Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-06-07
2002-06-04
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S069100, C435S091200, C435S190000, C435S320100, C435S325000, C536S025200, C536S023200
Reexamination Certificate
active
06399324
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to novel DNA sequences that encode for a branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex of an organism belong to the genus Streptomyces. It also relates to the production of a
Streptomyces avermitilis
branched-chain alpha-ketoacid dehydrogenase (bkd)-deficient mutant by genetic engineering technology. The bkd-deficient mutant lacks branched-chain alpha-ketoacid dehydrogenase activity, and is useful for the fermentative production of novel (non-natural) avermectins.
S. avermitilis
naturally produces eight distinct but closely related antiparasitic polyketide compounds named avermectins. The avermectin complex produced by
S. avermitilis
has four major components, A1a, A2a, B1a, and B2a, and four minor components, A1b, A2b, B1b, and B2b. The structure of the various components are depicted below.
Avermectin
R
1
R
2
X-Y
A1a
sec
-butyl
Me
CH═CH
A1b
Isopropyl
Me
CH═CH
A2a
sec
-butyl
Me
CH
2
—CH(OH)
A2b
Isopropyl
Me
CH
2
—CH(OH)
B1a
sec
-butyl
H
CH═CH
B1b
Isopropyl
H
CH═CH
B2a
sec
-butyl
H
CH
2
—CH(OH)
B2b
Isopropyl
H
CH
2
—CH(OH)
The avermectin polyketide structure is derived from seven acetate, five propionate molecules, and one alpha-branched-chain fatty acid molecule, which is either S(+)-2-methylbutyric acid or isobutyric acid. The designations “A” and “B” refer to avermectins wherein the 5-substituent is methoxy or hydroxy, respectively. The numeral “1” refers to avermectins wherein a double bond is present at the 22-23 position, and numeral “2” to avermectins having a hydrogen at the 22-position and hydroxy at the 23-position. Lastly, the C-25 has two possible substituents: the sec-butyl substituent (derived from the incorporation of S(+)-2-methylbutyric acid) is present in the avermectin “a” series, and the isopropyl substituent (derived from the incorporation of isobutyric acid) is present in the avermectin “b” series (for a review see Fisher, M. H. and Mrozik, H., 1984, “Macrolide Antibiotics”, Academic Press, chapter 14).
By “natural” avermectins is meant those avermectins produced by
S. avermitilis
wherein the 25-position substituent is, as mentioned above, either isopropyl or sec-butyl. Avermectins wherein the 25-position group is other than isopropyl or sec-butyl are referred to herein as novel or non-natural avermectins.
One metabolic route to the natural alpha-branched-chain fatty acids in their CoA form is from the alpha branched-chain amino acids isoleucine and valine through a branched-chain amino acid transaminase reaction followed by a branched-chain alpha-ketoacid dehydrogenase reaction. (Alternatively, branched-chain fatty acyl-CoA derivatives can arise from branched-chain alpha-ketoacids produced by de novo synthesis). These metabolic pathways are depicted below.
A mutant of
S. avermitilis
with no detectable branched-chain alpha-ketoacid dehydrogenase (BCKDH) activity in the last mentioned enzyme was previously isolated (Hafner et al., 1988, European Patent EP 284,176, which issued on Oct. 20, 1993). The mutant was isolated following standard chemical mutagenesis of
S. avermitilis
strain ATCC 31272 in a screen searching for the absence of
14
CO
2
production from
14
C-1 labeled 2-oxoisocaproic acid substrate (leucine analog). The mutant is unable to synthesize natural avermectins except when the S(+)-2-methylbutyric acid or isobutyric acid or a precursor bearing the isopropyl or sec-butyl (S-form) group is added to the medium in which the mutants are fermented. The mutant is also capable of producing novel (non-natural) avermectins when fermented under aqueous aerobic conditions in a nutrient medium containing an exogenously added alternative carboxylic acid, such as cyclohexane carboxylic acid (CHC), or a precursor thereof, as indicated above.
To clone the genes that encode the branched-chain alpha-ketoacid dehydrogenase complex of
S. avermitilis
is highly desirable. Manipulation of these genes through recombinant DNA techniques should facilitate the production of natural and novel avermectins. For certain strains, increased titer of natural avermectins would be anticipated by increasing the copy number of the bkd genes. In addition, generation of an irreversibly blocked bkd strain, having BCKDH activity permanently deleted or modified by gene replacement, would be an improved alternative to the bkd mutant which was obtained, as mentioned before, by chemical mutagenesis.
