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A-factor regulates streptomycin biosynthesis in S

As modular PKS contains multi-catalytic domains on a polypeptide, the size of the gene encoding modular PKS is quite large in comparison with genes encoding type-II or type-III PKSs and the entire biosynthetic gene clusters are at least 40-kb. We used an integrating BAC (Bacterial Artificial Chromosome) vector, pKU503, to clone the entire 75-kb gene cluster for macrocyclic lactone, pladienolide B (7) (), biosynthesis (). A similar technique was applied to clone the entire biosynthetic gene clusters for erythromycin (8), bafilomycin B1 (9), nemadectin α (10), and linear polyketides, aureothin (11) and leptomycin (12) (). A BAC clone, pKU503eryP4-A14, containing the entire 60-kb gene cluster for erythromycin biosynthesis () of Sacchropolyspora erythraea NRRL 2338, was isolated and the transformants carrying pKU503eryP4-A14 produced about 4 mg/L erythromycin A in the culture broth. Unfortunately, during several rounds of propagation, productivity was reduced and they accumulated the aglycone moiety of erythromycin, 6-deoxyerythronolide B. The entire 72 kb gene cluster for bafilomycin B1 biosynthesis () of Kitasatospora setae KM-6054 was cloned into BAC vector and the resultant BAC clone, pKU503bafP3-P20 (pBFM1), was introduced into S. avermitilis SUKA22. The transformants produced 16 mg/L bafilomycin B1 (9) and a trace amount of bafilomycin A1. Anthelmintic macrolide, nemadectin α(10), is structurally related to avermectin and the organization of each gene in the gene cluster was also quite similar to that in the avermectin gene cluster. The gene cluster for nemadectin biosynthesis () of S. cyaneogriseus subsp. noncyanogenus NRRL 15774 was cloned into BAC vector. The resultant BAC clone, pKU503nemP11-L5, containing the entire 80-kb gene cluster for nemadectin biosynthesis (accession No. AB363939), was introduced into S. avermitilis SUKA22 and the transformants produced 1.6 mg/L nemadectin α (10) and a trace amount of related components in the mycelium. Two entire biosynthetic gene clusters for linear polyketide compounds, aureothin (11) and leptomycin (12) were cloned into cosmid and BAC vectors, respectively. Transformants carrying a cosmid clone, pKU465aurP2-A6, carrying the entire 35-kb gene cluster for aureothin biosynthesis () of Streptomyces sp. MM 23 produced 116 mg/L aureothin (11) in the culture broth. Since the biosynthetic gene cluster for leptomycin was estimated to be larger than 40-kb, the entire biosynthetic gene cluster for leptomycin () of Streptomyces sp. EM52 was cloned by BAC vector and a BAC clone, pKU503lepP5-L8, containing the entire 80-kb gene cluster for leptomycin, was isolated. The resultant transformants produced 5 mg/L leptomycin (12) in the mycelium. Thus, large-deletion mutants of S. avermitilis could transform a ~100-kb BAC clone without detectable deletions involved in the highly homologous region of each module in the biosynthetic gene cluster.

Induction of streptomycin-inactivating enzyme by A-factor in Streptomyces griseus
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Transformants of S. avermitilis wild-type strain carrying the biosynthetic gene cluster for streptomycin (1) or cephamycin C (13) produced about half of the amount of each metabolite as the original producer S. griseus IFO 13350 or S. clavuligerus ATCC 27065. The productivity of these metabolites in S. avermitilis large-deletion mutant SUKA17 transformants was improved, being 4.5 to 7-fold higher than that of wild-type transformants and 2 to 2.5-fold higher than that of the original producers as described previously. Both transformants of S. avermitilis wild-type and its large deletion-mutant SUKA17 carrying biosynthetic gene cluster for deoxystreptamine-containing aminoglycoside, ribostamycin (2), of S. ribosidificus ATCC 21294 produced small amount of ribostamycin (; about 1.5 – 4% productivity of that of S. ribosidificus ATCC 21294). Similarly, transformants of S. avermitilis SUKA17 carrying the biosynthetic gene cluster for oxytetracycline produced about half of the amount of metabolite as the original producer S. rimosus NRRL 2234 and the productivity of S. avermitilis wild type transformants was inefficient (). These two biosynthetic gene clusters might be inefficiently expressed in S. avermitilis.

by A-factor in Streptomyces griseus

Genetic analysis of A-factor synthesis in Streptomyces coelicolor A3(2) and Streptomyces griseus
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A prominent property of members of the actinomycete microorganisms belonging to the order Actinomycetales is the ability to produce numerous secondary metabolites, including antibiotics and other biologically active compounds of proven value in human and veterinary medicine, agriculture, or useful as biochemical tools. These structurally diverse metabolites possess not only antibacterial, antifungal, antiviral and antitumor activity but also antihypertensive and immunosuppressant properties. Streptomyces are a rich source of pharmaceutical compounds in which common cellular intermediates, including sugars, fatty acids, amino acids and terpenes, are combined to give more complex structures by defined biochemical pathways. In this century, genome analysis of Streptomyces microorganisms, S. avermitilis MA 4680,, S. bingchenggensis BCW-1, S. cattleya NRRL 8057, S. clavuligerus ATCC 27064, S. coelicolor A3(2), S. griseus IFO 13350, S. scabiei 87.22, and S. venezuelae ATCC 10712, has revealed that these microorganisms each have a large linear chromosome that harbors over twenty secondary metabolic gene clusters encoding the biosynthesis of polyketides by polyketide synthases (PKSs), peptides by non-ribosomal peptide synthetases (NRPSs), bacteriocins, terpenoids, shikimate-derived metabolites, aminoglycosides, and other natural products.

Aminoglycoside antibiotics are synthesized from sugar pathway and a streptidine-containing aminoglycoside, streptomycin (1) (), has been produced by a S. avermitilis large-deletion derivative carrying the gene cluster for streptomycin biosynthesis () of S. griseus IFO 13350. The biosynthetic gene cluster for deoxystreptamine-containing aminoglycoside, ribostamycin (2) (), of S. ribosidificus ATCC 21294 was cloned by a cosmid vector and about 35-kb segment containing the entire set of the gene cluster () was subcloned into the integrating vector pKU465cos to generate pRSM1. The resultant transformants produced ribostamycin in the culture broth and their productivity was about 8 mg/L. In general, aminoglycoside antibiotics consist of one molecule of aminocyclitol and two or more molecules of neutral or amino sugars such as streptomycin and ribostamycin. Since kasugamycin (3) () consists of one amino sugar and one neutral cyclitol, myo-inositol, the size of the gene cluster was smaller than biosynthetic gene clustsers for other aminoglycoside antibiotic (about 20-kb). Transformants carrying the entire gene cluster for kasugamycin biosynthesis () of S. kasugaensis MB 273 (accession No. AB360380) produced about 5 mg/L kasugamycin in the culture broth. The expression of the kasugamycin biosynthetic genes was controlled by the gene product of regulatory gene kasT. When kasT was expressed in the multi-copy plasmid that gave the desired gene-dosage effect or by the alternative promoter, productivity was improved (7 to 12 mg/L).

A-factor of Streptomyces griseus ..

A σB-like factor responsible for carotenoid biosynthesis in Streptomyces ..
Photo provided by Flickr

ArpA, senses the gamma-butyrolactone A-factor in Streptomyces griseus (see below).
Photo provided by Flickr
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