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Dive into the research topics where Donald J. Merlo is active.

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Featured researches published by Donald J. Merlo.


Chemistry & Biology | 2001

Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa1

Clive Waldron; Patti Matsushima; Paul Robert Rosteck; Mary C. Broughton; Jan R. Turner; Krishnamurthy Madduri; Kathryn P. Crawford; Donald J. Merlo; Richard H. Baltz

BACKGROUND Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. RESULTS We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the spinosyn pathway. The spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. CONCLUSIONS Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.


Archive | 2001

Research PaperCloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa1

Clive Waldron; Patti Matsushima; Paul Robert Rosteck; Mary C. Broughton; Jan R. Turner; Krishnamurthy Madduri; Kathryn P. Crawford; Donald J. Merlo; Richard H. Baltz

BACKGROUND Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. RESULTS We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the spinosyn pathway. The spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. CONCLUSIONS Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.


Nature Biotechnology | 2003

Insect resistance conferred by 283-kDa Photorhabdus luminescens protein TcdA in Arabidopsis thaliana

Dong Liu; Stephanie L. Burton; Todd P. Glancy; Ze-Sheng Li; Ronnie Hampton; Thomas Meade; Donald J. Merlo

The tcdA gene of Photorhabdus luminescens encodes a 283-kDa protein, toxin A, that is highly toxic to a variety of insects, including some agriculturally important pests. We tested the efficacy of transgenic toxin A in Arabidopsis thaliana for control of feeding insects. Plants with toxin A expression above about 700 ng/mg of extractable protein were highly toxic to tobacco hornworm (Manduca sexta). Toxin A isolated from transgenic plants also strongly inhibited growth of the southern corn rootworm (Diabrotica undecimpunctata howardi). Addition of 5′ and 3′ untranslated regions of a tobacco osmotin gene (osm) increased toxin A production 10-fold and recovery of insect-resistant lines 12-fold. In the best line, high toxin A expression and insect resistance were maintained for at least five generations in all progeny. The intact tcdA mRNA represents the largest effective transgenic transcript produced in plants to date. These results may open a new route to transgenic pest control in agriculture.


Journal of Bacteriology | 2001

Rhamnose Biosynthesis Pathway Supplies Precursors for Primary and Secondary Metabolism in Saccharopolyspora spinosa

Krishnamurthy Madduri; Clive Waldron; Donald J. Merlo

Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in Saccharopolyspora spinosa are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of spinosyn fermentation products.


Journal of Industrial Microbiology & Biotechnology | 2001

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa.

Krishnamurthy Madduri; Clive Waldron; Patti Matsushima; Mary C. Broughton; Kathryn P. Crawford; Donald J. Merlo; Richard H. Baltz

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402.


Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology | 2000

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa.

Clive Waldron; Krishnamurthy Madduri; Kathryn P. Crawford; Donald J. Merlo; Patti Jean Treadway; Mary C. Broughton; Richard H. Baltz

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21–carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.


Synthetic insecticidal crystal protein gene. | 1989

Synthetic insecticidal crystal protein gene

Michael J. Adang; Thomas A. Rocheleau; Donald J. Merlo; Elizabeth E. Murray


Archive | 2005

Novel Herbicide Resistance Genes

Terry R. Wright; Justin M. Lira; Donald J. Merlo; Nicole L. Arnold


Archive | 1992

Insecticidal proteins and method for plant protection

Terence A. Walsh; Robert A. Houtchens; James A. Strickland; Gregory L. Orr; Donald J. Merlo


Archive | 1998

Insecticidal protein toxins from photorhabdus

Jerald C. Ensign; David J. Bowen; James K. Petell; Raymond Fatig; Sue Schoonover; Richard H. Ffrench-Constant; Thomas A. Rocheleau; Michael B. Blackburn; Timothy D. Hey; Donald J. Merlo; Gregory L. Orr; Jean L. Roberts; James A. Strickland; Lining Guo; Todd A. Ciche; Kitisri Sukhapinda

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