The overall architecture of the PKS gene cluster responsible for the biosynthesis of the 26-membered polyene macrolide pimaricin has been determined. Eleven additional tailoring genes have been cloned and analysed. The availability of the PKS cluster will facilitate the generation of designer pimaricins by combinatorial biosynthesis approaches. This work represents the extensive description of a second polyene macrolide biosynthetic gene cluster after the one for the antifungal nystatin.

The genetic organization of the left edge (ty/€DHFJ region) of the tylosin biosynthetic gene cluster from Streptomyces fradiae has been determined. Sequence analysis of a 12.9 kb region has revealed the presence of 11 ORFs, 10 of them belonging to the biosynthetic cluster. The putative functions of the proteins encoded by these genes are as follows: peptidase (ORF1, d d d ) , tylosin resistance determinant (ORFZ, tlrB), glycosyltransferase (ORF3, ty//U), methyltransferase (ORF4, tyl€), ketoreductase (ORF5, tylD), ferredoxin (ORF6, tylH2), cytochrome P450 (ORF7, tylH7), methyltransferase (ORF8, tylF), epimerase (ORF9, tylJ), acyl-CoA oxidase (ORF10, tylP) and receptor of regulatory factors (ORF11, tylQ). The functional identification of the genes in the proposed tylosin biosynthetic pathway has been deduced by database searches and previous genetic complementation studies performed with tylosin idiotrophic mutants blocked a t various stages in tylosin biosynthesis. The tlrB gene has been shown to be useful as a tylosin resistance marker in Streptomyces liwidans, Streptomyces parvulus and Streptomyces coelicolor and the effect of tylF on macrocin depletion has been confirmed. A pathway for the biosynthesis of 6-deoxy-~-allose, the unmethylated mycinose precursor, involving the genes tylD, tylJ and tylN is proposed.

We have demonstrated that PimD is the epoxidase responsible for the conversion of 4,5-deepoxypimaricin to pimaricin in S. natalensis. The metabolite accumulated by the recombinant mutant, in which the epoxidase has been knocked out, constitutes the first designer polyene obtained by targeted manipulation of a polyene biosynthetic gene cluster. This novel epoxidase could prove to be valuable for the introduction of epoxy substituents into designer macrolides.

The basidiomycete Hypholoma sublateritium produces the triterpenoid antitumor clavaric acid, an inhibitor of the human Ras-farnesyl transferase. The H. sublateritium squalene epoxidase gene (erg1) has been cloned and shown to encode a flavoprotein monooxygenase that requires FAD, NADPH, and P450 cofactors. Silencing of the erg1 gene in H. sublateritium using constructions expressed from the gdh promoter of Agaricus bisporus showed that the squalene epoxidase is involved in clavaric acid formation and in ergosterol biosynthesis; silenced expression of erg1 resulted in an ergosterol-dependent phenotype for full growth. Overexpression of erg1 gene resulted in up to 32% to 97% increment of clavaric acid production confirming its involvement in the biosynthesis of this antitumor product. Oxidosqualene (or dioxidosqualene) appears to be the branching point for primary metabolism (sterols) and secondary metabolites in basidiomycetes.

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (P gpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.

The tlrB gene from Streptomyces fradiae has been cloned and used to construct bifunctional Streptomyces-Escherichia coli shuttle vectors carrying the antibiotic resistance genes to kanamycin-neomycin, thiostrepton and tylosin as selection markers. In the same way, the tlrB gene was subcloned in plasmids including the apramycin resistance gene and the oriT sequence from the plasmid pSET152 to facilitate conjugation of Streptomyces spores. The usefulness of the tlrB gene as tylosin resistance marker was ascertained in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor, but not in Streptomyces clavuligerus. The tlrB gene constitutes a useful selection marker when high-frequency of conjugation/transformation is not required or as secondary marker in recombinant Streptomyces species where thiostrepton and kanamycin have been utilized for primary selection.

The enhancement of industrial antibiotic yield has been achieved through technological innovations and traditional strain improvement programs based on random mutation and screening. The development of recombinant DNA techniques and their application to antibiotic producing microorganisms has allowed yield increments and the design of biosynthetic pathways giving rise to new antibiotics. Genetic manipulations of the cephalosporin producing fungus Cephalosporium acremonium have included yield improvements, accomplished increasing biosynthetic gene dosage or enhancing oxygen uptake, and new biosynthetic capacities as 7‐aminocephalosporanic acid (7‐ACA) or penicillin G production. Similarly, in Penicillium chrysogenum, the industrial penicillin producing fungus, heterologous expression of cephalosporin biosynthetic genes has led to the biosynthesis of adipyl‐7‐aminodeacetoxycephalosporanic acid (adipyl‐7‐ADCA) and adipyl‐7‐ACA, compounds that can be transformed into the economically relevant 7‐ADCA and 7‐ACA intermediates. Escherichia coli expression of the genes encoding D‐amino acid oxidase and cephalosporin acylase activities has simplified the bioconversion of cephalosporin C into 7‐ACA, eliminating the use of organic solvents. The genetic manipulation of antibiotic producing actinomycetes has allowed productivity increments and the development of new hybrid antibiotics. A legal framework has been developed for the confined manipulation of genetically modified organisms. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 216–226, 1997.

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