Recombinant cells that highly express chromosomally-integrated heterologous genes

Abstract

Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis , recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

1 - 16 . (canceled)  
     
     
         17 . A process for producing a recombinant host cell strain that produces high levels of a desired polypeptide, 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, 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 produced in step (b) that express said selectable marker polypeptide at a first level;    (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.    
     
     
         18 . The process according to  claim 17 , wherein said strain is a strain of  Escherichia coli  and wherein further 
 (i) in step (b) said DNA molecule is a plasmid, wherein said plasmid comprises a replicon that is temperature-sensitive for replication;    (ii) in step (b) said transforming host cells further comprises introducing said plasmid into said host-cells and growing said host cells under conditions that select for cells that express said selectable marker gene at said first level and at a temperature that does not permit replication of said plasmid, resulting in integration of said plasmid into said host gene of said chromosome by homologous recombination; and    (iii) in step (c) said selecting for host cells further comprises    (1) growing said host cells that express said selectable marker gene, resulting in excision from said host gene of said temperature-sensitive replicon and of said plasmid, and wherein further    (2) said host cells are grown under said conditions at a second temperature that does not permit replication of said plasmid, resulting in host cells that retain said heterologous DNA molecule encoding said desired polypeptide in the absence of said plasmid.    
     
     
         19 . A process according to  claim 17 , wherein further 
 (i) in step (b) said DNA molecule comprises a closed circular DNA lacking an ability to replicate, and    (ii) in step (f) said testing host cells comprises selecting for host cells produced in step (d) or step (e) that express said selectable marker gene At a second level that is higher than said first level, and then screening said host cells that express said selectable marker gene at said second level for host cells that produce said desired protein at a level higher than said initial level.    
     
     
         20 . A process according to  claim 17 , wherein said selectable marker protein confers resistance to chloramphenicol on said host cell strain.  
     
     
         21 . A process according to  claim 20 , wherein said first level of expression of said selectable marker gene confers resistance to at least about 20 Ag/ml of chloramphenicol.  
     
     
         22 . A process according to  claim 20 , wherein said first level of expression of said selectable marker gene confers resistance to at least about 20/.tg/ml of chloramphenicol and said second level of expression of said selectable marker protein confers resistance to at least about 100 μg/ml of chloramphenicol.  
     
     
         23 . A process according to  claim 20 , wherein said coding region of the upstream genetic element of said heterologous DNA segment further comprises a second coding region encoding a second desired polypeptide.  
     
     
         24 . A cell strain according to  claim 17 , wherein said enteric bacterial host cell is selected from the group consisting of  Erwinia, Escherichia  and  Klebsiella.    
     
     
         25 . A process according to  claim 24 , wherein said enteric bacterial host cell strain is a strain of  Escherichia coli.    
     
     
         26 . A process according to  claim 23 , wherein said coding region for the first genetic element of said heterologous DNA molecule encodes an alcohol dehydrogenase and a pyruvate decarboxylase.  
     
     
         27 . A process according to  claim 26 , wherein said alcohol dehydrogenase and said pyruvate decarboxylase are encoded by genes from  Zymomonas mobilis.    
     
     
         28 . A process according to  claim 27 , wherein said strain is an  Escherichia coli  strain, and said strain is able to produce ethanol by fermentation of glucose or xylose with a theoretical yield corresponding to conversion of at least about 90% of added sugar to ethanol.  
     
     
         29 . A process according to  claim 28 , wherein said chromosome further comprises a mutation that impairs succinate production.  
     
     
         30 . A process according to  claim 29 , wherein said mutation that impairs succinate production comprises a mutation in a fumarate reductase (frd) gene.  
     
     
         31 . A process according to  claim 25 , wherein said chromosome further comprises a mutation that impairs recombination in said host cell strain.  
     
     
         32 . A process according to  claim 31 , wherein said mutation that impairs recombination comprises a mutation in a recA gene.  
     
     
         33 .- 39 . (canceled)  
     
     
         40 . A process according to  claim 27 , wherein said cell is able to produce ethanol by fermentation of glucose or xylose with a theoretical yield corresponding to conversion of at least about 90% of added sugar to ethanol.  
     
     
         41 . (canceled)  
     
     
         42 . A method according to  claim 26 , wherein said cell strain is designated K04 and ATCC #55123.  
     
     
         43 . A method according to  claim 26 , wherein said cell strain is designated K011 and ATCC* #55124.  
     
     
         44 . A method according to  claim 26 , wherein said cell strain is designated K012 and ATCC #55125.  
     
     
         45 . A method according to  claim 26 , wherein said cell strain is designated K020 and ATCC #55126.  
     
     
         46 . (canceled)  
     
     
         47 . A process according to  claim 27 , wherein said strain is a  Klebsiella oxytoca  strain, and said strain is able to produce ethanol by fermentation of glucose or cellobiose with a theoretical yield corresponding to conversion of at least about 90% of added sugar to ethanol.  
     
     
         48 .- 49 . (canceled)