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Microbial-catalyzed biotransformations have considerable potential for the generation of an enormous variety of structurally diversified organic compounds, especially natural products with complex structures like triterpenoids, flavonoids, steroids, steroidal saponins, and sesquiterpenoids. They offer efficient and economical ways to produce semisynthetic analogues and novel lead molecules. Microorganisms such as bacteria and fungi could catalyze chemo-, regio-, and stereospecific hydroxylations of diverse substrates that are extremely difficult to produce by chemical routes. During recent years, considerable research has been performed on the microbial transformation of bioactive compounds, in order to obtain biologically active molecules with diverse structural features. In green chemistry, biotransformations are an important chemical methodology toward more sustainable industrial processes.
chapter and author info
Laboratorio de Biotransformaciones y Química de Productos Naturales, L-314, Facultad de Estudios Superiores Zaragoza, UNAM, Mexico
Laboratorio de Biotransformaciones y Química de Productos Naturales, L-314, Facultad de Estudios Superiores Zaragoza, UNAM, Mexico
Iker S. Escalona-Torres
Laboratorio de Biotransformaciones y Química de Productos Naturales, L-314, Facultad de Estudios Superiores Zaragoza, UNAM, Mexico
Sección de Química Orgánica, Departamento de Ciencias Químicas, FES-Cuautitlán, UNAM, Mexico
*Address all correspondence to: email@example.com;, firstname.lastname@example.org
Microbial transformation is regarded as an enzymatic reaction by using the metabolic activities of microorganisms to modify the chemical structures of bioactive substrates for finding the new chemical derivatives with the potent bioactivities and physical-chemical characteristics. It has a number of advantages over chemical synthesis such as higher stereo- and regioselectivity but is also enantiospecific, allowing the production of chiral products from racemic mixtures. The conditions for biotransformations are mild, and in the majority of cases, they do not require the protection of pre-existing functional groups. Furthermore, some reactions that do not occur when using chemical approaches are easily carried out by microbial transformation. Microbial factories show advantages, for instance, growing rapidly and ease of large-scale production [1, 2, 3].
The use of microorganisms may be a highly efficient method of production of these compounds. The reactions involved in biotransformation of organic compounds by whole cells of various microorganisms include oxidation, reduction, hydroxylation, esterification, methylation, demethylation, isomerization, hydrolysis, glycosylation, and hydrogenation [4, 5].
Biotransformation may be carried out with isolated enzyme systems or with intact organism. Although isolated enzyme systems may be more specific and efficient for certain biotransformation, these reactions may involve isolating the enzyme system, and, for some classes of enzyme-catalyzed reaction, a recycling sequence may be required to regenerate the enzyme .
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 . 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, Mucorand Rhizopusare commonly used in the industry. Rhizopusstrains are important in citric acid production. Mucorstrains make a significant number of important lipases and catalyze the hydroxylation of a wide range of chemical compounds [2, 3, 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 stereospecificity is required; (7) maintenance of stock cultures of microorganisms is simpler and cheaper than the maintenance of cell or tissue cultures or laboratory animals; (8) ease of setup and manipulation; and (9) more reliable and reproducible [8, 9].
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 with different microorganisms
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 1through biological and chemical methodologies have been reported to yield more effective and water-soluble derivatives. A wide array of microbial transformations of 1involve 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 1usually 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).
3β-hydroxy-4,12-epoxy-1-deoxyartemisinin Artemisinin G 3,13-epoxyartemisinin 4α-hydroxy-1-deoxyartemisinin
Epoxidation, hydroxylation C-3β site Endoperoxide function reduction Breakdown of heterocyclic rings Epoxidation C-3 and C-13 Hydroxylation C-4, endoperoxide function reduction
Products obtained from the biotransformation of artemisinin (1) by different microorganisms.
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 activities, such as antibacterial, anti-allergic, antioxidative, anti-inflammation, and antitumor activities .
Microbial transformation of ursolic acid (2) by Bacillus megateriumCGMCC 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%) . The biotransformation studies of 2by Alternaria longipesAS 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-dihydroxy-ursolic 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 . Biotransformation of 2by A. alternataeight 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.
