Penicillium patulum

Griseofulvin Griseofulvin, 7-chloro-2, 4,6-trimethoxy-6 -methylspiro[benzofuran-2(3H),r-[2]-cyclohexen]-3, 4-dione (35.4.1), is an antibiotic produced by the mycelial fungus Penicillium patulum [44-51]. 

Patulin is an excellent example of an acetate-derived structure synthesized from an aromatic substrate via oxidative cleavage and subsequent modifications (Figure 3.44). Patulin is a potent carcinogen produced by Penicillium patulum, a common contaminant on apples. If mould-infected apples find their way into food products, e.g. 

As the polyketide chain is built up, any of the reductions or eliminations from fatty acid biosynthesis can occur at any stage. The simple metabolite 6-methyl salicylic acid (6-MSAJ is made in the microorganism Penicillium patulum, and it could come from the same intermediate as orsellinic acid with one reduction,... 

Cell-free systems capable of in vitro synthesis of 6-methylsalicylic acid (6-MS A) and a related tetraketide, orsellinic acid, were developed long before the advent of recombinant DNA technologies in the field of natural product biosynthesis [113-115] (Fig. 5). Since then, the biosynthetic mechanisms and molecular recognition features of 6-methylsalicylic acid synthase (6-MSAS) have been extensively studied. 6-MSAS initiates synthesis with an acetyl group derived from acetyl Co A, extends the polyketide chain to a tetraketide via three decar-boxylative condensations of malonyl CoA-derived extender units, and uses NADPH to specifically reduce one of resulting carbonyls to a hydroxyl group. In its natural producer, Penicillium patulum, the product, 6-MSA is subsequently glycosylated to form the antibiotic patulin [116]. 

Beck J, Ripka S, Siegner A, Schiltz E, Schweizer. The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases. Eur. J. Biochem. 1990 192 487-498. 

Child CJ, Spencer JB, Bhogal P, Shoolingin-Jordan PM. Structural similarities between 6-methylsalicylic acid synthase from Penicillium patulum and vertebrate type 1 fatty acid synthase evidence from thiol modification studies. Biochemistry 1996 35 12267-12274. 

Scheme 18 illustrates the proposed stages in 6-MSA biosynthesis in which the first and second condensation steps proceed with inversion to give the triketide (63). Ketoreduction gives the alcohol (64) and then elimination followed by a final malonyl condensation generates the tetraketide (65) which cyclises via an intramolecular condensation and enolises to give the aromatic nucleus of (66). In the first set of experiments (J )- and (S)-[l- C, H]nialonales were incubated separately with 6-MSA synthase purified from Penicillium patulum [56]. Isotope incorporations were determined by mass spectrometry. All the possible isotope patterns for retention or loss of the pro-J or pro-S hydrogens from C-3 and C-5 were permutated. Comparison with the actual spectra obtained demonstrated that opposite prochiral hydrogens were eliminated. The absolute stereochemistry was established in an analogous experiment [57] where the chiral malonates were incubated with acetoacetyl CoA rather than acetyl CoA. Subsequent mass spectral analysis showed that it is the Hr proton that is retained at C-3 of 6-MSA and so it can be deduced that the hydrogen at C-5 must be derived from the opposite prochiral hydrogen, Hg. The overall result is summarised in Scheme 18. In a recent collaborative study we have synthesised the triketide alcohol (64) as its NAC thioester and shown that it is indeed a precursor as, on incubation with 6-MSA synthase and malonyl CoA, 6-MSA production is observed [unpublished results]. Current work is aimed at synthesis of both enantiomers of (64) to study the overall stereochemistry of the ketoreduction and elimination reactions.

Triketides are relatively rare. Triacetic acid lactone (4.2) has been detected in Penicillium patulum. It is also produced by fatty acid synthase in the absence of the reductant NADPH. Radicinin (4.3) is a major phytotoxin isolated from Ahernaria radicina (Stemphyllium radicinum) which causes a black rot of carrots. It is also formed by other Ahernaria species. Its pyrano[4,3- ]pyran structure, the identification of which had eluded purely chemical degradative studies, was established in one of the earlier applications of NMR spectroscopy to natural product structure elucidation. The biosynthesis of radicinin from acetate units was studied in 1970 by both radio-isotope methods using carbon-14 and by carbon-13 enrichment studies with NMR methods of detection. This was one of the first applications of this NMR technique to biosynthetic problems. These results established the labelling pattern for radicinin shown in 4.3. 

Two interesting metabolites of Penicillium patulum are patulin and penicillic acid. Their biosynthesis involves the cleavage of an aromatic ring. These substances are mycotoxins and their activity in this context is discussed in Chapter 9. 

Bacterium Penicillium patulum Corn steep liquor... 

Micromonospora purpurea Gentamicin sulfate Micromonospora sagamiensis Micro nomicin Penicillium bacterium Penicillin 0 Penicillium patulum Griseofulvin Pseudomonas bacterium Salicylic acid Semliki Forest arborvirus Interferon Septomyxa affinis Fluprednisolone Methylprednisolone Sporidiobolus ruinenli Ubidecarenone Streptococcus fecalis Floxuridine... 

