5
CHAPTER I
INTRODUCTION
GENERAL PATHWAYS OF MICROBIAL DEGRADATION OF AROMATIC COMPOUNDS A number of microorganisms can perform various types of reactions (Fonken and Johnson, 1972; Beukers et al., 1972) such as oxidation, reduction, hydrolysis, esterification, phosphorylation etc. These enzymatic reactions enable the microorganism to either synthesize or degrade complex organic compounds with ease. Such studies have become important from both fundamental and applied considerations. The microorganisms which are frequently used are fungi or bacteria.
A great deal of work has been done on the microbiological transformation of steroids (Charney and Herzog, 1967) and a number of transformations have actually been exploited for commercial applications (Briggs and Brotherton, 1970). To cite an example, cortisone (l) an adrenocorticoid hormone which was effective against rheumatoid arthritis, a grave crippling disease was prepared by a sequence of chemical reaction from bile acids involving 32 steps
(Peterson, 1963; Briggs and Brotherton, 1970). The formidable step was the introduction of an oxygen function (Carbonyl group) in the position 11 of the steroidal nucleus. With the combined chemical and microbial methods, the sequence of steps was reduced to only 12.
Amongst steroids, the compounds studied are hormones of androstane
Biit ocidt
and pregnane series, estrogens, bile acids, cardenolides and bufadienolides. Studies have also been carried out on the microbial
transformation of terpenes (Ciegler, 1969; Abbott and Cledhill, 1971),
carbohydrates (Vezinae et al., 1968) and alkaloids (lizuka and Naito,
1967; Vining, 1969). Besides these compounds the microbial transforma
tion studies were also reported on antibiotics (Sebek and Perlman, 1971;
Sebek, 1974), herbicides (Kaufman and Kearney, 1976) and pesticides
(Bollag, 1974) etc. In recent years, there are also some reports on the microbial utilization of naturally occurring lignins (Crawford
and Crawford, 1980), tannins and flavonoids (Barz and Hosel, 1975).
In the present chapter attempt will be made to describe briefly the
bio-degradation of aromatic compounds by microorganisms. However, a more detailed summary on the microbiological transformations of
flavonoids which is the main subject of this dissertation is also included in this chapter.
DEGRADATION OF AROMATIC CONPOUNDS
Studies related to the microbiological transformation of aromatic compounds have grown considerably during the last thirty years. Degradation of aromatic compounds is a fascinating subject of research for several reasons. It provides an excellent example of the part played by the microbes in maintaining the balance of chemical compounds available to living systems. Microbes also
degrade the aromatic compounds that are used as hormones, herbicides, pesticides and detergents, thus avoiding serious ecological changes. Interest in the microbial degradation of aromatic compounds centers around:
(1) Studies on the metabolic intermediates (Abbott and
Cledhill, 1971; Evans, 1969; Dagley, 1971; Chapman, 1972;
Sugumaran and Vaidyanathan, 1978) in the degradation of
different aromatic substrates;
(ii) Enzymatic mechanisms of hydroxylation and ring fission
(Hayaishi, 1964; 1968; Gibson, 1968);
(ill) Investigations into the control of enzymes talcing part in
the metabolism of aromatic compounds (Stanier and Ornston,
1973).
Bacteria (Evans, 1969) are quite versatile in the breakdown of aromatic compounds, but several yeasts and fungi can also degrade a limited range of benzenoid structures. Among the eubacteria, representatives of the families Coccaceae, Mycpbacteriaceae, Pseudo- monadaceae, Splrillaceae, Bacteriaceae and Bacillaceae are able to utilize a large number of benzenoid compounds. Some of the yeasts like Oospora, Candida, Debaromyces, Pichia and Saccharomyces can utilize simple phenols as sole carbon source. While higher fungi like Aspergillus, PeniciIlium and Neurospora can attack benzenoid compounds. A variety of soil and wood-rotting fungi degrade the aromatic polymer of plant origin like flavonoids, tannins and lignins,
In all these cases of aromatic ring metabolism, molecular oxygen is an obligatory oxidant. DEGRADATION UNDER AEROBIC CONDITIONS
Degradation of substituted or polynuclear aromatic confounds to dihydroxyphenolst
Microbes must manipulate a large variety of aromatic
compounds into either ortho- or para-di hydroxy derivatives (Gibson,
1968), before cleavage can occur to give aliphatic compounds which
are energy yielding reactions. The most common intermediates in the microbial degradation of many aromatic compounds are catechol (2), and protocatechuic acid (3) which are ortho-dihydroxyphenols or gentisic
acid (4), which is a para-dihydroxyphenol. Microbes must degrade/ transform various kinds of aromatic compounds to the above dihydroxy compounds or their derivatives before ring cleavage occurs. Some typical examples of manipulation of aromatic compounds to dihydroxy- phenol derivatives by microbes are given below.
(i) Aromatic acidic compounds;
Aromatic acids can either be metabolised directly to catechol (2) or undergo hydroxylations, followed by decarboxylation
COOH aOH I COOH OH 2 3 4 to form protocatechuic acid (3) (Fig. 1.1). Thus salicylic acid (5) is metabolised (Katagiri et al., 1965) by a Pseudomonas sp. to PatudomoncLG •OH GP OH COOH
\j -
COOH /r/eb/'-sej/a. a.eromenta OH OH ! 0H OH
COOH COOH
OH
i> Wo HO COOH COOH COOH_ COOH S S^p—CCCH HO HO
COOH OH ! y —— y OH 10 OH <«• 1.1. The degradative pathways of aromatic acids by bacteria (Buswell and Clark, 1976; Channa Heddy et al., 1976; Katagiri et ai., 1965; Reiner, 1971; Reiner and Hegeman, 1971; Ribbons and Evans, 1960; WhaUs et al., 1967). catechol (2) by oxidative decarboxylation* Similarly* benzoic acid
(6) is transformed (Heiner, 1971; Seiner and Hegeman, 1971) by
•tt-lcaligenes eutrophus to catechol (2) via 3,5-cyclohexadiene-1,2- diol-1-carboxylic acid (7). *hile a Pseudomonaa Sp. (Wheelis, et al 1967) metabolized benzoic acid (6) to protocatechuic acid (3) by sequential hydroxylation. Similarly phthalic acid (8) undergoes hydroxylation
(Ribbons and Evans. 1960) by a feeudomonas Sp. to 4,5-dihydroxyphtbalate
(9) which is then decarboxylated to protocatechuic acid (3). However, a strain of Bacillus transformed p-hydroxybenzoic acid (10) to gentisic acid (4) by an unusual pathway in which the migration of carboxylic acid group to ortho position due to the hydr0xy8*tioa
(NIH shift) seems to have occurred (Buswell and Clark, 1976) (Fig. 1.1).
Shile protocatechuic acid (3) is decarboxylated to catechol (2) by
Klebisella aerogenee (Channa Reddy et al., 1976).
(ii) Polynuclear aromatic compoundst
Polynuclear aromatic compounds are first transformed to o-dihydroxy compounds which are further degraded finally to aliphatic compounds via catechol (Pig. 1.2). Thus benzene (11) was metabolised
(Gibson, 1968) by a Pseudomonas Sp. to 3»5-cyclohexadien-1,2-cis-diol
(12) through a cyclic peroxide intermediate. The intermediate (12) is then transformed to catechol (2) by enzymatic dehydrogenation. In a similar patheay, naphthalene (13) is first transformed (Jerina et al.,
1971) by a soil Pseudomonas Sp. to cis-1,2-dihydroxy-1»2-dihydro- naphthalene (14) which on dehydrogenation gave 1,2-dihydrOxynaphthalene
(15). The latter compound then undergoes ring cleavage (Davies and
Evans, 1964) by the disruption of bond between the angular carbon and JH 0 I c$ M-OH 0=~ U II
13
18 */• + /r ft CH5COC0OH IS
Vh %+ 25 Fig. 1.2. The pathways of degradation of benzene and other polycyclic aromatic compounds by soil Paeudomonas sp. (Daries and Evans, 1964; Evans et al., 1965; Gibson et al., 1968; Jerina et al., 1971). carbon-1 of the naphthalene nucleus to yield cis-o-hydroxybenzal- pyruvic acid (16) (anion form). The compound (16) is then cleaved to salicylaldehyde (18) and pyruvic acid (19) via i -hydroxy- r-o- hydroxyphenyl-oC-oxobutyric acid (17). Salicylaldehyde is then oxidatively decarboxylated to catechol (2) which is degraded further
(Fig. 1.2). In the same way anthracene (20) and phenanthrene (23J are transformed (Evans et al., 1965) by a Pseudomonas Sp. to 1,2- dihydroxy compounds (22) and (25) via 1,2-di hydro-1,2-di hydroxy compounds (21) and (24) respectively. The 1,2-dihydroxy confounds
(22) and (25) are degraded to catechol (2) by a similar pathway as described for the degradation of naphthalene (Fig. 1.2).