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-dihydroxy-urs-12-en-21-oxo-28-oic acid (26) which were afforded . Additionally, of the biotransformation of 2with by S. racemosumcompounds 27–30and 11,26-epoxy-3β-21β-dihydroxy-urs-12-en-28-oic acid were obtained (31), (Figure 1) .
The endophytic fungi Pestalotiopsis microsporaisolated from medical plant Huperzia serratacan transform 1to 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-urs-12-en-28-oic acid (34), and 30.
Microbial transformation of ursolic acid by Mucor spinosusAS 3.3450 were isolated and their structures were identified as 9, 22and 3β,7β-dihydroxy-ursolic acid-28-ethanone (35) (Figure 1) .
The gum resin Boswellia serratahas 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-β-boswellic acid (ABA). Microbial transformation 36by Cunninghamella blakesleeana(AS 3.970) yielded ten regioselective transformed products: 7β-hydroxy-11-keto-β-boswellic acid (37), 7β,15α-dihydroxy-11-keto-β-boswellic acid (38), 7β,16β-dihydroxy-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 36with Bacillus megateriumbased on a recombinant cytochrome P450 system yielded regio- and stereoselective 15α-hydroxylation (47) of substrate (Figure 2) .
18β-glycyrrhetinic acid (48) is the active form of glycyrrhizin which is the major pentacyclic triterpene found in licorice (Glycyrrhiza glabraL.). Glycyrrhetinic acid has been shown to possess several pharmacological activities, such as antiulcerative, anti-inflammatory, immunomodulating, antitumor, antiviral, antihepatitis effects, and anticancer. Biotransformation 48with a fungus C. blakesleeana(AS 3.970) yielded 3-oxo-7β-hydroxyglycyrrhetinic acid (49) and 7β-hydroxyglycyrrhetinic acid (50) , while of 48using 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 53on C-17 (Figure 3) .
Ginsenoside Rb1 (61) is the most predominant protopanaxadiol-type ginsenoside in Panaxspecies (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. Deglycosylation appears to be the major transformation pathway, and the intermediate and the final hydrolysis products of 61depended 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) .
Examples of biotransformed triterpenes (61–64) with different microorganisms.
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-β-glucopyranosyl 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 . Biotransformation of 58by C. muscaeyielded 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 .
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-seco-olean-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 affinisATCC 6737 and Glomerella fusarioidesATCC 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 76has 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 76were produced by C. elegansand Aspergillus nidulans. Microbial transformation of 76using white-rot fungus Coriolus versicolorafforded 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 .
The ability of different fungi to transform diosgenin (76).
AD androst-4-ene-3,17-dione; ADD androsta1,4-diene-3,17-dione; 16-AD androst-4-ene-3,16-dione.
Microbial transformation of 76using Cunninghamella blakesleeanaAS 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) , 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 76by Syncephalastrum racemosumafforded (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%) . C. echinulata(CGMCC3.2716) metabolized 76to 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) .
Microorganisms are able to hydroxylate steroids in different positions C-1 to C-21. These represent the most widespread type of steroid bioconversion carried 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 Rhizopusspecies . Microbial hydroxylation of 102by A. griseolaproduced two hydroxylated pregnane identified as 6β,14α-dihydroxyprogesterone (103) and 7α,14α-dihydroxyprogesterone (104). R. pusillusproduced 6β,11α-dihydroxyprogesterone (105) with excellent yield (65.5%) and 7α,14α-dihydroxyprogesterone (106) (Figure 7) .
Industry, which is carried by different microorganisms, such as different species of Curvulariaspp., Cunninghamellaspp. 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 lusitanicustransformed 5-en-3β-ol steroids (108and 109) into di- and trihydroxy products. The compound 108yielded 3β,7α,11α-trihydroxypregna-5-en-20-one (110, 46.4%), and 109afforded 111(3β,7α-dihydroxyandrost-5-en-17-one, 43.6%) (Figure 8) .
