**2. Microbiological transformations of some selected natural products with different microorganisms**

activities, such as antibacterial, anti-allergic, antioxidative, anti-inflammation, and

*N. corallina* Deoxyartemisinin Endoperoxide function reduction [11]

*Products obtained from the biotransformation of artemisinin (1) by different microorganisms.*

**Microorganism Products Action Reference**

Epoxidation, hydroxylation C-3β site Endoperoxide function reduction Breakdown of heterocyclic rings Epoxidation C-3 and C-13 Hydroxylation C-4, endoperoxide

[10]

[11, 12]

[13, 14]

[15]

[16]

[17]

function reduction

Hydroxylation of C-14 site Breakdown of heterocyclic rings Hydroxylation of C-4α site Endoperoxide function reduction

Hydroxylation C-7β site Epimerization C-9 Hydroxylation C-4α site Hydroxylation C-7β site Hydroxylation C-6β site Hydroxylation C-7α site Hydroxylation C-6β and C-7α sites

Acetylation of C-9β site Hydroxylation of C-9α site

Oxidation of C-9 site Hydroxylation of C-9α site Hydroxylation of C-9β site Hydroxylation of C-3α site

Endoperoxide function reduction Hydroxylation of C-1 site

*A. niger* 3β-hydroxy-4,12-epoxy-1 deoxyartemisinin Artemisinin G 3,13-epoxyartemisinin 4α-hydroxy-1 deoxyartemisinin

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

> 14-hydroxyartemisinin Artemisinin G

Deoxyartemisinin

α-hydroxy-1 deoxoartemisinin β-hydroxyartemisinin β-hydroxyartemisinin α-hydroxyartemisinin β,7α-dihydroxyartemisinin

4α-hydroxydeoxyartemisinin

7β-hydroxy-9α-artemisinin

9β-acetoxyartimisinin 9α-hydroxyartemisinin

1α-hydroxyartemisinin 10β-hydroxyartemisinin

9-artemisitone α-hydroxyartemisinin β-hydroxyartemisinin α-hydroxyartemisinin

*A. flavus* (MTCC

9167)

*C. elegans* (ATCC 9245)

*P.*

**Table 1.**

**277**

*simplicissimum*

*S. griseus* (ATCC 13273)

*R. stolonifer* Deoxyartemisinin

Microbial transformation of ursolic acid (**2**) by *Bacillus megaterium* CGMCC 1.1741 yielded five metabolites identified as 3-oxo-urs-12-en-28-oic acid (**3**, 6.2%); 1β*,*11α-dihydroxy-3-oxo-urs-12-en-28-oic acid (**4**, 13.5%); 1β-hydroxy-3-oxo-urs-12 en-28,13-lactone (**5**, 5.0%); 1β,3β,11α-trihydroxy-urs-12-en-28-oic acid (**6**, 26.9%); and 1β,11α-dihydroxy-3-oxo-urs-12-en-28-O-β-D-glucopyranoside (**7**, 8.6%) [19]. The biotransformation studies of **2** by *Alternaria longipes* AS 3.2875 have led to the isolation of six products of hydroxylation or glycosylation. Their structures were identified as 3-carbonyl-ursolic acid-28-O-β-D-glucopyranosyl ester (**8**), ursolic acid-3-O-β-D-glucopyranoside (**9**), ursolic acid-28-O-β-D-glucopyranosyl ester (**10**), 2α,3β-dihydroxy-ursolic acid-28-O-β-D-glucopyranosyl ester (**11**), 3β,21β-dihydroxyursolic acid-28-O-β-D-glucopyranosyl ester (**12**), and 3-O-(β-D-glucopyranosyl) ursolic acid-28-O-(β-D-glucopyranosyl) ester (**13**). Glycosylation reaction on

pentacyclic triterpenoid fulfilled with difficulty in the process of chemical synthesis is facile by microbial transformation [20]. Biotransformation of **2** by *A. alternata* eight metabolites were found to be 2α,3β-dihydroxyurs-12-en-28-oic acid (corosolic acid, **14**), urs-12-en-2α,3β,28-triol (**15**), 3β,23-dihydroxyurs-12-en-28-oic acid (**16**), 2α,3β,23-trihydroxyurs-12-en-28-oic acid (**17**), 2α,3β,23,24-tetrahydroxyurs-12-en-28-oic acid (**18**), 3β,28-dihydroxy-12-ursene (**19**), urs-12-en-3β-ol (**20**), and urs-12 en-2α,3β-diol (**21**). The reduction of the C-28 carboxyl group and hydroxylation at

C-2, 23, and 24 are steps in the metabolic pathway of **2** [21].

antitumor activities [18].