(iii) Alkoxy aromatic compounds*
Alkoxy aromatic compounds are dealkylated to give the parent phenol with the concomitant liberation of alkyl moiety as an aldehyde. Thus 4-methoxybenzoic acid (26) (Henderson, 1957), vanillic acid (28) (Cartwright and Smith, 1967; Henderson, 1961), the herbicide, 2,4-dichlorophenoxyacetic acid (29) (Tiedje and
Alexander, 1969; Evans et al., 1971a) and 4-chloro-2-methylphenoxy- acetic acid (31) (Gaunt and Evans, 1971) are transformed to
4-hydroxybenzoic acid (27), protocatechuic acid (3), 2,4-dichloro- phenol (30) and 5-chloro-o-cresol (32) respectively by soil bacteria
(Fig. 1.3). The phenols are further transformed to o-dihydroxy compounds which are degraded further to aliphatic compounds. OCH
OH
DM
HCMO
OH O-CHCOOM
GCHjCOOH
Fig. 1.3. The degradative pathways of metabolism of alkoxyaromatic compounds by soil bacteria (Cartwright and Smith, 1967; Evans, 1971; Henderson, 1957; 1961; Tiedje and Alexander, 1969). (iv) Alkyl aromatic compounds:
Alkyi aromatic compounds are either hydroxylated to catechol systems or undergo successive oxidation of alkyl group to give substituted benzoic acid which is degraded further either through catechol or protocatechuic pathway. Thus, o- and m- cresols (33, 35) are metabolised by Pseudomonas sp. to 3-methylcatechols (34). While p-cresol (36) is metabolised by the same bacteria to 4-methyl catechol
(37). These methylcatechoIs are degraded further (Fig. 1.4) (Ribbons,
1966; Bayly et al., 1966). While toluene (38), m- and p-xylenes (42,
44) degraded (Worsey and Williams, 1977) by Pseudomonas putida MT20 to benzoic acid (41), m-toluic acid (45) and p-toltdc acid (49) respectively by hydroxylation of methyl to hydroxymethyl group
(compounds 39, 43, 47), followed by successive oxidation via aldehydes
(compounds 40, 44, 48). The resulting benzoic acids are further trans formed to catechol (2), 3-raethyl- and 4-methyl catechols (34, 37) respectively which are degraded further. However, p-toluic acid (49) is transformed (Golovleva et al., 1978) by a Pseudomonas sp. to p-hydroxybenzoic acid (10) and then to protocatechuic acid (3) which is degraded further (Fig. 1.4).
(v) Halogen substituted aromatic compounds?
Halogen group can remain in tact in the microbial degrada tion or can be replaced directly by hydroxyl group. Thus 4-chloro- phenol (50) and 2,4-dichlorophenol (30) are transformed (Bollag et al.,
1968b) by Arthrobarter sp. to 4-chloroe.atechol (51) and 3,5-dichloro- catechol (52) respectively. Catechols are degraded further by CH8
34
CHS 0:°:
COOH COOH COOH 49 10 3 Pig. 1.4. The proposed oathwayt of degradation cf alkyl-aronatic coroounds by soil bacteria (Bayly et al., 1966; Golovleva et al., 1978; Ribbons, 1966; ;«otsey and Williams, 1977). 13
o-cleavage pathway (Bollag et al., 1968a) with elimination of chlorine at the level of muconic acids. Similarly* herbicides
4-chlorophenoxyacetic acid (53) (CPA) (Evans et al., 1971b) 4-chloro-
2-methylphenoxyacetic acid (31) (MCPA) (Gaunt and Evans, 1971),
2,4-dichlorophenoxyacetic acid (29) (2,4-D) (Evans et al., 1971a) are transformed by Pseudomonas sp. to 4-chloro-2-hydroxyphenoxy- acetic acid (54), 5-chloro-o-cresol (32), 2,4-dlchlorophenol (30) respectively. These transformation products further undergo hydroxy- lation to yield 4-chloroeatechol (55), 5-chloro-3-raethylcatechol (56) and 3,5-dichlorocatechol (57) which are degraded further (Fig. 1.5).
While, 2-fluorobenzoic acid (58) is transformed (Goldman et al., 1967) by a Pseudomonas sp. to 3-fluorocatechol (59) or by defluorination to catechol (2) directly (Fig. 1.5).
(vi) Nitro substituted aromatic compoundst
Nitro group can be replaced by hydroxy groups directly or after reduction to an amino group (Fig. 1.6). Thus Arthrobacter simplex degraded (Tewfik and Evans, 1966) 3,5-dinitro-o-cresol (DNOC)
(60) to 3-raethyl-5-nitrocatechol (61) and finally to 2,3,5-trihydroxy- toluene (62) by direct replacement of nitro group by hydroxyl group.
While DNOC (60) is transformed (Tewfik and Evans, 1966) by a
Pseudomonas sp. first to 3-amino-5-nitro-o-cresol (63) which is further metabolised to 3-oethyl-5-nitrocatechol (61) by the reduction of the nitro group, followed by its replacement by a hydroxyl group. Nitro group in the compound (61) is reduced further to yield 3-methyl-5- aminocatechol (64). The later unstable compound (64) is then un Arothrobocter Sp
Arothrobacfer 9p H^v
Peeudomonos Sp
OCH, COOH
Pseudomonas Sp
0CH2 COOH Pseudomonas Sp CI
p^V000" Pseudomonos Sp
58 59
COOH Pseudomonos Sp
Fig. 1.5. The pathways of degradation of halogenated aromatic compounds by soil bacteria (Bollag et al., 1968 a; 1968b; Evans et al., 1971a; 1971b; Gaunt and Evans, 1971, Goldman et al., 1967). OH I $>mp>e V^" - » L. y
60
OH
66 67
The pathways of degradation of nitroaromatic compounds by Fig. 1.6. bacteria (Cain and Cartwright, 1960; Tevfik and Evans, 1° X
transformed to 2,3,5-trihydroxytoluene (62) which is degraded
further to aliphatic confounds (Fig. 1.6). Similarly, p-nitrobenzoic
acid (65) and o-nitrebenzoic acid (67) are transformed (Cain and
Cartwright, 1960) to p-aminobenzoic acid (66) and anthranilic acid
(68) respectively by a Nocardia sp. These amino compounds are
degraded further in one of the above pathways (Fig. 1.6).
(vii) Amino substituted aromatic compoundst
Amino group in the arylamines is replaced by a hydroxyl
group or oxidatively replaced to give directly catechol system (Fig.
1.7). Thus p-aminobenzoic acid (66) (Durham, 1956) and 3-amino-5- nitro-o-cresol (63) (Tewfik and Evans, 1966) are transformed by
Pseudomonas sp. to p-hydroxyben zoic acid (8) and 3-methyl-5-nitro- catechol (61) respectively. While aniline (69) is directly converted
(Walker and Harris, 1969) to catechol (2) without prior formation of phenol. Similarly, anthranilic acid (68) is oxidatively deaminated
(Tanuichi et al., 1964) to yield catechol (2) by a Pseudomonas fluorescens sp. (Fig. 1.7).
