Polyketide synthase (PKS) genes have been cloned from several fungi, such as Penicillium patulum MSAS, Aspergillus terreus atX and TPKS, Colletotrichum lagenarium PKS1, Aspergillus nidulans wA and stcA, Aspergillus parasiticus pksA, Cochliobolus heterostrophus CH-PKS1. Their coded PKSs all belong to type I of multifunctional enzymes. In contrast to bacterial type I PKS of modular type, fungal PKSs are of iterative type and classified into three groups: A) single aromatic ring PKS, B) multi-aromatic ring PKS, and C) reduced complex-type PKS. To confirm actual function of fungal PKSs, atX, PKS1, and wA genes were expressed in heterologous fungi with the amyB controlled fungal expression system. Their product compounds were identified to be 6- methylsalicylic acid (1), 1,3,6,8-tetrahydroxynaphthalene (3), and naphthopyrone compound YWA1 (15), respectively. Modification of C-terminal region of WA PKS caused shift of product from YWA1 (15) to the same heptaketide compound citreoisocoumarin (14). This result indicated that domains previously designated as thioesterase of group B PKSs function as Claisen-type cyclase.
The 16S rRNA gene sequences of invalidly described actinomycetes were determined. Phylogenetic analysis revealed that “Microstreptospora cinerea” and “Trichotomospora caesia” belong to the genus Streptomyces, “Sebekia benihana” and “Cathayosporangium alboflavum” belong to the genus Microtetraspora, and “Streptomycoides glaucoflavus” and “Parvopolyspora pallida” belong to the genus Actinomadura. “Sarraceniospora aurea” is closely related to the genus Actinocorallia. “Asiosporangium albidum” and “Actinoalloteichus cyanogriseus” belong to the family Pseudonocardiaceae. On the basis of its chemotaxonomic properties and phylogenetic analysis, “Actinoalloteichus cyanogriseus” is considered to form a new taxon.
Screening of actinomycetes and fungi for genes involved in beta-lactam production can be carried out using a technique involving both PCR with conserved primers to the isopenicillin N synthase gene and a Southern dot blotting. This technique was successful with nine out of nine known beta-lactam producing Actinomycetes tested and three out of six known beta-lactam producing Fungi tested. It proved unable to detect the isopenicillin N synthase gene in the two Flexibacter beta lactam producing eubacteria strains tested. It also identified the presence of potential beta-lactam production genes in two known none beta-lactam producing actinomycetes and one out of fourteen novel newly isolated actinomycetes. This technique has potential for use in commercial screens for novel beta-lactam producers.
Shuttle vectors, pR4C10 and pR4C11, composed of Streptomyces cosmid and Escherichia coli plasmid were constructed. With both plasmids, Streptomyces lividans and Escherichia coli K12 strains were transformed at high frequencies, comparable with the original plasmids. A vector, pR4Cll, was shown to be encapsidated into particles of actinophage R4 in vivo, and transferred to Streptomyces lividans.
The nucleotide sequence of a 12-kilobase region covering afsA encoding a key enzyme for A- factor biosynthesis in Streptomyces griseus was determined and open reading frames were deduced by means of G+C plot analysis. Although this region about 150-kb from one end of the linear chromosome is known to be readily deleted, open reading frames crowd the region in a total of eleven, including afsA and sgaA involved in healthy growth on medium of high osmolality. These also include a pair of a sensor histidine kinase and a regulator which comprises a typical prokaryotic two-component regulatory system, proteins similar to O-methyltransferase, LysR-type regulator, 3-ketoacyl-acyl carrier protein reductase, and NADP-dependent alcohol dehydrogenase. In contrast to afsA-like genes in other Streptomyces strains, such as barX locating closely to the gene for the virginiae butanolide receptor in Streptomyces virginiae and farX locating closely to the IM-2-receptor gene in a Streptomyces sp., no open reading frames in this region in S. griseus show homology with the A-factor receptor protein, which suggests that the gene organization of receptor genes for γ-butyrolactone autoregulators differs from strain to strain.
We tried to isolate actinomycetes from fallen leaves by using three methods ; the topping, the agar layered and the extraction methods. A large number of rare actinomycetes were isolated from fallen leaves. The genus Microbispora was high frequency in the samples of bamboo (Sasa and Pleioblastus) and loosestrife (Lythrum).