazonensis and enhanced the immune response in infected mice [52]. The ethyl acetate fraction of Glycyrrhiza uralensis root extract inhibited TNF-α-induced activation of NF-κB in HepG2 cells [42]. The ethyl acetate fraction of Potentilla fulgens root extract containing ursane triterpenoids showed antioxidant activity by inhibiting the production of free radicals [58]. An alcohol extract and ethyl acetate fraction of Fragaria ananassa perianth containing various triterpenoids had a pronounced cytotoxic effect on B16-F10 melanoma cells and inhibited their melanogenesis by 79.1% and 80.2%, respectively [4]. The allelopathic effect (inhibition of seed maturation and root growth of nearby plants) of Alstonia scholaris was also due to high UA content (2.5 0.6 mg/g dry weight of leaves) [49]. ± Isolation of every triterpenic acid individually allows studying their bioactive properties in detail and explaining the pharmacological properties of some plants. The antibacterial property of birch bark was determined by a high content of pentacyclic triterpenoids, in particular, OA, which exhibits a pronounced antibacterial activity against S. aureus (minimal inhibitory concentration (MIC) 1.25%) and B. subtilis (MIC 0.625%) [3]. Antimicrobial activity of GA was manifested as the ability to reduce the motility of Pseudomonas aeruginosa cells and the level of biofilm formation. This can make a significant contribution to the development of effective antibiotic-free therapy for Pseudomonas infections [74]. UA was able to inhibit both the growth of Mycobacterium tuberculosis in vitro [75] and the replication Molecules 2020, 25, 5526 12 of 31

of rotavirus in a dose-dependent manner [76]. In addition, OA and UA were shown to inhibit the COVID-19 (SARS-CoV-2) main protease, a key enzyme of the virus replication, through in silico studies [77,78]. OA, as an antitumor agent, increased the sensitivity of sarcoma cells to chemotherapeutic drugs in human soft tissues [79]. UA had similar properties, significantly increased the effectiveness of colorectal cancer chemotherapy, and reduced its side effects in vitro and in vivo [80]. The cytotoxic effect of the extract of Fragaria ananassa perianth on B16-F10 melanoma cells was determined partly by the presence of cytotoxic UA that suppressed the melanin production by 40.2% [4]. Additionally, UA reduced the spread of human myeloma cells by inhibiting the deubiquitinating protease USP7 [81] and caused apoptosis of gastric cancer cells by activating the caspases poly (ADP-ribose) polymerase and by inducing the release of reactive oxygen species [6]. The OA hepatoprotective activity was shown to be related to its inhibitory effect against carboxylesterase (therapeutic target for hypertriglyceridemia) and the hepatitis C virus (HCV) [5,82]. GA exhibited the hepatoprotective effect by inhibiting NO formation in rat hepatocytes, iNOS suppression, and COX-2 expression and by decreasing the activity of NF-κB transcription factor in HepG2 cells [42]. The ability of GA to stimulate a neuroprotective property of microglia and to suppress the MAPK signaling pathway of the central nervous system caused a decrease in the severity of experimental autoimmune encephalomyelitis in mice [83]. UA could be an effective antidiabetic agent due to its ability to inhibit α-glucosidase activity [60]. OA and UA exhibited their inhibitory effects against lypopolysaccharide (LPS)-induced NO production in RAW 264.7 cells that determined their anti-inflammatory activity [7]. Along with their parent acids, the native derivatives also exhibit pronounced biological activities. Thus, the OA derivatives 22β-acetoxy-3,25-epoxy-3α-hydroxyolean-12-en-28-oic acid (26) and methyl Molecules 20203,25-epoxy-3, 25, x FORα-hydroxy-11-oxo-22 PEER REVIEW β-senecioyloxyolean-12-en-28-oate (27) isolated from Lantana camara 14 of 34 herb extract demonstrated antibacterial activity against a number of gram-positive and gram-negative Molecules 2020bacteria, 25, x [84 FOR]. PEER REVIEW 14 of 34 O O O CH OCH3 OC 3 COOHO COOH C C O H CH3 O CH O OCH3 O OC 3 COOH COOH C C HO H CH 26 HO 27 3 O O HO HO 26 27 Oleanane 27-carboxy derivatives (28‒30) isolated from Chrysosplenium carnosum exhibited pronouncedOleanane inhibitory 27-carboxy activities derivatives against (the28-30 mouse) isolated melanoma from Chrysosplenium cell lines carnosumB16F10 exhibitedand SP2/0 [85]. Oleanane 27-carboxy derivatives (28‒30) isolated from Chrysosplenium carnosum exhibited Natural pronounced2α-hydroxylated inhibitory OA activities derivative against (20 the, corosolic mouse melanoma acid) had cell linescytotoxic B16F10 effects and SP2 on/0 [85CaSki]. cells pronouncedNatural inhibitory 2α-hydroxylated activities OA derivative against (20the, corosolic mouse acid) melanoma had cytotoxic cell eff ectslines on CaSkiB16F10 cells and (human SP2/0 [85]. (human cervical cancer) by inducing apoptosis, by arresting the cell cycle in the G2/M phase, and by Natural cervical2α-hydroxylated cancer) by inducing OA derivative apoptosis, by ( arresting20, corosolic the cell acid) cycle in had the G2cytotoxic/M phase, andeffects by inhibiting on CaSki cells inhibiting the PI3K/Akt signaling pathway [86]. 2,3-seco-Derivative of the ursane type (31) from (human thecervical PI3K/ Aktcancer) signaling by inducing pathway [apoptosis,86]. 2,3-seco-Derivative by arresting of thethe ursanecell cycle type in (31 the) from G2/MSiphonodon phase, and by Siphonodoncelastrineus celastrineusshowed showed pronounced pronounced cytotoxic activity cytotoxic against activity MOLT-3 against cancer MOLT-3 cells [87]. cancer cells [87]. inhibiting the PI3K/Akt signaling pathway [86]. 2,3-seco-Derivative of the ursane type (31) from Siphonodon celastrineus showed pronounced cytotoxic activity against MOLT-3 cancer cells [87]. OH

1 2 3 HO O 28: R = O, R = βOH, R = H OH

1 2 3 COOH 2829:: RR1 = O,R =R β2 =OH, βOH, R =R H3 = H HO O

R1 31 30: R11 = R23 = βOH, R32 = H O COOH 29: R = R = βOH, R = H R2 R R 3 1 30: R1 = R3 = βOH, R2 = H O 31 R2 R Oleanane derivatives3 from Panax stipuleanatus (С3, С28-diglycosides 32‒34) and Astilbe rivularis (3β-trans-p-coumaroyloxy-olean-12-en-27-oic acid (35) and 6β-hydroxy-3-oxoolean-12-en-27-oic acid Oleanane derivatives from Panax stipuleanatus (С3, С28-diglycosides 32‒34) and Astilbe rivularis (36)) as well as oleanane and ursane polyhydroxylated derivatives (37‒40) from Rosa laevigata (3β-trans-p-coumaroyloxy-olean-12-en-27-oic acid (35) and 6β-hydroxy-3-oxoolean-12-en-27-oic acid exhibited anti-inflammatory activities [88–90] due to the suppression of TGFBIp-mediated (36)) as well as oleanane and ursane polyhydroxylated derivatives (37‒40) from Rosa laevigata hyperpermeability in vitro and in vivo as well [90]. Ursane derivatives from Durio zibethinus were exhibited anti-inflammatory activities [88–90] due to the suppression of TGFBIp-mediated shown to exhibit a more pronounced anti-inflammatory effect in LPS-induced NO production in hyperpermeability in vitro and in vivo as well [90]. Ursane derivatives from Durio zibethinus were RAW 264.7 cell inhibition tests compared to oleanane triterpenoids, for which the activity was shown to exhibit a more pronounced anti-inflammatory effect in LPS-induced NO production in reduced by C2 hydroxylation [7]. RAW 264.7 cell inhibition tests compared to oleanane triterpenoids, for which the activity was reduced by C2 hydroxylation [7].

R2O 32: R1 = COOCH3, R2 = Ara(f), R3 = H HO COOGlc R1 R2O 3233:: RR11 = COOCHCH2OH, 3R, R2 =2 =H, Ara(f), R3 = Xyl R3 = H O RHO3O R COOGlc O1 3334:: RR11 = CHCOOH,2OH, R R22 = = R H,3 = R H3 = Xyl O R3O O 34: R1 = COOH, R2 = R3 = H

O COOH COOH O O 35 O 36 COOH OH COOH HO O O OH 35 36 OH OH HO OH OH 38: R1 = βOH, R2 = OH O HO COOH O 37 HO O 3839:: RR11 = βαOH,OH, RR22 == OHH O COOH HO HO OH HO O 37 R1 40: R1 = αOH, R2 = OH O 39: R1 = αOH, R2 = H OH HO HOHO OH OH R2 R1 40: R1 = αOH, R2 = OH

OH HO OH R The pronounced inhibitory activity of Cecropia2 telentida root extracts against 11β-hydroxysteroid dehydrogenase could be determined by the presence of a new, probably 2α,20β-dihydroxylated UA The pronounced inhibitory activity of Cecropia telentida root extracts against 11β-hydroxysteroid dehydrogenase could be determined by the presence of a new, probably 2α,20β-dihydroxylated UA

Molecules 2020, 25, x FOR PEER REVIEW 14 of 34

O O O CH OCH3 OC 3 COOH COOH C C H CH3 O O HO HO 26 27

Oleanane 27-carboxy derivatives (28‒30) isolated from Chrysosplenium carnosum exhibited pronounced inhibitory activities against the mouse melanoma cell lines B16F10 and SP2/0 [85]. Natural 2α-hydroxylated OA derivative (20, corosolic acid) had cytotoxic effects on CaSki cells (human cervical cancer) by inducing apoptosis, by arresting the cell cycle in the G2/M phase, and by inhibiting the PI3K/Akt signaling pathway [86]. 2,3-seco-Derivative of the ursane type (31) from Siphonodon celastrineus showed pronounced cytotoxic activity against MOLT-3 cancer cells [87].

28: R1 = O, R2 = βOH, R3 = H HO O

1 2 3 COOH 29: R = R = βOH, R = H R 1 30: R1 = R3 = βOH, R2 = H O 31 R2 R3

Molecules 2020, 25, 5526 13 of 31 Oleanane derivatives from Panax stipuleanatus (С3, С28-diglycosides 32‒34) and Astilbe rivularis (3β-trans-p-coumaroyloxy-olean-12-en-27-oic acid (35) and 6β-hydroxy-3-oxoolean-12-en-27-oic acid (36)) as Oleananewell as oleanane derivatives and from ursaPanaxne stipuleanatus polyhydroxylated(C3, C28-diglycosides derivatives 32(37–34‒40) and) fromAstilbe Rosa rivularis laevigata exhibited(3β-trans anti-inflammatory-p-coumaroyloxy-olean-12-en-27-oic activities [88–90] acid (due35) and to 6βthe-hydroxy-3-oxoolean-12-en-27-oic suppression of TGFBIp-mediated acid hyperpermeability(36)) as well as oleanane in vitro and and ursane in vivo polyhydroxylated as well [90]. derivativesUrsane derivatives (37–40) from fromRosa Durio laevigata zibethinusexhibited were anti-inflammatory activities [88–90] due to the suppression of TGFBIp-mediated hyperpermeability shown to exhibit a more pronounced anti-inflammatory effect in LPS-induced NO production in in vitro and in vivo as well [90]. Ursane derivatives from Durio zibethinus were shown to exhibit a RAW 264.7 cell inhibition tests compared to oleanane triterpenoids, for which the activity was more pronounced anti-inflammatory effect in LPS-induced NO production in RAW 264.7 cell inhibition reducedtests by compared C2 hydroxylation to oleanane triterpenoids,[7]. for which the activity was reduced by C2 hydroxylation [7].

R2O 32: R1 = COOCH3, R2 = Ara(f), R3 = H HO R COOGlc 1 33: R1 = CH2OH, R2 = H, R3 = Xyl O R3O O 34: R1 = COOH, R2 = R3 = H

O COOH COOH O 35 O 36 OH HO OH OH

38: R1 = βOH, R2 = OH O HO COOH O 37 HO O 39: R1 = αOH, R2 = H HO HO OH R1 40: R1 = αOH, R2 = OH

OH HO OH R2 Molecules 2020, 25, x FOR PEER REVIEW 15 of 34 β The Thepronounced pronounced inhibitory inhibitory activity activity of of CecropiaCecropia telentidatelentidaroot root extracts extracts against against 11 11-hydroxysteroidβ-hydroxysteroid dehydrogenase could be determined by the presence of a new, probably 2α,20β-dihydroxylated UA dehydrogenasederivative (41, isoyarumic could be determined acid) [91]. Ursaneby the presence type 2,3-dihydroxy of a new, probably derivatives 2α,20 fulgicβ-dihydroxylated acids A (42) and UA B (43) derivativeisolated from (41, isoyarumic Potentilla acid)fulgens [91 exhibited]. Ursane typeantioxidant 2,3-dihydroxy effect derivatives by inhibiting fulgic the acids formation A (42) and of free B(43) isolated from Potentilla fulgens exhibited antioxidant eﬀect by inhibiting the formation of free radicals [58]. Antiviral, antibacterial, hepatoprotective, anti-inflammatory, and antitumor activities radicals [58]. Antiviral, antibacterial, hepatoprotective, anti-inﬂammatory, and antitumor activities of of root extracts of Bupleurum chinense and B. scorzonerifolium species that are widespread in China root extracts of Bupleurum chinense and B. scorzonerifolium species that are widespread in China were werepartially partially provided provided by theby presencethe presence of bioactive of bioactive oleanane oleanane saikosaponins saikosaponins (44–46)[92 (44]. ‒46) [92].

OH OH

COOH COOH 42: R = αOH HO HO 43: R = βOH HO 41 R

O 44: R1 = β-D-Glc-(1-3)-β-D-Fuc, R2 = βOH, R3 = OH

R2 45: R1 = β-D-Glc-(1-6)-[α-L-Rha-(1-4)]-β-D-Glc, R2 = βOH, R3 = H

R1O 46: R1 = β-D-Glc-(1-3)-β-D-Fuc, R2 = αOH, R3 = OH CH2R3

Due to the wide distribution and availability of the pentacyclic triterpenoids discussed in the review, they are often used to synthesize new semi-synthetic derivatives with antibacterial [93], antiviral [10,82,94], anti-inflammatory [95,96], hepatoprotective [5], antitumor [8,97–99], and other activities. Biological activities of oleanane and ursane semi-synthetic derivatives were studied in a number of reviews summarizing the data on derivative types [100–102] and biological activities [103– 105].

5. Biological Transformation Taking into account the relative availability of the discussed triterpenic acids in natural sources and their high bioactivity, it is interesting to assess the possibility of directed transformations of these compounds to expand the range of biologically active compounds and to increase their bioavailability. Chemical methods are currently the most tested and used to transform acids 1‒3. However, chemical methods often require extreme acidity and temperature values, expensive catalysts, or protective groups of molecule reactive centers [8–10,106]. In contrast, biological transformation processes do not use aggressive reagents and can occur under normal eco-friendly conditions. Moreover, microorganisms are able to catalyze a wide range of regio- and stereoselective reactions that are difficult to perform chemically [12]. One of the most promising ways to highlight the pharmacological potential of native pentacyclic triterpenoids is the functionalization of their molecules by polyhydroxylation. Such functionalized derivatives hydroxylated by plant P450-dependent monooxygenases [107] are widespread in nature but are usually found in trace amounts or as part of a difficult-to-separate mixture. Enzymatic activity of microorganisms used for transformation of pentacyclic triterpenoids allows for obtaining hydroxylated derivatives with high yield and regioselectivity. Moreover, microbial hydroxylation occurs not only in the A ring but also at hard-to-reach positions on the B, D, and E rings. In addition to hydroxylation, microbial functionalization of pentacyclic triterpenoids can occur by less frequent reactions of carboxylation, glycosylation, lactone formation, and others.

Molecules 2020, 25, 5526 14 of 31

Due to the wide distribution and availability of the pentacyclic triterpenoids discussed in the review, they are often used to synthesize new semi-synthetic derivatives with antibacterial [93], antiviral [10,82,94], anti-inﬂammatory [95,96], hepatoprotective [5], antitumor [8,97–99], and other activities. Biological activities of oleanane and ursane semi-synthetic derivatives were studied in a number of reviews summarizing the data on derivative types [100–102] and biological activities [103–105].