#### **2.1 Sesquiterpene lactone**

Artemisinin (**1**), a sesquiterpene lactone endoperoxide and an antimalarial drug, is effective against chloroquine-resistant parasites; but its toxicities and low solubility in water hamper its therapeutical use. Studies on modification of **1** through biological and chemical methodologies have been reported to yield more effective and water-soluble derivatives. A wide array of microbial transformations of **1** involve oxidation, reduction, and degradation reactions by different microorganisms, such as *Aspergillus niger*, *A. flavus*, *A. adametzi* (ATCC 10407), *Cunninghamella echinulata, Caenorhabditis elegans, Mucor polymorphous, M. rammanianus, Streptomyces griseus, Penicillium simplicissimum, P. chrysogenum, P. purpuresceus*, *Pestalotiopsis guepini* (P**-**8), *Eurotium amstelodami,Trichoderma viride* (T-58)*, Saccharomyces cerevisiae*, *and Pichia pastoris.* Biotransformation of **1** usually includes the processes such as hydroxylation of methyl, methine and methylene groups, deoxidation reactions, hydration and acetylation reactions, epimerization, and breakdown of heterocyclic rings (**Table 1**).

#### **2.2 Triterpene**

Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid, UA, **2**), a natural pentacyclic triterpene, is broadly used in food, cosmetics, and biomedical industries. As a ubiquitous constituent in the plant kingdom and the major component of many traditional medicine herbs, ursolic acid remarkably exhibits a lot of biological


*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

#### **Table 1.**

efficient for certain biotransformation, these reactions may involve isolating the enzyme system, and, for some classes of enzyme-catalyzed reaction, a recycling

animals; (8) ease of setup and manipulation; and (9) more reliable and

isms, such as *Aspergillus niger*, *A. flavus*, *A. adametzi* (ATCC 10407), *Cunninghamella echinulata, Caenorhabditis elegans, Mucor polymorphous, M. rammanianus, Streptomyces griseus, Penicillium simplicissimum, P. chrysogenum, P. purpuresceus*, *Pestalotiopsis guepini* (P**-**8), *Eurotium amstelodami,Trichoderma viride* (T-58)*, Saccharomyces cerevisiae*, *and Pichia pastoris.* Biotransformation of **1** usually includes the processes such as hydroxylation of methyl, methine and methylene groups, deoxidation reactions, hydration and acetylation reactions, epimerization,

The objective of this review is to highlight the importance of microorganisms or enzymes isolated from them in the biotransformation process of natural products or xenobiotic compounds, according to green chemistry or white biotechnology.

**2. Microbiological transformations of some selected natural products**

Artemisinin (**1**), a sesquiterpene lactone endoperoxide and an antimalarial drug, is effective against chloroquine-resistant parasites; but its toxicities and low solubility in water hamper its therapeutical use. Studies on modification of **1** through biological and chemical methodologies have been reported to yield more effective and water-soluble derivatives. A wide array of microbial transformations of **1** involve oxidation, reduction, and degradation reactions by different microorgan-

Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid, UA, **2**), a natural pentacyclic triterpene, is broadly used in food, cosmetics, and biomedical industries. As a ubiquitous constituent in the plant kingdom and the major component of many traditional medicine herbs, ursolic acid remarkably exhibits a lot of biological

reproducible [8, 9].

*Microorganisms*

**with different microorganisms**

and breakdown of heterocyclic rings (**Table 1**).

**2.1 Sesquiterpene lactone**

**2.2 Triterpene**

**276**

Fungi are playing a prominent role in the catalysis of organic compounds and in the production of commercially and industrially important compounds, because of their ability to catalyze novel reactions [7]. Fungi are commonly used in the industry for production of fermented beverages, foods, physiologically active substances, solvents, organic acids, polysaccharides, antibiotics, etc. Of the zygomycota, *Mucor* and *Rhizopus* are commonly used in the industry. *Rhizopus* strains are important in citric acid production. *Mucor* strains make a significant number of important lipases and catalyze the hydroxylation of a wide range of chemical compounds [2–4]. The use of the microbial model offers a number of advantages over the use of animals in metabolism studies, mainly: (1) simple, easy, and can be prepared at low cost; (2) screening for a large number of strains is a simple repetitive process; (3) the large number of metabolites formed allows easier detection, isolation, and structural identification; (4) newer metabolites can be isolated; (5) utilized for synthetic reactions involving many steps; (6) useful in cases where *regio*- and *stereo*specificity is required; (7) maintenance of stock cultures of microorganisms is simpler and cheaper than the maintenance of cell or tissue cultures or laboratory

sequence may be required to regenerate the enzyme [6].