Microbial transformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (112) is by four filamentous fungi, Cunninghamella elegans(113–119), Macrophomina phaseolina(115, 117, 120–122), Rhizopus stolonifer(113, 123), and Gibberella fujikuroi(115–117, 123). These metabolites were obtained as a result of biohydroxylation of 112at C-6β, 7β, 11α, 14α, 15β, 16β, and 17α positions (Figure 9) .
The 11α-, 11β-, 15α, and 16α-hydroxylations are currently established processes in the steroid industry mainly for the production of adrenal cortex hormones and their analogues. 11α-, 11β-, and 16α-hydroxylations are usually performed using Rhizopusspp. or Aspergillusspp., Curvulariaspp. or Cunninghamellaspp. and Streptomycesspp., respectively (Figure 10) (Table 4) .
Some examples of steroid hydroxylation reactions promoted by microorganisms and their applications.
Boldenone (124) is an important steroid hormone drug which is the derivative of testosterone. Biotransformation of 124by Arthrobacter simplexand recombinant Pichia pastoriswith 17β-hydroxysteroid dehydrogenase from Saccharomyces cerevisiaeproduces BD (124) from androst-4-ene-3,17-dione (79, AD) efficiently . Many microorganisms such as Mucor racemosus, Nostoc muscorum, and Arthrobacter oxydanscan utilize androst-1,4-diene-3,17-dione (80, ADD) as substrate to produce testosterone through 17β-carbonyl reduction reactions (Table 5). The ability of microorganisms to reduce 17-keto- to 17β-hydroxysteroids was evidenced for a wide variety of substrates and microorganisms of different taxonomy: bacteria, fungi, and yeast [54, 56, 57, 66].
Actinomucor elegans Agaricus silvaticus A. pantherina A. spissa Armillaria mellea Corticium centrifugum Fusarium spp. Gibberella saubinetti Mucorspp. Penicilliumspp. Aphanocladium album Aspergillus chevalieri A. flavus A. oryzae A. tamarii B. obtusa C. aphidicola Ceratocystis paradoxa C. lunatus Colletotrichum musae C. radicicola Exophiala jeanselmeivar. lecaniicorni
Fusarium culmorum F. oxysporumvar. cubense F. solani Mucor piriformis M. spinosus P. chrysogenum P. crustosum P. blakesleeanus R. stolonifer Septomyxa affinis T. piriforme Trichoderma viride Zygodesmussp.
Candida albicans C. pelliculosa C. pseudotropicalis C. robusta C. tropicalis C. utilis Cryptococcus albidus C. laurentii C. tsukubaensis Debaryomyces hansenii D. kloeckeri D. nicotianae D. subglobosus D. vini Hansenula anomala H. califórnica H. schnegii H. suaveolens Kloeckera jensenii Saccharomyces carlsbergensis S. cerevisiae S. fragilis S. lactis S. oviformis S. turbidans S. validus Pichia farinosa P. membranaefaciens Torulopsisspp. Hortaea werneckii Phaeotheca triangularis P. herbarum P. ostreatus Rhodotorula aurantiaca R. mucilaginosa
B. stearothermophilus Bacteroides fragilis Brevibacterium sterolicum Clostridium paraputrificum Comamonas testosteroni Lactobacillus bulgaricus Mycobacteriumspp. B. stearothermophilus Bacteroides fragilis Brevibacterium sterolicum Clostridium paraputrificum Comamonas testosteroni (syn. Pseudomonas testosteroni) Lactobacillus bulgaricus Pediococcus cerevisiae Sarcina lutea Staphylococcus aureus Streptomyces globisporus S. sphaeroides S. viridochromogenes S. hydrogenans S. lavendulae
Reduction of the C-17 carbonyl group of steroids by (17βHSDs) different microorganisms.