5. Biological Transformation Taking into account the relative availability of the discussed triterpenic acids in natural sources and their high bioactivity, it is interesting to assess the possibility of directed transformations of these compounds to expand the range of biologically active compounds and to increase their bioavailability. Chemical methods are currently the most tested and used to transform acids 1–3. However, chemical methods often require extreme acidity and temperature values, expensive catalysts, or protective groups of molecule reactive centers [8–10,106]. In contrast, biological transformation processes do not use aggressive reagents and can occur under normal eco-friendly conditions. Moreover, microorganisms are able to catalyze a wide range of regio- and stereoselective reactions that are diﬃcult to perform chemically [12]. One of the most promising ways to highlight the pharmacological potential of native pentacyclic triterpenoids is the functionalization of their molecules by polyhydroxylation. Such functionalized derivatives hydroxylated by plant P450-dependent monooxygenases [107] are widespread in nature but are usually found in trace amounts or as part of a diﬃcult-to-separate mixture. Enzymatic activity of microorganisms used for transformation of pentacyclic triterpenoids allows for obtaining hydroxylated derivatives with high yield and regioselectivity. Moreover, microbial hydroxylation occurs not only in the A ring but also at hard-to-reach positions on the B, D, and E rings. In addition to hydroxylation, microbial functionalization of pentacyclic triterpenoids can occur by less frequent reactions of carboxylation, glycosylation, lactone formation, and others.

5.1. Fungal Transformation The described biotransformation processes of the compounds discussed in this review often occur using mycelial fungi of various species from the phyla Ascomycota (orders Glomerellales, Hypocreales) and Mucoromycota (order Mucorales). Fungal conversions of these compounds are accompanied by the formation of derivatives with hydroxyl groups at C1, C7, C15, C21, C24, or C30; oxo groups at C3, C7, or C21; glucopyranoside groups at C3, C28, or C30; lactone groups at C28/C13 or C3/C4, etc. as well as by the A ring fragmentation. The acid concentration used in biotransformation experiments usually ranges from 0.02 g/L to 1.0 g/L. The yield of transformation products (1.0% to 77.5%) and the duration of the processes (2 to 20 days) vary depending on the fungal catalyst characteristics (Table3). Abundantly found in nature, Rhizomucor miehei CECT 2749 partially metabolized OA (approximately 0.5 g/L) for 13 days to form of 1β,30-dihydroxy-OA (47), 7β,30-dihydroxy-OA (48), and 30-hydroxy-OA (25) in equivalent amounts (5.0-6.0%) [108]. Compound 25, known as queretaroic acid, was ﬁrst isolated from Lemaireocereus queretaroensis and L. beneckei endemic to Mexico [109]. This compound was shown to exhibit a moderate antitumor activity against HeLa cells [110]. Queretaroic acid 25 (3.3%) was obtained by the 24-h transformation of OA (0.2 g/L) by Escherichia coli cells expressing Nonomuraea recticatena CYP450 moxA and Pseudomonas redox partner camAB. At the same time, the use of cell-free reaction systems allowed to increase the yield of compound 25 to 17.0% [111]. Transformation of OA methyl ester (approximately 0.3 g/L) by R. miehei CECT 2749 during 13 days was accompanied by 7,30-dihydroxylation and, in addition, a 9(11),12-diene moiety formation in the C ring, producing 15.0% methyl 3β,7β,30-trihydroxy-oleane-9(11),12-dien-28-oate (49)[112]. Molecules 2020, 25, x FOR PEER REVIEW 16 of 34 or С3/С4, etc. as well as by the A ring fragmentation. The acid concentration used in biotransformation experiments usually ranges from 0.02 g/L to 1.0 g/L. The yield of transformation products (1.0% to 77.5%) and the duration of the processes (2 to 20 days) vary depending on the fungal catalyst characteristics (Table 3). Abundantly found in nature, Rhizomucor miehei CECT 2749 partially metabolized OA (approximately 0.5 g/L) for 13 days to form of 1β,30-dihydroxy-OA (47), 7β,30-dihydroxy-OA (48), and 30-hydroxy-OA (25) in equivalent amounts (5.0‒6.0%) [108]. Compound 25, known as queretaroic acid, was first isolated from Lemaireocereus queretaroensis and L. beneckei endemic to Mexico [109]. This compound was shown to exhibit a moderate antitumor activity against HeLa cells [110]. Queretaroic acid 25 (3.3%) was obtained by the 24-h transformation of OA (0.2 g/L) by Escherichia coli cells expressing Nonomuraea recticatena CYP450 moxA and Pseudomonas redox partner camAB. At the same time, the use of cell-free reaction systems allowed to increase the yield of compound 25 to 17.0% [111]. Transformation of OA methyl ester (approximately 0.3 g/L) by R. miehei CECT 2749 during 13 days was accompanied by 7,30-dihydroxylation and, in addition, a 9(11),12- diene moiety formation in the С ring, producing 15.0% methyl 3β,7β,30-trihydroxy-oleane-9(11),12- Molecules 2020, 25, 5526 15 of 31 dien-28-oate (49) [112].

R3 OH H

R1 47: R1 = R3 = OH, R2 = H COOH COOMe 48: R1 = H, R2 = R3 = OH

HO R2 49 HO OH

Colletotrichum lini AS 3.4486 was shown to catalyze the C15 hydroxylation of OA to Colletotrichumform 15α-hydroxy-OA lini AS 3.4486 (50)[ 113was]. shown In turn, to catalyze ascomycete theTrichothecium C15 hydroxylation roseum (M of 95.56)OA to [114form] 15α- hydroxy-OAand mucoromycete (50) [113]. InCircinella turn, ascomycete muscae AS Trichothecium 3.2695 [15] catalyzed roseum the(M 95.56) oxidation [114] of and OA mucoromycete (0.08 g/L Circinellaand muscae 0.02 g /ASL, respectively) 3.2695 [15] catalyzed to form 7β the,15α oxidation-dihydroxy-3-oxo-olean-12-en-28-oic of OA (0.08 g/L and 0.02 acid g/L, (51 respectively)) on day to form 76β,15 (7.5%)α-dihydroxy-3-oxo-olean-12-en-28-oic and day 7 (6.1%), respectively. At acid the same(51) on time, day an 6 intermediate(7.5%) and (6.25%) day 7 of(6.1%), respectively.the dihydroxylation At the same process—15 time, anα -hydroxy-3-oxo-olean-12-en-28-oicintermediate (6.25%) of the dihydroxylation acid (52)—was also process—15 isolated α- hydroxy-3-oxo-olean-12-en-28-oicfrom the culture medium of T.acid roseum (52)—was(M 95.56) also [ 114isolated]. C. frommuscae theAS culture 3.2695 medium simultaneously of T. roseum catalyzed a wide variety of hydroxylation and glycosylation reactions with the formation of (M 95.56) [114]. C. muscae AS 3.2695 simultaneously catalyzed a wide variety of hydroxylation and 7β-hydroxy-OA (53), 7β,21β-dihydroxy-OA (54), 7α,21β-dihydroxy-OA (55), 7β,15α-dihydroxy-OA glycosylation(56), 7β-hydroxy-3-oxo-olean-12-en-28-oic reactions with the formation acid of (57 7),β 7-hydroxy-OAβ,15α-dihydroxy-OA (53), 28-7βO,21-ββ-d-dihydroxy-OA-glucopyranosyl (54), 7α,21βester-dihydroxy-OA (58), 21β-hydroxy-OA (55), 7β,15 28-Oα-dihydroxy-OA-β-d-glucopyranosyl (56 ester), 7β (59-hydroxy-3-oxo-olean-12-en-28-oic), and OA 28-O-β-d-glucopyranosyl acid (57), 7esterβ,15 (α60-dihydroxy-OA) ranging from 3.1% 28- toO- 5.8%β-D-glucopyranosyl [15]. C7 hydroxylation ester and (58 C28), glycosylation21β-hydroxy-OA presumably 28-O-β-D- glucopyranosylcontributed ester to an (59 increase), and inOA the 28- anti-inﬂammatoryO-β-D-glucopyranosyl activity of ester derivatives, (60) ranging while C21from hydroxylation 3.1% to 5.8% [15]. C7 hydroxylationled to a decreased and ability C28 ofglycosylation compounds to presumably inhibit the release contributed of LPS-induced to an nitric increase oxide byin RAW the anti- inflammatory264.7 cells activity [15]. of derivatives, while C21 hydroxylation led to a decreased ability of Molecules 2020, 25, x FOR PEER REVIEW 17 of 34 compounds to inhibit the release of LPS-induced nitric oxide by RAW 264.7 cells [15].

R4 H H COOH O

R5 OH HO 50 R3 R1 R2

53: R1 = R2 = βOH, R3 = R4 = H, R5 = OH H 54: R1 = R2 = R4 = βOH, R3 = H, R5 = OH COOH 55: R1 = R4 = βOH, R2 = αOH, R3 = H, R5 = OH

R2 O R 1 56: R1 = R2 = βOH, R3 = αOH, R4 = H, R5 = OH 51: R1 = βOH, R2 = αOH 57: R1 = O, R2 = βOH, R3 = R4 = H, R5 = OH

52: R1 = H, R2 = αOH 58: R1 = R2 = βOH, R3 = αOH, R4 = H, R5 = O-β-D-glucopyranosyl

59: R1 = R4 = βOH, R2 = R3 = H, R5 = O-β-D-glucopyranosyl

60: R1 = βOH, R2 = R3 = R4 = H, R5 = O-β-D-glucopyranosyl

CC.. muscae ASAS 3.2695 3.2695 was was also also active active against against GA (approximately GA (approximately 0.06 g/L) 0.06 and g initiated/L) and oxidation, initiated oxidation,acetylation, acetylation, and glycosylation and glycosylation reactions with reactions the formation with theof metabolites formation (yield of metabolites did not exceed (yield 2.4%) did noton day exceed 7. The 2.4%) metabolites on day 7. included The metabolites 7β-hydroxy-GA included (61 7β), -hydroxy-GA15α-hydroxy-GA (61), (62 15),α -hydroxy-GA7β,15α-dihydroxy- (62), 7GAβ,15 (α63-dihydroxy-GA), 3,11-dioxo-7β-hydroxy-18 (63), 3,11-dioxo-7β-olean-12-en-30-oicβ-hydroxy-18 acidβ-olean-12-en-30-oic (64), 7β,15α-dihydroxy-3,11-dioxo-18 acid (64), 7β,15αβ-- dihydroxy-3,11-dioxo-18olean-12-en-30-oic acidβ -olean-12-en-30-oic(65), 7β-hydroxy-11-oxo-18 acid (65), 7ββ-hydroxy-11-oxo-18 -olean-12-en-30-oicβ -olean-12-en-30-oic acid 3-О-β-D- d acidglucopyranoside 3-O-β- -glucopyranoside (66), 7β-hydroxy-11-oxo-18 (66), 7β-hydroxy-11-oxo-18β-olean-12-en-30-oicβ-olean-12-en-30-oic acid 3-О-β-D acid-6′-О-acetyl- 3-O-β- d d glucopyranoside-60-O-acetyl-glucopyranoside (67), 15α-hydroxy-11-oxo-18 (67), 15α-hydroxy-11-oxo-18β-olean-12-en-30-oicβ-olean-12-en-30-oic acid 3-О-β-D-glucopyranoside acid 3-O-β- - (68), 15α-hydroxy-11-oxo-18β-olean-12-en-30-oic acid 3-О-β-D-6′-О-acetyl-glucopyranoside (69), and 7β-hydroxy-GA 30-О-β-D-glucopyranoside (70) [115]. The above GA derivatives inhibited LPS- induced NO release by RAW 264.7 cells to different extents. Moreover, compounds 61 and 64 were shown to exhibit antimicrobial activity against the antibiotic-resistant strain Enterococcus faecalis [116], while compound 63 exhibited antioxidant and hepatoprotective properties [117].

COOH HO 61: R1 = R2 = βOH, R3 = H HO H O O 62: R1 = βOH, R2 = H, R3 = αOH HO O 63: R1 = R2 = βOH, R3 = αOH OH O

R3 1 2 3 H R R 64: R = O, R = βOH, R = H O 1 2 65: R1 = O, R2 = βOH, R3 = αOH

66: R1 = O-β-D-glucopyranosyl, R2 = βOH, R3 = H HO OH 70

67: R1 = O-β-D-6′-О-acetyl-glucopyranosyl, R2 = βOH, R3 = H

68: R1 = O-β-D-glucopyranosyl, R2 = H, R3 = αOH

69: R1 = O-β-D-6′-О-acetyl-glucopyranosyl, R2 = H, R3 = αOH

The same authors reported Rhizopus arrhizus AS 3.2893 to perform oxidative transformation of GA (approximately 0.06 g/L) at С3, С7, and С15 for 7 days with the formation of 7β-hydroxy-GA (61), 15α-hydroxy-GA (62), 7β,15α-dihydroxy-GA (63), 3β-acetoxy-7β-hydroxy-11-oxo-18β -olean-12-en- 30-oic acid (71), 7-oxo-GA (72), 7α-hydroxy-GA (73), and 15α-hydroxy-7-oxo-GA (74) (the yield of each compound did not exceed 2.8%). They also exhibited anti-inflammatory effects in the LPS- induced NO production inhibition test in RAW 264.7 cells [115].

Molecules 2020, 25, x FOR PEER REVIEW 17 of 34

R4 H H

COOH O

R5 OH HO 50 R3 R1 R2

53: R1 = R2 = βOH, R3 = R4 = H, R5 = OH H 54: R1 = R2 = R4 = βOH, R3 = H, R5 = OH COOH 55: R1 = R4 = βOH, R2 = αOH, R3 = H, R5 = OH

R2 O R 1 56: R1 = R2 = βOH, R3 = αOH, R4 = H, R5 = OH 51: R1 = βOH, R2 = αOH 57: R1 = O, R2 = βOH, R3 = R4 = H, R5 = OH

52: R1 = H, R2 = αOH 58: R1 = R2 = βOH, R3 = αOH, R4 = H, R5 = O-β-D-glucopyranosyl

59: R1 = R4 = βOH, R2 = R3 = H, R5 = O-β-D-glucopyranosyl

60: R1 = βOH, R2 = R3 = R4 = H, R5 = O-β-D-glucopyranosyl

C. muscae AS 3.2695 was also active against GA (approximately 0.06 g/L) and initiated oxidation, acetylation, and glycosylation reactions with the formation of metabolites (yield did not exceed 2.4%) on day 7. The metabolites included 7β-hydroxy-GA (61), 15α-hydroxy-GA (62), 7β,15α-dihydroxy- GA (63), 3,11-dioxo-7β-hydroxy-18β-olean-12-en-30-oic acid (64), 7β,15α-dihydroxy-3,11-dioxo-18β- olean-12-en-30-oicMolecules 2020, 25, 5526 acid (65), 7β-hydroxy-11-oxo-18β -olean-12-en-30-oic acid 3-16О of-β 31-D- glucopyranoside (66), 7β-hydroxy-11-oxo-18β-olean-12-en-30-oic acid 3-О-β-D-6′-О-acetyl- glucopyranoside (67), 15α-hydroxy-11-oxo-18β-olean-12-en-30-oic acid 3-О-β-D-glucopyranoside glucopyranoside (68), 15α-hydroxy-11-oxo-18β-olean-12-en-30-oic acid 3-O-β-d-60-O-acetyl- (68glucopyranoside), 15α-hydroxy-11-oxo-18 (69), andβ 7-olean-12-en-30-oicβ-hydroxy-GA 30-O acid-β-D-glucopyranoside 3-О-β-D-6′-О-acetyl-glucopyranoside (70)[115]. The above (69 GA), and 7βderivatives-hydroxy-GA inhibited 30-О-β LPS-induced-D-glucopyranoside NO release (70) by [115]. RAW The 264.7 above cells toGA different derivatives extents. inhibited Moreover, LPS- inducedcompounds NO release61 and by64 RAWwere shown264.7 cells to exhibit to different antimicrobial extents. activityMoreover, against compounds the antibiotic-resistant 61 and 64 were shownstrain toEnterococcus exhibit antimicrobial faecalis [116 activity], while against compound the antibiotic-resistant63 exhibited antioxidant strain Enterococcus and hepatoprotective faecalis [116], whileproperties compound [117]. 63 exhibited antioxidant and hepatoprotective properties [117].