*Products obtained from the biotransformation of artemisinin (1) by different microorganisms.*

activities, such as antibacterial, anti-allergic, antioxidative, anti-inflammation, and antitumor activities [18].

Microbial transformation of ursolic acid (**2**) by *Bacillus megaterium* CGMCC 1.1741 yielded five metabolites identified as 3-oxo-urs-12-en-28-oic acid (**3**, 6.2%); 1β*,*11α-dihydroxy-3-oxo-urs-12-en-28-oic acid (**4**, 13.5%); 1β-hydroxy-3-oxo-urs-12 en-28,13-lactone (**5**, 5.0%); 1β,3β,11α-trihydroxy-urs-12-en-28-oic acid (**6**, 26.9%); and 1β,11α-dihydroxy-3-oxo-urs-12-en-28-O-β-D-glucopyranoside (**7**, 8.6%) [19]. The biotransformation studies of **2** by *Alternaria longipes* AS 3.2875 have led to the isolation of six products of hydroxylation or glycosylation. Their structures were identified as 3-carbonyl-ursolic acid-28-O-β-D-glucopyranosyl ester (**8**), ursolic acid-3-O-β-D-glucopyranoside (**9**), ursolic acid-28-O-β-D-glucopyranosyl ester (**10**), 2α,3β-dihydroxy-ursolic acid-28-O-β-D-glucopyranosyl ester (**11**), 3β,21β-dihydroxyursolic acid-28-O-β-D-glucopyranosyl ester (**12**), and 3-O-(β-D-glucopyranosyl) ursolic acid-28-O-(β-D-glucopyranosyl) ester (**13**). Glycosylation reaction on pentacyclic triterpenoid fulfilled with difficulty in the process of chemical synthesis is facile by microbial transformation [20]. Biotransformation of **2** by *A. alternata* eight metabolites were found to be 2α,3β-dihydroxyurs-12-en-28-oic acid (corosolic acid, **14**), urs-12-en-2α,3β,28-triol (**15**), 3β,23-dihydroxyurs-12-en-28-oic acid (**16**), 2α,3β,23-trihydroxyurs-12-en-28-oic acid (**17**), 2α,3β,23,24-tetrahydroxyurs-12-en-28-oic acid (**18**), 3β,28-dihydroxy-12-ursene (**19**), urs-12-en-3β-ol (**20**), and urs-12 en-2α,3β-diol (**21**). The reduction of the C-28 carboxyl group and hydroxylation at C-2, 23, and 24 are steps in the metabolic pathway of **2** [21].

Biotransformation of UA by *S. racemosum* (3.2500) yielded five metabolites 3β,7β,21β-trihydroxy-urs-12-en-28-oic acid (**22**); 3β,21β-dihydroxy-urs-11-en-28-oic acid-13-lactone (**23**); 1β,3β,21β-trihydroxy-urs-12-en-28-oic acid (**24**); 3β,7β,21β-trihydroxy-urs-1-en-28-oic acid-13-lactone (**25**); and 1β,3β-dihydroxyurs-12-en-21-oxo-28-oic acid (**26**) which were afforded [22]. Additionally, of the biotransformation of **2** with by *S. racemosum* compounds **27–30** and 11,26-epoxy-3β-21β-dihydroxy-urs-12-en-28-oic acid were obtained (**31**), (**Figure 1**) [23].

The endophytic fungi *Pestalotiopsis microspora* isolated from medical plant *Huperzia serrata* can transform **1** to afforded 3-oxo-15β,30-dihydroxy-urs-12-en-28 oic acid (**32**), 3β,15β-dihydroxy-urs-12-en-28-oic acid (**33**), 3β,15β,30-trihydroxyurs-12-en-28-oic acid (**34**), and **30** [24].

Microbial transformation of ursolic acid by *Mucor spinosus* AS 3.3450 were isolated and their structures were identified as **9**, **22** and 3β,7β-dihydroxy-ursolic acid-28-ethanone (**35**) (**Figure 1**) [25].

