Patent ID: 6107093
Filing Date: 2000-08-22
Classification: C12N,C12P,G06T,H04N,Y02E

Abstract:
A recombinant host cell strain that is the product of a process comprising the steps of:(a) providing a culture comprised of enteric bacterial host cells comprising a pyruvate formate lyase promoter which is endogenous to said host cells and a DNA encoding a pyruvate formate lyase gene under transcriptional control of said promoter;(b) transforming host cells in said culture with a heterologous DNA molecule comprising(i) two genetic elements assembled such that the coding regions of both elements are translated in the same direction, wherein the downstream genetic element comprises a selectable marker gene which confers chloramphenicol resistance on said host cell strain, a promoter that controls the transcription of said selectable marker gene, and a transcription termination sequence, and wherein the upstream genetic element comprises one or more promoterless coding regions encoding at least one desired polypeptide followed by a transcription termination sequence, and(ii) sequences that flank said genetic elements and are oriented such that their direction of translation is the same as that of the two heterologous genetic elements, and(iii) sequences that flank said genetic elements and are sufficiently homologous to said pyruvate formate lyase gene to enable integration by homologous recombination,whereby integration of said genetic elements into said pyruvate formate lyase gene results by means of homologous recombination;(c) selecting for host cells produce in step (b) that express said selectable marker polypeptide at first and second levels, wherein said first level of expression of said selectable marker gene confers resistance to at least about 20 .mu.g/ml of chloramphenicol and said second level of expression of said selectable marker protein confers resistance to at least about 100 .mu.g/ml of chloramphenicol;(d) screening host cells obtained in step (c) to obtain host cells that produce said desired polypeptide at an initial level;(e) optionally exposing host cells identified in step (d) to a mutagen under conditions such that mutations are created in said DNA; and then(f) testing host cells produced in step (d) or step (e) for host cells that produce said marker polypeptide at a level higher than said initial level, to obtain host cells having a mutation that causes increased expression of the upstream genetic element resulting in an increase in production by said host cells of all polypeptides encoded by said heterologous DNA molecule compared to said production of all polypeptides encoded by said heterologous DNA molecule by said host cells in the absence of said mutation, wherein said increased expression is retained in the absence of conditions that select for cells having said increased expression.