COOH HO 61: R1 = R2 = βOH, R3 = H HO H O O 62: R1 = βOH, R2 = H, R3 = αOH HO O 63: R1 = R2 = βOH, R3 = αOH OH O

R3 1 2 3 H R R 64: R = O, R = βOH, R = H O 1 2 65: R1 = O, R2 = βOH, R3 = αOH

66: R1 = O-β-D-glucopyranosyl, R2 = βOH, R3 = H HO OH 70

67: R1 = O-β-D-6′-О-acetyl-glucopyranosyl, R2 = βOH, R3 = H

68: R1 = O-β-D-glucopyranosyl, R2 = H, R3 = αOH

69: R1 = O-β-D-6′-О-acetyl-glucopyranosyl, R2 = H, R3 = αOH

Rhizopus arrhizus TheThe same same authors authors reported reported Rhizopus arrhizusAS AS 3.2893 3.2893 to performto perform oxidative oxidative transformation transformation of GA of (approximately 0.06 g/L) at C3, C7, and C15 for 7 days with the formation of 7β-hydroxy-GA GA (approximately 0.06 g/L) at С3, С7, and С15 for 7 days with the formation of 7β-hydroxy-GA (61), (61), 15α-hydroxy-GA (62), 7β,15α-dihydroxy-GA (63), 3β-acetoxy-7β-hydroxy-11-oxo-18β 15α-hydroxy-GA (62), 7β,15α-dihydroxy-GA (63), 3β-acetoxy-7β-hydroxy-11-oxo-18β -olean-12-en- -olean-12-en-30-oic acid (71), 7-oxo-GA (72), 7α-hydroxy-GA (73), and 15α-hydroxy-7-oxo-GA (74) (the 30-oic acid (71), 7-oxo-GA (72), 7α-hydroxy-GA (73), and 15α-hydroxy-7-oxo-GA (74) (the yield of yield of each compound did not exceed 2.8%). They also exhibited anti-inﬂammatory eﬀects in the each compound did not exceed 2.8%). They also exhibited anti-inflammatory effects in the LPS- MoleculesLPS-induced 2020, 25, x NOFOR productionPEER REVIEW inhibition test in RAW 264.7 cells [115]. 18 of 34 induced NO production inhibition test in RAW 264.7 cells [115]. COOH COOH

1 2 H H 72: R = O, R = H O O 73: R1 = αOH, R2 = H

74: R1 = O, R2 = αOH 71 R2 AcO OH HO R1

BiologicalBiological hydroxylation hydroxylation of ofGA GA at atconcentrations concentrations of of 0.25 0.25 g/L g/L (48 (48 h) h) and and 1.0 1.0 g/L g/L (14 (14 days) days) at C7 and atC15 C7 producing and C15 producingthe main metabolite the main metabolite 7β,15α-dihydroxy-GA 7β,15α-dihydroxy-GA (63) was (also63) wascatalyzed also catalyzed by C. lini AS 3.4486by C[118]. lini andAS 3.4486Absidia [ 118pseudocylinderospora] and Absidia pseudocylinderospora ATCC 24169 ATCC[117], 24169respectively. [117], respectively. Moreover, A. pseudocylinderosporaMoreover, A. pseudocylinderospora ATCC 24169, duringATCC 24169,long-term during cultivation long-term cultivation(14 days), catalyzed (14 days), catalyzed the formation the of formation of more than 18.0% of this compound (63) possessing antioxidant, hepatoprotective [117], more than 18.0% of this compound (63) possessing antioxidant, hepatoprotective [117], and anti- and anti-inﬂammatory [115] activities. inflammatory [115] activities. Preparative biotransformation of 0.1 g/L GA by Cunninghamella blakesleana CGMCC 3.970 forPreparative 7 days resulted biotransformation in a mixture ofof 150.1α ,24-dihydroxy-GAg/L GA by Cunninghamella (75), 15α,24-dihydroxy-3,11-dioxo-18 blakesleana CGMCC 3.970β for 7 days-olean-12-en-30-oic resulted in a mixture acid of 15 (76α),,24-dihydroxy-GA 7β,24-dihydroxy-3,11-dioxo-18 (75), 15α,24-dihydroxy-3,11-dioxo-18β-olean-12-en-30-oic acidβ -olean-12- (77), en-30-oic3,11-dioxo-7 acid β,15(76α),,24-trihydroxy-18 7β,24-dihydroxy-3,11-dioxo-18β-olean-12-en-30-oicβ-olean-12-en-30-oic acid (78), and 7αacid,24-dihydroxy-3,11 (77), 3,11-dioxo- 7β,15-dioxo-18α,24-trihydroxy-18β-olean-12-en-30-oicβ-olean-12-en-30-oic acid (79). The yield acid of(78 each), and acid 7 didα,24-dihydroxy-3,11 not exceed 1.3% [14 ].-dioxo-18 Compoundsβ-olean- 12-en-30-oic75, 78, and acid79 were (79). found The yield to eﬀectively of each inhibit acid did LPS-induced not exceed NO 1.3% production [14]. Compounds in mouse microglia 75, 78 cells, and 79 werewith found IC50 tovalues effectively of 0.76 mmol inhibit/L, 0.94LPS-induced mmol/L, and NO 0.16 production mmol/L, respectively in mouse [14 microglia]. Interestingly, cells whenwith IC50 valuesC. blakesleana of 0.76 mmol/L,AS 3.970 was0.94 used, mmol/L, an increase and in0.16 GA mmol/L, concentration respectively to 0.3 g/L led[14]. to accumulationInterestingly, of when two C. blakesleanamain products AS 3.970 7β was-hydroxy-GA used, an (increase61, 30.0%) in and GA 3,11-dioxo-7 concentrationβ-hydroxy-18 to 0.3 g/Lβ -olean-12-en-30-oicled to accumulation acid of two main products 7β-hydroxy-GA (61, 30.0%) and 3,11-dioxo-7β-hydroxy-18β-olean-12-en-30-oic acid (64, 25.0%) with pronounced antibacterial activity after 5 days [116]. In the case of C. elegans TSY- 0865, only 2.5% of 7β-hydroxy-GA (61) was formed during 8 days [119].

COOH COOH

H H O O 76: R1 = H, R3 = αOH 77: R1 = βOH, R3 = H 78: R1 = βOH, R3 = αOH OH HO R2 75 OR1 79: R1 = αOH, R3 = H OH OH

UA biotransformation processes include oxidation and lactone formation reactions. Thus, Syncephalastrum racemosum CGMCC 3.2500 transformed approximately 0.1 g/L UA to 7β,21β- dihydroxy-UA (80, 12.9%), 1β,21β-dihydroxy-UA (81, 3.9%), 1β-hydroxy-21-oxo-UA (82, 12.1%), 3β,21β-dihydroxy-urs-11-en-28,13-olide (83, 3.4%), and 3β,7β,21β-trihydroxy-urs-11-en-28,13-olide (84, 2.9%) within 10 days [13]. Similar bioconversion processes have been previously demonstrated using another strain S. racemosum AS 3.264 [120]. UA derivatives with a rare lactone moiety were shown to exhibit moderate inhibitory activity against HCV [13].

R3 OH

R1 H COOH O O

H H

HO R2 HO R

80: R1 = H, R2 = R3 = βOH 83: R = H 81: R1 = R3 = βOH, R2 = H 84: R = βOH

82: R1 = βOH, R2 = H, R3 = O

Molecules 2020, 25, x FOR PEER REVIEW 18 of 34

Molecules 2020, 25, x FOR PEER REVIEW 18 of 34 COOH COOH

1 2 H COOH H COOH 72: R = O, R = H O O 1 2 H H 7273: R: R 1= =O, α OH,R = HR2 = H O O 7374: R: R1 1= =α O,OH, R2 R =2 α=OH H 71 R2 AcO OH HO R1 74: R1 = O, R2 = αOH 71 R2 AcO OH HO R1

Biological hydroxylation of GA at concentrations of 0.25 g/L (48 h) and 1.0 g/L (14 days) at C7 andBiological C15 producing hydroxylation the main ofmetabolite GA at concentrations 7β,15α-dihydroxy-GA of 0.25 g/L (63 (48) was h) and also 1.0 catalyzed g/L (14 days)by C. liniat C7 AS and3.4486 C15 producing[118] and theAbsidia main pseudocylinderosporametabolite 7β,15α-dihydroxy-GA ATCC 24169 ( 63[117],) was alsorespectively. catalyzed Moreover,by C. lini AS A. 3.4486pseudocylinderospora [118] and Absidia ATCC pseudocylinderospora24169, during long-term ATCC cultivation 24169 (14[117], days), respectively. catalyzed theMoreover, formation A. of pseudocylinderosporamore than 18.0% of ATCC this compound24169, during (63 long-term) possessing cultivation antioxidant, (14 days),hepatoprotective catalyzed the [117], formation and anti- of moreinflammatory than 18.0% [115] of activities.this compound (63) possessing antioxidant, hepatoprotective [117], and anti- inflammatoryPreparative [115] biotransformation activities. of 0.1 g/L GA by Cunninghamella blakesleana CGMCC 3.970 for 7 daysPreparative resulted in biotransformationa mixture of 15α,24-dihydroxy-GA of 0.1 g/L GA by ( 75Cunninghamella), 15α,24-dihydroxy-3,11-dioxo-18 blakesleana CGMCC β3.970 -olean-12- for 7 daysen-30-oic resulted acid in a mixture(76), 7β of,24-dihydroxy-3,11-dioxo-18 15α,24-dihydroxy-GA (75),β 15-olean-12-en-30-oicα,24-dihydroxy-3,11-dioxo-18 acid (77), β -olean-12-3,11-dioxo- en-30-oic7β,15α,24-trihydroxy-18 acid (76), 7ββ,24-dihydroxy-3,11-dioxo-18-olean-12-en-30-oic acid (78β),-olean-12-en-30-oic and 7α,24-dihydroxy-3,11 acid ( 77-dioxo-18), 3,11-dioxo-β-olean- 7β12-en-30-oic,15α,24-trihydroxy-18 acid (79). Theβ-olean-12-en-30-oic yield of each acid acid did ( 78not), andexceed 7α ,24-dihydroxy-3,111.3% [14]. Compounds -dioxo-18 75, 78β,-olean- and 79 12-en-30-oicwere found acid to effectively (79). The yieldinhibit of LPS-inducedeach acid did NO not productionexceed 1.3% in [14]. mouse Compounds microglia 75cells, 78 ,with and IC7950 werevalues found of 0.76 to effectivelymmol/L, 0.94inhibit mmol/L, LPS-induced and 0.16 NO mmol/L, production respectively in mouse [14]. microglia Interestingly, cells with when IC 50C . valuesblakesleana of 0.76 AS mmol/L,3.970 was 0.94 used, mmol/L, an increase and in0.16 GA mmol/L, concentration respectively to 0.3 g/L [14]. led Interestingly, to accumulation when of twoC. blakesleanamainMolecules products2020 AS, 25 3.970, 5526 7β-hydroxy-GA was used, an increase(61, 30.0%) in GAand concen 3,11-dioxo-7trationβ to-hydroxy-18 0.3 g/L ledβ to-olean-12-en-30-oic accumulation17 of 31 of two acid main(64, 25.0%)products with 7β -hydroxy-GApronounced antibacterial (61, 30.0%) andactivity 3,11-dioxo-7 after 5 daysβ-hydroxy-18 [116]. In theβ-olean-12-en-30-oic case of C. elegans acidTSY- 0865, only 2.5% of 7β-hydroxy-GA (61) was formed during 8 days [119]. (64(64, 25.0%), 25.0%) with with pronouncedpronounced antibacterial antibacterial activity activity after 5after days 5 [ 116days]. In[116]. the case In oftheC .caseelegans of TSY-0865,C. elegans TSY- 0865,only only 2.5% 2.5% of 7β -hydroxy-GAof 7β-hydroxy-GA (61) was (61 formed) was duringformed 8 during days [119 8 ].days [119]. COOH COOH

H COOH H COOH O O 76: R1 = H, R3 = αOH H H 77: R1 = βOH, R3 = H O O 76: R1 = H, R3 = αOH 78: R1 = βOH, R3 = αOH OH 77: R1 = βOH, R3 = H R2 HO OR 1 3 75 1 7879: R: R1 = =β αOH,OH, R R3 = =α HOH OH R HO OH 2 75 OROH 1 79: R1 = αOH, R3 = H OH OH UA biotransformation processes include oxidation and lactone formation reactions. Thus, UA biotransformation processes include oxidation and lactone formation reactions. SyncephalastrumUA biotransformation racemosum processesCGMCC include3.2500 transformedoxidation and approximately lactone formation 0.1 g/L reactions. UA to 7Thus,β,21β - dihydroxy-UAThus, Syncephalastrum (80, 12.9%), racemosum 1β,21CGMCCβ-dihydroxy-UA 3.2500 transformed (81, 3.9%), approximately 1β-hydroxy-21-oxo-UA 0.1 g/L UA (82 to, 12.1%), Syncephalastrum7β,21β-dihydroxy-UA racemosum (80, 12.9%), CGMCC 1β,21β -dihydroxy-UA3.2500 transformed (81, 3.9%), approximately 1β-hydroxy-21- oxo-UA0.1 g/L( 82UA, 12.1%), to 7β,21β- 3β,21β-dihydroxy-urs-11-en-28,13-olide (83, 3.4%), and 3β,7β,21β-trihydroxy-urs-11-en-28,13-olide dihydroxy-UA3β,21β-dihydroxy-urs-11-en-28,13-olide (80, 12.9%), 1β,21β-dihydroxy-UA (83, 3.4%), and 3(β81,7β, ,213.9%),β-trihydroxy-urs-11-en-28,13-olide 1β-hydroxy-21-oxo-UA (82 (84, ,12.1%), (84, 2.9%) within 10 days [13]. Similar bioconversion processes have been previously demonstrated 3β2.9%),21β-dihydroxy-urs-11-en-28,13-olide within 10 days [13]. Similar bioconversion (83, 3.4%), processes and have 3β,7 beenβ,21 previouslyβ-trihydroxy-urs-11-en-28,13-olide demonstrated using (84usinganother, 2.9%) another strain withinS strain. racemosum10 days S. racemosum [13].AS 3.264Similar [120AS bioconversio]. 3.264 UA derivatives [120]. nUA processes with derivatives a rare have lactone with been moiety a previously rare were lactone shown demonstrated moiety to were usingshownexhibit another moderateto exhibit strain inhibitory moderate S. racemosum activity inhibitory against AS 3.264activity HCV [120]. [13 against]. UA HCVderivatives [13]. with a rare lactone moiety were shown to exhibit moderate inhibitory activity against HCV [13].

R3 OH

R3 OH R1 H COOH O O

R1 H H COOH H O O

HO R2 HO R H H HO R HO R 80: R1 = H, R2 = 2R3 = βOH 83: R = H 80: R1 = H, R2 = R3 = βOH 81: R1 = R3 = βOH, R2 = H 8384: R: R = =H β OH Molecules 2020, 25, x FOR PEER REVIEW 19 of 34 8182: R: R1 1= =R β3 OH,= βOH, R2 =R H,2 = RH3 = O 84: R = βOH

For 20 days, 82Gliocladium: R1 = βOH, roseum R2 = H, CGMCC R3 = O 3.3657 catalyzed oxidative transformation of the A ring of UAFor (0.1 20g/L) days, by BaeyerGliocladium–Villiger-type roseum CGMCC reaction 3.3657and C21 catalyzed oxidation oxidative to form transformation21β-hydroxy-3-oxo-urs- of 12-en-3,4-olide-28-oicthe A ring of UA (0.1acid g(/85L), by8.0%), Baeyer–Villiger-type 3,21-dioxo-urs-12-en-3,4-olide-28-oic reaction and C21 oxidation acid (86 to, 6.25%), form 21β- 21β-hydroxy-3-oxo-urs-12-en-3,4-olide-28-oic acid (85, 8.0%), 3,21-dioxo-urs-12-en-3,4-olide-28-oic acid hydroxy-3,4-seco-ursane-4(23),12-dien-3,28-dioic acid (87, 1.5%), and 21-oxo-3,4-seco-ursane- (86, 6.25%), 21β-hydroxy-3,4-seco-ursane-4(23),12-dien-3,28-dioic acid (87, 1.5%), and 21-oxo-3,4- 4(23),12-dien-3,28-dioic acid (88, 1.0%). Derivatives 86 and 88 containing a 21-oxo group showed the seco-ursane-4(23),12-dien-3,28-dioic acid (88, 1.0%). Derivatives 86 and 88 containing a 21-oxo group mostshowed pronounced the most pronounced anti-HCV anti-HCVactivity [121]. activity [121].

R R H H

COOH COOH

H H O 85: R = βOН HOOC 87: R = βOН O 86: R = O 88: R = O

β WhenWhen MucorMucor spinosus AS AS 3.3450 3.3450 was was used used to bioconvertto bioconvert UA, UA, three three metabolites, metabolites, 7 -hydroxy-UA 7β-hydroxy-UA 28-ethanone (89, 5.04%), 7β,21β-dihydroxy-UA (81, 1.64%), and 21β-hydroxy-urs-12-en-28-oic acid 28-ethanone (89, 5.04%), 7β,21β-dihydroxy-UA (81, 1.64%), and 21β-hydroxy-urs-12-en-28-oic acid 3- 3-O-β-D-glucopyranoside (90, 2.13%), were formed within 96 h. Compound 89 was shown to exhibit O-β-D-glucopyranoside (90, 2.13%), were formed within 96 h. Compound 89 was shown to exhibit pronounced (higher than that of UA) cytotoxic activity against HeLa, K562, and KB tumor cell pronouncedlines [122]. (higher than that of UA) cytotoxic activity against HeLa, K562, and KB tumor cell lines [122].