(viii) Arvlsulphonate compounds!
Sulphonic acid substituent is degraded by the removal of the sulphonic acid moiety as bisulfite or bisulfate and having in its place a hydrogen atom or hydroxyl group (Fig. 1.8). The resulting compound is further hydroxylated to yield catechols which are degraded further. Thus benzenesulphonic acid (70) and p-toluenesulphonic acid
(72) are degraded (Cain and Farr, 1968) by Pseudomonas sp. to catechol (2) Peeudomonae Sp
OH
Pteudomonoi Sp
N02
Pseudomonqt Sp
69
NH2 COOH Peeudomonoe 6 fluorescens 68 *
Kg, 1»7* ffo ttoqradailve pstkmr* of mt*bo2l9m of arylam(t
«"XE •f Mg S03 ?2 37
CHS CM« CHj cH3 OH OH OH
OH 0*H" OH 3-
S09M 50fH S03M H»SO» 72 ?3
Fig. 1.8. The pathways of degradation of arylsulphonic acids by Pseudomonas aeruginosa (Cain & Farr, 1968; Focht & Williams, 1970).
OH OH
OM
74
OH
OH *0" -"O7€> 75
Fig. 1.9. The metabolism of phenol and resorcinol by Trichosporon
cutaneum (Gaal and Neujahr, 1979). and 4-methylcatechol (37) respectively with release of sulphonate as
sulphite (71). While another Pseudomonas sp. transforms (Focht and
Williams, 1970) p-toluenesulphonic acid (72) to 3-methylcatechol (34)
with the release of sulphonate as sulfate (73).
(ix) Hydroxy aroroatlc compounds:
Hydroxyaromatic compounds (phenols) axe transformed to
catechol derivatives by hydroxylation, which are degraded further
(Fig. 1.9). Thus phenol (74) and resorcinol (75) are metabolised
(Gaal and Neujahr, 1979) by a yeast Triehospprum cutaneum to catechol
(2) and 1,2,4-tri hydroxy benzene (76) which are degraded further.
DEGRADATION OF DIHfDROXVPHENOLS TO ALIPHATIC COWOUNDS
Dihydroxy aromatic compounds are cleaved to aliphatic acids by the following pathways:
a) Ortho-cleavage or /?-ketoadipate pathway (1,2-di hydroxy compounds):
In ortho cleavage pathway (Omston and Stanier, 1966)
catechol (2) is oxidatively degraded by aerobic bacteria by the
fission of bond between carbon atoms bearing hydroxyl groups to produce cis, cis-muconic acid (77). The latter compound is lactonized
to muconolactone (78), which is then isomerized to /G-oxoadipic acid
eno1-lactone (1 -carboxymethyl- ^ -butenolide) (79). The compound
(79) undergoes lactone ring hydrolysis to form /i-oxoadipic acid (80)
which is cleaved after activation to yield acetyl coenzyme A and
succinic acid. In a similar fashion protocatechuic acid (3) is 3 2
\ I
COOH CCOOM t^^COOH 77 I I
COOH ^C=0 C=0 S2 78 >>COOH VCOi
679 - 79 I °
Fig. 1.10. The pathways for the degradation of catechol and proto- catechuic acid in Paeudomonas putida (Ornston and Stanier, 1966). cleaved first to /^-carboxy-cis, cisHcnuconic acid (81) which then undergoes lactonization to form Y-carboxymuconolactone (82). The latter compound is decarboxylated to the common intermediate /^-oxoadipic acid enol-lactone (79) which is further cleaved to succinic acid and acetyl coenzyme A as described for catechol (Fig. l.lo). b) Meta-cleavage or t^-muconlc semialdehyde pathway:
In meta cleavage catechol (2) is metabolized by the fission of the bond between the carbon atom carrying a hydroxyl group and its adjacent carbon that carries a hydrogen atom to yield highly reactive cC-hydroxymuconic semialdehyde (83), which is degraded further in two ways. The compound (83) in the first pathway undergoes hydrolysis
(Bayly and Dagley, 1969; Dagley and Gibson, 1965) to formic acid (84) and 2-ketopent-4-enoic acid (85). While in the second pathway
(Nishizuka et al., 1962$ Sala-Trepat and Evans, 1971; Catterall et al.,
1971) the compound (83) is oxidised to » -oxalocrotonic acid (88) which on further decarboxylation (Williams et al., 1971) yielded common intermediate 2-ketopent-4-enoic acid (85). The latter compound (85) undergoes hydration of double bond to yield 2-keto-4-hydroxyvaleric acid (86) which is finally degraded to acetaldehyde (87) and pyruvic acid (19)* In a similar way protocatechuic acid (3) is first degraded
(Dagley et al., 1960J Hegeman, 1967) to » -carboxy-oC-hydroxymuconic semialdehyde (89) which on hydrolytic elimination of formic acid (84) gave compound (90). The latter compound (90) is hydrated to V~-hydroxy-
1 -methyl-OC-ketoglutaric acid (91) which undergoes aldolase cleavage to yield pyruvic acid (19). However, the second pathway, degradation COOH
OH
OH }*• L c=o COOH <^OH 83 HCOOH 8l»
COOH COOH COOH OH HOCC ^90 HOOC T~°* U HOOC 88 85 *2 rkA-H^ D jr COCOO' H COOH i COOH COO1 H co_ HOOC z^-o Cx HO .1*HOOC 1 86 HOOC 91 93 I I I
CH« CHs CH5 CH? :OOH CH, t • CrO + CrO C-0 -\ C=0 c=o + c-o COOH COO H toOH CH» COOH COOH 87 19 1? 19 94 19
Fig. 1.11. The meta fission pathways of catechol and protocatechuic acid
by bacteria (Bayly and Dagley, 1969; Dagley et al., i960;
Dagley and Gibson, 1965; Hegeman, 1967; Nishizuka et al., 1962;
Sala-Trepat and Evans, 1971). of protocatechuic acid (3) is different to those reported for catechol degradation. In this pathway the intermediate oC-hydroxy-Y-carboxy- mueonic semi aldehyde (89) is transformed to a tricarboxylic acid (92) which is hydrated to provide hydroxy keto acid (93). The latter compound on aldolase cleavage gave oxaloacetic acid (94) and pyruvic acid (19). The characteristic feature of meta fission is the intense yellow colour of the culture broth in neutral and basic solution which disappears upon acidification. The general pathways of degrada tion of catechol and protocatechuic acid by met a fission pathway is shown in Fig. 1.11. c) Homogentislc and gentisic acid pathway (l»4-Dlhydroxy compounds)»
Homogentisic acid (95) is an intermediate in the bacterial metabolism of tyrosine, phenylalanine and phenylacetic acid. It is degraded (Chapman and Dagley, 1962) first to maleylacetoacetic acid (96) by the cleavage at the carbon-carbon bond between a hydroxyl and carboxymethyl side chain. The intermediate (96) is further degraded in two different pathways. In the first pathway maleylaceto- acetic acid (96) is isomer!zed to fumarylacetoacetic acid (97) which is further hydrolysed to yield acetoacetic acid (98) and fumaric acid (99). While in the second pathway the intermediate (96) without isomerization hydrolyzed to yield acetoacetic acid (98) and maleic acid (100).
