Biosynthesis of Phenazines

Using ether-treated cells of P. aureofaciens, dicarbonyl-14C2 phenazme-l,6-dicarboxylic acid las (121)1 was found to be an efficient and specific precursor for phenazine-1-carboxylic acid (123), and also for 2-hydroxyphenazine-l-carboxylic acid (124). The rate of growth of the organism appeared to be important, because an incorporation was also recorded of the labelled (121) into (123), albeit at a lower level, with cultures that had been grown rapidly. The position of phenazine-1,6-dicarboxylic acid (121) as a universal intermediate in the biosynthesis of phenazines now seems secure. The previously reported failure of dimethyl phenazine-1,6-dicarboxylate (122) to act as a precursor of phenazines cf. Vol. 10, p. 28 Vol. 9, p. 29) has been confirmed with ether-treated cells of P. aureofaciens. Efficient hydrolysis of (125) to (123) did, however, occur.101... 

Simulation of biosynthesis of phenazine pigments through the dimerization of substituted anilines by symmetrical carbon carbon pairing leads to substituted phenazines. Thus, oxidation of anthranilic acid with manganese(IV) oxide or lead(IV) oxide affords phenazine-l,6-di-carboxylic acid in 16% yield. ... 

Shikimic acid, an intermediate in the biosynthesis of phenazine derivatives (e.g., iodinine, pyocyanin) can act as the sole carbon source in the formation of the phenazine skeleton. 

Scheme 2. Proposed Alternate Pathway in the Biosynthesis of Phenazine-1,6-dicarboxylic Acid ...

Mavrodi, D.V. et al., Functional analysis of genes for biosynthesis of pyocianin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAOl, J- Bacterial, 183, 6454, 2001. 

Mavrodi DV, RE Bonall, SMK Delaney, MJ Soule, G Phillips, LS Thomashow (2001) Eunctional analysis of genes for biosynthesis of pyocyanin and phenazine-l-carboxamide from Pseudomonas aeruginosa PAGE J Bacterial 183 6454-6465. 

The more recently discovered and most unusual structures include the dimeric phenazine derivatives esmeraldin A (52) and esmeraldin B (53), which are produced by Streptomyces antibioticus Tii 2706 together with 49 [54]. Esmeraldins exhibit no antibacterial activity but 53 is effective against tumor cells. Much effort has been directed to the elucidation of the biosynthesis of the esmeraldins [55]. Some rare L-quinovose esters (55a-d) of saphenic acid have... 

Phenazines.—Results on the biosynthesis of microbial phenazines from shikimic acid (previously published in preliminary form cf. Vol. 5, p. 44 and Vol. 7, p. 27) are now available in full papers.53 Additional results are that 2,3-dihydro-3-hydroxyanthranilic acid (140) was not a precursor for iodinin (141), nor was... 

The accumulation of phenazine-1,6-dicarboxylic acid (154) by mutants of Pseudomonas phenazinium148 which normally produce hydroxy-phenazine derivatives supports a role for (154) in phenazine biosynthesis. In further studies143 with Ps. phenazinium the sequence of hydroxylative steps leading to the various phenazines has been deduced143 148 to be that illustrated in Scheme 15 the biosynthesis deduced for iodinin (156) is in agreement with earlier conclusions about its formation in cultures of another organism (Brevibacterium iodinum ).146... 

H]shikimic acid were exclusively at C-2 and C-7, it followed that the biosynthesis of iodinin (135), and most probably other phenazines, was according to pattern (136) and not pattern (137). This accords with the structures of other naturally occurring phenazines like the griseoluteins, e.g. griseolutein A (140). 

Biosynthesis, synthetic analogs, and biological activity of phenazine natural products 04CRV1663. 

Phenazines and Phenoxazlnones.— A preliminary report (cf. Vol. 9, p.28) concerning the incorporation of phenazine-1,6-di-carboxylic acid (113) and its methyl ester (114) into phenazine-1-carboxylic acid (115) in Pseudomonas aureofaciens, and of (113) into lomofungin (112) in Streptomyces lomodensis, has appeared in full (cf. Vol. 10, p.28 Vol. 12, p.29). In addition,(111) has been isolated from an extract of S, lomodensis cultures after the extract had been treated with excess diazomethane. The (111) incorporated label from radioactive (113). It is reasonable to conclude from this that (110) (or a methyl ester ) is produced from (113) as an intermediate in the biosynthesis of lomofungin (112). 

The above results do not allow one to decide whether one or two molecules of shikimic acid are involved in the biosynthesis of the phenazine nucleus but there is other evidencethat the number of molecules involved is two. If this is accepted there are two ways in which the shikimic acid units can be arranged, (181) and (182) (181) is preferred, for such an arrangement of C-, units is to be seen in phenazine-1,6-dicarboxylic acid (183) and in the griseoluteins, e.g. griseolutein A (184). ... 

Where incorporations are favourable, deuterium can provide more information than tritium, one example being the biosynthesis of microbial phenazines, where the analysis of deuterium incorporation from [2- H]shikimic acid has allowed clear definition of the way in which shikimic acid is used in the construction of the phenazine ring system. ... 