> 18β-glycyrrhetinic acid (**48**) is the active form of glycyrrhizin which is the major pentacyclic triterpene found in licorice (*Glycyrrhiza glabra* L.). Glycyrrhetinic acid has been shown to possess several pharmacological activities, such as antiulcerative, anti-inflammatory, immunomodulating, antitumor, antiviral, antihepatitis effects, and anticancer. Biotransformation **48** with a fungus *C. blakesleeana* (AS 3.970) yielded 3-oxo-7β-hydroxyglycyrrhetinic acid (**49**) and 7β-hydroxyglycyrrhetinic acid (**50**) [27], while of **48** using *Absidia pseudocylindrospora* (ATCC 24169), *Gliocladium viride* (ATCC 10097) and *Cunninghamella echinulata* (ATCC 8688a) afforded seven derivatives: **51**, **52**, 7β,15α-dihydroxy-18β-glycyrrhetinic acid (**53**), 15α-hydroxy-18β-glycyrrhetinic acid (**54**), 1α-hydroxy-18β-glycyrrhetinic acid (**55**) and 13β-hydroxy-7α,27-oxy-12-dihydro-18β-glycyrrhetinic acid (**56**), and the epi-

Ginsenoside Rb1 (**61**) is the most predominant protopanaxadiol-type ginsenoside in *Panax* species (ginseng). Several microbial transformations of this substrate (Ginsenoside Rb1) have been accomplished with an ample and varied

group of microorganisms, all of these having β-glucosidase activities.

mer of compound **53** on C-17 (**Figure 3**) [28].

*Biotransformation products of 11-keto-b-boswellic acid (***36***).*

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

*Biotransformation products of 18β-glycyrrhetinic acid (***48***).*

**Figure 3.**

**279**

**Figure 2.**

The gum resin *Boswellia serrata* has been used for the treatment of inflammatory and arthritic diseases. Its major active constituents are ursane triterpenoids, which include 11-keto-β-boswellic acid (KBA, **36**), β-boswellic acid (BA), and acetyl-βboswellic acid (ABA). Microbial transformation **36** by *Cunninghamella blakesleeana* (AS 3.970) yielded ten regioselective transformed products: 7β-hydroxy-11-keto-βboswellic acid (**37**), 7β,15α-dihydroxy-11-keto-β-boswellic acid (**38**), 7β,16βdihydroxy-11-keto-β-boswellic acid (**39**), 7β,16α-dihydroxy-11-keto-β-boswellic acid (**40**), 7β,22β-dihydroxy-11-keto-β-boswellic acid (**41**), 7β,21β-dihydroxy-11 keto-β-boswellic acid (**42**), 7β,20β-dihydroxy-11-keto-β-boswellic acid (**43**), 7β,30 dihydroxy-11-keto-β-boswellic acid (**44**), 3α,7β-dihydroxy-11-oxours-12-en,24,30 dioic acid (**45**), and 3α,7β-dihydroxy-30-(2-hydroxypropanoyloxy)-11-oxours-12 en, 24-oic acid (**46**). Bioconversion of **36** with *Bacillus megaterium* based on a recombinant cytochrome P450 system yielded *regio*- and *stereo*selective 15αhydroxylation (**47**) of substrate (**Figure 2**) [26].

**Figure 1.** *Biotransformation products of ursolic acid (2).*

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

Biotransformation of UA by *S. racemosum* (3.2500) yielded five metabolites 3β,7β,21β-trihydroxy-urs-12-en-28-oic acid (**22**); 3β,21β-dihydroxy-urs-11-en-28-oic

3β,7β,21β-trihydroxy-urs-1-en-28-oic acid-13-lactone (**25**); and 1β,3β-dihydroxyurs-12-en-21-oxo-28-oic acid (**26**) which were afforded [22]. Additionally, of the biotransformation of **2** with by *S. racemosum* compounds **27–30** and 11,26-epoxy-3β-21β-dihydroxy-urs-12-en-28-oic acid were obtained (**31**), (**Figure 1**) [23]. The endophytic fungi *Pestalotiopsis microspora* isolated from medical plant *Huperzia serrata* can transform **1** to afforded 3-oxo-15β,30-dihydroxy-urs-12-en-28 oic acid (**32**), 3β,15β-dihydroxy-urs-12-en-28-oic acid (**33**), 3β,15β,30-trihydroxy-

Microbial transformation of ursolic acid by *Mucor spinosus* AS 3.3450 were isolated and their structures were identified as **9**, **22** and 3β,7β-dihydroxy-ursolic

