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Overproduction hosts for biosynthesis of polyketides

a polyketide and overproduction technology, applied in biochemistry apparatus and processes, microorganisms, bacteria, etc., can solve the problems of slow progress, low production level of many genetically modified pkss, and limited current understanding, so as to achieve rapid introduction and expression

Inactive Publication Date: 2005-12-29
KOSAN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides a method for producing a first polyketide by expressing polyketide synthase genes in a cell that has been optimized for the production of a second polyketide. The method involves introducing genes that express a PKS that produces the first polyketide into the overproducing cell or deleting or rendering inactive genes that produce the second polyketide. The overproducing cell produces the second polyketide at a high level and the first polyketide at a high level. The invention also provides a generic overproducing host cell that can be easily modified to produce the first polyketide."

Problems solved by technology

However, one of the current challenges to the construction of very large compound libraries (>1000 compounds) is the decline in production levels associated with many genetically modified PKSs, particularly those in which multiple domains have been modified (PCT Pub.
While it is desirable to use PKS structure-activity knowledge to help guide more optimal engineering of PKSs, due to the complexity and size of these enzymes, the current understanding is relatively limited, and progress has been slow.
Although this conventional approach to strain improvement could be applied to strains carrying engineered PKSs, it is a labor-intensive process, especially given the potentially large numbers of mutant strains that could be generated by combinatorial biosynthesis.

Method used

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  • Overproduction hosts for biosynthesis of polyketides
  • Overproduction hosts for biosynthesis of polyketides
  • Overproduction hosts for biosynthesis of polyketides

Examples

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Effect test

example 1

Strains, Culture Conditions, and DNA Manipulation

[0099] DNA manipulations were performed in Escherichia coli XL1-Blue (Stratagene). The Saccharopolyspora erythraea overproduction strain produced erythromycins at up to 7 g / L (strain K1). Transformation of this strain was performed according to standard Streptomyces protoplast methods except TSB media was used in place of YEME. Transformants were selected with thiostrepton (50 μg / mL) or apramycin (100 μg / mL) overlay on R2YE or R5 regeneration plates.

example 2

Construction of a Saccharopolyspora erythraea KS1 Null Mutant Strain

[0100] Plasmid pKOS40-57 was constructed by introducing a 4.0 kb NdeI-EcoRV fragment containing a DEBS KS1 null mutation (specific replacement the active-site cysteine of DEBS KS1 domain with alanine; see Kao et al., 1996, Biochemistry 35: 12363-12368, incorporated herein by reference) into a suicide vector (a pUC18 derivative containing apramycin resistance conferring gene) as the delivery vector. Plasmid pKOS40-57 was transformed into Saccharopolyspora erythraea K1. Apramycin-resistant strains (K1-1) were grown in TSB medium containing apramycin (100 μg / ml) to confirm resistance, and integration was confirmed by PCR analysis of chromosomal DNA. For resolution of a double-crossover, transformants were passaged twice in TSB medium without apramycin. Protoplasts were regenerated on R2YE plates. Individual colonies arising on the regeneration plates were then screened for sensitivity to apramycin. A colony containing...

example 3

Production and Analysis of Erythromycin Derivatives

[0101]Saccharopolyspora erythraea strains described herein were grown in 25 mL of R2YE for 5 days at 30° C. The cultures were analyzed by HPLC / MS for the erythromycin derivatives. Structure determination was based primarily on the agreement between the structure predicted for the engineered strains and the mass spectrum and HPLC profile. An S. erythraea wild-type strain was used as the reference for the mass spectrum and HPLC. Further structural validation by NMR spectroscopy on 15-methylerythromycin A was also performed, as described in Example 4. Biological activity was assessed by the agar plate diffusion assay with Bacillus subtilis as an indicator strain and incubated overnight at 37° C. to visualize zones of inhibition.

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Abstract

Generic overproduction host cells can be used to produce any polyketide and obviate the need for performing conventional strain improvement.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. Ser. No. 09 / 847,526 filed May 1, 2001, now allowed, which claims the benefit of U.S. provisional patent application 60 / 201,287, filed May 2, 2000. The entire contents of these documents are incorporated herein by reference.STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH [0002] This invention was made with United States government support in the form of a grant (R43 AI47567) from the NIH; the United States government has certain rights to this invention.TECHNICAL FIELD [0003] The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology. BACKGROUND ART [0004] Polyketides are an important class of natural products responsible for the development ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N1/21C12N15/52C12P19/62C12N15/09C12R1/01
CPCC12P19/62C12N15/52
Inventor SANTI, DANIELMCDANIEL, ROBERTTANG, LIKHOSLA, CHAITAN
Owner KOSAN BIOSCI