The above negative results with (152) and its dimethyl ester were obtained in Pseudomonas and closely related species for metabolites bearing a single aryl-Ci substituent or none. It was still an attractive possibility that (152) could be a precursor for phenazines bearing two aryl-Ci substituents, e.g. lomofungin (155), and indeed it was found to be ineorporated into this metabolite in Streptomyces lomodensis with an efficiency which indicated that it is an intermediate in lomofungin biosynthesis. It is not yet clear whether this positive result was obtained because (152) is transported across the cell walls of Streptomyces but not Pseudomonas species or because (152) is only a precursor for phenazines with two aryl-Ci substituents. Experiments with another Streptomyces species (5. luteoreticuli) which produces the methyl ester of phenazine-1-carboxylic acid (153) should allow resolution of the problem. 

Dihydroxyphenazine-l,6-dicarboxylic acid (157) has been isolated as its dimethyl ester from Pseudomonas cepacia Although the acid (157) has been proposed as an intermediate in phenazine biosynthesis before phenazine-1,6-dicarboxylic acid (152), the necessary loss in vivo of two phenolic hydroxy-groups in the formation of (152) or, e.g., (153), makes this highly unlikely, and preliminary testing " of the hypothesis supports this view. The dihydroxy-acid (157) could, however, be an intermediate, and more reasonably so, in the biosynthesis of compounds like lomofungin (155) at a stage after (152). 

Studies on the biosynthesis of FNQ I are related to the isolation of the gene cluster from 5. cinnamonensis DSM 1042, which was reported in 2006 [291]. Actually, this strain produces two classes of secondary metabolites of mixed isoprenoid/nonisoprenoid origin FNQ I, a polyketide-isoprenoid compound, and several prenylated phenazines, mainly phenazine A 83. 

McDonald, M. and Mavrodi, D. V. 2001. Phenazine biosynthesis in Pseudomonas fluorescens Branchpoint from the primary shikimate biosynthetic pathway and role of phenazine-... 

In the aromatic amino acid biosynthesis, chorismic acid (51) is converted into anthranilic acid, which would be a potential precursor providing both nitrogens for the ring as well as the aromatic system of phenazines. However, anthranilic acid and other proposed intermediates like quinic acid, tryptophan, tyrosine, and phenylalanine have been questioned on the basis of studies of mutants of phenazine producing organisms with blocked catabolism of these various possible intermediates. 

The biosynthesis of saphenic acid (27) and derivatives thereof is based on addition of a one-carbon unit to phenazine-1,6-carboxylic acid (Iq, Scheme 3). The transfer of a methyl group from C2 of acetate is a well-known biosynthetic transformation and occurs when the thioester of acetyl coenzyme A is converted by acetyl-CoA carboxylase to malonyl-CoA. Malonyl-CoA undergoes a decarboxylative Claisen condensation with a mono-CoA thioester of phenazine-1,6-... 

Precursors with stable isotopic labels can provide further information not available with radioactive labels. Thus in the biosynthesis of microbial phenazines [as 2.29) [2- H]shikimic acid (2.28) gave iodinin [as 2.29)] some molecules of which were dilabelled as... 

Tryptophan (7.25) serves as a precursor for a number of microbial metabolites. A notable example is the ergot alkaloids (Section 7.5.1). Other aromatic amino acids are involved in the biosynthesis of various metabolites. Their origins are through shikimic acid 7,78) (see Section 5.1). Shikimic acid itself, or a close metabolic relative, serves as a source for some metabolites, e.g. phenazines (Section 7.5.5) and the ansamycins (Section 7.6.1). For compounds like the latter, acetate and propionate are also important starting materials for biosynthesis. Another important part source for many metabolites is mevalonate, invariably as a C5 (dimethylallyl) unit. 

Phenazines.—Shikimic acid (134) is clearly implicated as a precursor for microbial phenazines, e.g. iodinin (135), and it can act as the sole source of the carbon skeleton. Essential proof that two molecules of shikimic acid are involved in phenazine biosynthesis was provided when it was shown that on incorporation of DL-[1,6- C2, 2- H]shikimic acid [as (134)] into iodinin (135) in Brevibacterium iodinum, some (7.5%) of the molecules of (135) produced were dideuteriated. [The shikimic acid was incorporated with the usual high efficiency (similar values for and H) and the deuterium label was confined to the expected positions (see below).]... 

It follows from this result and the C labelling studies " that phenazine biosynthesis proceeds from two shikimic acid units linked as in (136) or (137). By determining that the sites of deuterium labelling in the iodinin (135) derived from... 

Further support for the conclusion that two molecules of shikimic acid are involved in phenazine biosynthesis comes from the incorporation of >-[1,6,7,- CaJshikimic acid [as (134)] into phenazine-1-carboxylic acid (138) in Pseudomonas aureofaciens with close to a fifth of the activity present in the carboxy-group, as required if two molecules of shikimic acid are involved [however, the same result would have been obtained if only one molecule of shikimic add was implicated provided that a symmetrical intermediate of type (139) was also involved in the elaboration of (138)]. 

Two molecules of shikimic acid (87) are used for the construction of the phenazine ring system diversion from the shikimate pathway to phenazine biosynthesis seems to occur at... 

Both l-carboxy-5-methyl[6,7,8,9- H4]phenazinium betaine (as 171) and 1-carboxy[6,7,8,9- H4]phenazine (as 170) were incorporated efficiently into pyocyanin (173) without loss of deuterium. Hydroxylation of (171) thus occurs, as a specific decarboxylative reaction. This specificity, together with the high level of incorporation of these two compounds, strongly suggested that they are normal intermediates in pyocyanin biosynthesis and since 1-hydroxy-... 

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