The gum resin *Boswellia serrata* has been used for the treatment of inflammatory and arthritic diseases. Its major active constituents are ursane triterpenoids, which include 11-keto-β-boswellic acid (KBA, **36**), β-boswellic acid (BA), and acetyl-βboswellic acid (ABA). Microbial transformation **36** by *Cunninghamella blakesleeana* (AS 3.970) yielded ten regioselective transformed products: 7β-hydroxy-11-keto-βboswellic acid (**37**), 7β,15α-dihydroxy-11-keto-β-boswellic acid (**38**), 7β,16βdihydroxy-11-keto-β-boswellic acid (**39**), 7β,16α-dihydroxy-11-keto-β-boswellic acid (**40**), 7β,22β-dihydroxy-11-keto-β-boswellic acid (**41**), 7β,21β-dihydroxy-11 keto-β-boswellic acid (**42**), 7β,20β-dihydroxy-11-keto-β-boswellic acid (**43**), 7β,30 dihydroxy-11-keto-β-boswellic acid (**44**), 3α,7β-dihydroxy-11-oxours-12-en,24,30 dioic acid (**45**), and 3α,7β-dihydroxy-30-(2-hydroxypropanoyloxy)-11-oxours-12 en, 24-oic acid (**46**). Bioconversion of **36** with *Bacillus megaterium* based on a recombinant cytochrome P450 system yielded *regio*- and *stereo*selective 15α-

acid-13-lactone (**23**); 1β,3β,21β-trihydroxy-urs-12-en-28-oic acid (**24**);

urs-12-en-28-oic acid (**34**), and **30** [24].

*Microorganisms*

acid-28-ethanone (**35**) (**Figure 1**) [25].

hydroxylation (**47**) of substrate (**Figure 2**) [26].

**Figure 1.**

**278**

*Biotransformation products of ursolic acid (2).*

**Figure 2.** *Biotransformation products of 11-keto-b-boswellic acid (***36***).*

18β-glycyrrhetinic acid (**48**) is the active form of glycyrrhizin which is the major pentacyclic triterpene found in licorice (*Glycyrrhiza glabra* L.). Glycyrrhetinic acid has been shown to possess several pharmacological activities, such as antiulcerative, anti-inflammatory, immunomodulating, antitumor, antiviral, antihepatitis effects, and anticancer. Biotransformation **48** with a fungus *C. blakesleeana* (AS 3.970) yielded 3-oxo-7β-hydroxyglycyrrhetinic acid (**49**) and 7β-hydroxyglycyrrhetinic acid (**50**) [27], while of **48** using *Absidia pseudocylindrospora* (ATCC 24169), *Gliocladium viride* (ATCC 10097) and *Cunninghamella echinulata* (ATCC 8688a) afforded seven derivatives: **51**, **52**, 7β,15α-dihydroxy-18β-glycyrrhetinic acid (**53**), 15α-hydroxy-18β-glycyrrhetinic acid (**54**), 1α-hydroxy-18β-glycyrrhetinic acid (**55**) and 13β-hydroxy-7α,27-oxy-12-dihydro-18β-glycyrrhetinic acid (**56**), and the epimer of compound **53** on C-17 (**Figure 3**) [28].

Ginsenoside Rb1 (**61**) is the most predominant protopanaxadiol-type ginsenoside in *Panax* species (ginseng). Several microbial transformations of this substrate (Ginsenoside Rb1) have been accomplished with an ample and varied group of microorganisms, all of these having β-glucosidase activities.

**Figure 3.** *Biotransformation products of 18β-glycyrrhetinic acid (***48***).*

**Figure 4.** *Triterpenic acid: ginsenoside Rb1 (***61***), oleanolic acid (***62***), betulinic (***63***) and betulonic acid (***64***).*

Deglycosylation appears to be the major transformation pathway, and the intermediate and the final hydrolysis products of **61** depended on the microorganisms used. The biotransformation of various triterpenes, such as **61–64**, has been described in the literature. For each triterpenoid, the transforming microorganism together with the type and site of the reaction catalyzed is given in **Table 2** (**Figure 4**) [29].

