Products obtained from the biotransformation of artemisinin (1) by different microorganisms.
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.
- microbial transformation
- natural products
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, 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, 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 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).
|Epoxidation, hydroxylation C-3β site|
Endoperoxide function reduction
Breakdown of heterocyclic rings
Epoxidation C-3 and C-13
Hydroxylation C-4, endoperoxide function reduction
|A. flavus (MTCC 9167)||14-hydroxyartemisinin|
|Hydroxylation of C-14 site|
Breakdown of heterocyclic rings
Hydroxylation of C-4α site
Endoperoxide function reduction
|C. elegans (ATCC 9245)||7β-hydroxy-9α-artemisinin|
|Hydroxylation C-7β site|
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
|Endoperoxide function reduction|
Hydroxylation of C-1 site
|S. griseus (ATCC 13273)||9-artemisitone|
|Oxidation of C-9 site|
Hydroxylation of C-9α site
Hydroxylation of C-9β site
Hydroxylation of C-3α site
|N. corallina||Deoxyartemisinin||Endoperoxide function reduction|||
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 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-
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 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) .
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-urs-12-en-28-oic acid (34), and 30 .
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-β-boswellic acid (ABA). Microbial transformation 36 by 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 36 with Bacillus megaterium based 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 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) , 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) .
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. 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) .
|Ginsenoside Rb1 (61)||A. niger (KTC 6909)||Deglycosylation at the C-3 and C-20 sites|||
|A. niger (AS 3.1858)||Deglycosylation at the C-3 and C-20 sites|||
|A. usamii (KTC 6956)||Deglycosylation at the C-3 and C-20 sites|||
|F. sacchari||Deglycosylation at the C-3 and C-20 sites|||
|P. oxalicum||Deglycosylation at the C-3 site|||
|C. lunata (AS 3.1109)||Deglycosylation at the C-20 site, hydration Δ24(25)|
Formation of tertiary alcohol
|R. stolonifer (AS 3.822)||Deglycosylation at the C-3 and C-20 sites|||
|Oleanolic acid (62)||C. blakesleeana||Diverse hydroxylation at the C-1β, C-7β, C-13β sites|||
|F. lini||Dehydrogenation C-13 and C-18. oleanderolide formation|||
|P. chrysogenum||Hydroxylation on C-21. Oxidation of the hydroxyl group in C-3|||
|C. phomoides||Hydroxylation in C-6β|||
|A. ochraceus (NG 1203)||Hydroxylation in C-11α|||
|Chaetomium longirostre||Oxidative ring A cleavage, hydroxylation at the C-21β sites|||
|Nocardia sp. (NRRL 5646)||Methyl esterification of the C-28 carboxyl group|||
|R. miehei (CECT 2749)||Hydroxylation of the C-7β, C-15α and C-30 sites|
|Betulinic acid (63)||B. megaterium (ATCC 14581)||Dehydrogenation of the C-3 secondary alcohol group, hydroxylation at the C-6α and C-7β sites|||
|B. megaterium (ATCC 13368)||Dehydrogenation of the C-3 secondary alcohol group, hydroxylation at the C-7β and C-15α sites|||
|C. elegans (ATCC 9244)||Hydroxylation at the C-1β and C-7β sites|||
|Cunninghamella sp.||Introduction of a β-glucopyranosyl at the C-28 carboxylic acid group|||
|Betulonic acid (64)||B. megaterium (ATCC 13368)||Ketone α-hydroxylation at the C-2 site|||
|Ch. longirostris||Oxidative ring A cleavage, hydroxylation, decarboxylation|||
|C. lunata (ATCC 13432)||Hydroxylations at the C-7β and (or) C-15β sites||[4, 40]|
Biotransformation of oleanolic acid (62) with Bacillus subtilis (ATCC 6633) resulted in five more polar metabolites as 28-O-β-
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 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 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 .
|Fungi||Diosgenona (77)||AD (79)||ADD (80)||Progesterone (102)||16-AD|
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) , 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%) . 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) .
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 Rhizopus species . 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-dihydroxyprogesterone (106) (Figure 7) .
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 108 yielded 3β,7α,11α-trihydroxypregna-5-en-20-one (110, 46.4%), and 109 afforded 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 112 at 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 Rhizopus spp. or Aspergillus spp., Curvularia spp. or Cunninghamella spp. and Streptomyces spp., respectively (Figure 10) (Table 4) .
|C-7α||Fusarium sp., Gibberella sp., Nigrospora sp., Acremonium sp., Phycomyces sp.||Production of bile acids and drugs for neuropsychiatry and immunology||[55, 56]|
|C-7β||Mortierella sp.||Obtaining drugs for prostate cancer||[56, 57]|
|11β||Curvularia sp., Absidia sp., Cunninghamella sp., Trichoderma sp., Cochliobolus sp.||Obtaining anti-inflammatory drugs, like hydrocortisone, prednisone acetate, dexamethasone||[56, 57, 58, 59, 60]|
|11α||Aspergillus sp., Rhizopus sp.||Obtaining of anti-inflammatory, immunosuppressive, anti-allergic drugs, and production of contraceptive drugs||[56, 61]|
|15β||Bacillus sp.||[56, 62]|
|16α||Streptomyces sp.||[63, 64]|
Boldenone (124) is an important steroid hormone drug which is the derivative of testosterone. Biotransformation of 124 by Arthrobacter simplex and recombinant Pichia pastoris with 17β-hydroxysteroid dehydrogenase from Saccharomyces cerevisiae produces BD (124) from androst-4-ene-3,17-dione (79, AD) efficiently . Many microorganisms such as Mucor racemosus, Nostoc muscorum, and Arthrobacter oxydans can 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].
