title: Gene disruption and replacement in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253.
abstract: A system for gene disruption and replacement based on a streptomycete temperate phage vector was developed to introduce DNA in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253. This will be useful in attempts to produce, through genetic manipulation, novel forms of the therapeutically important immunosuppressive drug rapamycin. Recombinant phages were constructed from the phi C31 phage derivative KC515 (C+ attp) carrying a thiostrepton or viomycin resistance gene along with segments of the S. hygroscopicus chromosome. Each of the cloned segments also contained the aphll neomycin/kanamycin resistance gene to enable gene replacement by loss of the phage-derived DNA. Specific deletion of the entire polyketide synthase (PKS) believed to govern rapamycin biosynthesis resulted in the loss of rapamycin production. In contrast, disruption or deletion of a region predicted to encode four PKS open reading frames, or another region predicted to encode another PKS plus a cytochrome P450 hydroxylase and ferredoxin, had no effect on the production of rapamycin or nigericin, a polyether antibiotic also produced by S. hygroscopicus. Therefore, S. hygroscopicus may have the capacity to produce polyketides additional to rapamycin and nigericin.
authors.affiliation: School of Pharmacy, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Natalie
authors.initials: N
authors.lastname: Lomovskaya
authors.affiliation: School of Pharmacy, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Leonid
authors.initials: L
authors.lastname: Fonstein
authors.affiliation: Antiinfective Discovery Research, Abbott Labs, Abbott Park, IL 60064, USA.
authors.forename: Xiaoan
authors.initials: X
authors.lastname: Ruan
authors.affiliation: Antiinfective Discovery Research, Abbott Labs, Abbott Park, IL 60064, USA.
authors.forename: Diane
authors.initials: D
authors.lastname: Stassi
authors.affiliation: Antiinfective Discovery Research, Abbott Labs, Abbott Park, IL 60064, USA.
authors.forename: Leonard
authors.initials: L
authors.lastname: Katz
authors.affiliation: Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA.|School of Pharmacy, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: C Richard
authors.initials: CR
authors.lastname: Hutchinson
mesh_terms: D001435:Bacteriophages / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011090:Polyenes / Q000378:metabolism*; D020123:Sirolimus; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004279:DNA, Viral; D011090:Polyenes; D020123:Sirolimus