The alpha-ketoacici dehydrogenase multienzyme complexes—the branched—chain alpha-ketoacid dehydrogenase (BCKDH) complex, the pyruvate dehydrogenase (PDH) complex, and the alpha-ketoglutarate dehydrogenase (KGDH) complex catalyze the oxidative decarboxylations of branched-chain alpha-ketoacids, pyruvate, and alpha-ketoglutarate, respectively, releasing CO2 and generating the corresponding Acyl-CoA and NADH (Perham, R. N., 1991
, Biochemistry
, 30: 8501-8512). Each complex consists of three different catalytic enzymes: decarboxylase (E1), dihydrolipoamide acyltransferase transacylase (E2), and dihydrolipoamide dehydrogenase (E3).
Branched-chain alpha-ketoacid dehydrogenase (BCKDH) is a multienzyme complex composed of three functional components, E1, the decarboxylase, E2, the transacylase, and E3, the lipoamide dehydrogenase. The purified complexes from
Pseudomonas putida, Pseudomonas aeruginosa
, and
Bacillus subtilis
, are composed of four polypeptides. The purified mammalian complexes also consist of four polypeptides, E1alpha, E1beta, E2, and E3. An alpha-ketoacid dehydrogenase complex has been isolated from
Bacillus subtilis
which has both pyruvate and branched-chain alpha-ketoacid dehydrogenase activities. This dual function complex oxidizes both pyruvate branched-chain alpha-ketoacids for membrane phospholipids.
Cloning of prokaryotic branched-chain alpha-ketoacid dehydrogenase genes has been reported for Pseudomonas and Bacillus. In these systems it was found that the genes encoding the BCKDH were clustered in an operon. The genes of the BCKDH complex of
Pseudomonas putida
have been cloned and the nucleotide sequence of this region determined (Sykes et al., 1987
, J. Bacteriol
., 169:1619-1625, and Burns et al., 1988
, Eur. J. Biochem
, 176:165-169, and 176:311-317). The molecular weight of E1alpha is 45289, of E1beta is 37138, of E2 is 45134, and of E3 is 48164. The four genes are clustered in the sequence: E1alpha , E1beta, E2, and E3. Northern blot analysis indicated that expression of these four genes occurs from a single mRNA and that these genes constitute an operon. There is a typical prokaryotic consensus promoter immediately preceding the start of the E1alpha coding region that permits the constitutive expression of the Pseudomonas bkd genes. The initiator codon for the E1beta coding region is located only 40 nucleotides downstream from the end of the E1alpha open reading frame (ORF). In contrast, there is no intergenic space between the E1beta and E2 ORFs since the stop codon for the E1beta ORF is the triplet immediate preceding the initiator codon of the E2 ORF. The intergenic space between the E2 and the E3 ORFs is reduced to only 2 nucleotides. Therefore, the Pseudomonas bkd genes are tightly linked.
Similarly, the operon coding for the
Bacillus subtilis
BCKDH/PDH dual complex has been cloned (Hemila et al., 1990
, J. Bacteriol
., 172:5052-5063). This operon contains four ORFs encoding four proteins of 42, 36, 48, and 50 kilodaltons (kDa) in size, shown to be highly homologous to the E1alpha, E1beta, E2, and E3 subunits of the Pseudomonas bkd cluster. The genes encoding the alpha and beta subunits of the E1 component of the dual BCKDH/PDH multienzyme complex from
Bacillus stearothermophilus
have also been cloned and sequenced (estimated molecular weights of the alpha and beta subunits are approximately 41,000 and 35,000, respectively) (Hawkins et al., 1990
, Eur. J. Biochem
., 191:337-346).
Additionally, the sequences of a number of eukaryotic E1 alpha and beta BCKDH subunits (human, bovine, and rat) have been disclosed. Recently, an amino ac
Denoya Claudio D.
Stutzman-Engwall Kim J.
Benson Gregg C.
Low Christopher S. F.
Pfizer Inc.
Richardson Peter C.
Robinson Hope A.
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