The pathway of gentisic acid (101) degradation is analogous to that of homogentisic acid (95). Gentisic acid (101) is oxidatively cleaved by the fission of carbon carrying a hydroxyl group and its COOH
COOH
fJ*| COO H HoA^ 102 s^GSH U-HgO COOH COOH COOH s C=0 I ..COOH COOH ^COOH C 0 , COOH OOH COOH I HOOC^ CH3 89 99 Fig. 1.12. The pathways of degradation of homogentisic and gentisic acid by bacteria (Chapman and Dagley, 1962; Clark and Buswell, 1979; Crawford, 1975; Hopper et al., 1968; 1970; Lack, 1969; 1961; Tanaka et al., 1957). adjacent carbon that carries a carboxyl group to form maleylpyruvic acid (102). The latter compound can degrade by two different pathways. In the first pathway (Lack, 1969; 1961; Tanaka et al., 1957; Clark and Buswell, 1969) maleylpyruvic acid (102) is isomer!zed to furaaryl- pyruvic acid (103) which is hydrolytically cleaved to pyruvic acid (19) and fumaric acid (99). In the second pathway (Hopper et al., 1968; 1970; Cvawford, 1975; Clark and Buswell, 1979) maleylpyruvic acid (102) is hydrolyzed without isomerization to pyruvic acid (19) and maleic acid (100). The general pathway of degradation of homo- gentisic and gentisic acid by bacteria is shown in Fig. 1.12. DEGRADATION UNDER ANAEROBIC CONDITIONS Aromatic compounds could also be degraded by certain bacteria under anaerobic conditions (Evans, 1977). Thus Rhodo- pseudomonas palustris growing photosynthetically (Dutton and Evans, 1969; Guyer and Hegeman, 1969) with benzoic acid (6) reduced it to cyclohex-1-ene carboxylic acid (105) probably via cyclohexanecarboxylic acid (104). The intermediate (105) is further hydrated to 2-hydroxy- cyclohexanecarboxylic acid (106) which on dehydrogenation gave 2-oxDcyclohexanecarboxylic acid (107) and finally on ring cleavage to pimelic acid (108) (Fig. 1.13). However, Moraxella sp. degraded (Williams and Evans, 1975) benzoic acid anaerobically using nitrate or nitrite as electron acceptor to adipic acid (110) instead of pimelic acid (108). Authors proposed that adipic acid (HO) is formed from cyclohexanone (109) which in turn is formed from the intermediate 2-oxocyclohexanecarboxylic acid (107) by decarboxylation (Fig. 1.13). COOH COOH COOH COOH COOH OH COOH {^^&° C COOH. ^METABOLIC 109 no Fig. 1J3. The proposed pathways for the anaerobic metabolism of benzoic acid by Rhodopseudomonas palustris (Dalton and Evans, 1969; Guyer and Hageman, 1969) and by Mpraxella sp. (Williams and Evans, 1975). RtOv OH w 'rO- OH ill , R ' RuHnos* (f 4-, Rj « Rhamnoglucos* 112, R » Rhamnos* f15 / R|» H 113, R • H H . CHS — 0 ) V H.OH ^ H\ •/ OH OH 119 MICROBIAL TRANSFORMATIONS OF FLAVONOIDS Microbial utilization studies have been carried out on a few flavonoid compounds during the last decade or so. The confounds extensively cited in these studies are rutin (ill), quercetrin (112), quercetin (113), naringin (114) and naringenin (115). Recently studies on the microbial transformation of flavonoids have been intensified due to the isolation of compounds belonging to iso- flavonoid group from the diseased plants. These compounds which are called "phyto alexins" may possess antibiotic properties whose behaviour on plant pathogens is currently attracting active attention of research workers. Microbial degradation of flavonoids is generally carried out by molds, yeasts and bacteria. Studies on the utilization of flavonoids with these organisms are described below. Bio-degradation by moldsi Hattori and Noguchi (1958, 1959) were the first to report the results of microbial transformation of a flavonoid and showed that the organism Pullularia fermentans var Candida degraded rutin (111) to protocatechuic acid (3), phloroglucinol (116) and a depside (117) (Fig. 1.14). During the same period Sakamoto (1960) reported (Fig. 1.15) that Penicillium brevi-compactum metabolized rutin (ill) and quercetin (113) to protocatechuic acid (3) and phloroglucinol- carboxylic acid (118). They further observed that Penicillium link completely degraded rutin only in the presence of added glucose. Wakimoto et al (i960) also reported the degradation of rutin and quercetin by Pirjculaija oryzae to protocatechuic acid (3) and OM OH ORu'linos© Fig. 1»14. The pathways of degradation of rutin by Pullularla fencentans (Hattori and Noguchi, 1958j 1959). CO OH COOH HO -OH y> \ >>- OH tf OH OH IIS 113, R = H 111, n - Ru1i?i©$e Fig. 1.15. The degradative pathways of metabolism of quercetin and rutin ^ PeniciIlium bravicoeft actum (Sakamoto, 1S59? I960). 'J phloroglucinol (116). They further observed that the fungi Cochliobolus miyabeanus degraded rutin and quercetin completely. While Tokodi (1960) reported the degradation of quercetin by Streptomyces riroosus to hydroxybenzoic acid and dihydroxyphenylacetic acid. However, from these earlier studies it is not possible to give a stepwise mechanism for the degradation of rutin and quercetin by microbes. Simpson et al carried out a systematic study on the microbial degradation of rutin (111) and quercetin (113). These authors (Westtake et al., 1959; Westlake and Simpson, 1961) screened a number of molds, yeasts and bacteria and found that several of Aspergillus sp. (Aspergillus flavus PRL72, A.flavus PRL 1720 and A.niger PRL 18) were able to utilize rutin to the maximum extent. They have further shown (Westlake et al., 1959$ Simpson et al., 1960) that rutin was degraded by the extracellular enzyme systems of the Aspergillus sp. to rutinose (6-o-oC-rhamnosyl-D- glucose) (119), phloroglucinolcarboxylic acid (118), protocatechuic acid (3) and the depside (117). The depside (117) was the first stable intermediate to be identified. These products accounted for 26 carbon atoms out of 27 carbon atoms of the rutin molecule. These authors (Simpson et al., I960) on further manometric and radioactive studies showed that the missing carbon was released as carbon monoxide from C-3 position of the rutin. From the enzymatic studies they were able to give two probable mechanisms of degradation of rutin (Fig. 1.16). In the first mechanism the initial step was the removal of rutinose from rutin by a y^-glucosidase (rutinase) yielding quercetin. The latter compound further attacked by an enzyme 'quercetinase' between carbon-2 and carbon-3 to give unstable intermediate from which carbon RUTIN -£5-OH -OH OH + --jtl'oKi 113 4?3 0'; ! quarcet'nase' >H V&- + C0 + RufJnote 119 Fig. 1.16. The probable pathways of degradation of rutin by Aspergillus, «p. (Sinpson et al.» I960). 21 monoxide was released to yield the depside phloroglucinol carboxylic acid (117). Finally the depside (117) was hydrolyzed by an 'esterase* to give phloroglucinol carboxylic acid and protocatechuic acid. In the second mechanism, rutin was cleaved between carbon 2 and 3 to give compound (120) which was further degraded to depside (117), carbon monoxide and rutinose (119). The depside (117) was cleaved to the acids (118) and (3) as above (Fig. 1.16). From their further work (Westlake and Simpson, 1961; Simpson et al., 1963; Hay et al., 1961) on isolated adaptive enzymes 'rutinase', 'quercetinase' and 'esterase* these authors were able to come to the following conclusions. These adaptive enzywes could be isolated from Aspergillus sp. grown on both flavonoid glycosides or aglycones. The adaptive enzyme 'rutinase' was not specific and was able to hydrolyse a number of flavonoid glycosides to their corresponding aglycones. Enzyme 'quercetinase' (Simpson et al., 1963; Krishnamurthy and Simpson, 1970; Oka and Simpson, 1971; 1972; Oka et al., 1971; 1972) was shown to be a dioxygenase in nature and was able to degrade a number of flavonols. The last isolated enzyme 'esterase' (Oka and Simpson, 1971; Child et al., 1963; 1971) was able to attack only on depside linkages, but not the aliphatic esters. Westlake et al (1961) from their further work on the micro bial utilization of a number of flavonoids by Aspergillus flavus PRL 1805 and Fusarium sp. PRL 30 have come to the following conclusions. Utilization of naringin (114), apigenin (121) and taxifolin (125) by molds suggested that the presence of double bond between C-2 and C-3 F»3 R4 V HO s-0 v -V V OH rrJ kj r^•RC2 1 0 Ri (3.1, fig • R3 » R4 * H. Rt » OH R H 125, R, » R2. R3 . 