Biotransformation of oleanolic acid (**62**) with *Bacillus subtilis* (ATCC 6633) resulted in five more polar metabolites as 28-O-β-D-glucopyranosyl oleanic acid (**63**), 3β-O-β-D-glucopyranosyl oleanic acid (**64**), 3-O-(β-D-glucopyranosyl)-oleanic acid-28-O-β-D-glucopyranoside (**61**), 24-hydroxyl-oleanolic acid (**62**), and 3β-24-dihydroxy-olean-12-en-28-O-β-D-glucopyranosyl-oic acid (**63**), while echinocystic acid (**64**, 250 mg) was metabolized to three more polar metabolites as 28-O-β-D-glucopyranosyl echinocystic acid (**65**), 3-O-(β-D-glucopyranosyl) echinocystic acid-28-O-β-D-glucopyranoside (**66**), and 24-hydroxyl-28-O-βglucopyranosyl echinocytic acid (**67**), and then biotransformation of betulinic acid (**68**) contributed four metabolites as 28-O-β-D-glucopyranosyl betulinic acid (**69**), 3-O-(β-D-glucopyranosyl)-betulinic acid-28-O-β-D-glucopyranoside (**70**), 23-hydroxy-betulinic acid (**71**), and 23-hydroxy-28-O-D-β-glucopyranosyl betulinic acid (**72**). In this way there were two types of reactions in the biotransformation of triterpenic acids **58**, **64**, and **68**: hydroxylation and glycosylation [41]. Biotransformation of **58** by *C. muscae* yielded nine hydroxylated and glycosylated metabolites. The specific hydroxylation (7β, 15α, and 21β) was main reaction type. In addition, the selective glycosylation at C-28 was another main reaction type. It was also observed that the 3β-OH group was selectively dehydrogenated into carbonyl group [42].

A C-3 oxidized derivative of oleanolic acid **73** (3-oxoolean-12-en-28-oic acid) was transformed by the *Chaetomium longirostre* (RF-1095) into 4-hydroxy-3,4-secoolean-12-ene-3,28-dioic acid (**74**) and the corresponding 21-hydroxylated derivative (**75**). Analogous ring-A cleavage oxidation reactions have been observed in the biotransformation of triterpenoid substrates with the fungi *Septomyxa affinis* ATCC 6737 and *Glomerella fusarioides* ATCC 9552. (**Figure 5**) [4, 43].

### **2.3 Steroidal saponins**

Diosgenin [(25R)-spirost-5-en-3β-ol, **76**] is an important natural starting material in the pharmaceutical industry to produce steroid drugs and hormones since the last century. In recent years, a wide array of new biological activities of **76** has been disclosed. Diosgenin was subjected to several structural modification studies to secure new derivatives via microbial transformation. Several microorganisms have been found to be capable of degrading **76**, *Bacillus megaterium*, *Corynebacterium mediolanum*, *Mycobacterium fortuitum*, *M. phlei*, *Nocardia rhodochrous,* and *F. solani*.

Three major products were accumulated, diosgenone (**77**), 1-dehydrodiosgenone (**78**), androst-4-en-3,17-dione (AD, **79**), and androsta-1,4-diene-3,17-dione

(ADD, **80**) (**Table 3**) [44, 45]. In addition, two side-chain cleavage intermediates of **76** were produced by *C*. *elegans* and *Aspergillus nidulans*. Microbial transformation

**Triterpenoid Microorganism Reaction Reference**

*F. sacchari* Deglycosylation at the C-3 and C-20 sites [31] *P. oxalicum* Deglycosylation at the C-3 site [32]

*C. blakesleeana* Diverse hydroxylation at the C-1β, C-7β, C-13β sites [4]

*C. phomoides* Hydroxylation in C-6β [4]

Formation of tertiary alcohol

*F. lini* Dehydrogenation C-13 and C-18. oleanderolide

Deshidrogenation Δ9(11)

carboxylic acid group

*Ch. longirostris* Oxidative ring A cleavage, hydroxylation, decarboxylation

*Examples of biotransformed triterpenes (***61–64***) with different microorganisms.*

*P. chrysogenum* Hydroxylation on C-21. Oxidation of the hydroxyl

formation

group in C-3

sites

Deglycosylation at the C-20 site, hydration Δ24(25)

Deglycosylation at the C-3 and C-20 sites [30]

Deglycosylation at the C-3 and C-20 sites [30]

Deglycosylation at the C-3 and C-20 sites [30]

Deglycosylation at the C-3 and C-20 sites [33]

Hydroxylation in C-11α [4]

Methyl esterification of the C-28 carboxyl group [4]

Oxidative ring A cleavage, hydroxylation at the C-21β

Hydroxylation of the C-7β, C-15α and C-30 sites

hydroxylation at the C-6α and C-7β sites

hydroxylation at the C-7β and C-15α sites

Introduction of a β-glucopyranosyl at the C-28

Dehydrogenation of the C-3 secondary alcohol group,

Dehydrogenation of the C-3 secondary alcohol group,

Hydroxylation at the C-1β and C-7β sites [4]

Ketone α-hydroxylation at the C-2 site [37]

Hydroxylations at the C-7β and (or) C-15β sites [4, 40]

[33]

[4]

[4]

[34]

[35]

[36]

[37]

[38]

[39]

Ginsenoside Rb1 (**61**)

Oleanolic acid (**62**)

Betulinic acid (**63**)

Betulonic acid (**64**)

**Table 2.**

**281**

*A. niger* (KTC 6909)

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

> *A. niger* (AS 3.1858)

*A. usamii* (KTC 6956)

*C. lunata* (AS 3.1109)

*R. stolonifer* (AS 3.822)

*A. ochraceus* (NG

*Chaetomium longirostre*

*Nocardia* sp. (NRRL 5646)

*R. miehei* (CECT

*B. megaterium* (ATCC 14581)

*B. megaterium* (ATCC 13368)

*Cunninghamella*

*B. megaterium* (ATCC 13368)

*C. lunata* (ATCC 13432)

*C. elegans* (ATCC 9244)

sp.

