Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-23
2003-02-18
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S252300, C435S325000, C435S254110, C435S419000, C536S023200
Reexamination Certificate
active
06521406
ABSTRACT:
SUMMARY OF THE INVENTION
The present invention provides novel biosynthetic genes, vectors incorporating the biosynthetic genes,
Saccharopolyspora spinosa
strains transformed with the biosynthetic genes, methods using these genes to increase production of spinosyn insecticidal macrolides, and methods using the genes or fragments thereof to change the products produced by spinosyn-producing strains of
Saccharopolyspora spinosa.
BACKGROUND OF THE INVENTION
As disclosed in U.S. Pat. No. 5,362,634, fermentation product A83543 is a family of related compounds produced by
Saccharopolyspora spinosa
. The known members of this family have been referred to as factors or components, and each has been given an identifying letter designation. These compounds are hereinafter referred to as spinosyn A, B, etc. The spinosyn compounds are useful for the control of arachnids, nematodes and insects, in particular Lepidoptera and Diptera species, and they are quite environmentally friendly and have an appealing toxicological profile. Tables 1 and 2 identify the structures of a variety of known spinosyn compounds:
TABLE 1
Factor
R
1′
R
2′
R
3′
R
4′
R
5′
R
6′
R
7′
spinosyn A
H
CH
3
C
2
H
5
CH
3
CH
3
CH
3
spinosyn B
H
CH
3
C
2
H
5
CH
3
CH
3
CH
3
spinosyn C
H
CH
3
C
2
H
5
CH
3
CH
3
CH
3
spinosyn D
CH
3
CH
3
(a)
C
2
H
5
CH
3
CH
3
CH
3
spinosyn E
H
CH
3
(a)
CH
3
CH
3
CH
3
CH
3
spinosyn F
H
H
(a)
C
2
H
5
CH
3
CH
3
CH
3
spinosyn G
H
CH
3
C
2
H
5
CH
3
CH
3
CH
3
spinosyn H
H
CH
3
(a)
C
2
H
5
H
CH
3
CH
3
spinosyn J
H
CH
3
(a)
C
2
H
5
CH
3
H
CH
3
spinosyn K
H
CH
3
(a)
C
2
H
5
CH
3
CH
3
H
spinosyn L
CH
3
CH
3
(a)
C
2
H
5
CH
3
H
CH
3
spinosyn M
H
CH
3
(b)
C
2
H
5
CH
3
H
CH
3
spinosyn N
CH
3
CH
3
(b)
C
2
H
5
CH
3
H
CH
3
spinosyn O
CH
3
CH
3
(a)
C
2
H
5
CH
3
CH
3
H
spinosyn P
H
CH
3
(a)
C
2
H
5
CH
3
H
H
spinosyn Q
CH
3
CH
3
(a)
C
2
H
5
H
CH
3
CH
3
spinosyn R
H
CH
3
(b)
C
2
H
5
H
CH
3
CH
3
spinosyn S
H
CH
3
(a)
CH
3
H
CH
3
CH
3
spinosyn T
H
CH
3
(a)
C
2
H
5
H
H
CH
3
spinosyn U
H
CH
3
(a)
C
2
H
5
H
CH
3
H
spinosyn V
CH
3
CH
3
(a)
C
2
H
5
H
CH
3
H
spinosyn W
CH
3
CH
3
(a)
C
2
H
5
CH
3
H
H
spinosyn Y
H
CH
3
(a)
CH
3
CH
3
CH
3
H
spinosyn A 17-
H
CH
3
H
C
2
H
5
CH
3
CH
3
CH
3
Psa
spinosyn D 17-
CH
3
CH
3
H
C
2
H
5
CH
3
CH
3
CH
3
Psa
spinosyn E 17-
H
CH
3
H
CH
3
CH
3
CH
3
CH
3
Psa
spinosyn F 17-
H
H
H
C
2
H
5
CH
3
CH
3
CH
3
Psa
spinosyn H 17-
H
CH
3
H
C
2
H
5
H
CH
3
CH
3
Psa
spinosyn J 17-
H
CH
3
H
C
2
H
5
CH
3
H
CH
3
Psa
spinosyn L 17-
CH
3
CH
3
H
C
2
H
5
CH
3
H
CH
3
Psa
TABLE 2
Factor
R
1′
R
2′
R
3′
R
4′
R
5′
spinosyn A 9-Psa
H
CH
3
C
2
H
5
H
spinosyn
CH
3
CH
3
(a)
C
2
H
5
H
D 9-Psa
spinosyn
H
CH
3
H
C
2
H
5
H
A
Aglycone
spinosyn
CH
3
CH
3
H
C
2
H
5
H
D
Aglycone
The naturally produced spinosyn compounds consist of a 5,6,5-tricylic ring system, fused to a 12-membered macrocyclic lactone, a neutral sugar (rhamnose) and an amino sugar (forosamine) (see Kirst et al. (1991). If the amino sugar is not present the compounds have been referred to as the pseudoaglycone of A, D, etc., and if the neutral sugar is not present then the compounds have been referred to as the reverse pseudoaglycone of A, D, etc. A more preferred nomenclature is to refer to the pseudoaglycones as spinosyn A 17-Psa, spinosyn D 17-Psa, etc., and to the reverse pseudoaglycones as spinosyn A 9-Psa, spinosyn D 9-Psa, etc.