0H i27, COOH 129,R*QIUCOM 128 and/or hydroxyl at C-3 was not the only criterion for biodegradation. Thus 3-hydroxyflavanoids having unsaturation between carbon-2 and carbon-3 (flavonols) viz. quercetin (113), kaeropferol (122), fisetin (123) and raorin (124) and other 3-hydroxyflavonoids having saturation at carbon-2 and carbon-3 (flavonols) viz. taxifolin (125) and fustin (126) were utilized by the molds. In these transformations, one of the products was carbon monoxide. Further, the compounds like apigenin (121) and hesperetin (127) without hydroxyl group at C-3 position were also degraded by the mold. However, absence of the product, carbon monoxide in these transformations further suggested that hydroxyl grouping at C-3 position was responsible for the release of carbon monoxide, it was also found that the position of functional group such as methoxyl group interferes in the degradation. However, catechin (128) where the position C-4 is occupied by a methylene, was not at all degraded, thus suggesting that an active center at carbon-4 is necessary for the microbial attack. There are some reports on the microbial hydrolysis of flavonoid glycosides. Thus quercetrin (112) was hydrolysed (Westlake et al., 1961) by a number of molds like Alternaria. Fusarium, PeniciIlium, Pullularia and bacteria Pseudomonas. Corvnebacterium to the corresponding aglycone quercetin (113) which was further degraded in the usual manner. Hydrolysis of naringin (114) to naringenin (115) by an isolated enzyme system 'naringenase* was reported by a number of authors (Bram and Solomon, 1965; Iizuka et al., 1964; Hara and Koaze, 1965; Fukumoto and Okada, 1972; Bai et al., 1978). Stepwise hydrolysis of flavonoid glycoside has been reported by Ciegler et al 23 (1971). These authors showed that naringin (114) was hydrolyzed first to prunin (naringenin-7-glucoside) (129) and then to the aglycone, naringenin (115). The organisms which brought about these reactions were Penicillium charlesli NRRL 1887, P.nigricans NRRL 91% Helminthosporium sativum NRRL 3356 and Cephalothecium roseum NRRL 1665, Aspergillus flavus NRRL 3357, Wojnowlcla grand nis NRRL 2472. When the incubation period was more than four days these organisms degraded naringin (114) rapidly to water soluble products which were not identified. However, naringenin (115) the aglycone itself was not utilized by these organisms which might be due to its insolubility in the medium. Barz (1971) reported that Fusarium oxyporum was able to degrade kaempferol (122) and quercetin (113) in a similar manner that was reported for Aspergillus sp. Thus hesperetin (127) was degraded by Fusarium oxysporum to isovanillic acid (130). While Padron et al (1960) reported that extracellular enzyme system from Aspergillus sp. was able to degrade only flavonol amongst other flavonoids. Recently Baltabaeve and Kanaeva (1977) have reported the degradation of rutin by Aspergillus oryzae to quercetin (113) and protocatechuic acid (3). Decomposition of flavonoids by molds is reported by a number of authors. Pickard and Westlake (1969, 1970) have reported that Polyporua versicolor PRL 572 decomposes rutin (ill) to dark brown pigments, but without the evolution of carbon monoxide* However* these authors were able to isolate an extracellular enzyme 'polyphenol 24 oxidase' (laccase) from the same organism, which attacked both dihydroflavonols and flavonols. The enzyme 'laccase' required phenyl group at carbon-2 and a hydroxyl group at carbon-3. Lewis and Papavizas (1969) also reported the decomposition of rutin (111), quexcetin (113), phloroglucinol (116), protocatechuic acid (3) by a strain of Fusarium to water soluble products. Cserjesi (1969) reported the decomposition of dihydroquercetin (Taxifolin) (125) by white-rot and soft-rot fungi to water soluble products. The author suggested that the presence of 'polyphenol oxidase' in the white-rot fungi was responsible for the degradation of the substitute. However, dihydroquercetin was not degraded by the brown-rot fungi suggesting that the degradation products might be more toxic than dihydroquercetin itself. There are also some reports on the microbial degradation of flavonoids in which the product formed were different than those reported for the degradation of rutin by Aspergillus sp. Thus, Armand-Fraysse and Lebreton (1969) reported that a wood destroying fungus Coniophora puteana transformed rutin (111) to isoquercitrin (Glucosyl-3-quercetin) (131), quercetin (113), 4'-ribosylquercetin (132) and 4«,7-diribosylquercetin (133). These results (Fig. 1.17) suggested a novel glycosidic capability of the organism. Barz (1969) has also reported an isolated organism 'Cicer M}' which degraded rutin (111) and quercetin (113) to water soluble products which were different from those reported for Aspergillus sp. Haluk and Metche (1970) have found that a strain of Aspergillus niqer degraded quercetin to phenolic acids and identified a new metabolite 7,3'-dimethylquercetin (134) from the culture broth. While Kunaeva and coworkers (Kunaeva, 1970; Kunaeva and Klyshev, 1971) reported that the molds Aspergillus orvzae and A.awarl growing on rutin, degraded the later compound (111) to expected products viz. quercetin (113), rhamnose (135) and proto- catechuic acid (3). However, from the culture filtrates two new products viz. cinnamic acid (136) and benzoic acid (6) could also be isolated. H H CH.CHCOOH HON IS4 135 186 The first report of hydroxylation of a flavonoid was by Jeffrey et al (1972b) who observed that taxifolin (125) was hydro xy- lated at carbon-8 position by Pseudomonas sp. NCIB 9940 to give dihydrogossypetin (137). The same authors isolated 'taxifolin hydroxylase' enzyme from this organism grown on catechln, which hydroxylated taxifolin to dihydrogossypetin (137). Later Jeffrey et al (1972a) reported the degradation of the dihydrogossypetin (137) by the cell-free extracts from Pseudomonas sp. and obtained oxaloacetic acid (138) and 5-(3,4-dihydroxyphenyl) 4-hydroxy-3-oxovalero-0-lactone (139). From the tracer experiments they have further established that by cleaving ring A, oxaloacetic acid was formed from C-5, C-6, OH OH oRiaosu \3Z 133 Fig. 1.17. The pathways of metabolism of rutin by Coniophora puteana (Aimand- Fraysse and Lebreton, 1969). HO Fig. 1.18. The pathways of degradation of toxifolin by enzyme systems from Pseudomonas sp. (Jeffrey et al., 1972b). C-7 and C-8 unit. This is the first report where ring A in such a system is degraded by the microbes (Fig. 1.18). Recently there is another interesting report on the microbial degradation of an 8-lsopropenyl substituted flavanone glucoside by Sakai (1977). He reported that phellamurin (140) (3,4',5,7-tetra- hydroxy-8-isoprenylflavanone-7-o-glucoside) on Incubation with Aspergillus nlger gave a number of neutral and acidic transformation products (140 - 147, 116, 118 & 10). From a study on the formation of various products as well as from the 'resting culture* experiments in which intermediates are used as substrates, the authors proposed the following mechanism for the formation of various products from phellamurin