Corticium centrifugum Fusarium spp.
Exophiala jeanselmei var. lecaniicorni
F. oxysporum var. cubense
Comamonas testosteroni (syn. Pseudomonas testosteroni)
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 (Bacillus sp.), and C15 (A. glauca, Aspergillus fumigatus, B. obtusa) [54, 56, 57, 67, 68, 69]. The biotransformation of 79 with different microorganisms is shown. Compound 79 is 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 128 with 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 128 by Curvularia lunata yielded 3-ketoesclareolide (129), 1β-hydroxysclareolide (130), 3β-hydroxysclareolide (131), 1α,3β-dihydroxysclareolide (133), and 1β,3β-dihydroxysclareolide (134) . The incubation of 128 with Cunninghamella elegans afforded 129, 131, 133, and 135–137 . C blakesleeana metabolized 128 to afford 129, 135, 134, and 138–140. Biotransformation of 128 with C. echinulata yielded 5-hydroxysclareolide (141) and 7β-hydroxysclareolide (142) . Fermentation of 148 with A. niger using a nutrient-rich culture medium yielded 141 and 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 Aspergillus strains 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 147-152 were regioselectively cyclized to flavanones (Figure 14). Hydrogenation of flavonoids was only reported on transformation of chalcones to dihydrochalcones (Figure 14) [92, 93].
Aspergillus niger is one of the most applied microorganisms in the flavonoids’ biotransformation; for example, A. niger can 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 170 to 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. echinulata and C. militaris [96, 97].
Incubation of Absidia coerulea (AM93) with prenylnaringenin (178) led to metabolite 179 (8-prenylnaringenin 7-O-β-
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 glauca AM177, A. coerulea AM93, Rhizopus nigricans UPF701, Beauveria bassiana AM278, and B. bassiana AM446 are able to conjugate sugar moiety to chalcones, flavanones, and isoflavanones with high regioselectivity. Therefore, it is possible to use Beauveria and Absidia for 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. coerulea with 185 resulted in the isolation of xanthohumol 4′-O-β-
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 185 in 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. tricinctum reduced α,β-double bond of 185 to give 4,2′,4′-trihydroxy-6′-methoxy-3-prenyl-α,β-dihydrochalcone (194). Penicillium albidum (AM79) oxidized 185 at the double bond of prenyl group to xanthohumol H (195) . The culture of the yeast, Rhodotorula marina (AM 77), converted 185 and 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. bassiana AM278 and Absidia glauca AM177 converted isoxanthohumol (189) into glucoside derivatives (200, 201), whereas Fusarium equiseti AM15 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].
|Baking, brewing, starch liquefaction|
Clarification of fruit juice
High glucose and high fructose syrups
|Lactase (β-galactosidase)||Kluyveromyces lactis|
|Lactose intolerance reduction in people|
Prebiotic food ingredients
C. cylindraceae Ay30
|Cheese flavor development|
Use in cosmetics
Use as biosensors
Use in biodegradation
|Phospholipases||Fusarium oxysporum||Cheese flavor development|
|Esterases||Bacillus licheniformis||Enhancement of flavor and fragrance in fruit juice|
|Clarification of fruit juice|
Beer quality improvement
|Glucose oxidase||A. niger|
|Food shelf life important|
Food flavor improvement
|Polyphenol removal from wine|
|Clarification of fruit juice|
Removal of H2O2 from milk prior to cheese production
|Peroxidase||Streptomyces viridosporus||Development of flavor, color and nutritional quality of food|
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) .
Thermoproteus texas Hyperthermus butylicus
|Aldolase||Stereoselective C-C bond formation||Pharmaceutical industry|||
|Pyrococcus furiosus||Hydrogenase||Final stage of glucose oxidation by oxidative pentose phosphate cycle||Enhanced production of biohydrogen|||
|Geobacillus thermoleovorans||Carboxylesterase||Carboxyl ester hydrolysis||Agriculture, food, and pharmaceutical industries|||
|Bacillus pumilus||Acidic thermostable lipase||Degradation of palm oil||Treatment of palm oil-containing wastewater|||
|Geobacillus sp.||Lipase||Hydrolysis of diver’s lipid substrates||Biofuel, cosmetics, or perfume production, leather and pulp industries|||
|Microbial community from solid-state|
|Protease||Degradation of hair waste from tannery||Leather industry|||
|Sulfolobus tokodaii||Chitinase||Hydrolysis of β-(1, 4)-glycosidic bonds in chitin||Biomedical, pharmaceutical, food, and environmental|||
|Acidothermus cellulolyticus||Endoxylanase||β-(1,4)-xylan cleavage||Biofuel production from lignocellulose|||
|Thermotoga neapolitana||Pullulanase||Hydrolysis of α-(1, 6)-glucosidic linkages||Biofuel production|||
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.
The authors thank Carrera de Biología, FES-Zaragoza, UNAM, Al Departamento de Química Orgánica, FES-Cuautitlán, UNAM.
Conflict of interest
The authors report no conflicts of interest.