CH3 O OH C OH C O O OH HO OH OH O 89 90

5.2. Bacterial Transformation The literature describes a few cases of pentacyclic triterpenoid bioconversion using gram- positive bacteria of the genera Bacillus, Nocardia, and Streptomyces and accompanied by the formation of С1, С2, С7, С11, С21, С24, or С29 hydroxylated derivatives, derivatives with a methyl ester group at C28; oxogroup at C3; additional carboxyl groups at С29 or С30; glucopyranoside groups at С3, С28, or С30; lactone group at С28/С13; and derivatives with a fragmented A ring. In biotransformation experiments, the compounds are usually used in concentrations ranging from 0.04 g/L to 0.3 g/L, and the yield of derivatives ranges from 5.0% to 60.0%. The duration of bioconversion is 3 to 5 days; only in the case of using Nocardia, it reached 13 days (Table 3). Actinobaceria of the genus Nocardia were capable of selective methylation of the C28-carboxylic group of pentacyclic triterpenoids [123]. The use of resting or immobilized cells of N. iowensis DSM 45197 as biocatalysts of the OA (approximately 0.3 g/L) transformation process for 13 days resulted in the formation of methyl OA (91) as the main bioconversion product (more than 60.0%), small amounts (≤5.0%) of methyl 3-oxo-olean-12-en-28-oat (92), and metabolite 93 unidentified by the authors [16]. 3-oxo-OA (92) was shown to have pronounced antimelanoma [124], antileishmanial, and antitrypanosomal effects [125]. Despite numerous successful examples to increase the efficiency of the biotransformation process by immobilizing microbial cells [126], the use of fixed Nocardia cells in alginate carriers led to a decrease in their catalytic activity, as confirmed by a 10-fold decrease in the formation of compound 91 and only a short-term occurrence of compound 93 in the culture medium [16]. The ability of Nocardia sp. to transform UA by methylation, by C3 oxidation, and by formation of the enone moiety in the A ring was previously shown. It was noted that the biotransformation process did not depend on the composition of the culture medium used, while the temperature increase (from 28 °С to 36 °С) for actinobacteria cultivation contributed to a 2-fold increase in the reaction rate [127].

Molecules 2020, 25, x FOR PEER REVIEW 19 of 34

For 20 days, Gliocladium roseum CGMCC 3.3657 catalyzed oxidative transformation of the A ring of UA (0.1 g/L) by Baeyer–Villiger-type reaction and C21 oxidation to form 21β-hydroxy-3-oxo-urs- 12-en-3,4-olide-28-oic acid (85, 8.0%), 3,21-dioxo-urs-12-en-3,4-olide-28-oic acid (86, 6.25%), 21β- hydroxy-3,4-seco-ursane-4(23),12-dien-3,28-dioic acid (87, 1.5%), and 21-oxo-3,4-seco-ursane- 4(23),12-dien-3,28-dioic acid (88, 1.0%). Derivatives 86 and 88 containing a 21-oxo group showed the most pronounced anti-HCV activity [121].

R R H H

COOH COOH

H H O 85: R = βOН HOOC 87: R = βOН O 86: R = O 88: R = O

When Mucor spinosus AS 3.3450 was used to bioconvert UA, three metabolites, 7β-hydroxy-UA 28-ethanone (89, 5.04%), 7β,21β-dihydroxy-UA (81, 1.64%), and 21β-hydroxy-urs-12-en-28-oic acid 3- O-β-D-glucopyranoside (90, 2.13%), were formed within 96 h. Compound 89 was shown to exhibit Molecules 2020, 25, 5526 18 of 31 pronounced (higher than that of UA) cytotoxic activity against HeLa, K562, and KB tumor cell lines [122].

CH3 O OH C OH C O O OH HO OH OH O 89 90

5.2.5.2. Bacterial Bacterial TransformationTransformation The literatureliterature describes describes a few a casesfew ofcases pentacyclic of pentac triterpenoidyclic triterpenoid bioconversion bioconversion using gram-positive using gram- positivebacteria ofbacteria the genera of theBacillus genera, Nocardia Bacillus, and, NocardiaStreptomyces, and Streptomycesand accompanied and accompanied by the formation by ofthe C1, formation ofC2, С C7,1, С C11,2, С7, C21, С11, C24, С21, or C29С24, hydroxylated or С29 hydroxylated derivatives, derivatives, derivatives with deri avatives methyl with ester a group methyl at C28; ester group atoxogroup C28; oxogroup at C3; additional at C3; carboxyladditional groups carboxyl at C29 groups or C30; glucopyranosideat С29 or С30; groupsglucopyranoside at C3, C28, or groups C30; at С3, Сlactone28, or group С30; at C28lactone/C13; andgroup derivatives at С with28/С a13; fragmented and derivatives A ring. In biotransformation with a fragmented experiments, A ring. In biotransformationthe compounds are usuallyexperiments, used in the concentrations compounds ranging are usually from used 0.04 g in/L concentrations to 0.3 g/L, and the ranging yield of from 0.04 g/Lderivatives to 0.3 g/L, ranges and from the 5.0%yield to of 60.0%. derivatives The duration ranges of from bioconversion 5.0% to 60.0%. is 3 to 5 The days; duration only in the of casebioconversion of using Nocardia, it reached 13 days (Table3). is 3 to 5 days; only in the case of using Nocardia, it reached 13 days (Table 3). Actinobaceria of the genus Nocardia were capable of selective methylation of the C28-carboxylic Actinobaceria of the genus Nocardia were capable of selective methylation of the C28-carboxylic group of pentacyclic triterpenoids [123]. The use of resting or immobilized cells of N. iowensis DSM group45197 asof biocatalystspentacyclic of triterpenoids the OA (approximately [123]. The 0.3 use g/ L)of transformationresting or immobilized process for cells 13 days of N resulted. iowensis DSM 45197in the as formation biocatalysts of methyl of the OA OA (91 (approximately) as the main bioconversion 0.3 g/L) transformation product (more process than 60.0%), for 13 smalldays resulted inamounts the formation ( 5.0%) ofof methylmethyl 3-oxo-olean-12-en-28-oat OA (91) as the main bioconversion (92), and metabolite product93 unidentified (more than by 60.0%), the small ≤ amountsauthors [ 16(≤].5.0%) 3-oxo-OA of methyl (92) was 3-oxo-olean-12-en-28-oat shown to have pronounced (92 antimelanoma), and metabolite [124], 93 antileishmanial, unidentified by the authorsand antitrypanosomal [16]. 3-oxo-OA effects (92 [)125 was]. Despite shown numerous to have successfulpronounced examples antimelanoma to increase the[124], effi ciencyantileishmanial, of andthe biotransformationantitrypanosomal process effects by [125]. immobilizing Despite microbialnumerous cells successful [126], the examples use of fixed toNocardia increasecells the in efficiency ofalginate the biotransformation carriers led to a decrease process in by their immobilizing catalytic activity, microbial as confirmed cells [126], by a 10-foldthe use decrease of fixed inNocardia the cells information alginate of carriers compound led91 toand a decrease only a short-term in their occurrence catalytic ofactivity, compound as confirmed93 in the culture by a medium 10-fold [ 16decrease]. in The ability of Nocardia sp. to transform UA by methylation, by C3 oxidation, and by formation of the the formation of compound 91 and only a short-term occurrence of compound 93 in the culture enone moiety in the A ring was previously shown. It was noted that the biotransformation process did medium [16]. The ability of Nocardia sp. to transform UA by methylation, by C3 oxidation, and by not depend on the composition of the culture medium used, while the temperature increase (from formation of the enone moiety in the A ring was previously shown. It was noted that the Molecules28 ◦C 2020 to 36, 25◦, C)x FOR for actinobacteriaPEER REVIEW cultivation contributed to a 2-fold increase in the reaction rate [127].20 of 34 biotransformation process did not depend on the composition of the culture medium used, while the temperature increase (from 28 °С to 36 °С) for actinobacteria cultivation contributed to a 2-fold increase in the reaction rate [127]. O O H H O O + 1/2 O2 R 91: R = OH HO

92: R = O 93

TheThe bacterial bacterial culture culture ofofStreptomyces Streptomyces griseus griseusATCC ATCC 13273 catalyzed13273 catalyzed hydroxylation hydroxylation and site-selective and site- selectiveoxidation oxidation of the C29of the methyl C29 methyl group of group OA (0.04 of OA g/L) (0 to.04 the g/L) carboxyl to the groupcarboxyl within group 5 days within to form 5 days to 3β-hydroxy-olean-12-ene-28,29-dioic acid (94, 21.9%), 3β,24-dihydroxy-olean-12-ene-28,29-dioic acid form 3β-hydroxy-olean-12-ene-28,29-dioic acid (94, 21.9%), 3β,24-dihydroxy-olean-12-ene-28,29- (95, 32.7%), and 3β,21β,24-trihydroxy-olean-12-ene-28,29-dioic acid (96, 5.9%). Hydroxylation at C21 dioic acid (95, 32.7%), and 3β,21β,24-trihydroxy-olean-12-ene-28,29-dioic acid (96, 5.9%). was shown to increase the anti-inﬂammatory activity of OA derivatives [128]. Using the same strain, Hydroxylationbiotransformation at C21 of was OA shown (approximately to increase 0.05 the g/L) anti with-inflammatory the formation activity of derivatives of OA94 derivativesand 96 was [128]. Usingpreviously the same described strain, by biotransformation Y. Zhu et al. [129]. of OA (approximately 0.05 g/L) with the formation of derivatives 94 and 96 was previously described by Y. Zhu et al. [129].

HOOC R1 94: R1 = R2 = H COOH 95: R1 = OH, R2 = H

HO 96: R1 = OH, R2 = βOH

In addition to OA, S. griseus ATCC 13273 cells can also transform UA (0.04 g/L) by catalyzing site-selective oxidation of the C30 methyl group to the carboxyl one and C24-hydroxylation within 3 days to produce 3β-hydroxy-urs-12-ene-28,30-dioic acid (97) and 3β,24-dihydroxy-urs-12-ene-28,30- dioic acid (98), with the product yield exceeding 30.0%. Transformation of UA derivatives 3-oxo-UK (99) and 2α-hydroxy-UA (20, corosolic acid) at a concentration of 0.04 g/L by this actinobacterial strain also occurred by selective C30-oxidation and hydroxylation, that led to a mixture of 3-oxo-urs-12- ene-28,30-dioic acid (100, 24.1%) and 24-hydroxy-3-oxo-urs-12-ene-28,30-dioic acid (101, 45.9%) in the former case and a mixture of 2α,3β-dihydroxy-urs-12-ene-28,30-dioic acid (102, 29.0%) and 2α,3β,24- trihydroxy-urs-12-ene-28,30-dioic acid (103, 15.9%) in the latter case [17].

COOH R2 COOH

H H

COOH COOH COOH HO

HO HO 97: R = H O 99: R1 = R2 = H 102: R = H

R R R 98: R = OH 1 100: R1 = H, R2 = COOH 103: R = OH

101: R1 = OH, R2 = COOH

A gram-positive Bacillus megaterium CGMCC 1.1741 was able to transform UA (0.2 g/L), generating the main derivative 1β,11α-dihydroxy-UA (104, 26.87%) and minor derivatives (5.03– 13.50%) 3-oxo-urs-12-en-28-oic acid (99), 1β,11α-dihydroxy-3-oxo-urs-12-en-28-oic acid (105), 1β- hydroxy-3-oxo-urs-12-en-28,13-olide (106), and 1β,11α-dihydroxy-3-oxo-urs-12-en -28-O-β-D- glucopyranoside (107) over 4 days. Derivatives 105 and 106 were shown to effectively inhibit LPS- induced NO release in RAW 264.7 cells (IC50 1.71 µmol and 1.24 µmol, respectively) [18], and derivative 99 was shown to inhibit cathepsin L-like rCPB2.8 protease of Leishmania mexicana [130].

Molecules 2020, 25, 5526 19 of 31

Table 3. Biological activity of pentacyclic triterpenoid products derived by biotransformation.

Microorganism Initial Compound, Concentration Derivative, Yield Biological Activity Reference Fungi 1β,30-dihydroxy-OA (47), 5.0% - * OA, 0.5 g/L 7β,30-dihydroxy-OA (48), 6.0% - [108] Rhizomucor miehei CECT 2749 30-hydroxy-OA (25, queretaroic acid), 5.0% Antitumor OA methyl ester, 0.3 g/L Methyl 3β,7β,30-trihydroxy-oleane-9(11),12-dien-28-oate (49), 15.0% -[112] 7β,15α-dihydroxy-3-oxo-olean-12-en-28-oic acid (51), 7.5% Anti-inflammatory Trichothecium roseum (M 95.56) OA, 0.08 g/L [114] 15α-hydroxy-3-oxo-olean-12-en-28-oic acid (52), 6.25% - 7β,15α-dihydroxy-3-oxo-olean-12-en-28-oic acid (51), 6.1% Anti-inflammatory 7β-hydroxy-OA (53), 5.8% Anti-inflammatory 7β,21β-dihydroxy-OA (54), 4.2% - 7α,21β-dihydroxy-OA (55), 3.1% - OA, 0.02 g/L 7β,15α-dihydroxy-OA (56), 4.9% Anti-inflammatory [15] 7β-hydroxy-3-oxo-olean-12-en-28-oic acid (57), 3.8% - 7β,15α-dihydroxy-OA 28-O-β-d-glucopyranosyl ester (58), 4.7% Anti-inflammatory 21β-hydroxy-OA 28-O-β-D-glucopyranosyl ester (59), 5.1% Anti-inflammatory OA 28-O-β-D-glucopyranosyl ester (60), 5.5% Anti-inflammatory Anti-inflammatory 7β-hydroxy-GA (61), 1.5% Antimicrobial 15α-hydroxy-GA (62), 1.9% Anti-inflammatory Circinella muscae AS 3.2695 Anti-inflammatory 7β,15α-dihydroxy-GA (63), 1.8% Antioxidant Hepatoprotective Anti-inflammatory 3,11-dioxo-7β-hydroxy-18β-olean-12-en-30-oic acid (64), 1.7% Antimicrobial 7β,15α-dihydroxy-3,11-dioxo-18β-olean-12-en-30-oic acid (65), 2.2% Anti-inflammatory GA, 0.06 g/L [115] 7β-hydroxy-11-oxo-18β-olean-12-en-30-oic acid Anti-inflammatory 3-O-β-d-glucopyranoside (66), 1.8% 7β-hydroxy-11-oxo-18β-olean-12-en-30-oic acid Anti-inflammatory 3-O-β-d-60-O-acetyl-glucopyranoside (67), 1.1% 15α-hydroxy-11-oxo-18β-olean-12-en-30-oic acid Anti-inflammatory 3-O-β-d-glucopyranoside (68), 2.5% 15α-hydroxy-11-oxo-18β-olean-12-en-30-oic acid Anti-inflammatory 3-O-β-d-60-O-acetyl-glucopyranoside (69), 1.8% 7β-hydroxy-GA 30-O-β-d-glucopyranoside (70), 2.2% Anti-inflammatory Molecules 2020, 25, 5526 20 of 31

Table 3. Cont.

Microorganism Initial Compound, Concentration Derivative, Yield Biological Activity Reference Anti-inflammatory 7β-hydroxy-GA (61), 2.8% Antimicrobial 15α-hydroxy-GA (62), 2.2% Anti-inflammatory Anti-inflammatory 7β,15α-dihydroxy-GA (63), 1.7% Antioxidant Rhizopus arrhizus AS 3.2893 GA, 0.06 g/L [115] Hepatoprotective 3β-acetoxy-7β-hydroxy-11-oxo-18β-olean-12-en-30-oic acid (71), 1.7% Anti-inflammatory 7-oxo-GA (72), 1.3% Anti-inflammatory 7α-hydroxy-GA (73), 2.6% Anti-inflammatory 15α-hydroxy-7-oxo-GA (74), 1.3% Anti-inflammatory Anti-inflammatory Colletotrichum lini AS 3.4486 GA, 0.25 g/L 7β,15α-dihydroxy-GA (63), NR ** Antioxidant [118] Hepatoprotective Anti-inflammatory Absidia pseudocylinderospora ATCC GA, 1.0 g/L 7β,15α-dihydroxy-GA (63), 18.0% Antioxidant [117] 24169 Hepatoprotective 15α,24-dihydroxy-GA (75), 0.11% Anti-inflammatory 15α,24-dihydroxy-3,11-dioxo-18β-olean-12-en-30-oic acid (76), 0.75% - Cunninghamella blakesleana 7β,24-dihydroxy-3,11-dioxo-18β-olean-12-en-30-oic acid (77), 0.16% - GA, 0.1 g/L [14] CGMCC 3.970 3,11-dioxo-7β,15α,24-trihydroxy-18β-olean-12-en-30-oic acid Anti-inflammatory (78), 0.29% 7α,24-dihydroxy-3,11-dioxo-18β-olean-12-en-30-oic acid (79), 1.27% Anti-inflammatory Anti-inflammatory 7β-hydroxy-GA (61), 30.0% C. blakesleana CGMCC 3.970 GA, 0.3 g/L Antimicrobial [116] Anti-inflammatory 3,11-dioxo-7β-hydroxy-18β-olean-12-en-30-oic acid (64), 25.0% Antimicrobial Anti-inflammatory C. elegans TSY-0865 GA, 0.4 g/L 7β-hydroxy-GA (61), 2.5% [119] Antimicrobial 7β,21β-dihydroxy-UA (80), 12.9% - 1β,21β-dihydroxy-UA (81), 3.9% - Syncephalastrum racemosum UA, 0.1 g/L 1β-hydroxy-21-oxo-UA (82), 12.1% - [13] GCMCC 3.2500 3β,21β-dihydroxy-urs-11-en-28,13-olide (83), 3.4% Antihepatitis 3β,7β,21β-trihydroxy-urs-11-en-28,13-olide (84), 2.9% Antihepatitis 21β-hydroxy-3-oxo-urs-12-en-3,4-olide-28-oic acid (85), 8.0%) - 3,21-dioxo-urs-12-en-3,4-olide-28-oic acid (86), 6.25% Antihepatitis Gliocladium roseum CGMCC 3.3657 UA, 0.1 g/L [121] 21β-hydroxy-3,4-seco-ursane-4(23),12-diene-3,28-dioic acid (87), 1.5% - 21-oxo-3,4-seco-ursane-4(23),12-diene-3,28-dioic acid (88), 1.0% Antihepatitis Molecules 2020, 25, 5526 21 of 31

Table 3. Cont.