by A.njger. First, the substrate (140) is attacked by a '/"-glucosidase' enzyme of A.njger to give neophellanuretin (3,4',5,7- tetrahydroxy-8-isopentylflavanone) (141). Further by the addition of two molecules of water to neophellamuretin (141), the formation of an unidentified intermediate to yield 3,4,,5,7-tetrahydroxy-8-j?Q,'f - dihydroxyisovaleryl) flavanone (142) is implied. Compound (142) on simultaneous dehydration and ring formation involving the side chain gave 5M-hydroxyisopropyl-4H,5"-dihydrofurano^~2",3,,-hj7-3,4' ,5-tri- hydroxyflavanone (143). The latter compound is further isomer!zed to the corresponding chalcone viz. 5"-hydroxyisopropyl-4",5,,-dihydro- furano /"2",3«^d_7-2,,4,6,,dC-tetrahydroxychalcone (144) through fission of the heterocyclic ring. Chalcone (144) is degraded further by the cleavage of the C-C bond between carbonyl and carbon at the £-position to p-hydroxymandelic acid (146) (derived from ring B) and compound (145) (derived from ring A), p-Hydroxymandelic acid (146) OH 0 140 R = Giucose HjCN /CHj H3C CH3 CO OH C-OH C-OH I CH2 H-6— CH2 HC—CHo f 0 J^ .0. ^r ' >r-Or H H OH ^N >-COOH OH r OH 0 OH \+3 ! i r OH OHO OH HOOC- OH k 'I HOOC-C ;M 6H OH OH 14-7 10 11 ;-i 116 14-6 Fig. 1J9. The probable pathways for the degradation of phellamwrin by Aspergillus niger (Sakai, 1977). 27 is further degraded to p -hydroxy ben zoic acid (10) via p-hydroxy- benzaldehyde (147) by an enzyme of mandelate pathway (Hegeman, 1966). While side chain of compound (145) is cleaved to yield phloroglucinol carboxylic acid (118) which is subsequently decarboxylated to form phloroglucinol (116). The probable pathways of metabolism of pnellaraurin (140) by Aspergillus niqer is shown in Fig. 1.19. Another recent report on the degradation of A ring of flavanoid is by Schultz et al (1974) who isolated Pseudomonas putida. growing on quercetin as a sole carbon source. They have been able to show that the bacteria P.putida is able to degrade quercetin (113) to protocatechuic acid (3) through a series of intermediates (Fig. 1.20). Based on further work with the "cell free" enzyme system they have been able to propose a degradative pathway for the utilization of quercetin. Quercetin is first hydroxylated at carbon-8 by a mixed function oxygenase called '7-hydroxyflavone-8-hydroxylase' that required NADH to yield gossypetin (148). As gossypetin can undergo autooxidation and is also difficult to purify, authors are able to show 8-hydroxylation properties of this enzyme system on the simpler substrates like chrysin (153) and 7-hydroxyflavone (156) which contained hydroxyl group at carbon-7. These substrates on incubation with '7-hydroxyflavone-8- hydroxylase' enzyme from P.putida yielded norwogonin (154) and 7,8- dihydroxyflavone (157) respectively (Fig. 1.21). Further this enzyme is shown to have the properties of a typical flavoprotein and can act only on flavones or flavanones with 7-hydroxyl group. Thus the enzyme is unable to degrade 7-methylflavone (158), 7-raethoxyl compound viz., techtochrysin (159) and 3-hydroxyflavone (160) in which 7-hydroxvl OH H HO OH %JP ° + o OH NADH NAD OH 0 H3 oo^ HO /CCCK H0 c for. TV °v° 149 »° ^£« + 0H T > fY 0 V: H20 H ° >^* HOOC-C-CH2COOH Q OH H20 9 + A5Z 151 Fig. 1.20. The proposed pathway of degradation of c^ercetin by the enzyme systems fmmPseudomonas putida (Schultz et al., 1974). 154 UOt HOvjcft -0, J3fi CflrO 0 156 The metabolism of chrysin and 7-hydroxyflavone by the enzyme system of Pseudomonas putida (Schultz et al., 1974) 28 group is absent. Enzyme is unable to hydroxylate 8-hydroxyflavones to yield inverse hydroxylated product at 7-position. It is surprising that the enzyme is incapable of hydroxylation of a 7-hydroxy confound viz. rutin (ill). The author attributes the presence of a bulky group at carbon-3 for difference in behaviour of the enzyme. Furthermore, the enzyme is shown to be substrate specific as it is incapable of utilizing 2,,4,,4-trihydroxychalcone (161) and the isoflavone, Biochanin A (162). H!C HO. -// y--OCH, OH 0 The second step in the pathway is the oxidative fission of the aromatic ring occurring between C-8 and the flavonoid C ring by 7,8-dihydroxyflavone dioxygenase. The cleavage is of extradiol type and not a intradiol cleavage which is confirmed from the following ^y considerations. It is knovm that if 5-hydroxyl group is present, extradiol cleavage will yield 2-pyrone due to rearrangement. While intradiol cleavage between carbon-7 and carbon-6 of flavone ring could give 4-pyrone derivative. Formation of 2-pyrone derivative confirmed from the study on the product formed from the norwogenin (154) by the second enzyme system. From the detailed mass spectral fragmentation studies of the product as well as its deuterated product, it is shown that the degradation product of norwogenin is 3-(l,3- diketo-4-carboxybutyl)-4-hydroxy-6-phenyl-2-pyrone (or a keto-enol tautomer of this compound) (155) thus substantiating an extradiol cleavage (Fig. 1.21). Enzyme can act only if the substrate contain a hydroxyl at carbon-3 position and having double bond between C-2 and C-3 of the substrate. Thus taxifolin (125) which is 3-hydroxy- flavanone derivative is not a substrate for this enzyme. Further the formation of diol in the C ring at C-3 and C-4 is also dependent upon the isomer!zation of the product of the ring cleavage to a 2-pyrone. This process Can occur only if there is a hydro xyl at C-5. Thus fisetin (123) which has C-3 hydroxyl group but lacks a C-5 hydroxyl group is not a substrate for this enzyme. However, morin (124) which contained a 5-hydroxyl group could not be degraded which probably may be due to steric considerations viz. the presence of 2'-hydroxyl group. The intermediate, 2-pyrone-4,5-diol (150) formed in the quercetin degradation by P.putida enzyme system, is further cleaved between C-5 and C-6 by yet another dioxygenase. However, cleavage can occur between the two hydroxyls or to either side of the two hydroxyls. Of these, cleavage between C-5 and C-6 is favoured resulting in the formation of product (151). The latter compound on hydrolysis yielded unsaturated aliphatic compound (152) and proto- catechuic acid (3). The former compound is further hydrolysed to give two molecules of oxaloacetic acid (94) which is probably degraded further to simpler aliphatic acids by the known pathways and thus resulting in the accumulation of protocatechuic acid (3) in the medium. Proposed pathway of degradation of quercetin (113) by the enzyme system of Pseudomonas putida is shown in Fig. 1.20. From a similar study authors have also shown the degradation of kaempferol (122) to p-hydroxybenzoic acid (10) by the above enzyme systems. There are some reports on the microbial utilization of isoflavones, dihydrochalcones and catechins by molds, and bacteria which are described below. Barz and coworkers (Barz, 1970; Barz et al., 1970) studied the bio-degradation of isoflavones and flavones and have shown that an unidentified bacterium ('Cicer My) isolated from the roots of deer arietinum degraded formononetin (163) and daidzein (164) to carbon dioxide and water soluble products. While isoflavones having substi- tuents in 2,2'- and 8 positions did not degrade. Barz claimed that the bacterium 'Cicer M' was the first example of a microorganism to degrade both isoflavonolds and flavonoids. Batkai et al (1973) were the first to report the demethylation of isoflavonolds by microorganisms. During their studies they demethylated methoxyisoflavones (165) and (167) to (166) and (168) respectively by using Penicillium cycloplum Westling 1911 (Fig. 1.22). Other examples of microbial degradation of isoflavonolds are described separately in the end of this chapter under 'phytoalexins'. 