The naturally produced spinosyn compounds may be produced via fermentation from cultures NRRL 18395, 18537, 18538, 18539, 18719, 18720, 18743 and 18823. These cultures have been deposited and made part of the stock culture collection of the Midwest Area Northern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, Ill., 61604.
U.S. Pat. No. 5,362,634 and corresponding European Patent Application No. 375316 A1 disclose spinosyns A, B, C, D, E, F, G, H, and J. These compounds are disclosed as being produced by culturing a strain of the novel microorganism
Saccharopolyspora spinosa
selected from NRRL 18395, NRL 18537, NRRL 18538, and NRRL 18539.
WO 93/09126 disclosed spinosyns L, M, N, Q, R, S, and T. Also disclosed therein are two spinosyn J producing strains: NRRL 18719 and NRRL 18720, and a strain that produces spinosyns Q, R, S, and T: NRRL 18823.
WO 94/20518 and U.S. Pat. No. 5,6704,486 disclose spinosyns K, O, P, U, V, W, and Y, and derivatives thereof. Also disclosed is spinosyn K-producing strain NRRL 18743.
A challenge in producing spinosyn compounds arises from the fact that a very large fermentation volume is required to produce a very small quantity of spinosyns. It is highly desired to increase spinosyn production efficiency and thereby increase availability of the spinosyns while reducing their cost. A cloned fragment of DNA containing genes for spinosyn biosynthetic enzymes would enable duplication of genes coding for rate limiting enzymes in the production of spinosyns. This could be used to increase yield in any circumstance when one of the encoded activities limited synthesis of the desired spinosyn. A yield increase of this type was achieved in fermentations of
Streptomyces fradiae
by duplicating the gene encoding a rate-limiting methyltransferase that converts macrocin to tylosin (Baltz et al., 1997).
Cloned biosynthetic genes would also provide a method for producing new derivatives of the spinosyns which may have a different spectrum of insecticidal activity. New derivatives are desirable because, although known spinosyns inhibit a broad spectrum of insects, they do not control all pests. Different patterns of control may be provided by biosynthetic intermediates of the spinosyns, or by their derivatives produced in vivo, or by derivatives resulting from their chemical modification in vitro. Specific intermediates (or their natural derivatives) could be synthesized by mutant strains of
S. spinosa
in which certain genes encoding enzymes for spinosyn biosynthesis have been disrupted. Such strains can be generated by integrating, via homologous recombination, a mutagenic plasmid containing an internal fragment of the target gene. Upon plasmid integration, two incomplete copies of the biosynthetic gene are formed, thereby eliminating the enzymatic function it encoded. The substrate for this enzyme, or some natural derivative thereof, should accumulate upon fermentation of the mutant strain.
Such a strategy was used effectively to generate a strain of
Saccharopolyspora erythraea
producing novel 6-deoxyerythromycin derivatives (Weber & McAlpine, 1992).
Novel intermediates could also be synthesized by mutant strains of
S. spinosa
in which parts of certain genes encoding enzymes for spinosyn biosynthesis have been replaced with parts of the same gene which have been specifically mutated in vitro, or with corresponding parts of genes from other organisms. Such strains could be generated by swapping the target region, via double homologous recombination, with a mutagenic plasmid containing the new fragment between non-mutated sequences which flank the target region. The hybrid gene would produce protein with altered functions, either lacking an activity or performing a novel enzymatic transformation. A new derivative would accumulate upon fermentation of the mutant strain. Such a strategy was used to generate a strain of
Saccharopolyspora erythraea
producing a novel anhydroerythromycin derivative (Donadio et al., 1993).
Biosynthesis of spinosyns proceeds via stepwise condensation and modification of 2- and 3-carbon carboxylic acid precursors, generating a linear polyketide that is cyclized and bridged to produce the tetracyclic aglycone. Pseudoaglycone (containing tri-O-methylated rhamnose) is formed next, then di-N-methylated forosamine is added to complete the biosynthesis (Broughton et al., 1991). Other macrolides, such as the antibiotic erythromycin, the antiparasitic avermectin and the immunosuppressant rapamycin, are synthesized in a similar fashion. In the bacteria producing these compounds, most of the macrolide biosynthetic genes are c
Baltz Richard H.
Broughton M. Christine
Crawford Kathryn P.
Madduri Krishnamurthy
Merlo Donald J.
Achutamurthy Ponnathapu
Dow AgroSciences LLC
Kerr Kathleen
Stuart Donald R.
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