Inventors:
Colin Manoil - Chestnut Hill MA
Jonathan Beckwith - Cambridge MA
Michael Syvanen - Brookline MA
Ralph R. Isberg - Menlo Park CA
Charles S. Hoffman - Wenham MA
Andrew Wright - West Newton MA
International Classification:
C12P 2100
C12P 2102
C12N 1200
Abstract:
An export sequence of export DNA can be identified by transforming a population of cells with a vector having a transposon that includes a structural gene encoding a detectable compound, positioned between insertion sequences. The structural gene encodes a detectable compound that, in wild-type organisms, is translated with an export peptide effective to export the compound. Since the transposon lacks DNA coding for an export sequence capable of exporting the detectable gene product, transformants that export the gene product include a DNA fusion of the transposon to export DNA from the parent cell, in a position to allow expression of the fused DNA. The transformants are analyzed to locate the export DNA or a gene comprising it, and the position of the export DNA in the cell's genome is determined; the orientation of the insertion also is determined. Having identified cell genes that naturally contain export DNA, the export DNA is cloned and fused to a gene encoding a product whose production and export are desired. For example, a cell is engineered to produce and export a desired product, to enable easier substance recovery and to improve cell tolerance for high levels of the desired product.