0 163 164 -OCH, OCM: 166 • CC °VCH3 OCHj OCH3 0 67 166 Fig. 1.22. The demethylation of methoxyisoflavone by FeniciIlium cyclopium Westling 1911 (Batkai et al., 1973). The dittydrochalcone, phloridzin (169) a constituent of plant belonging to Mai us species had attracted attention because of its implication in a "soil sickness** associated with old orchard sites. Jayasankar et al (1969) found that various organisms were able to degrade phloridzin to phloretin (170), phloretitt acid (171) phloroglucinol (116), and p-hydroxybenzoic acid (10). These authors have reported that Aspergillus njger degraded phloridzin (169) to phloroglucinol only in growing culture fluids, while A.njger 814 and Its cell free enzyme preparation (phloretin hydrolase) when used were able to degrade phloretin to phloretic acid and phloroglucinol. The authors have also shown that phloretic acid in the presence of Aspergillus nlger degraded to p-hydroxybenzoic acid which was further metabolised. The overall sequence of the fungal degradation of phloridzin is shown in Fig. 1.23. Chatterjee and Gibbins (1969) also reported that the yellow bacteria Erwinia herblcola Y46 isolated from diseased apple and pear trees cleaved phloridzin (169) to phloretin (170) which was further hydrolytically cleaved to phloro glucinol (116) and phloretic acid (171). Some of the strains of E.herblcola possessed ^-glucosidase activity which liberated glucose from phloridzin. These authors were able to isolate enzyme 'phloretin hydrolase' from the cells of E.herblcola Y46 which degraded phloretin to phloroglucinol and phloretic acid. The catechins having the general structure (128, 172 - 174) that are present in the tea leaves have been studied by Vartapetyan and Bogdanova (1963, 1964) using soil organism Penlcilllum expansum. They inferred the following sequence of transformation. The complex HO. OH OH y -o- 7f OR 0 169, H ' Glucoit HO-v<^N^OH A OH + Qlucost vOH v0 170 COOH H0> OH ~r HO-H/ CH2-CH2~-COOH *» "*• xz: OH 171 11G Fig. 1.23. The pathways of microbial degradation of phlonidzin, phloretin and phloretic acid by Aspergillus niger (Jayasankar et al., 1969). catechins were first degraded by hydrolytic splitting of the ester bond of the complex resulting in a decrease in the galloylated catechins and gallic acid, d,1,-Catechin and its gallate disappeared first from the nutrient medium. The degraded simple catechins and gallic acid underwent a gradual and complete oxidative degradation to give acidic compounds. Farr and Evans (1972) observed that (+) catechin (128) was aerobically metabolised to taxifolin by an organism isolated from rat faeces. Latter compound was further metabolised to protocatechuic acid (3). From the tracer experiments it was shown that protocatechcic acid arose from the B ring of the catechin. 128, R = H 173, R = H 174, R = GdMoyi 172, R = GaMoy1 33 Bio-degradation by yeasts: Westlake and Spencer (1966) were able to show that the cell free extracts of Pullularia pullulans, Cryptococcus albidus and Cryptococcus diffluens, degraded rutin in the same pathway as that reported for the mold Aspergillus sp. However, in this experiment they were unable to detect rutinose (119), quercetin (113) and depside (117), thus suggesting the presence of a very active enzyme system in these yeasts. Spencer and Gorin (1971) have shown that the yeasts species belonging to Trichosporo" cutaneum, Cryptococcus albidus, C.diffluens, C.terreus, C.laurentii, Rhodotorula glutinis, R.minuta, R.rubra and Candida humicola isolated from the citrus orchards, soils and citrus waste disposal areas, utilized flavonoids such as rutin (111), quercetin (113), naringin (114), naringenin (115) and hesperidin (175). Amongst flavonoid substrates, glycosides were used better than aglycones by these yeasts. OH 0CH3 XCHO 0 ^ l75. R, Eutinoaide Bio-degradation under anaerobic conditions* Simpson et al (1969) were the first to report that rutin (111), quercetin (l13)» hesperidin (l75)t naringin (114), quercetrin (112) were degraded by the microflora of the bovine rumen fluid anaerobically to water soluble products. Hilorogltteinol (116) was detected as a transitory intermediate in the fsi-mentation of rutin and quercetin* These authors (Chang et *l.t 1969; Simpson et al*f 1969; Krishnamurthy et al*. 1970) latter reported that one of the bacteria strain Butyrivibrio sp* C isolated from bovine fluid was able to utilise rutin (ill) and degraded it to phloroglucinol (116), 3.4-dihydroxybenzaldehyde (176), 3,4-dihydroxyphenylacetic acid (1T7), carbon dioxide and 3,4-dihydroxyphenylacetic ethyl ester (178)* They suspected that the compound (178) might be an artefact resulting from the condensation of 3»4-dihydroxyphenylacetic acid (177) and ethanol during the fermentation or extraction with ethyl acetate under acidic conditions* They have also shown that the intermediates phloroglucinol (116) and 3»4-dihydroxyphenylacetic acid (177) were not further metabolised by the organism even in the presence of glucose* It was presumed that 3»4-dihydroxyben«aldehyde (176) must have arisen directly from the postulated intermediate rather than the /decarboxylation of dihydroxyphenylacetic acid* The postulated pathway for anaerobic degradation of rutin by Butvrivibrio sp* C^ is shown in Pig. 1*24* Similarly queroitrin was degraded by the same bacteria to the corresponding water soluble products* However* Butvrivibrio sp* C was unable to degrade quercetin, even in the presence of free glucose, rhamnose, or rutinose in the media* They GIUCOM + Rhamnot. CHO H ZC - COOR I HO OH + C02 + ->S. QH S^-^O H OH OM 1** OH R • H 116 R • C2H9 Fig. 1.24. The probable mechanism for the anaerobic degradation of rutin by Butyrivibrio sp. C3 (Cheng et al., 1969; Krishnamurty et al., 1970; Simpson et al., 1969). explained that this might be due to the insolubility of aglycone and thus non-utilization of quercetin by the bacteria. Further, same authors (Cheng et al., 1971) reported the degradation of naringin (114) by Butyrivibrio sp. C3 anaerobically to naringenin (115) and neohesperidose (2-o-oC-L-rhamnosyl-Drglucose) (179). The former was further cleaved reductively to phloroglucinol (116) and phloretic acid (171). However, phloretin (170) and free monosaccharides were not found in the naringin (114) fermentation liquor. Postulated pathway of anaerobic degradation of naringin (114) by Butyri vibrio sp. C3 is given in Fig. 1.25. Smith and Griffiths (1970) reported OH HO O'H O 180, RsRHAMNOSE . 182 0CH3 IftS 184 RO OH 114, R*NEOHESPERlDOSE J-—-0 H/H >HOH HO\OH H //~\ V -OH H H ,?9 k ! I ~:.:' 7 HO/f° ° /CH n 3 OVA o H 115 i 1/ H OH OH OH 7 HO N> "V > OH \— + H20 1OH V f?0 I HO OH HX OH ; OH HO~C=^ o° 171 tit g. 1.25. The mechanism for the anaerobic degradation of naringin Butyrivibrio sp. C„ (Cheng et al., 1971). 