Microorganism Initial Compound, Concentration Derivative, Yield Biological Activity Reference 7β-hydroxy-UA 28-ethanone (89), 5.04% Antitumor UA, 0.3 g/L 7β,21β-dihydroxy-UA (81), 1.64% - Mucor spinosus AS 3.3450 [122] 21β-hydroxy-urs-12-en-28-oic acid 3-O-β-d-glucopyranoside (90), - 2.13% OA, 0.2 g/L 7β,21β-dihydroxy-OA (54), 53.75% - Rhizopus chinensis CICC 40335 Anti-inflammatory [34] GA, 0.2 g/L 7β-hydroxy-GA (61), 77.5% Antimicrobial Bacteria Methyl 3β-hydroxy-olean-12-en-28-oat (91), 63.0% Antitumor Nocardia iowensis (DSM 45197, Methyl 3-oxo-olean-12-en-28-oat (92, oleanonic acid methyl ester), Antitumor OA, 0.3 g/L [16] NRRL 5646) 5.0% Antiprotozoal Metabolite 93 with hydroxyl and methyl groups, 2.0% - 3β-hydroxy-olean-12-ene-28,29-dioic acid (94), 21.9% - Streptomyces griseus ATCC 13273 OA, 0.04 g/L 3β,24-dihydroxy-olean-12-ene-28,29-dioic acid (95), 32.7% - [128] 3β,21β,24-trihydroxy-olean-12-ene-28,29-dioic acid (96), 5.9% Anti-inflammatory 3β-hydroxy-urs-12-ene-28,30-dioic acid (97), 30.4% - UA, 0.04 g/L 3β,24-dihydroxy-urs-12-ene-28,30-dioic acid (98), 31.6% - 3-oxo-urs-12-ene-28,30-dioic acid (100), 24,1% - S. griseus ATCC 13273 3-oxo-UA, 0.04 g/L [17] 24-hydroxy-3-oxo-urs-12-ene-28,30-dioic acid (101), 45.9% - 2α,3β-dihydroxy-urs-12-ene-28,30-dioic acid (102), 29.0% - 2α-hydroxy-UA, 0.04 g/L 2α,3β,24-trihydroxy-urs-12-ene-28,30-dioic acid (103), 15.9% - 1β,11α-dihydroxy-UA (104), 26.87% - 3-oxo-urs-12-en-28-oic acid (99), 6.23% Anti-leishmania Bacillus megaterium 1β,11α-dihydroxy-3-oxo-urs-12-en-28-oic acid (105), 13.5% Anti-inflammatory CGMCC UA, 0.2 g/L [18] 1.1741 1β-hydroxy-3-oxo-urs-12-en-28,13-olide (106), 5.03% Anti-inflammatory 1β,11α-dihydroxy-3-oxo-urs-12-en-28-O-β-d-glucopyranoside (107), - 8.57% 7β,21β-dihydroxy-olean-12-en-28-oic acid 3-O-β-d-glucopyranoside 7β,21β-dihydroxy-OA, 0.2 g/L - (108), 46.5% B. subtilis ATCC 6633 GA, 0.2 g/L GA 30-O-β-d-glucopyranoside (111), 27.5% - [34] 7β-hydroxy-GA, 0.2 g/L 7β-hydroxy-GA 30-O-β-d-glucopyranoside (68), 44.0% - 7β,21β,29-trihydroxy-OA (109), 26.0% - S. griseus ATCC 13273 7β,21β-dihydroxy-OA, 0.2 g/L 3β,7β,21β-trihydroxy-olean-12-ene-28,29-dioic acid (110), 15.0% Neuroprotective Note: * Biological activity of the compound has not yet been detected. ** The value is not reported. GA, glycyrrhetinic acid; OA, oleanolic acid; UA, ursolic acid. Molecules 2020, 25, x FOR PEER REVIEW 20 of 34 Molecules 2020, 25, x FOR PEER REVIEW 20 of 34

O O H O H O H H O O O O + 1/2 O2 R 91: R = OH HO + 1/2 O2 R 91: R = OH HO 93 92: R = O 92: R = O 93

The bacterial culture of Streptomyces griseus ATCC 13273 catalyzed hydroxylation and site- The bacterial culture of Streptomyces griseus ATCC 13273 catalyzed hydroxylation and site- selective oxidation of the C29 methyl group of OA (0.04 g/L) to the carboxyl group within 5 days to selective oxidation of the C29 methyl group of OA (0.04 g/L) to the carboxyl group within 5 days to form 3β-hydroxy-olean-12-ene-28,29-dioic acid (94, 21.9%), 3β,24-dihydroxy-olean-12-ene-28,29- form 3β-hydroxy-olean-12-ene-28,29-dioic acid (94, 21.9%), 3β,24-dihydroxy-olean-12-ene-28,29- dioic acid (95, 32.7%), and 3β,21β,24-trihydroxy-olean-12-ene-28,29-dioic acid (96, 5.9%). dioic acid (95, 32.7%), and 3β,21β,24-trihydroxy-olean-12-ene-28,29-dioic acid (96, 5.9%). Hydroxylation at C21 was shown to increase the anti-inflammatory activity of OA derivatives [128]. Hydroxylation at C21 was shown to increase the anti-inflammatory activity of OA derivatives [128]. Using the same strain, biotransformation of OA (approximately 0.05 g/L) with the formation of UsingMolecules the2020 same, 25, 5526 strain, biotransformation of OA (approximately 0.05 g/L) with the formation22 of of 31 derivatives 94 and 96 was previously described by Y. Zhu et al. [129]. derivatives 94 and 96 was previously described by Y. Zhu et al. [129].

HOOC HOOC R1 R1 94: R1 = R2 = H 94: R1 = R2 = H COOH COOH 95: R1 = OH, R2 = H 95: R1 = OH, R2 = H

HO 96: R1 = OH, R2 = βOH HO 96: R1 = OH, R2 = βOH R2 R2 In addition to OA,S S.griseus griseus ATCC 13273 cells can also transform UA (0.04 g/L) by catalyzing In addition to OA, S.. griseus ATCC 13273 cells can also transform UA (0.04 g/L) g/L) by catalyzing site-selective oxidation of the C30 methyl group to the carboxyl one and C24-hydroxylation within 3 site-selective oxidationoxidation of of the the C30 C30 methyl methyl group group to theto the carboxyl carboxyl one one and and C24-hydroxylation C24-hydroxylation within within 3 days 3 days to produceβ 3β-hydroxy-urs-12-ene-28,30-dioic acid (97)β and 3β,24-dihydroxy-urs-12-ene-28,30- daysto produce to produce 3 -hydroxy-urs-12-ene-28,30-dioic 3β-hydroxy-urs-12-ene-28,30-dioic acid (acid97) and(97) 3and,24-dihydroxy-urs-12-ene-28,30-dioic 3β,24-dihydroxy-urs-12-ene-28,30- dioic acid (98), with the product yield exceeding 30.0%. Transformation of UA derivatives 3-oxo-UK dioicacid (acid98), ( with98), with the productthe product yield yield exceeding exceeding 30.0%. 30.0%. Transformation Transformation of of UA UA derivativesderivatives 3-oxo-UK (99) and 2αα-hydroxy-UA (20, corosolic acid) at a concentration of 0.04 g/L by this actinobacterial strain (99) and and 2 2α-hydroxy-UA-hydroxy-UA (20 (20, corosolic, corosolic acid) acid) at a at concentration a concentration of 0.04 of g/L 0.04 by g/ thisL by actinobacterial this actinobacterial strain also occurred by selective C30-oxidation and hydroxylation, that led to a mixture of 3-oxo-urs-12- alsostrain occurred also occurred by selective by selectiveC30-oxidation C30-oxidation and hydroxylation, and hydroxylation, that led to a that mixture led toof a3-oxo-urs-12- mixture of ene-28,30-dioic acid (100, 24.1%) and 24-hydroxy-3-oxo-urs-12-ene-28,30-dioic acid (101, 45.9%) in the ene-28,30-dioic3-oxo-urs-12-ene-28,30-dioic acid (100, 24.1%) acid and (100 24-hydroxy-3-oxo-u, 24.1%) and 24-hydroxy-3-oxo-urs-12-ene-28,30-dioicrs-12-ene-28,30-dioic acid (101, 45.9%) acid in ( 101the, former case and a mixture of 2α,3β-dihydroxy-urs-12-ene-28,30-dioicα β acid (102, 29.0%) and 2α,3β,24- former45.9%) incase the and former a mixture case and of 2 aα,3 mixtureβ-dihydroxy-urs-12-ene-28,30-dioic of 2 ,3 -dihydroxy-urs-12-ene-28,30-dioic acid (102, 29.0%) acid and (102 2α,,3 29.0%)β,24- trihydroxy-urs-12-ene-28,30-dioicα β acid (103, 15.9%) in the latter case [17]. trihydroxy-urs-12-ene-28,30-dioicand 2 ,3 ,24-trihydroxy-urs-12-ene-28,30-dioic acid (103, 15.9%) acid in (103 the, latter 15.9%) case in the[17]. latter case [17].

COOH R2 COOH COOH R2 COOH

H H H H COOH COOH COOH COOH COOH HO COOH HO

HO 97: R = H O 1 2 HO HO 99: R = R = H HO 102: R = H 97: R = H O 99: R1 = R2 = H 102: R = H R R1 R R 98: R = OH R 100: R1 = H, R2 = COOH R 103: R = OH 98: R = OH 1 100: R1 = H, R2 = COOH 103: R = OH 101: R1 = OH, R2 = COOH 101: R1 = OH, R2 = COOH A gram-positive Bacillus megaterium CGMCC 1.1741 was able to transform UA A gram-positive Bacillus megaterium CGMCC 1.1741 was able to transform UA (0.2 g/L), (0.2A g /L),gram-positive generating Bacillus the main megaterium derivative CGMCC 1β,11 α1.1741-dihydroxy-UA was able to (104 transform, 26.87%) UA and (0.2 minorg/L), generating the main derivative 1β,11α-dihydroxy-UA (104, 26.87%) and minor derivatives (5.03– generatingderivatives the (5.03–13.50%) main derivative 3-oxo-urs-12-en-28-oic 1β,11α-dihydroxy-UA acid (99 (104), 1β, ,1126.87%)α-dihydroxy-3-oxo-urs-12-en-28-oic and minor derivatives (5.03– 13.50%) 3-oxo-urs-12-en-28-oic acid (99), 1β,11α-dihydroxy-3-oxo-urs-12-en-28-oic acid (105), 1β- 13.50%)acid (105 3-oxo-urs-12-en-28-oic), 1β-hydroxy-3-oxo-urs-12-en-28,13-olide acid (99), 1β,11α-dihydroxy-3-oxo-urs-12-en-28-oic (106), and 1β,11α-dihydroxy-3-oxo-urs-12-en acid (105), 1β- hydroxy-3-oxo-urs-12-en-28,13-olide (106), and 1β,11α-dihydroxy-3-oxo-urs-12-en -28-O-β-D- -28-hydroxy-3-oxo-urs-12O-β-D-glucopyranoside-en-28,13-olide (107) over (106 4 days.), and Derivatives 1β,11α-dihydroxy-3-oxo-urs-12-en105 and 106 were shown to-28- eﬀectivelyO-β-D- glucopyranoside (107) over 4 days. Derivatives 105 and 106 were shown to effectively inhibit LPS- glucopyranosideinhibit LPS-induced (107 NO) over release 4 days. in RAW Derivatives 264.7 cells 105 (IC and50 1.71106 wereµmol shown and 1.24 to µeffectivelymol, respectively) inhibit LPS- [18], Moleculesinduced 2020 NO, 25 ,release x FOR PEER in RAWREVIEW 264.7 cells (IC50 1.71 µmol and 1.24 µmol, respectively) [18],21 ofand 34 inducedand derivative NO release99 was in shown RAW to 264.7 inhibit cells cathepsin (IC50 1.71 L-like µmol rCPB2.8 and protease1.24 µmol, of Leishmania respectively) mexicana [18],[ 130and]. derivative 99 was shown to inhibit cathepsin L-like rCPB2.8 protease of Leishmania mexicana [130]. derivative 99 was shown to inhibit cathepsin L-like rCPB2.8 protease of Leishmania mexicana [130].

OH R3 O R1 OH OH COOH O O O Glc R2 O O 106 107 104: R1 = R2 = βOH, R3 = αOH

105: R1 = βOH, R2 = O, R3 = αOH

Xu et al. (2020) have recently used the tandem biotransformation of oleanane-type pentacyclic Xu et al. (2020) have recently used the tandem biotransformation of oleanane-type pentacyclic triterpenoids using the fungal strain Rhizopus chinensis CICC 40335 and bacterial strains Bacillus subtilis triterpenoids using the fungal strain Rhizopus chinensis CICC 40335 and bacterial strains Bacillus ATCC 6633 and Streptomyces griseus ATCC 13273 [34]. The primary transformation of OA (0.2 g/L) subtilis ATCC 6633 and Streptomyces griseus ATCC 13273 [34]. The primary transformation of OA (0.2 using R. chinensis CICC 40335 occurred within 4 days by the formation of 7β,21β-dihydroxy-OA (54, g/L) using R. chinensis CICC 40335 occurred within 4 days by the formation of 7β,21β-dihydroxy-OA 53.75%) previously obtained using the fungal strains Mucor rouxii NRRL 1894 [131] and Circinella (54, 53.75%) previously obtained using the fungal strains Mucor rouxii NRRL 1894 [131] and Circinella muscae AS 3.2695 [15]. Further 4-day biotransformation of the obtained compound (54) led to the formation of 7β,21β-dihydroxy-olean-12-en-28-oic acid 3-O-β-D-glucopyranoside (108, 46.5%) using B. subtilis ATCC 6633 cells and a mixture of 7β,21β,29-trihydroxy-OA (109, 26.0%) and 3β,7β,21β- trihydroxy-olean-12-ene-28,29-dioic acid (110, 15.0%) using S. griseus ATCC 13273 cells [34]. Bioconversion of GA (0.2 g/L) using R. chinensis CICC 40335 occurred by selective oxidation with the formation of 7β-hydroxy-GA (61, 77.5%) on day 4 [34]. Note that the C7-hydroxylation process is typical for many cultures, for example, C. muscae AS 3.2695, Rhizopus arrhizus AS 3.2893 [115], and representatives of the genus Cunninghamella [116,119]. Further addition of GA or compound 61 in the culture medium of B. subtilis ATCC 6633 led to the formation of 30-O-β-D-glucopyranoside derivatives (111 (27.5%) and 68 (44.0%), respectively) previously obtained by B. Fan et al. [115]. Assessment of the neuroprotective potential of the obtained OA and GA derivatives revealed that glycosylation significantly contributed to a decrease in the neuroprotective activity of compounds while carboxylation led to a significant increase in the neuroprotective effect of OA derivatives [34].