1203)

2749)

## *Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*


#### **Table 2.**

Deglycosylation appears to be the major transformation pathway, and the intermediate and the final hydrolysis products of **61** depended on the microorganisms used. The biotransformation of various triterpenes, such as **61–64**, has been described in the literature. For each triterpenoid, the transforming microorganism together with the type and site of the reaction catalyzed is given in **Table 2** (**Figure 4**) [29]. Biotransformation of oleanolic acid (**62**) with *Bacillus subtilis* (ATCC 6633) resulted in five more polar metabolites as 28-O-β-D-glucopyranosyl oleanic acid (**63**), 3β-O-β-D-glucopyranosyl oleanic acid (**64**), 3-O-(β-D-glucopyranosyl)-oleanic acid-28-O-β-D-glucopyranoside (**61**), 24-hydroxyl-oleanolic acid (**62**), and 3β-24-dihydroxy-olean-12-en-28-O-β-D-glucopyranosyl-oic acid (**63**), while

*Triterpenic acid: ginsenoside Rb1 (***61***), oleanolic acid (***62***), betulinic (***63***) and betulonic acid (***64***).*

echinocystic acid (**64**, 250 mg) was metabolized to three more polar metabolites as 28-O-β-D-glucopyranosyl echinocystic acid (**65**), 3-O-(β-D-glucopyranosyl) echinocystic acid-28-O-β-D-glucopyranoside (**66**), and 24-hydroxyl-28-O-βglucopyranosyl echinocytic acid (**67**), and then biotransformation of betulinic acid (**68**) contributed four metabolites as 28-O-β-D-glucopyranosyl betulinic acid (**69**),

23-hydroxy-betulinic acid (**71**), and 23-hydroxy-28-O-D-β-glucopyranosyl betulinic acid (**72**). In this way there were two types of reactions in the biotransformation of triterpenic acids **58**, **64**, and **68**: hydroxylation and glycosylation [41]. Biotransformation of **58** by *C. muscae* yielded nine hydroxylated and glycosylated metabolites. The specific hydroxylation (7β, 15α, and 21β) was main reaction type. In addition, the selective glycosylation at C-28 was another main reaction type. It was also observed that the 3β-OH group was selectively dehydrogenated into carbonyl

A C-3 oxidized derivative of oleanolic acid **73** (3-oxoolean-12-en-28-oic acid) was transformed by the *Chaetomium longirostre* (RF-1095) into 4-hydroxy-3,4-secoolean-12-ene-3,28-dioic acid (**74**) and the corresponding 21-hydroxylated derivative (**75**). Analogous ring-A cleavage oxidation reactions have been observed in the biotransformation of triterpenoid substrates with the fungi *Septomyxa affinis* ATCC

Diosgenin [(25R)-spirost-5-en-3β-ol, **76**] is an important natural starting material in the pharmaceutical industry to produce steroid drugs and hormones since the last century. In recent years, a wide array of new biological activities of **76** has been disclosed. Diosgenin was subjected to several structural modification studies to secure new derivatives via microbial transformation. Several microorganisms have been found to be capable of degrading **76**, *Bacillus megaterium*, *Corynebacterium mediolanum*, *Mycobacterium fortuitum*, *M. phlei*, *Nocardia rhodochrous,* and *F. solani*.

3-O-(β-D-glucopyranosyl)-betulinic acid-28-O-β-D-glucopyranoside (**70**),

6737 and *Glomerella fusarioides* ATCC 9552. (**Figure 5**) [4, 43].

group [42].