38 that a mixed inoculum of microorganism;obtained by the sterile section of rat caecum when incubated with myricitrin (180) in a thio glycol late medium under anaerobic condition, degraded it to myricttin (181) which was further degraded to 3,5-dihydroxyphenylacetic acid (182) and 3,4,5-trihydroxyphenylacetic acid (183). Later, Griffiths and Smith (1972) reported that two more substrates delphinidin (184) and tricin (185) were also degraded by the intestinal microbes of rat. The products obtained in all these experiments ware the same as those obtained in urine following the oral administration of respective substrates suggesting that the microbes in the intestine are respon sible for the degradation of flavonoids. Metabolism of phytoalexins by microbes: Phytoalexins are antibiotic chemicals synthesized by the plants which are invaded by incompatible plant pathogens like fungus, virus or bacteria (Greisebach and Ebel, 1978). These belong to the class of isoflavonoids, terpenoids or hydrocarbons. Of these, iso- flavonoids (Van Etten and Pueppke, 1976) are the most commonly produced phytoalexins. As these compounds are antibacterial (Gnanamanickam and Patil, 1977; Mitscher et al., 1978; Ravise and Chopin, 1978; Wyman and Van Etten, 1978), antifungal (Vibhute and Wadje, 1976) and antiviral (Wacker and Eilmes, 1978) investigations on phytoalexins have featured prominently in recent years. However, it was revealed (Van Den Heuvel and Van Etten, 1973; Van Etten and Stein, 1978; Bailey and Burden, 1973) that some of the phytoalexins are in fact the transformation products which are formed by metabolism 37 of parent compounds to yield less toxic compounds by a mechanism which is known as 'detoxification mechanism' by the plant pathogens. These transformation products had less antifungal activity and are formed by the modification of parent compounds by hydroxylation, methylation, demethylation and even ring fission of the molecules (Ingham, 1976). Various isoflavonoid phy to alexins formed by the attack of plants by pathogens are shown in the table 1.1. Some of the results on the microbial transformation of isoflavonoid phytoalexins are described below. The isoflavonoid phytoalexin phaseollin (186) is the most extensively studied member of the series as far as its behaviour vis-a-vis microbes is concerned. Earlier studies revealed that phaseollin is degraded by plant pathogen Fusarium solani f.sp. (Van Den Heuvel and Van Etten, 1973), Stemphylium botryosum (Heath and Higgins, 1973), S.loti and nonpathogen Colletotrichum phomoides (Higgins and Millar, 1970) to water soluble products. While Christenson (1969) reported the total degradation of pisatin (212) by two pathogen fungi Fusarium solani f.sp. pisi and Ascochyta pisi and partial degradation by two non-pathogen Fusarium solani f.sp. phaseoli and Monillnia fructicola. Similarly medicarpin (198) is found (Higgins and Miller, 1969; Sakuma and Millar, 1972) to be degraded rapidly by three pathogens of alfalfa Stemphylium botryosum, S.sarci- naeforme, Ascochyta imperfecta and also by a non-pathogen Fusarium solani f.phaseoli. Further work (Bailey et al., 1977) revealed that phaseollin is also utilized rapidly by Colletotrichum lindemu- thlanum, C.lagenarium, C.gleosporiodes. moderately by Septoria nodorum. 38 f* o» & in r- ri n 97 4 ON t ON »-• • r-1 +» w c r-»*»* Ou "—' « TJ ofl • C ~« r-l a « * h r-l .» 10 o» A O c r-l •H «A ?0> x: ~* X •5 E c lug ££ x; is 5O C«O r-1 r-l ON rri ; c •H «H •H A •ss o> (0 r-l fc ~l « S2 <0 as «5 o^ a. c 09 <*TON 8 ON «H -o C I 0 C c e f- • 4* •» *-» oo»-» >-» • oo re 5* i-» o \Q r-l «~l *-» iO """' <-* <—»ON *a *»—% +> 0 w O^SPO , «o rs a> A ON OQ>-^«*< ON eQ -' * C O § A o Oi r-l •H 6 § S S x: C N A o re Al 3 solan ! A TJx\ •s •H c e «H u A 1 ct o to t 1 o eo l • xt re » •s ?, r-l c •H o % 3 •H X! •s a. u. icu 8 r-l o: 5! 0 •fi re****- % o e rH re ? * 1 c •0 i-C A 3 r-l oe s '8 « £ 2 A •H £ Fr e o >- A re •H x: a. o. e> 39 +» •H I w c 1 2 I 00 «-* CO g e TJ -O T> -O >«>. I T u f x: x: r^- b- |T « • (0 Itf 10 (0 II ! I fl § 9 u •a o 1 o § i 8 c ^ J8 « « •p t 0 * CU B c o 5 •a +» o £ 3 5 6i I mi m fc •H° «+ u ta tH o § 0J «D a C 3 •H ! •8 3 U "O +O» •rl .-» +S>> »-* HO* ^V°>> HO Y^V^ I! =* \J • I! OH 0 OH 0 HOy /-^ OH 0 19-> is;: 133 LOH L j! J.OH O ^>OH 0 k 195 135 1 0 0- ^ OCH, 6 ^V " 0CH3 138 x%^r^y^ 201 202 203 Alternaria brassicicola while Golletotrichuro coffeanum, Cladosporlum cucumerlnum, Ascochyta fabae, Fusarium culmorum, Aspergillus niger and Botrytis cinerea poorly utilized phaseolin. First report of microbial isolation of transformation of phytoalexins is by Higgins et al (1974), who found that phaseollin (186) is transformed by Stemphylium botryosum Waller to phaseollin- isoflavan (204) by the reductive ring opening. It is interesting to note here that 2'-hydroxyisoflavans though prepared by a synthetic route involving hydrogenolysis (Wong, 1970) can also be formed by fungal transformations. Further work by Van Den Heuvel et al (1974) and Coffen and Williams (1974) revealed that mold Fusarium solani f. sp. phaseoli metabolised phaseollin to la-hydroxyphaseollone (205). While Burden et al (1974) reported that phaseollin is transformed to 6a-hydroxyphaseollin (206) and 6a,7-dihydroxyphaseollin (207) by the mold Colletotrichum lindemuthianum. These transformation products disappeared from culture media on incubation for longer period. Recently Bailey et al (1977) found that phaseollin is transformed by a nonpathogen of bean Septoria nodorum to cis (208 or 209) and trans (210 or 211) isomers of 12,13-dihydrodihydroxyphaseollin. The pathways of metabolism of phaseollin by various molds is shown in Fig. 1.26. Another isoflavonoid phytoalexin pisatin (212) is meta bolised (Van Etten et al, 1975) by Fusarium solani f.sp. pisi a pathogen of pea to 3,6a-dihydroxy-8,9-methylenedioxypterocarpan (213) which indicated the demethylation of pisatin at 3. While Lappe and Barz (1978) reported . similar results by different species viz., Fig. 1.26* The metabolism of phaseollin by Steaphyliua botryoaun (Higgins et al., 1974)» Fuaarium f sp pbaseoli (Van Den Heuvel et al., 1974)» Colletotri- chum lindemutaianua (Burden et al., 1974), Septoria nodorua (Bailey et al., 1977). 42 Fusarium anguiodes and F.averraceum. The transformation product is found to be less toxic (Fig. 1.27). While raaackiain (3-hydroxy-8,9-methylenedioxypterocarpan) (199) is degraded (Higgins, 1975) by Stemphylium botryosum an alfalfa pathogen to dihydromaackiain (7,2,-dihyd^oxy-4,,5'-methylene dioxy- isoflavan) (214) which is further degraded (Fig. 1.27). Phytoalexin medicarpin ^"(-)3-hydroxy-9-methoxypterocarpan_7 (198) is degraded (Steiner and Millar, 1974) by Stemphylium botryosum to vestitol (7,2,-dihydroxy-4,-methoxyisoflavan) (215). The latter compound degraded to water soluble products on further incubation for longer period. Further studies by Ingham (1976) showed that medicarpin is also metabolised by three fungi Botrytis cinerea, Colletotrichum llndemuthianum. C.coffeanum to a number of products (200, 202, 216 - 222) which are formed as a result of hydroxylation (200, 202, 220) C-3 methylation (221) C-9 demethylation (222) and ring fission of the substrate (Fig. 1.28). However, it is found Helminthosporium carbonum lacked this ability of degrading medicarpin. Similarly, Kievitone (189) is also transformed (Kuhn et al., 1977) by Fusarium solani f.sp.phaseoli to less toxic phytoalexin 5,7,2',4'. -tetrahydroxy-8(3H-hydroxy-3"-methylbutyl) isoflavanone (223) which involves hydroxylation of side chain (Fig. 1.29). These results clearly showed that invading pathogen detoxifies the phyto- alexins by a variety of enzymatic reactions. Very recently Gnanamanickam (1979) reported the isolation of phytoalexin from Phaseolus vulgaris (French bean) seeds infected by Rhizoctonia solani. OH Fusarlutn Soloni t K / • Sp Pi Si" 2J3 •Wi, * &p o —b^M-* > 21 & tm bot*.vjosuno > > 199 214 Fig. 1.27. Metabolism of Pisatin (Vanetten et al, 1975) and Maackiain (Higgins, 1975) by molds. OH \/ OH OH 21* 18$ Fig. 1.20. The metabolism of kievitone by Fusarium soIani phaseoli (Kuhn st al, 1977). This is an important result as it was thought earlier that phytoalexin are only produced in plants. Author suggested that the contamination of human consumable seeds by pathogen can lead to the formation of toxic phytoalexins. Further interesting results from similar studies are expected in coming years.