Anslow WK, Raistrick H, Smith G (1943) Antifungal Substances From Molds. I. Patulm (Anhydro-3-hydroxymethylenetetrahydro-l,4-pyrone-2-carboxylic acid), a Metabolic Product of Penicillium patulum, Bainier, and Penicillium expansum. J Soc Chem bid 62 236... 

Few areas of natural products chemistry have seen as many major advances in the study of biosynthetic pathways as have occurred in polyketide compounds. Birch and Donovan (1953) demonstrated that a wide range of structural types are derived from acetate (later shown to be acetate and malonate). In experiments witfi deuterated precursors, acetate serves preferentially as a starter unit for the formation of 6-methylsalicylic acid in Penicillium griseofulvum (Simpson, 1983). Thus, polyketides are derived from the same precursors as fatty acids and the initial step seems to be similar (Fig. 5.1). Extensive purifrcation of 6-methylsalicylate synthetase from Penicillium patulum has been performed. This enzyme system is distinct and separable from the co-occurring fatty acid synthetase and has a molecular weight approximately half that of the former enzyme. NADPH is required as a coenzyme for methylsalicylate synthetase from this source (O Hagan, 1990 Packter, 1980). 

Formation of 6-methylsalicylic acid involves condensation of an acetyl-CoA unit with three units of malonyl-CoA. The entire sequence is enzyme bound and intermediates are not released. Furthermore, attempts to introduce exogenous intermediates in feeding studies failed. As mentioned above, extensive purification of 6-methylsalicylate synthetase from Penicillium patulum yielded an enzyme complex distinct and separable from fatty acid synthetase and approximately half in molecular weight. NADPH is required for production of 6-methylsalicylic acid (Packter, 1980). The application of NMR spectroscopy to study of the biosynthetic steps in the formation of 6-methylsalicylic acid and other polyketides has been reviewed (Simpson, 1987). 

Polyketides 6-Methylsalicylic acid, patulin (D 3.3.1) Penicillium patulum A, B... 

Me Master WJ, Scott AI, Trippett S (1960) Metabolic products of Penicillium patulum, J Chem Soc 4628-4631 Meyappan A, Neelakantan S, Ramesh P (1981) t-Butanolysis of lecanoric acid. Curr Sci 50 1028-1029 Mietzsch E, Lumbsch HT, Elix JA (1992) Wintabolites (mactabolites for windows) users manual. Essen Minami K (1944) Synthesis of glomellin. Yakugaku Zasshi 64 315-317... 

The biosynthesis of patulin (10) has been studied extensively, since the molecule represents a relatively simple model system in which to examine the detailed enzymology of polyketide biosynthesis. Patulin is biosynthesized by the fungus Penicillium patulum via an oxidative pathway from 6-methylsalicylic acid (188) which is synthesized from acetyl-CoA and malonyl-CoA. The major pathway from (188) and the biosynthetic relationships of the phenolic secondary metabolites of P. patulum are... 

Scott, A. L, and L. Beadling Biosynthesis of Patulin. Dehydrogenase and Dioxygenase Enzymes of Penicillium patulum. Bioorganic Chem. 3, 281 (1974). 

F. Lynen, H. Engeser, J. Friedrich, W. Schindlbeck, R. Seyffert, and F. Wieland, Fatty acid synthetase of yeast and 6-methylsalicylate synthetase of Penicillium patulum - two multienzyme complexes, iji "Microenvironments and Metabolic Conpartmentation," P.A. Srere and R.W. Estabrook, eds.. Academic Press, New York (1978). 

Patulin (52X Although the conversion of 6-methylsalicylic acid (51) into patulin (52) by Penicillium patulum was known, the nature of the aromatic intermediates on the pathway to the lactone was obscure. The discovery of several phenolic co-metabolites, m-cresol (53a R = H), m-hydroxybenzyl alcohol (54), and toluquinol (55) in this organism suggested that (53) is a likely intermediate, as shown in Scheme IS. Administration of 3-methyl[2,4,6- H3]phenol (53b R = H) to P. patultim afforded an enriched patulin. The [M + 2)... 

Bassett, E. W., and S. W. Tanenbaum The metabolic products of Penicillium patulum and their probable interrelationship. Experientia 14, 38 (1958). 

Birkinshaw, J. H., A. Bracken, and H. Raistrick Studies in the biochemistry of microorganisms. 72. Gentisyl alcohol, a metabolic product of Penicillium patulum Bainier. Biochem. J. 37, 726 (1943). 

Tanenbaum, S. W., and E. W. Bassett The biosynthesis of patulin. I. Related aromatic substances from Penicillium patulum, strain 2159 A II, the general physiology of several strains of P. patulum. Biochim. et Biophys. Acta 28, 21, 247 (1958b). 

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