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Title: NOVEL SYSTEM FOR THE SEQUENTIAL, DIRECTIONAL CLONING OF MULTIPLE DNA SEQUENCES AND ITS USE IN METABOLIC ENGINEERING

Author
item JONES, JAMES - NORTHWESTERN UNIV
item HOHN, THOMAS - SYNGENTA BIOTECHNOL
item Leathers, Timothy

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 12/3/2002
Publication Date: 12/3/2002
Citation: Jones, J.D., Hohn, T.M., Leathers, T.D. 2002. Novel system for the sequential, directional cloning of multiple DNA sequences and its use in metabolic engineering [abstract]. Metabolic Profiling: Pathways in Discovery. Poster #10.

Interpretive Summary:

Technical Abstract: Metabolic engineering of biosynthetic pathways may require the cloning and expression of multiple genes. Often this is technically challenging due to the limited number of available restriction sites and selectable markers. Furthermore, directional ligation of multiple DNA sequences into vectors is often hindered by the inability to force the orientation of inserts. To address these problems, a convenient general method was developed which combines the use of PCR and restriction enzymes that cleave internally degenerate recognition sites to allow the sequential, directional cloning of multiple DNA sequences. Using this method, the isoprenoid pathway of the fungus Fusarium sporotrichioides was redirected toward the synthesis of carotenoids of commercial interest. A gene disrupted mutant was transformed with three- and four-gene cassettes containing the genes necessary for the synthesis of lycopene and beta-carotene, respectively. Although culture conditions for carotenoid production have not yet been optimized, preliminary experiments provided yields of approximately 0.5 mg/g dry wt for lycopene and 5.0 mg/g dry wt for beta-carotene.