The oxidation of 17β-hydroxyl group was observed along with hydroxylation of steroids at C5 (Penicillium crustosum, P. chrysogenum), C6 (Bacillus stearothermophilus, B. obtusa, P. blakesleeanus), C7 (α/β) (A. coerulea, Botrytis cinerea, B. obtusa, P. blakesleeanus, Rhizopus stolonifer), C10 (Absidia glauca), C11 (α/β) (A. coerulea, B. obtusa, Cephalosporium aphidicola, R. stolonifer), C12 (A. glauca, B. obtusa), C14 (Bacillussp.), and C15 (A. glauca, Aspergillus fumigatus, B. obtusa) [54, 56, 57, 67, 68, 69]. The biotransformation of 79with different microorganisms is shown. Compound 79is an endogenous weak androgen steroid hormone and intermediate in the biosynthesis of estrone and of testosterone from dehydroepiandrosterone (DHEA) . DHEA is an endogenous steroid hormone. It functions as a metabolic intermediate in the biosynthesis of the androgen and estrogen sex steroids. Various microorganisms have had the ability to biotransform steroidal compounds such as AD (79) , DHEA (125) [54, 55, 70, 71, 72, 73, 76, 77, 80, 81], testosterone [54, 55, 74, 75, 76, 81], cortexolone (126) [78, 79], and prednisone (127) (Figure 11a–d) [54, 55, 82].
Sclareolide (128) is a natural product isolated from several plant species which displays phytotoxic and cytotoxic activities against several human tumor cells lines. This compound has also been used as starting material for the synthesis of various bioactive products. Regarding the biotransformation of the 128with different microorganisms, mono- (130, 131, 135, 140–142) and dihydroxylation (132–134, 136, 139, 143, 146), oxidation (129, 144), hydroxylation/oxidation (145), epimerization (137), and cyclization (138) products have been obtained . The microbial transformation of 128by Curvularia lunatayielded 3-ketoesclareolide (129), 1β-hydroxysclareolide (130), 3β-hydroxysclareolide (131), 1α,3β-dihydroxysclareolide (133), and 1β,3β-dihydroxysclareolide (134) . The incubation of 128with Cunninghamella elegansafforded 129, 131, 133,and 135–137. C blakesleeanametabolized 128to afford 129, 135, 134, and 138–140. Biotransformation of 128with C. echinulatayielded 5-hydroxysclareolide (141) and 7β-hydroxysclareolide (142) . Fermentation of 148with A. nigerusing a nutrient-rich culture medium yielded 141and 144–146(Figure 12) .
As most important phytochemicals in food, the dietary flavonoids exert a wide range of benefits for human health. Recent researches have explored diverse biological and pharmacological activities of natural flavonoids—antioxidant activity, anti-inflammatory activity, anti-Alzheimer’s disease, antibacterial activity, antifungal activity, anti-HIV activity, anticoagulant activity, antileishmanial activity, and anti-obesity activity [87, 88, 89, 90, 91]. Microbial biotransformation strategies for production of flavonoids have attracted considerable interest because they allow yielding novel flavonoids, which do not exist in nature.
The main reactions during microbial biotransformation are hydroxylation, dehydroxylation, O-methylation, O-demethylation, glycosylation, deglycosylation, dehydrogenation, hydrogenation, C ring cleavage of the benzo-γ-pyrone system, cyclization, and carbonyl reduction. Cunninghamella, Penicillium, and Aspergillusstrains are very popular to biotransform flavonoids, and they can perform almost all the reactions with excellent yields (Figure 13). Isoflavones are usually hydroxylated at the C-3′ position of the B ring by microorganisms. Chalcones 147-152were regioselectively cyclized to flavanones (Figure 14). Hydrogenation of flavonoids was only reported on transformation of chalcones to dihydrochalcones (Figure 14) [92, 93].
Aspergillus nigeris one of the most applied microorganisms in the flavonoids’ biotransformation; for example, A. nigercan transfer flavanone to flavan-4-ol, 2′-hydroxydihydrochalcone, flavone, 3-hydroxyflavone, 6-hydroxyflavanone, and 4′-hydroxyflavanone. The hydroxylation of flavones by microbes usually happens on the ortho position of the hydroxyl group on the A ring and C-4′ position of the B ring, and microbes commonly hydroxylate flavonols at the C-8 position. Natural flavonoids, such as naringenin (166), hesperetin (167), chrysin (168), apigenin (169), and luteolin (170) were subjected to microbiological transformations by Rhodotorula glutinis(KCh 735). Yeast was able to regioselectively C-8 hydroxylate 167, 168, 169, and 170to generate 171(17%), 172(31%), 173(12.9%), and 174(25%), respectively. Naringenin (166) was transformed to carthamidin (175) and isocarthamidin (176) in a ratio of 1:19, respectively (Figure 15) .
