Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-09-07
2001-03-06
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S076000, C435S183000, C435S069100, C435S320100, C435S252300, C435S254110, C435S419000, C435S325000, C435S253500
Reexamination Certificate
active
06197591
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention is directed to compositions and methods for producing avermectins, and is primarily in the field of animal health. More particularly, the present invention relates to the identification and characterization of two novel genes, herein referred to as the aveR1 and aveR2 genes, that are involved in regulating avermectin polyketide synthase (PKS) expression and avermectin biosynthesis in
Streptomyces avermitilis
. The present invention is based on the discovery that inactivation of these genes results in an increase in the amount of avermectin produced by
S. avermitilis.
2. BACKGROUND OF THE INVENTION
Streptomyces species produce a wide variety of secondary metabolites, including the avermectins, which comprise a series of eight related sixteen-membered macrocyclic lactones with potent anthelmintic and insecticidal activity. The eight distinct but closely related compounds are referred to as A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b. The “a” series of compounds refers to the natural avermectin wherein the substituent at the C25 position is (S)sec-butyl, and the “b” series refers to those wherein the substituent at the C25 position is isopropyl. The designations “A” and “B” refer to avermectins wherein the substituent at C5 is methoxy and hydroxy, respectively. The numeral “1” refers to avermectins wherein a double bond is present at the C22, 23 position, and the numeral “2” refers to avermectins having a hydrogen at the C22 position and a hydroxy at the C23 position. Among the related avermectins, the B1 type of avermectin is recognized as having the most effective antiparasitc and pesticidal activity, and is therefore the most commercially desirable avermectin.
The avermectins and their production by aerobic fermentation of strains of
S. avermitilis
are described, among other places, in U.S. Pat. Nos. 4,310,519 and 4,429,042.
The avermectin (ave) genes, like many genes involved in the production of secondary metabolites and other Streptomyces antibiotics, are found clustered together on the bacterial chromosome. The ave gene cluster for avermectin biosynthesis spans a 95 kb genomic fragment of DNA which includes DNA encoding the avermectin polyketide synthase (PKS) (MacNeil et al., 1992, Gene 115:119-125).
The regulation of antibiotic biosynthesis in Streptomyces is perhaps best characterized in the species
Streptomyces coelicolor
. Four antibiotics produced by
S. coelicolor
include actinorhodin (Act), undecylprodigiosin (Red), calcium-dependent antibiotic (CDA), and methylenomycin (Mmy). Each of these antibiotics is encoded by a different cluster of genetically distinct genes. Genes have been identified that are linked to either the Act gene cluster or the Red gene cluster that encode products which specifically regulate the expression of the Act biosynthetic gene cluster or the Red biosynthetic gene cluster, respectively. A number of loci containing genes that globally regulate more than one of the antibiotic biosynthetic gene clusters have also been identified. For example, mutations in two independent loci, absA and absB, have been shown to block the synthesis of all four antibiotics in
S. coelicolor
(Brian et al., 1996, J. Bact. 178:3221-3231). The absA locus has been cloned and characterized, and its gene products have been shown to be involved in a signal transduction pathway which normally acts as a global negative regulator of antibiotic synthesis in
S. coelicolor
(Brian et al., 1996, above).
U.S. Pat. No. 5,876,987 to Champness et al. relates to hyperproduction of antibiotic in Streptomyces spp. as a result of interruption of the absA locus.
U.S. Pat. No. 5,707,839 to Denoya, and U.S. Pat. No. 5,728,561 to Denoya et al. relate to DNA sequences encoding branched-chain alpha-ketoacid dehydrogenase complexes of Streptomyces and methods for enhancing the production of novel avermectins.
Understanding the mechanism by which Type I polyketide synthase expression is regulated in
S. avermitilis
will permit genetic manipulation of the ave genes to increase the production of avermectins.
3. SUMMARY OF THE INVENTION
The present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an aveR1 gene product from
S. avermitilis
. In a preferred embodiment, the aveR1 gene product comprises the amino acid sequence of SEQ ID NO:2. In a non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of the aveR1 ORF of
S. avermitilis
as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317. In a further non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:1.
The present invention further provides an isolated polynucleotide molecule that is homologous to a polynucleotide molecule comprising the nucleotide sequence of the aveR1 ORF of
S. avermitilis
as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide having an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO:2.
The present invention further provides an isolated polynucleotide molecule consisting of a nucleotide sequence that is a substantial portion of any of the aforementioned aveR1-related polynucleotide molecules of the present invention. In a preferred embodiment, the substantial portion of the aveR1-related polynucleotide molecule consists of a nucleotide sequence that encodes a peptide fragment of a
S. avermitilis
aveR1 gene product or aveR1-related homologous polypeptide of the present invention. In a specific though non-limiting embodiment, the present invention provides a polynucleotide molecule consisting of a nucleotide sequence encoding a peptide fragment consisting of a sub-sequence of the amino acid sequence of SEQ ID NO:2.
The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that naturally flank the aveR1 ORF of
S. avermitilis
in situ. Such flanking sequences can be selected from the nucleotide sequence of SEQ ID NO:1 from about nt 1 to about nt 1111, and from about nt 2318 to about nt 5045. The present invention further provides an isolated polynucleotide molecule comprising one or more nucleotide sequences that are homologous to nucleotide sequences that naturally flank the aveR1 ORF of
S. avermitilis
in situ. Each flanking sequence, or homolog thereof, in the isolated polynucleotide molecule of the present invention is preferably at least about 200 nt in length. In a non-limiting embodiment, the present invention provides an isolated polynucleotide molecule comprising one or more of the aforementioned nucleotide sequences that naturally flank the aveR1 ORF of
S. avermitilis
in situ, or that are homologous to such nucleotide sequences, and further comprising one of the aforementioned aveR1-related nucleotide sequences of the present invention such as, e.g., the nucleotide sequence of the aveR1 ORF of
S. avermitilis
as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317 or substantial portion thereof.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding an aveR2 gene product from
S. avermitilis
. In a preferred embodiment, the aveR2 gene product comprises the amino acid sequence of SEQ ID NO:4. In a non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of the aveR2 ORF of
S. avermitilis
as shown in SEQ ID NO:3 (note: SEQ ID NO:3 is identical to SEQ ID NO:1) from about nt 2314 to about nt 3021. In a further non-limiting embodiment, the isolated polynucleotide molecule of the present invention comprises the nucleotide sequence of SEQ ID NO:3.
The present invention further provides an isolated polynucleotide molecule that is homologous to a polynucleotide molecule comprising the nucleotide sequence o
Price Brenda S.
Stutzman-Engwall Kim J.
Achutamurthy Ponnathapu
Ginsburg Paul H.
Kerr Kathleen
Koller Alan L.
Pfizer Inc.
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