Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Reexamination Certificate
2002-11-27
2004-12-21
Kerr, Kathleen (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C530S350000
Reexamination Certificate
active
06833263
ABSTRACT:
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 polynucleotide molecules comprising nucleotide sequences encoding an AveC gene product, which can be used to modulate the ratio of class 2:1 avermectins produced by fermentation of cultures of
Streptomyces avermitilis
, and to compositions and methods for screening for such polynucleotide molecules. The present invention further relates to vectors, transformed host cells, and novel mutant strains of
S. avermitilis
in which the aveC gene has been mutated so as to modulate the ratio of class 2:1 avermectins produced.
BACKGROUND OF THE INVENTION
2.1. Avermectins
Streptomyces
species produce a wide variety of secondary metabolites, including the avermectins, which comprise a series of eight related sixteen-membered macrocyclic lactones having 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 where the substituent at the C25 position is (S)-sec-butyl, and the “b” series refers to those compounds where the substituent at the C25 position is isopropyl. The designations “A” and “B” refer to avermectins where the substituent at the C5 position is methoxy and hydroxy, respectively. The numeral “1” refers to avermectins where 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 antiparasitic 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 in U.S. Pat. Nos. 4,310,519 and 4,429,042. The biosynthesis of natural avermectins is believed to be initiated endogenously from the CoA thioester analogs of isobutyric acid and S-(+)-2-methyl butyric acid.
A combination of both strain improvement through random mutagenesis and the use of exogenously supplied fatty acids has led to the efficient production of avermectin analogs. Mutants of
S. avermitilis
that are deficient in branched-chain 2-oxo acid dehydrogenase (bkd deficient mutants) can only produce avermectins when fermentations are supplemented with fatty acids. Screening and isolation of mutants deficient in branched-chain dehydrogenase activity (e.g.,
S. avermitilis
, ATCC 53567) are described in European Patent (EP) 276103. Fermentation of such mutants in the presence of exogenously supplied fatty acids results in production of only the four avermectins corresponding to the fatty acid employed. Thus, supplementing fermentations of
S. avermitilis
(ATCC 53567) with S-(+)-2-methylbutyric acid results in production of the natural avermectins A1a, A2a, B1a and B2a; supplementing fermentations with isobutyric acid results in production of the natural avermectins A1b, A2b, B1b, and B2b; and supplementing fermentations with cyclopentanecarboxylic acid results in the production of the four novel cyclopentylavermectins A1, A2, B1, and B2.
If supplemented with other fatty acids, novel avermectins are produced. By screening over 800 potential precursors, more than 60 other novel avermectins have been identified. (See, e.g., Dutton et al., 1991, J. Antibiot. 44:357-365; and Banks et al., 1994, Roy. Soc. Chem. 147:16-26). In addition, mutants of
S. avermitilis
deficient in 5-O-methyltransferase activity produce essentially only the B analog avermectins. Consequently,
S. avermitilis
mutants lacking both branched-chain 2-oxo acid dehydrogenase and 5-O-methyltransferase activity produce only the B avermectins corresponding to the fatty acid employed to supplement the fermentation. Thus, supplementing such double mutants with S-(+)-2-methylbutyric acid results in production of only the natural avermectins B1a and B2a, while supplementing with isobutyric acid or cyclopentanecarboxylic acid results in production of the natural avermectins B1b and B2b or the novel cyclopentyl B1 and B2 avermectins, respectively. Supplementation of the double mutant strain with cyclohexane carboxylic acid is a preferred method for producing the commercially important novel avermectin, cyclohexylavermectin B1 (doramectin). The isolation and characteristics of such double mutants, e.g.,
S. avermitilis
(ATCC 53692), is described in EP 276103.
2.2. Genes Involved in Avermectin Biosynthesis
In many cases, genes involved in production of secondary metabolites and genes encoding a particular antibiotic are found clustered together on the chromosome. Such is the case, e.g., with the
Streptomyces
polyketide synthase gene cluster (PKS) (see Hopwood and Sherman, 1990, Ann. Rev. Genet. 24:37-66). Thus, one strategy for cloning genes in a biosynthetic pathway has been to isolate a drug resistance gene and then test adjacent regions of the chromosome for other genes related to the biosynthesis of that particular antibiotic. Another strategy for cloning genes involved in the biosynthesis of important metabolites has been complementation of mutants. For example, portions of a DNA library from an organism capable of producing a particular metabolite are introduced into a non-producing mutant and transformants screened for production of the metabolite. Additionally, hybridization of a library using probes derived from other
Streptomyces
species has been used to identify and clone genes in biosynthetic pathways.
Genes involved in avermectin biosynthesis (ave genes), like the genes required for biosynthesis of other
Streptomyces
secondary metabolites (e.g., PKS), are found clustered on the chromosome. A number of ave genes have been successfully cloned using vectors to complement
S. avermitilis
mutants blocked in avermectin biosynthesis. The cloning of such genes is described in U.S. Pat. No. 5,252,474. In addition, Ikeda et al., 1995
, J. Antibiot
. 48:532-534, describes the localization of a chromosomal region involving the C22,23 dehydration step (aveC) to a 4.82 Kb BamHI fragment of
S. avermitilis
, as well as mutations in the aveC gene that result in the production of a single component B2a producer. Since ivermectin, a potent anthelmintic compound, can be produced chemically from avermectin B2a, such a single component producer of avermectin B2a is considered particularly useful for commercial production of ivermectin.
Identification of mutations in the aveC gene that minimize the complexity of avermectin production, such as, e.g., mutations that decrease the B2:B1 ratio of avermectins, would simplify production and purification of commercially important avermectins.
3. SUMMARY OF THE INVENTION
The present invention provides an isolated polynucleotide molecule comprising the complete aveC ORF of
S. avermitilis
or a substantial portion thereof, which isolated polynucleotide molecule lacks the next complete ORF that is located downstream from the aveC ORF in situ in the
S. avermitilis
chromosome. The isolated polynucleotide molecule of the present invention preferably comprises a nucleotide sequence that is the same as the
S. avermitilis
AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or that is the same as the nucleotide sequence of the aveC ORF of
FIG. 1
(SEQ ID NO:1), or substantial portion thereof. The present invention further provides an isolated polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO:1 or a degenerate variant thereof.
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the
S. avermitilis
AveC gene product-encoding sequence of plasmid pSE186 (ATCC 209604), or to the nucleotide sequence of the aveC ORF presented in
FIG. 1
(SEQ ID NO:1) or substantial portion thereof.
The present invention furth
Katoh Yoshihiro
McArthur Hamish
Stutzman-Engwall Kim J.
Benson Gregg C.
Kerr Kathleen
Munchhof Martha G.
Pfizer Inc.
Richardson Peter C.
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