R3 HO R2 HO O H O HO OH COOH O

O R1 OH

108: R1 = O-β-D-glucopyranoside, R2 = βOH, R3 = H HO 111 109: R1 = R2 = βOH, R3 = OH

110: R1 = R2 = βOH, R3 = COOH

Molecules 2020, 25, x FOR PEER REVIEW 21 of 34

OH R3 O R1 OH OH COOH O O O Glc R2 O O 106 107 104: R1 = R2 = βOH, R3 = αOH

105: R1 = βOH, R2 = O, R3 = αOH

Xu et al. (2020) have recently used the tandem biotransformation of oleanane-type pentacyclic triterpenoids using the fungal strain Rhizopus chinensis CICC 40335 and bacterial strains Bacillus

Moleculessubtilis2020 ATCC, 25, 55266633 and Streptomyces griseus ATCC 13273 [34]. The primary transformation of OA23 of (0.2 31 g/L) using R. chinensis CICC 40335 occurred within 4 days by the formation of 7β,21β-dihydroxy-OA (54, 53.75%) previously obtained using the fungal strains Mucor rouxii NRRL 1894 [131] and Circinella muscaemuscaeAS AS 3.26953.2695 [[15].15]. Further Further 4-day 4-day biotransformation biotransformation of of the the obtained obtained compound compound (54 ()54 led) led to tothe theformation formation of 7 ofβ,21 7ββ,21-dihydroxy-olean-12-en-28-oicβ-dihydroxy-olean-12-en-28-oic acid acid 3-O- 3-β-OD--glucopyranosideβ-D-glucopyranoside (108, (46.5%)108, 46.5%) using usingB. subtilisB. subtilis ATCCATCC 6633 cells 6633 and cells a mixture and a mixture of 7β,21 ofβ,29-trihydroxy-OA 7β,21β,29-trihydroxy-OA (109, 26.0%) (109 and, 26.0%) 3β,7β and,21β- 3βtrihydroxy-olean-12-ene-28,29-dioic,7β,21β-trihydroxy-olean-12-ene-28,29-dioic acid (110 acid, 15.0%) (110 using, 15.0%) S. usinggriseusS .ATCCgriseus 13273ATCC cells 13273 [34]. cells [34]. BioconversionBioconversion of of GAGA (0.2 g/L) g/L) using using RR. .chinensischinensis CICCCICC 40335 40335 occurred occurred by byselective selective oxidation oxidation with withthe formation the formation of 7β of-hydroxy-GA 7β-hydroxy-GA (61, 77.5%) (61, 77.5%) on day on 4 [34]. day Note 4 [34 ].that Note the thatC7-hydroxylation the C7-hydroxylation process is processtypical is for typical many for cultures, many cultures, for example, for example, C. muscaeC. muscae AS 3.2695,AS 3.2695, RhizopusRhizopus arrhizus arrhizus AS 3.2893AS 3.2893 [115], [115 and], andrepresentatives representatives of the of thegenus genus CunninghamellaCunninghamella [116,119].[116,119 Further]. Further addition addition of GA of or GA compound or compound 61 in 61the inculture the culture medium medium of B of. subtilisB. subtilis ATCCATCC 6633 6633 led led to to the formationformation ofof 30- 30-OO-β--βd--glucopyranosideD-glucopyranoside derivativesderivatives ( 111(111(27.5%) (27.5%) and and68 68(44.0%), (44.0%), respectively) respectively) previouslypreviously obtainedobtained byby B.B. FanFan etet al.al. [115[115].]. AssessmentAssessment of of the the neuroprotective neuroprotective potential potential of of the the obtained obtained OA OA and and GA GA derivatives derivatives revealed revealed that that glycosylationglycosylation significantly significantly contributed contributed to to a a decrease decrease in in the the neuroprotective neuroprotective activity activity of of compounds compounds whilewhile carboxylation carboxylation led led to to a a significant significant increase increase in in the the neuroprotective neuroprotective eff effectect of of OA OA derivatives derivatives [34 [34].].

R3 HO R2 HO O H O HO OH COOH O

O R1 OH

108: R1 = O-β-D-glucopyranoside, R2 = βOH, R3 = H HO 111 109: R1 = R2 = βOH, R3 = OH

110: R1 = R2 = βOH, R3 = COOH

6. Conclusions Triterpenoids are secondary metabolites of plants, fungi, marine invertebrates, and algae that are formed during cyclization of an acyclic triterpene squalene [132–138]. According to the number of cycles, triterpenes and triterpenoids are divided into several groups; the most numerous are pentacyclic triterpenic derivatives [139]. In nature, this group is most widely represented by compounds of oleanane (OA and GA) and ursane (UA) types, which in large quantities can accumulate in various parts of higher plants [39,41,46]. In addition, the biosynthesis of these compounds can be carried out in microbial cells able to catalyze the MVA pathway and to be genetically modified using plant genes [64,68–70]. The main difficulties of microbial biosynthesis are generally considered to be complexity and long duration of processes of searching for terpenoid synthesis genes of plants, their isolation, and the preparation of genetically modified microorganisms. The rapid development of bioinformatics methods, sequencing techniques, and de novo DNA synthesis significantly simplified the abovementioned processes and gave a new impetus to research in this field [140]. With the close cooperation of biochemists, microbiologists, and genetic scientists, microbial biosynthesis can become a promising technology for obtaining valuable pentacyclic triterpenoids. The compounds discussed in the review exhibit antitumor, antiviral, hepatoprotective, neuroprotective, and other activities [5,7,42,60,74,82,83]. Despite the wide range of known biological properties, the use of pentacyclic triterpenoids in pharmacology and medicine is limited because of their high hydrophobicity. The solution to this problem might be the synthesis of triterpenic derivatives with increased bioactivity, solubility, and bioavailability [5,82,93,94,141]. Studies of the possibility of obtaining new OA, GA, and UA derivatives by directed biotransformations should be considered a promising area. Over 20 examples of biotransformations of these compounds using fungal and bacterial cultures most often catalyzing hydroxylation have been described since 2013. Less frequently, the literature describes processes of deeper oxidation of triterpenoids as well as their glycosylation, esterification, acetylation, or carboxylation. Biocatalytic formation of triterpenic lactones or their derivatives with fragmented C–C bond was reported only Molecules 2020, 25, 5526 24 of 31 in a few cases using UA [13,121]. In the biotransformation processes employing fungi, the degree of triterpenic acid conversion usually ranges from 2.6% to 77.5%, with an initial concentration of 0.02 g/L to 1.0 g/L, whereas in bacterial transformations, the degree of conversion reaches 27.5-70.0%, with an initial concentration of 0.04-0.3 g/L. When analyzed, the data showed that the biotransformation of oleanane and ursane pentacyclic triterpenoids led to derivatives with antioxidant, anti-inflammatory, antiviral, antitumor, antiparasitic, antimicrobial, neuroprotective, and hepatoprotective properties (Table3). Provided more active development as an interdisciplinary tool, this method of obtaining biologically active compounds and their intermediates seems to be a promising strategy to design new medicinal agents against cancer and neurodegenerative diseases as well as potent antibacterial drugs against antibiotic-resistant pathogenic strains of microorganisms. By combining methods of microbial synthesis of native pentacyclic triterpenoids and their subsequent microbial transformations into bioavailable compounds, the industrial microbiology could provide a cycle of production of valuable biologically active substances. However, it should be noted that the described microbial catalysts have significant drawbacks. Fungi usually demonstrate mycelial growth type and form spores and mycotoxins, whereas few bacterial catalysts described are mainly represented by species, with their individual strains being pathogens. In this context, it is essential to conduct further in-depth studies of the processes of biological transformation of pentacyclic triterpenoids and to search for new nonpathogenic bacterial strains able to carry out highly effective synthesis of triterpenic derivatives with pronounced biological activities.

Author Contributions: All authors made equal contributions to preparing this manuscript. Conceptualization, I.B.I., V.V.G., and N.A.L.; writing—original draft preparation, V.V.G. and N.A.L.; writing—review and editing, I.B.I., V.V.G., and N.A.L.; funding acquisition, I.B.I. and V.V.G. All authors have read and agreed to the published version of the manuscript. Funding: The work was funded by the Russian Foundation for Basic Research (grant No. 20-34-90104), by the Russian Science Foundation (grant number 18-14-00140), and by the Russian Federation Ministry of Education and Science (State Assignments AAAA-A19-119112290008-4, AAAA-A18-118030790037-7, and AAAA-A19-119112290010-7). Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Calixto, J.B. The role of natural products in modern drug discovery. An. Acad. Bras. Ciênc. 2019, 91, e20190105. [CrossRef][PubMed] 2. Kumar, D.; Dubey, K.K. Hybrid approach for transformation for betulin (an anti-HIV molecule). In New and Future Developments in Microbial Biotechnology and Bioengineering; Gupta, P., Pandey, A., Eds.; Elsevier BV: Amsterdam, The Netherlands, 2019; pp. 193–203. [CrossRef] 3. Duric, K.; Kovac-Besovic, E.; Niksic, H.; Sofic, E. Antibacterial activity of methanolic extracts, decoction and isolated triterpene products from different parts of birch, Betula pendula, Roth. J. Plant Stud. 2013, 2, 61–70. [CrossRef] 4. Song, N.Y.; Cho, J.G.; Im, D.; Lee, D.Y.; Wu, Q.; Seo, W.D.; Kang, H.C.; Lee, Y.H.; Baek, N.I. Triterpenoids from Fragaria ananassa calyx and their inhibitory effects on melanogenesis in B16-F10 mouse melanoma cells. Nat. Prod. Res. 2013, 27, 2219–2223. [CrossRef][PubMed] 5. Zou, L.-W.; Dou, T.-Y.; Wang, P.; Lei, W.; Weng, Z.-M.; Hou, J.; Wang, D.-D.; Fan, Y.-M.; Zhang, W.-D.; Ge, G.; et al. Structure-activity relationships of pentacyclic triterpenoids as potent and selective inhibitors against human carboxylesterase 1. Front. Pharmacol. 2017, 8, 435. [CrossRef] 6. Zhang, J.; Liu, F.; Zhang, X. Inhibition of proliferation of SGC7901 and BGC823 human gastric cancer cells by ursolic acid occurs through a caspase-dependent apoptotic pathway. Med Sci. Monit. 2019, 25, 6846–6854. [CrossRef] 7. Feng, J.; Yi, X.; Huang, W.; Wang, Y.; He, X. Novel triterpenoids and glycosides from durian exert pronounced anti-inflammatory activities. Food Chem. 2018, 241, 215–221. [CrossRef] 8. Alho, D.P.S.; Salvador, J.A.R.; Cascante, M.; Marin, S. Synthesis and antiproliferative activity of novel heterocyclic glycyrrhetinic acid derivatives. Molecules 2019, 24, 766. [CrossRef] Molecules 2020, 25, 5526 25 of 31

9. Chouaïb, K.; Hichri, F.; Nguir, A.; Daami-Remadi, M.; Elie, N.; Touboul, D.; Ben Jannet, H.; Hamza, M.A. Semi-synthesis of new antimicrobial esters from the natural oleanolic and maslinic acids. Food Chem. 2015, 183, 8–17. [CrossRef] 10. Grishko, V.V.; Galaiko, N.V.; Tolmacheva, I.A.; Kucherov, I.I.; Eremin, V.F.; Boreko, E.I.; Savinova, O.V.; Slepukhin, P.A. Functionalization, cyclization and antiviral activity of A-secotriterpenoids. Eur. J. Med. Chem. 2014, 83, 601–608. [CrossRef] 11. Wu, P.P.; Zhang, B.J.; Cui, X.P.; Yang, Y.; Jiang, Z.Y.; Zhou, Z.H.; Zhong, Y.Y.; Mai, Y.Y.; Ouyang, Z.; Chen, H.S.; et al. Synthesis and biological evaluation of novel ursolic acid analogues as potential α-glucosidase inhibitors. Sci. Rep. 2017, 7, 45578. [CrossRef] 12. Shah, S.A.A.; Tan, H.L.; Sultan, S.; Faridz, M.A.B.M.; Shah, M.A.B.M.; Nurfazilah, S.; Hussain, M. Microbial-catalyzed biotransformation of multifunctional triterpenoids derived from phytonutrients. Int. J. Mol. Sci. 2014, 15, 12027–12060. [CrossRef][PubMed] 13. Fu, S.B.; Yang, J.-S.; Cui, J.L.; Sun, D.-A. Biotransformation of ursolic acid by Syncephalastrum racemosum CGMCC 3.2500 and anti-HCV activity. Fitoterapia 2013, 86, 123–128. [CrossRef] 14. Ma, Y.; Liu, J.M.; Chen, R.; An, X.Q.; Dai, J.G. Microbial transformation of glycyrrhetinic acid and potent neural anti-inflammatory activity of the metabolites. Chin. Chem. Lett. 2017, 28, 1200–1204. [CrossRef] 15. Yan, S.; Lin, H.; Huang, H.-L.; Yang, M.; Xu, B.; Chen, G.-T. Microbial hydroxylation and glycosidation of oleanolic acid by Circinella muscae and their anti-inflammatory activities. Nat. Prod. Res. 2018, 33, 1849–1855. [CrossRef][PubMed] 16. Ludwig, B.; Geib, D.; Haas, C.; Steingroewer, J.; Bley, T.; Muffler, K.; Ulber, R. Whole-cell biotransformation of oleanolic acid by free and immobilized cells of Nocardia iowensis: Characterization of new metabolites. Eng. Life Sci. 2015, 15, 108–115. [CrossRef] 17. Xu, S.H.; Zhang, C.; Wang, W.W.; Yu, B.Y.; Zhang, J. Site-selective biotransformation of ursane triterpenes by Streptomyces griseus ATCC 13273. RSC Adv. 2017, 7, 20754–20759. [CrossRef] 18. Zhang, C.; Xu, S.-H.; Ma, B.-L.; Wang, W.-W.; Yu, B.; Zhang, J. New derivatives of ursolic acid through the biotransformation by Bacillus megaterium CGMCC 1.1741 as inhibitors on nitric oxide production. Bioorg. Med. Chem. Lett. 2017, 27, 2575–2578. [CrossRef][PubMed] 19. Grishko, V.V.; Tarasova, E.V.; Ivshina, I.B. Biotransformation of betulin to betulone by growing and resting cells of the actinobacterium Rhodococcus rhodochrous IEGM 66. Process. Biochem. 2013, 48, 1640–1644. [CrossRef] 20. Tarasova, E.V.; Grishko, V.V.; Ivshina, I.B. Cell adaptations of Rhodococcus rhodochrous IEGM 66 to betulin biotransformation. Process. Biochem. 2017, 52, 1–9. [CrossRef] 21. Laskin, A.I.; Grabowich, P.; Fried, J.; Meyers, C.D.L. Transformations of eburicoic acid. V. Cleavage of ring A by the fungus Glomerella fusarioides. J. Med. Chem. 1964, 7, 406–409. [CrossRef] 22. Canonica, L.; Jommi, G.; Pagnoni, U.M.; Pelizzoni, F.; Ranzi, B.M.; Scolastico, C. Microbiological oxidation of triterpenoids. I. 7β-Hydroxyglycyrrhetic acid. Gazz. Chim. Ital. 1966, 96, 820–831. 23. Canonica, L.; Ferrari, M.; Jommi, G.; Pagnoni, U.M.; Pelizzoni, F.; Ranzi, B.M.; Maroni, S.; Nencini, G.; Salvatori, T. Microbiological oxidation of triterpenoids. II. 15α-Hydroxyglycyrrhetic and 7β,15α-dihydroxyglycyrrhetic acids. Gazz. Chim. Ital. 1967, 97, 1032–1051. 24. Hikino, H.; Nabetani, S.; Takemoto, T. Microbial transformation of oleanolic acid. Yakugaku Zasshi 1969, 89, 809–813. [CrossRef] 25. Ferrari, M.; Pagnoni, U.M.; Pelizzoni, F.; Ranzi, B.M.; Salvatori, T. Microbiological oxidation of triterpenoids. III. Behavior of 18α-glycyrrhetic, liquiritic, and 18α-liquiritic acids. Gazz. Chim. Ital. 1969, 99, 848–862. 26. Sakano, K.-I.; Ohshima, M. Structures of conversion products formed from 18β-glycyrrhetinic acid by Streptomyces sp. G-20. Agric. Biol. Chem. 1986, 50, 763–766. [CrossRef] 27. Sakano, K.-I.; Ohshima, M. Microbial conversion of 18β-glycyrrhetinic acid and 22α-hydroxy- 18β-glycyrrhetinic acid by Chainia antibiotic. Agric. Biol. Chem. 1986, 50, 1239–1245. [CrossRef] 28. Collins, D.O.; Ruddock, P.L.D.; De Grasse, J.C.; Reynolds, W.F.; Reese, P.B. Microbial transformation of cadina-4,10(15)-dien-3-one, aromadendr-1(10)-en-9-one and methyl ursolate by Mucor plumbeus ATCC 4740. Phytochemistry 2002, 59, 479–488. [CrossRef] 29. Cheng, Z.; Yu, B.-Y.; Cordell, G.A.; Qiu, S.X. Biotransformation of quinovic acid glycosides by microbes: Direct conversion of the ursane to the oleanane triterpene skeleton by Nocardia sp. NRRL 5646. Org. Lett. 2004, 6, 3163–3165. [CrossRef] Molecules 2020, 25, 5526 26 of 31