**280**

**Figure 4.**

*Microorganisms*

**2.3 Steroidal saponins**

*Examples of biotransformed triterpenes (***61–64***) with different microorganisms.*

Three major products were accumulated, diosgenone (**77**), 1-dehydrodiosgenone (**78**), androst-4-en-3,17-dione (AD, **79**), and androsta-1,4-diene-3,17-dione (ADD, **80**) (**Table 3**) [44, 45]. In addition, two side-chain cleavage intermediates of **76** were produced by *C*. *elegans* and *Aspergillus nidulans*. Microbial transformation

#### **Figure 5.**

*Biotransformation products of oleanolic acid (***62***), echnocystic acid (***68***) and betulinic acid (***63***).*


out by fungi. The commercialized microbial process in the steroid field was in the production of 11α-hydroxyprogesterone. This process was realized for the first time by Peterson and Murray (1952), which patented this process of 11α-hydroxylation of progesterone (**102**) by *Rhizopus* species [50]. Microbial hydroxylation of **102** by *A. griseola* produced two hydroxylated pregnane identified as 6β,14α-dihydroxyprogesterone (**103**) and 7α,14α-dihydroxyprogesterone (**104**). *R. pusillus* produced 6β,11α-dihydroxyprogesterone (**105**) with excellent yield (65.5%) and 7α,14α-

Industry, which is carried by different microorganisms, such as different species of *Curvularia* spp., *Cunninghamella* spp. and fungi *Trichoderma hamatum*, *Cochliobolus lunatus*. Structural transformation of steroidal compounds through microorganisms has emerged as an important application in the steroidal drug industry. Microbial conversions of steroids generally involve dehydrogenation, esterification, halogenation, isomerization, methoxylation, and side-chain modification of steroidal skeleton. Recently, *Mucor circinelloides lusitanicus* transformed 5-en-3β-ol steroids (**108** and **109**) into di- and trihydroxy products. The compound

dihydroxyprogesterone (**106**) (**Figure 7**) [51].

*Biotransformation products of diosgenin (***76***).*

*Microorganisms as Biocatalysts and Enzyme Sources DOI: http://dx.doi.org/10.5772/intechopen.90338*

**Figure 6.**

**Figure 7.**

**283**

*Biotransformation products of progesterone (***102***).*

#### **Table 3.**

*The ability of different fungi to transform diosgenin (***76***).*

of **76** using white-rot fungus *Coriolus versicolor* afforded eight polyhydroxylated steroids, 7β-hydroxydiosgenin (**81**), (25R)-spirost-5-en-3β,7β,21-triol (**82**), (25R) spirost-5-en-3β,7β,12β-triol (**83**), (25R)-spirost-5-en-3β,7α,15α,21-tetraol (**84**), (25R)-spirost-5-en-3β,7β,12β,21-tetraol (**85**), (25R)-spirost-5-en-3β,7α,12β,21 tetraol (**86**), and (25R)-spirost-5-en-3β,7β,11α,21-tetraol (**87**). The 3β-hydroxyl group and double bond in the B-ring of 76 were found to be important structural determinants for their activity [46].

Microbial transformation of **76** using *Cunninghamella blakesleeana* AS 3.970 afforded polyhydroxylated derivatives, such as (25R)-spirost-5-en-3β,7α,12β-triol (**88**), (25R)-spirost-5-en-3β,7α,12β,15α,21-pentaol (**89**), (25R)-spirost-5-en-3β,7α,12β,18-tetraol (**90**), (25R)-spirost-5-en-3β,7α,12β,15α-tetraol (**91**), (25R) spirost-5-en-3β,7α,11α,21-tetraol (**92**), (25R)-spirost-5-en-3β,7β,15α,21-tetraol (**93**), and (25R)-spirost-5-en-3β,7β,12β,18-tetraol (**94**) [47], specifically, the hydroxylation, ketonization, and methoxylation by *Cunninghamella blakesleeana*, *C. elegans, Helicostylum piriforme*, and *Streptomyces virginiae*, at C-7, C-9, C-11, C-12, and C-25 positions of **76**. Biotransformation of **76** by *Syncephalastrum racemosum* afforded (25R)-spirost-5-en-3β,7α,9α-triol (**95,** 1%), (25R)-spirost-5-en-3β,9α,12α-triol-7 one (**96**, 2%), (25R)-spirost-5-en-3β,9α-diol-7,12-dione (**97**, 1.5%), (25R)-spirost-4 en-9α,12β,14α-triol-3-one (**98**, 0.66%), and (25S)-spirost-4-en-9α,14α,25β-triol-3 one (**99**, 0.66%) [48]. *C. echinulata* (CGMCC3.2716) metabolized **76** to afford **81** (0.9%), **83** (7.7%), (25R)-spirost-5-en-3β,7β-diol-11-one (**100**, 7, 1.5%), and (25R) spirost-5-en-3β,7β,11α-triol (**101**, 6.2%) (**Figure 6**) [49].