The microorganisms tend to hydroxylate flavanones at the C-5, 6, and 4′ positions; however, for prenylated flavanones, dihydroxylation often takes place on the Δ4(5) double bond on the prenyl group (the side chain of A ring), although cyclization of the prenyl group to dihydrofurane derivatives is rather common biotransformation pathway of prenylated flavonoids. Prenylated flavanones are a unique class of naturally occurring flavonoids characterized by the presence of a prenylated side chain (prenyl, geranyl) in the flavonoid skeleton . The prenyl chain generally refers to the 3,3-dimethylallyl substituent (3,3-DMA), geranyl and lavandulyl. It is proposed that the prenyl-moiety makes the backbone compound more lipophilic, which leads to its high affinity with cell membranes. The prenylation brings the flavonoids with enhancement of antibacterial, anti-inflammatory, antioxidant, cytotoxicity, larvicidal, as well as estrogenic activities. Figure 16 demonstrated the microbial biotransformation of kurarinone (177) using C. echinulataand C. militaris[96, 97].
Incubation of Absidia coerulea(AM93) with prenylnaringenin (178) led to metabolite 179(8-prenylnaringenin 7-O-β-d-glucopyranoside, 49.3%), while B. bassianatransformed 178into 180(8-prenylnaringenin 7-O-β-d-4″-O-methyl-glucopyranoside, 32.9%); the metabolites 179and 180originated in Sabouraud medium. In the absence of glucose in the culture of A. coerulea, the sulfation of substrate 178(8-prenylnaringenin-7-sulfate, 181, 31.1%) occurs, while B. bassianainto the same product (180). The capacity of some fungi—Cunninghamella elegans, Streptomyces fulvissimus, Mucor ramannianus, and B. bassiana—in the sulfation of certain phenolic compounds has been reported (Figure 17) .
Regioselective glycosylation of biologically active flavonoid aglycones catalyzed by microorganisms is an interesting and desired reaction, which significantly increases the water solubility of the compound and, therefore, may improve bioavailability of flavonoids. Absidia glaucaAM177, A. coeruleaAM93, Rhizopus nigricansUPF701, Beauveria bassianaAM278, and B. bassianaAM446 are able to conjugate sugar moiety to chalcones, flavanones, and isoflavanones with high regioselectivity. Therefore, it is possible to use Beauveriaand Absidiafor the microbial transformation of simple or prenylated flavonoids by glycosidation reactions [97, 99].
Bavachinin (182) is one kind compound of flavanones and isolated from the aerial parts and dried fruits of Psoralea corylifolia, and bavachinin displays a broad range of biological activities, such as antioxidant, antibacterial, antifungal, anti-inflammatory, antitumor, anti-pyretic, and analgesic properties [100, 101]. Bavachinin (182) was subject to biotransformation by cultured cells of A. flavus(ATCC 30899); C. elegans(CICC 40250) afforded the same product 183[(S)-6-((R)-2,3-dihydroxy-3-methylbutyl)-2-(4-hydroxyphenyl)-7-methoxychromen-4-one]. On the other hand, one major product 184[(2S,4R)-2-(4-hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)-chromen-4-ol] was obtained by P. raistrickii(ATCC 10490) by the reduction at the position of ketone group of the C-ring (Figure 18) .