30. Parra, A.; Rivas, F.; Garcia-Granados, A.; Martinez, A. Microbial transformation of triterpenoids. Mini Rev. Org. Chem. 2009, 18, 307–320. [CrossRef] 31. Bhatti, H.N.; Khera, R.A. Biotransformations of diterpenoids and triterpenoids: A review. J. Asian Nat. Prod. Res. 2013, 16, 70–104. [CrossRef] 32. Mutafova, B.; Fernandes, P.; Mutafov, S.; Berkov, S.; Pavlov, A. Microbial transformations of plant secondary metabolites. In Bioprocessing of Plant In Vitro Systems; Pavlov, A., Bley, T., Eds.; Springer Science and Business Media LLC: New York, NY, USA, 2018; pp. 85–124. [CrossRef] 33. Azerad, R. Microbial transformations of pentacyclic triterpenes. In Green Biocatalysis; Patel, R.N., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2016; pp. 675–714. [CrossRef] 34. Xu, S.H.; Chen, H.L.; Fan, Y.; Xu, W.; Zhang, J. Application of tandem biotransformation for biosynthesis of new pentacyclic triterpenoid derivatives with neuroprotective effect. Bioorg. Med. Chem. Lett. 2020, 30, 126947. [CrossRef] 35. Grishko, V.V.; Nogovitsina, Y.M.; Ivshina, I.B. Bacterial transformation of terpenoids. Russ. Chem. Rev. 2014, 83, 323–342. [CrossRef] 36. Hill, R.A.; Connolly, J.D. Triterpenoids. Nat. Prod. Rep. 2012, 29, 780–818. [CrossRef] 37. Thimmappa, R.; Geisler, K.; Louveau, T.; O’Maille, P.; Osbourn, A. Triterpene Biosynthesis in plants. Annu. Rev. Plant Biol. 2014, 65, 225–257. [CrossRef] 38. Wu, H.; Li, G.; Liu, S.; Liu, D.; Chen, G.; Hu, N.; Suo, Y.; You, J. Simultaneous determination of six triterpenic acids in some Chinese medicinal herbs using ultrasound-assisted dispersive liquid–liquid microextraction and high-performance liquid chromatography with fluorescence detection. J. Pharm. Biomed. Anal. 2015, 107, 98–107. [CrossRef] 39. Moldoveanu, S.C.; Scott, W.A. Analysis of four pentacyclic triterpenoid acids in several bioactive botanicals with gas and liquid chromatography and mass spectrometry detection. J. Sep. Sci. 2015, 39, 324–332. [CrossRef] 40. Peragón, J. Time course of pentacyclic triterpenoids from fruits and leaves of olive tree (Olea europaea L.) cv. Picual and cv. Cornezuelo during ripening. J. Agric. Food Chem. 2013, 61, 6671–6678. [CrossRef] 41. Kalani, K.; Agarwal, J.; Alam, S.; Khan, F.F.; Pal, A.; Srivastava, S.K. In silico and in vivo anti-malarial studies of 18β-glycyrrhetinic acid from Glycyrrhiza glabra. PLoS ONE 2013, 8, e74761. [CrossRef] 42. Chen, H.-J.; Kang, S.-P.; Lee, I.-J.; Lin, Y.-L. Glycyrrhetinic acid suppressed NF-κB activation in TNF-α-induced hepatocytes. J. Agric. Food Chem. 2014, 62, 618–625. [CrossRef] 43. Li, J.; Lee, Y.S.; Choi, J.S.; Sung, H.Y.; Kim, J.K.; Lim, S.S.; Kang, Y.H. Roasted licorice extracts dampen high glucose-induced mesangial hyperplasia and matrix deposition through blocking Akt activation and TGF-β signaling. Phytomedicine 2010, 17, 800–810. [CrossRef] 44. Ko, B.S.; Jang, J.S.; Hong, S.M.; Sung, S.R.; Lee, J.E.; Lee, M.Y.; Jeon, W.K.; Park, S. Changes in components, glycyrrhizin and glycyrrhetinic acid, in raw Glycyrrhiza uralensis Fisch, modify insulin sensitizing and insulinotropic actions. Biosci. Biotechnol. Biochem. 2007, 71, 1452–1461. [CrossRef][PubMed] 45. Wo´zniak,Ł.; Sk ˛apska,S.; Marszałek, K. Ursolic acid—A pentacyclic triterpenoid with a wide spectrum of pharmacological activities. Molecules 2015, 20, 20614–20641. [CrossRef][PubMed] 46. Guinda, A.; Rada, M.; Delgado, T.; Gutiérrez-Adánez, P.; Castellano, J.M. Pentacyclic triterpenoids from olive fruit and leaf. J. Agric. Food Chem. 2010, 58, 9685–9691. [CrossRef][PubMed] 47. Romero, C.; García, A.; Medina, E.; Ruíz-Méndez, M.V.; De Castro, A.; Brenes, M. Triterpenic acids in table olives. Food Chem. 2010, 118, 670–674. [CrossRef] 48. Pensec, F.; P ˛aczkowski,C.; Grabarczyk, M.; Wo´zniak,A.; Bénard-Gellon, M.; Bertsch, C.; Chong, J.; Szakiel, A. Changes in the triterpenoid content of cuticular waxes during fruit ripening of eight grape (Vitis vinifera) cultivars grown in the upper rhine valley. J. Agric. Food Chem. 2014, 62, 7998–8007. [CrossRef][PubMed] 49. Wang, C.M.; Chen, H.T.; Li, T.C.; Weng, J.H.; Jhan, Y.L.; Lin, S.X.; Chou, C.H. The role of pentacyclic triterpenoids in the allelopathic effects of Alstonia scholaris. J. Chem. Ecol. 2014, 40, 90–98. [CrossRef] 50. Alvarado, H.L.; Abrego, G.; Garduño-Ramirez, M.L.; Clares, B.; García, M.L.; Calpena, A.C. Development and validation of a high-performance liquid chromatography method for the quantification of ursolic/oleanic acids mixture isolated from Plumeria obtusa. J. Chromatogr. B 2015, 983, 111–116. [CrossRef][PubMed] 51. Hương, N.T.N.; Hung, T.; Dẹp, T.T. A study on morphogenesis of roots of Panax stipuleanatus HT Tsai et KM Feng in vitro and preliminary determination of oleanolic acid in roots. Vietnam. J. Biotechnol. 2016, 14, 49–54. [CrossRef] Molecules 2020, 25, 5526 27 of 31

52. Yamamoto, E.S.; Campos, B.L.S.; Laurenti, M.D.; Lago, J.H.G.; Grecco, S.D.S.; Corbett, C.E.P.; Passero, L.F.D. Treatment with triterpenic fraction purified from Baccharis uncinella leaves inhibits Leishmania (Leishmania) amazonensis spreading and improves Th1 immune response in infected mice. Parasitol. Res. 2014, 113, 333–339. [CrossRef] 53. Falev, D.I.; Kosyakov, D.S.; Ul’Yanovskii, N.V.; Ovchinnikov, D.V. Rapid simultaneous determination of pentacyclic triterpenoids by mixed-mode liquid chromatography–tandem mass spectrometry. J. Chromatogr. A 2020, 1609, 460458. [CrossRef] 54. Jang, S.-E.; Jeong, J.-J.; Hyam, S.R.; Han, M.J.; Kim, D.-H. Ursolic acid isolated from the seed of Cornus officinalis ameliorates colitis in mice by inhibiting the binding of lipopolysaccharide to toll-like receptor 4 on macrophages. J. Agric. Food Chem. 2014, 62, 9711–9721. [CrossRef][PubMed] 55. Zhang, S.; Sun, Y.; Sun, Z.; Wang, X.; You, J.; Suo, Y. Determination of triterpenic acids in fruits by a novel high performance liquid chromatography method with high sensitivity and specificity. Food Chem. 2014, 146, 264–269. [CrossRef][PubMed] 56. Fu, Q.; Zhang, L.; Cheng, N.; Jia, M.; Zhang, Y. Extraction optimization of oleanolic and ursolic acids from pomegranate (Punica granatum L.) flowers. Food Bioprod. Process. 2014, 92, 321–327. [CrossRef] 57. Oancea, I.A.; Van Staden, J.; Koos, F.; Oancea, E.; Ungureanu, E.-M. Electrochemical detection of ursolic acid from spruce (Picea abies) essential oils using modified amperometric microsensors. Anal. Lett. 2019, 52, 2214–2226. [CrossRef] 58. Choudhary, A.; Mittal, A.K.; Radhika, M.; Tripathy, D.; Chatterjee, A.; Banerjee, U.C.; Singh, I.P. Two new stereoisomeric antioxidant triterpenes from Potentilla fulgens. Fitoterapia 2013, 91, 290–297. [CrossRef] [PubMed] 59. Li, H.H.; Su, M.H.; Yao, D.H.; Zeng, B.Y.; Chang, Q.; Wang, W.; Xu, J. Anti-hepatocellular carcinoma activity of tormentic acid derived from suspension cells of Eriobotrya japonica (Thunb.) Lindl. Plant Cell Tissue Organ Cult. 2017, 130, 427–433. [CrossRef] 60. Wang, Z.W.; Wang, J.; Luo, J.; Kong, L. α-Glucosidase inhibitory triterpenoids from the stem barks of Uncaria laevigata. Fitoterapia 2013, 90, 30–37. [CrossRef] 61. Wu, X.D.; He, J.; Li, X.Y.; Dong, L.B.; Gong, X.; Gao, X.; Song, L.D.; Li, Y.; Peng, L.Y.; Zhao, Q. Triterpenoids and steroids with cytotoxic activity from Emmenopterys henryi. Planta Med. 2013, 79, 1356–1361. [CrossRef] 62. Zhu, M.; Wang, C.; Sun, W.; Zhou, A.; Wang, Y.; Zhang, G.; Zhou, X.; Huo, Y.; Li, C. Boosting 11-oxo-β-amyrin and glycyrrhetinic acid synthesis in Saccharomyces cerevisiae via pairing novel oxidation and reduction system from legume plants. Metab. Eng. 2018, 45, 43–50. [CrossRef] 63. Krivoruchko, A.; Nielsen, J. Production of natural products through metabolic engineering of Saccharomyces cerevisiae. Curr. Opin. Biotechnol. 2015, 35, 7–15. [CrossRef] 64. Lu, C.; Zhang, C.; Zhao, F.; Li, D.; Lu, W. Biosynthesis of ursolic acid and oleanolic acid in Saccharomyces cerevisiae. AIChE J. 2018, 64, 3794–3802. [CrossRef] 65. Zhao, Y.; Fan, J.; Wang, C.; Feng, X.; Li, C. Enhancing oleanolic acid production in engineered Saccharomyces cerevisiae. Bioresour. Technol. 2018, 257, 339–343. [CrossRef][PubMed] 66. Dai, Z.; Wang, B.; Liu, Y.; Shi, M.; Wang, D.; Zhang, X.; Liu, T.; Huang, L.; Zhang, X. Producing aglycons of ginsenosides in bakers’ yeast. Sci. Rep. 2014, 4, 3698. [CrossRef][PubMed] 67. Fukushima, E.O.; Seki, H.; Esawai, S.; Suzuki, M.; Ohyama, K.; Saito, K.; Muranaka, T. Combinatorial biosynthesis of legume natural and rare triterpenoids in engineered yeast. Plant Cell Physiol. 2013, 54, 740–749. [CrossRef][PubMed] 68. Dale, M.P.; Moses, T.; Johnston, E.J.; Rosser, S.J. A systematic comparison of triterpenoid biosynthetic enzymes for the production of oleanolic acid in Saccharomyces cerevisiae. PLoS ONE 2020, 15, e0231980. [CrossRef] [PubMed] 69. Li, D.; Wu, Y.; Wei, P.; Gao, X.; Li, M.; Zhang, C.; Zhou, Z.; Lu, W. Metabolic engineering of Yarrowia lipolytica for heterologous oleanolic acid production. Chem. Eng. Sci. 2020, 218, 115529. [CrossRef] 70. Dai, Z.; Liu, Y.; Sun, Z.; Wang, D.; Qu, G.; Ma, X.; Fan, F.; Zhang, L.; Li, S.; Zhang, X. Identification of a novel cytochrome P450 enzyme that catalyzes the C-2α hydroxylation of pentacyclic triterpenoids and its application in yeast cell factories. Metab. Eng. 2019, 51, 70–78. [CrossRef] 71. Moses, T.; Pollier, J.; Almagro, L.; Buyst, D.; Van Montagu, M.; Pedreño, M.A.; Martins, J.C.; Thevelein, J.M.; Goossens, A. Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16 hydroxylase from Bupleurum falcatum. Proc. Natl. Acad. Sci. USA 2014, 111, 1634–1639. [CrossRef] Molecules 2020, 25, 5526 28 of 31

72. Moses, T.; Thevelein, J.M.; Goossens, A.; Pollier, J. Comparative analysis of CYP93E proteins for improved microbial synthesis of plant triterpenoids. Phytochemistry 2014, 108, 47–56. [CrossRef] 73. Zhang, G.; Cao, Q.; Liu, J.; Liu, B.; Li, J.; Li, C. Refactoring β-amyrin synthesis in Saccharomyces cerevisiae. AIChE J. 2015, 61, 3172–3179. [CrossRef] 74. Kannan, S.; Sathasivam, G.; Marudhamuthu, M. Decrease of growth, biofilm and secreted virulence in opportunistic nosocomial Pseudomonas aeruginosa ATCC 25619 by glycyrrhetinic acid. Microb. Pathog. 2019, 126, 332–342. [CrossRef] 75. Jyoti, A.; Zerin, T.; Kim, T.-H.; Hwang, T.-S.; Jang, W.S.; Nam, K.-W.; Song, H.-Y. In vitro effect of ursolic acid on the inhibition of Mycobacterium tuberculosis and its cell wall mycolic acid. Pulm. Pharmacol. Ther. 2015, 33, 17–24. [CrossRef][PubMed] 76. Tohmé, M.; Giménez, M.; Peralta, A.; Colombo, M.; Delgui, L.R. Ursolic acid: A novel antiviral compound inhibiting rotavirus infection in vitro. Int. J. Antimicrob. Agents 2019, 54, 601–609. [CrossRef] 77. Kumar, A.; Choudhir, G.; Shukla, S.K.; Sharma, M.; Tyagi, P.; Bhushan, A.; Rathore, M. Identification of phytochemical inhibitors against main protease of COVID-19 using molecular modeling approaches. J. Biomol. Struct. Dyn. 2020, 1–11. [CrossRef][PubMed] 78. Sampangi-Ramaiah, M.H.; Vishwakarma, R.; Shaanker, R.U. Molecular docking analysis of selected natural products from plants for inhibition of SARS-CoV-2 main protease. Curr. Sci. 2020, 118, 1087–1092. [CrossRef] 79. Villar, V.H.; Vögler, O.; Barceló, F.; Gómez-Florit, M.; Martínez-Serra, J.; Obrador-Hevia, A.; Martín-Broto, J.; Ruíz-Gutierrez, V.; Alemany, R. Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein. J. Nutr. Biochem. 2014, 25, 429–438. [CrossRef] 80. Shan, J.; Xuan, Y.; Zhang, Q.; Zhu, C.; Liu, Z.; Zhang, S. Ursolic acid synergistically enhances the therapeutic effects of oxaliplatin in colorectal cancer. Protein Cell 2016, 7, 571–585. [CrossRef] 81. Jing, B.; Liu, M.; Yang, L.; Cai, H.Y.; Chen, J.B.; Li, W.; Kou, X.; Wu, Y.Z.; Qin, D.J.; Zhou, L.; et al. Characterization of naturally occurring pentacyclic triterpenes as novel inhibitors of deubiquitinating protease USP7 with anticancer activity in vitro. Acta Pharmacol. Sin. 2018, 39, 492–498. [CrossRef] 82. Yu, F.; Wang, Q.; Zhang, Z.; Peng, Y.; Qiu, Y.; Shi, Y.; Zheng, Y.; Xiao, S.; Wang, H.; Huang, X.; et al. Development of oleanane-type triterpenes as a new class of HCV entry inhibitors. J. Med. Chem. 2013, 56, 4300–4319. [CrossRef] 83. Zhou, J.; Cai, W.; Jin, M.; Xu, J.; Wang, Y.; Xiao, Y.; Hao, L.; Wang, B.; Zhang, Y.; Han, J.; et al. 18β-Glycyrrhetinic acid suppresses experimental autoimmune encephalomyelitis through inhibition of microglia activation and promotion of remyelination. Sci. Rep. 2015, 5, 13713. [CrossRef] 84. Ayub, A.; Begum, S.; Ali, S.N.; Siddiqui, B.S. Triterpenoids from the aerial parts of Lantana camara. J. Asian Nat. Prod. Res. 2017, 21, 141–149. [CrossRef] 85. Lu, M.Y.; Liao, Z.X.; Ji, L.J.; Sun, H.F. Triterpenoids of Chrysosplenium carnosum. Fitoterapia 2013, 85, 119–124. [CrossRef] 86. Xu, Y.Q.; Zhang, J.H.; Yang, X.S. Corosolic acid induces potent anti-cancer effects in CaSki cervical cancer cells through the induction of apoptosis, cell cycle arrest and PI3K/Akt signalling pathway. Bangladesh J. Pharmacol. 2016, 11, 453. [CrossRef] 87. Kaweetripob, W.; Mahidol, C.; Prawat, H.; Ruchirawat, S. Lupane, friedelane, oleanane, and ursane triterpenes from the stem of Siphonodon celastrineus Griff. Phytochemistry 2013, 96, 404–417. [CrossRef] 88. Liang, C.; Ding, Y.; Song, S.B.; Kim, J.A.; Nguyen, M.C.; Ma, J.Y.; Kim, Y.H.; Cuong, N.M. Oleanane-triterpenoids from Panax stipuleanatus inhibit NF-κB. J. Ginseng Res. 2013, 37, 74–79. [CrossRef] 89. Yan, M.; Zhu, Y.; Zhang, H.J.; Jiao, W.H.; Han, B.N.; Liu, Z.X.; Qiu, F.; Chen, W.S.; Lin, H.W. Anti-inflammatory secondary metabolites from the leaves of Rosa laevigata. Bioorg. Med. Chem. 2013, 21, 3290–3297. [CrossRef] 90. Kang, H.; Ku, S.K.; Kim, J.; Chung, J.; Kim, S.C.; Zhou, W.; Na, M.; Bae, J.S. Anti-vascular inflammatory effects of pentacyclic triterpenoids from Astilbe rivularis in vitro and in vivo. Chem. Interact. 2017, 261, 127–138. [CrossRef] 91. Mosquera, C.; Panay, A.J.; Montoya, G. Pentacyclic triterpenes from Cecropia telenitida can function as inhibitors of 11β-hydroxysteroid dehydrogenase type 1. Molecules 2018, 23, 1444. [CrossRef] 92. Yang, F.; Dong, X.; Yin, X.; Wang, W.; You, L.; Ni, J. Radix Bupleuri: A review of traditional uses, botany, phytochemistry, pharmacology, and toxicology. BioMed Res. Int. 2017, 2017, 7597596. [CrossRef] Molecules 2020, 25, 5526 29 of 31