The biotransformation of xanthohumol (185), a prenylated chalcone isolated from hops by selected fungi, Absidia coerulea(AM93), Rhizopus nigricans(UPF701), Mortierella mutabilis(AM404), and Beauveria bassiana(AM446), was investigated. The incubation of A. coeruleawith 185resulted in the isolation of xanthohumol 4′-O-β-d-glucopyranoside (186, 29%). This metabolite was also produced by R. nigricans(186, 14.2%). Biotransformation of 185with B. bassianaand M. mutabilisyielded xanthohumol 7-O-β-D(4‴-O-methyl)-glucopyranoside (187, 23%) and isoxanthohumol 7-O-β-glucopyranoside (188, 49%), respectively (Figure 19) . The compounds 188(9.3%) and 186(12%) were also observed as products of 185transformation by Cunninghamella elegans. Another way to obtain 188is by the transformation of isoxanthohumol (189, 61.6%) with Absidia glauca; although the efficiency of this process was high (61.6% yield), it required the chemical isomerization of 185to 189, prior to biotransformation .
2″-(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″:3′,4′]-4,2′-dihydroxy-6′-methoxychalcone (190), mixture of diastereoisomers of (2S, 2″S) and (2S, 2″R) 2″-(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″:7,8]-4′-hydroxy-5-methoxyflavanone (191), and (Z)-2″-(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″-6,7]-3′,4′-dihydroxy-4-methoxyaurone (192) were obtained by transformation of 185in Aspergillus ochraceus(AM 465) culture (Figure 19) .
Incubation of xanthohumol (185) both with Fusarium avenaceum(AM11) and F. oxysporum(AM727) gave a single metabolite 2″-(2″-hydroxyisopropyl)-dihydrofurano-[4″,5″:3′,4′]-4′,2-dihydroxy-6′-methoxy-α,β-dihydrochalcone (193), which turned out to be the product of the prenyl group cyclization and α,β-double bond reduction. F. tricinctumreduced α,β-double bond of 185to give 4,2′,4′-trihydroxy-6′-methoxy-3-prenyl-α,β-dihydrochalcone (194). Penicillium albidum(AM79) oxidized 185at the double bond of prenyl group to xanthohumol H (195) . The culture of the yeast, Rhodotorula marina(AM 77), converted 185and 4-methoxychalcone (196) to α,β-dihydroxanthohumol (197) and 4-methoxydihydrochalcone (198) with the yields of 18% and 20%, respectively . Penicillium albidum(AM79) dihydroxylated the Δ2″(3″) double bond of xanthohumol to produce 3′-[3″-hydroxy-3″-methylbutyl]-4,2′,4′-trihydroxy-6′-methoxychalcone (199, 22.84%) (Figure 19).
B. bassianaAM278 and Absidia glaucaAM177 converted isoxanthohumol (189) into glucoside derivatives (200, 201), whereas Fusarium equisetiAM15 transformed it into (2R)-2-(2-hydroxyisopropyl)-dihydrofurano-[2,3:7,8]-4-hydroxy5-methoxyflavanone (202) (Figure 20) [95, 106].
C. echinulata(ATCC 9244) sulfated silybin (203) to silybin-7-sulfate (204) and 2,3-dehydrosylibin-7-sulfate (205). Sulfonation at the C-7 position of silybin significantly decreased the DPPH free radical scavenging potential; however, further dehydrogenation Δ2(3) to 2,3-dehydrosilbyn-7-sulfate (206) drastically enhanced the DPPH free radical scavenging potential activity  (Figure 21).
2.7 Enzymes isolated from microorganisms and their application
Enzymes are the most proficient catalysts, offering much more competitive processes than chemical catalysts. A number of enzyme-based processes have been commercialized for producing several valuable products. During the 1980s and 1990s, engineering of enzymes based on structural information allowed extension of their substrate ranges, enabling the synthesis of unusual intermediates. Accordingly, the use of enzymes has been expanded to the manufacture of pharmaceutical intermediates and fine chemicals . Microorganisms and enzymes (biocatalysts) are highly enantio-, chemo-, and regioselective in a wide range of reaction conditions. Selectivity is extremely desirable in the synthesis of different synthesis products, since it offers advantages such as minimizing the side reactions that do not require protection and deprotection steps, which allows for shorter synthesis. Biocatalysis provides a technology that is environmentally safer, and it effectively reduces the level of waste and even eliminates the waste generation rather than remediation and disposal of wastes at the end of the process. In addition to, biocatalysts have many attractive features in the context of green chemistry and sustainable development. Various enzymes used in different industrial processes have been described in the literature. Table 6 indicates some enzymes, their source, and some applications [111, 112, 113].
Bacillus amyloliquefaciens B. stearothermophilus B. licheniformis
Baking, brewing, starch liquefaction Clarification of fruit juice Textile industry Paper industry
Aspergillus niger A. awamori Rhizopus oryzae
Beer production High glucose and high fructose syrups
Kluyveromyces lactis K. fragilis
Lactose intolerance reduction in people Prebiotic food ingredients
Candida antarctica C. cylindraceae Ay30 Helvina lanuginosa Pseudomonassp. Geotrichum candidum
Cheese flavor development Textile indutry Medicinal applications Use in cosmetics Use as biosensors Use in biodegradation
Cheese flavor development
Enhancement of flavor and fragrance in fruit juice
Streptomycessp. Bacillussp. Pseudomonassp.
Clarification of fruit juice Beer quality improvement
A. niger Metarhizium anisopliae Psychrobacter piscatorri
Food preservation Removal of H2O2 from milk prior to cheese production
Development of flavor, color and nutritional quality of food
Enzymes, source, and some applications.
A very interesting research area in biology and biotechnology is the of extremophile microorganisms. Extremophiles can be divided into group according to (i) temperature tolerance, (ii) salt concentration, (iii) pH range, or (iv) pressure conditions. Enzymes from extremophilic microorganisms offer versatile tools for sustainable developments in a variety of industrial applications as they show important environmental benefits due to their biodegradability, specific stability under extreme conditions, improved use of raw materials, and decreased amount of waste products. Although major advances have been made in the last decade, our knowledge of the physiology, metabolism, enzymology, and genetics of this fascinating group of extremophilic microorganisms and their related enzymes is still limited [114, 115, 116].
The outstanding properties of thermozymes are suited to industries that employ elevated temperatures, such as the pulp and paper, food, brewing, and feed processing industries. Thermophiles are often highly resistant to harsh conditions such as chemical denaturing agents, wide pH ranges, and/or nonaqueous solvents. Examples of such enzymes are cellulases, xylanases, pectinases, chitinases, amylases, pullulanases, proteases, lipases, glucose isomerases, alcohol dehydrogenases, and esterases. Thermophilic enzymes have played important roles not only at the industrial level but also in pharmaceutical applications requiring use of specific aldolases for the synthesis of enantiopure compounds (Table 7) .
Sulfolobus solfataricus S. acidocaldarius Thermoproteus texas Hyperthermus butylicus
Due to microorganisms’ abundant multienzyme systems, microbial transformation possesses advantages against chemosynthesis of environmental friendliness, mild reaction conditions, and high stereo-, regio, and chemo-selectivities as well as in improving conversion rates and reducing cost. Thus, microbial transformation technique is being increasingly used to structurally modify natural and synthetic compounds.
The hydrolytic and reductive capabilities of microorganisms have been known and are currently used in preparative and industrial reactions. Various classes of bioactive organic compounds have been subjected to enzymatic transformation to obtain more active and less toxic substances or to elucidate their metabolic pathways.
For example, biotransformation-derived steroids are used for a wide range of pharmacotherapeutic purposes, such as anti-inflammatory, immunosuppressive, progestational, diuretic, anabolic, as neurosteroids, and as contraceptive. Researchers continue to discover more useful steroid compounds and to isolate microorganisms that can perform the structural transformations desired. New technologies such as genomics, metanogenomics, gene shuffling, and DNA evolution provide valuable tools for improving or adapting enzyme properties to the desired requirements.
An alternative may be extremophilic microorganisms such as biocatalysts for countless future industrial applications that are more environmentally friendly.
Arturo Cano-Flores, Javier Gómez, Iker S. Escalona-Torres and Benjamín Velasco-Bejarano (January 16th 2020). Microorganisms as Biocatalysts and Enzyme Sources, Microorganisms, Miroslav Blumenberg, Mona Shaaban, Abdelaziz Elgaml, IntechOpen, DOI: 10.5772/intechopen.90338. Available from:
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Introductory Chapter: Melanin, a Versatile Guardian
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