93. Huang, L.R.; Luo, H.; Yang, X.S.; Chen, L.; Zhang, J.X.; Wang, D.P.; Hao, X.J. Enhancement of anti-bacterial and anti-tumor activities of pentacyclic triterpenes by introducing exocyclic α,β-unsaturated ketone moiety in ring A. Med. Chem. Res. 2014, 23, 4631–4641. [CrossRef] 94. Liang, S.; Li, M.; Yu, X.; Jin, H.; Zhang, Y.; Zhang, L.; Zhou, D.; Xiao, S. Synthesis and structure-activity relationship studies of water-soluble β-cyclodextrin-glycyrrhetinic acid conjugates as potential anti-influenza virus agents. Eur. J. Med. Chem. 2019, 166, 328–338. [CrossRef][PubMed] 95. Lee, J.Y.; Choi, J.K.; Jeong, N.-H.; Yoo, J.; Ha, Y.S.; Lee, B.; Choi, H.; Park, P.-H.; Shin, T.-Y.; Kwon, T.K.; et al. Anti-inflammatory effects of ursolic acid-3-acetate on human synovial fibroblasts and a murine model of rheumatoid arthritis. Int. Immunopharmacol. 2017, 49, 118–125. [CrossRef][PubMed] 96. Ishida, T.; Miki, I.; Tanahashi, T.; Yagi, S.; Kondo, Y.; Inoue, J.; Kawauchi, S.; Nishiumi, S.; Yoshida, M.; Maeda, H.; et al. Effect of 18β-glycyrrhetinic acid and hydroxypropyl γcyclodextrin complex on indomethacin-induced small intestinal injury in mice. Eur. J. Pharmacol. 2013, 714, 125–131. [CrossRef] [PubMed] 97. Wiemann, J.; Heller, L.; Csuk, R. Targeting cancer cells with oleanolic and ursolic acid derived hydroxamates. Bioorg. Med. Chem. Lett. 2016, 26, 907–909. [CrossRef] 98. Yoon, Y.; Lim, J.W.; Kim, J.; Kim, Y.; Chun, K.H. Discovery of ursolic acid prodrug (NX-201): Pharmacokinetics and in vivo antitumor effects in PANC-1 pancreatic cancer. Bioorg. Med. Chem. Lett. 2016, 26, 5524–5527. [CrossRef] 99. Mendes, V.I.S.; Bartholomeusz, G.A.; Ayres, M.; Gandhi, V.; Salvador, J.A.R. Synthesis and cytotoxic activity of novel A-ring cleaved ursolic acid derivatives in human non-small cell lung cancer cells. Eur. J. Med. Chem. 2016, 123, 317–331. [CrossRef] 100. Xu, B.; Wu, G.R.; Zhang, X.Y.; Yan, M.M.; Zhao, R.; Xue, N.N.; Fang, K.; Wang, H.; Chen, M.; Guo, W.B.; et al. An overview of structurally modified glycyrrhetinic acid derivatives as antitumor agents. Molecules 2017, 22, 924. [CrossRef] 101. Ayeleso, T.B.; Matumba, M.G.; Mukwevho, E. Oleanolic acid and its derivatives: Biological activities and therapeutic potential in chronic diseases. Molecules 2017, 22, 1915. [CrossRef] 102. Mlala, S.; Oyedeji, A.O.; Gondwe, M.; Oyedeji, O. Ursolic acid and its derivatives as bioactive agents. Molecules 2019, 24, 2751. [CrossRef] 103. Hodon, J.; Borkova, L.; Pokorny, J.; Kazakova, A.; Urban, M. Design and synthesis of pentacyclic triterpene conjugates and their use in medicinal research. Eur. J. Med. Chem. 2019, 182, 111653. [CrossRef] 104. Isah, M.B.; Ibrahim, M.A.; Mohammed, A.; Aliyu, A.B.; Masola, B.; Coetzer, T.H.T. A systematic review of pentacyclic triterpenes and their derivatives as chemotherapeutic agents against tropical parasitic diseases. Parasitology 2016, 143, 1219–1231. [CrossRef][PubMed] 105. Ren, Y.; Kinghorn, A.D. Natural product triterpenoids and their semi-synthetic derivatives with potential anticancer activity. Planta Med. 2019, 85, 802–814. [CrossRef][PubMed] 106. Parida, P.K.; Sau, A.; Ghosh, T.; Jana, K.; Biswas, K.; Raha, S.; Misra, A.K. Synthesis and evaluation of triazole linked glycosylated 18β-glycyrrhetinic acid derivatives as anticancer agents. Bioorg. Med. Chem. Lett. 2014, 24, 3865–3868. [CrossRef][PubMed] 107. Ghosh, S. Triterpene structural diversification by plant cytochrome P450 enzymes. Front. Plant Sci. 2017, 8, 1886. [CrossRef][PubMed] 108. Martinez, A.; Rivas, F.; Perojil, A.; Parra, A.; Garcia-Granados, A.; Fernandez-Vivas, A. Biotransformation of oleanolic and maslinic acids by Rhizomucor miehei. Phytochemistry 2013, 94, 229–237. [CrossRef][PubMed] 109. Djerassi, C.; Henry, J.A.; Lemin, A.J.; Rios, T.; Thomas, G.H. Terpenoids. XXIV. The structure of the cactus triterpene queretaroic acid. J. Am. Chem. Soc. 1956, 78, 3783–3787. [CrossRef] 110. Kinoshita, K.; Yang, Y.; Koyama, K.; Takahashi, K.; Nishino, H. Inhibitory effect of some triterpenes from cacti on 32Pi-incorporation into phospholipids of HeLa cells promoted by 12-O-tetradecanoylphorbol-13-acetate. Phytomedicine 1999, 6, 73–77. [CrossRef] 111. Fujii, Y.; Hirosue, S.; Fujii, T.; Matsumoto, N.; Agematu, H.; Arisawa, A. Hydroxylation of oleanolic acid to queretaroic acid by cytochrome P450 from Nonomuraea recticatena. Biosci. Biotechnol. Biochem. 2006, 70, 2299–2302. [CrossRef] 112. Martinez, A.; Perojil, A.; Rivas, F.; Parra, A.; Garcia-Granados, A.; Fernández-Vivas, A. Biotransformation of oleanolic and maslinic methyl esters by Rhizomucor miehei CECT 2749. Phytochemistry 2015, 117, 500–508. [CrossRef] Molecules 2020, 25, 5526 30 of 31

113. Sun, H.; Liu, T.; Shen, Y.-J.; Zhang, L.-M.; Wang, M. Preparation and crystal structure of 15α-hydroxyl-oleanolic acid. Jiegou Huaxue 2010, 29, 1789–1801. 114. Gong, T.; Zheng, L.; Zhen, X.; He, H.-X.; Zhu, H.-X.; Zhu, P. Microbial transformation of oleanolic acid by Trichothecium roseum. J. Asian Nat. Prod. Res. 2014, 16, 383–386. [CrossRef] 115. Fan, B.; Jiang, B.; Yan, S.; Xu, B.; Huang, H.; Chen, G.-T. Anti-inflammatory 18β-glycyrrhetinin acid derivatives produced by biocatalysis. Planta Med. 2019, 85, 56–61. [CrossRef] 116. Qin, Y.J.; Feng, B.; Song, X.B.; Zhou, W.B.; Yu, H.S.; Zhao, L.L.; Yu, L.Y.; Ma, B.P. Biotransformation of glycyrrhetinic acid by Cunninghamella blakesleeana. Chin. J. Nat. Med. 2010, 8, 373–381. [CrossRef] 117. Maatooq, G.T.; Marzouk, A.M.; Gray, A.I.; Rosazza, J.P. Bioactive microbial metabolites from glycyrrhetinic acid. Phytochemistry 2010, 71, 262–270. [CrossRef] 118. He, C.J.; Yang, Y.M.; Wu, K.Y. Microbial transformation of glycyrrhetinic acid by Colletotrichum lini AS3.4486. Adv. Mater. Res. 2015, 1120, 877–881. [CrossRef] 119. Choudhary,M.I.; Siddiqui, Z.A.; Nawaz, S.A. Atta-Ur-Rahman Microbial transformation of 18β-glycyrrhetinic acid by Cunninghamella elegans and Fusarium lini, and lipoxygenase inhibitory activity of transformed products. Nat. Prod. Res. 2009, 23, 507–513. [CrossRef] 120. Huang, F.X.; Yang, W.Z.; Ye, F.; Tian, J.Y.; Hu, H.B.; Feng, L.M.; Guo, D.A.; Ye, M. Microbial transformation of ursolic acid by Syncephalastrum racemosum (Cohn) Schroter AS 3.264. Phytochemistry 2012, 82, 56–60. [CrossRef] 121. Fu, S.; Meng, Q.; Yang, J.; Tu, J.; Sun, D.-A. Biocatalysis of ursolic acid by the fungus Gliocladium roseum CGMCC 3.3657 and resulting anti-HCV activity. RSC Adv. 2018, 8, 16400–16405. [CrossRef] 122. Zhang, S.S.; He, L.S.; Zhao, Y.M.; Fu, S.; Liu, D.L.; Yu, Z.H.; Liu, B.C. Three new triterpenoids transformed from ursolic acid by Mucor spinosus AS3.3450 and their cytotoxicity. Phytochem. Lett. 2019, 32, 33–37. [CrossRef] 123. Zhang, J.; Cheng, Z.H.; Yu, B.Y.; Cordell, G.A.; Qiu, S.X. Novel biotransformation of pentacyclic triterpenoid acids by Nocardia sp. NRRL 5646. Tetrahedron Lett. 2005, 46, 2337–2340. [CrossRef] 124. Huang, D.; Ding, Y.; Li, Y.; Zhang, W.; Fang, W.-S.; Chen, X. Anti-tumor activity of a 3-oxo derivative of oleanolic acid. Cancer Lett. 2006, 233, 289–296. [CrossRef][PubMed] 125. Funari, C.S.; De Almeida, L.; Passalacqua, T.G.; Martinez, I.; Ambrósio, D.L.; Cicarelli, R.M.B.; Silva, D.H.S.; Graminha, M.A.S. Oleanonic acid from Lippia lupulina (Verbenaceae) shows strong in vitro antileishmanial and antitrypanosomal activity. Acta Amaz. 2016, 46, 411–416. [CrossRef] 126. Krivoruchko, A.V.; Kuyukina, M.S.; Ivshina, I.B. Advanced Rhodococcus biocatalysts for environmental biotechnologies. Catalysts 2019, 9, 236. [CrossRef] 127. Leipold, D.; Wünsch, G.; Schmidt, M.; Bart, H.-J.; Bley, T.; Neuhaus, H.E.; Bergmann, H.; Richling, E.; Muffler, K.; Ulber, R. Biosynthesis of ursolic acid derivatives by microbial metabolism of ursolic acid with Nocardia sp. strains—Proposal of new biosynthetic pathways. Process. Biochem. 2010, 45, 1043–1051. [CrossRef] 128. Xu, S.H.; Wang, W.W.; Zhang, C.; Liu, X.F.; Yu, B.Y.; Zhang, J. Site-selective oxidation of unactivated C–H sp 3 bonds of oleanane triterpenes by Streptomyces griseus ATCC 13273. Tetrahedron 2017, 73, 3086–3092. [CrossRef] 129. Zhu, Y.Y.; Qian, L.W.; Zhang, J.; Liu, J.H.; Yu, B. New approaches to the structural modification of olean-type pentacylic triterpenes via microbial oxidation and glycosylation. Tetrahedron 2011, 67, 4206–4211. [CrossRef] 130. De Sousa, L.R.F.; Silva, J.A.D.; Vieira, P.C.; Costa, M.B.; Dos Santos, M.L.; Sbardelotto, A.B.; Pessoa, C.D.Ó.; De Moraes, M.O.; Menezes, A.C.S. Chemical constituents of the stem bark of Vochysia thyrsoidea Pohl. (Vochysiaceae) and evaluation of their cytotoxicity and inhibitory activity against cathepsins B and K. Química Nova 2014, 37, 288–292. [CrossRef] 131. Capel, C.S.; De Souza, A.C.D.; De Carvalho, T.C.; De Sousa, J.P.B.; Ambrósio, S.R.; Martins, C.H.G.; Cunha, W.R.; Hernández-Galán, R.; Furtado, N.A.J.C. Biotransformation using Mucor rouxii for the production of oleanolic acid derivatives and their antimicrobial activity against oral pathogens. J. Ind. Microbiol. Biotechnol. 2011, 38, 1493–1498. [CrossRef] 132. Chianese, G.; Golin-Pacheco, S.D.; Taglialatela-Scafati, O.; Collado, J.A.; Munoz, E.; Appendino, G.; Pollastro, F. Bioactive triterpenoids from the caffeine-rich plants guayusa and maté. Food Res. Int. 2019, 115, 504–510. [CrossRef] Molecules 2020, 25, 5526 31 of 31

133. Sit, N.W.; Chan, Y.; Lai, S.; Lim, L.; Looi, G.; Tay, P.; Tee, Y.; Woon, Y.; Khoo, K.; Ong, H. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. J. Mycol. Med. 2018, 28, 561–567. [CrossRef] 134. Su, H.G.; Peng, X.R.; Shi, Q.Q.; Huang, Y.J.; Zhou, L.; Qiu, M.H. Lanostane triterpenoids with anti-inﬂammatory activities from Ganoderma lucidum. Phytochemistry 2020, 173, 112256. [CrossRef][PubMed] 135. Tian, M.; Zhao, P.-J.; Li, G.; Zhang, K.-Q. In depth natural product discovery from the basidiomycetes Stereum species. Microorganisms. 2020, 8, 1049. [CrossRef][PubMed] 136. Ying, Y.M.; Yu, H.F.; Tong, C.P.; Shan, W.G.; Zhan, Z.J. Spiroinonotsuoxotriols A and B, two highly rearranged triterpenoids from Inonotus obliquus. Org. Lett. 2020, 22, 3377–3380. [CrossRef][PubMed] 137. Shi, Q.Q.; Huang, Y.J.; Su, H.G.; Gao, Y.; Lu, S.Y.; Peng, X.R.; Li, X.N.; Zhou, L.; Qiu, M.H. Structurally diverse lanostane triterpenoids from medicinal and edible mushroom Ganoderma resinaceum Boud. Bioorg. Chem. 2020, 100, 103871. [CrossRef][PubMed] 138. Nursid, M.; Marraskuranto, E.; Chasanah, E. Cytotoxicity and apoptosis induction of sea cucumber Holothuria atra extracts. Pharmacogn. Res. 2019, 11, 41. [CrossRef] 139. Xu, R.; Fazio, G.C.; Matsuda, S.P.T. On the origins of triterpenoid skeletal diversity. Phytochemistry 2004, 65, 261–291. [CrossRef] 140. Chubukov, V.; Mukhopadhyay, A.; Petzold, C.J.; Keasling, J.D.; Martin, H.G. Synthetic and systems biology for microbial production of commodity chemicals. NPJ Syst. Biol. Appl. 2016, 2, 16009. [CrossRef] 141. Heller, L.; Schwarz, S.; Perl, V.; Köwitsch, A.; Siewert, B.; Csuk, R. Incorporation of a Michael acceptor enhances the antitumor activity of triterpenoic acids. Eur. J. Med. Chem. 2015, 101, 391–399. [CrossRef]

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