Method of improving the production of biomass or a desired product from a cell

Abstract

The production of biomass or a desired product from a cell can be improved by inducing conversion of ATP to ADP without primary effects on other cellular metabolites or functions which is achieved by expressing an uncoupled ATPase activity in said cell and incubating the cell with a suitable substrate to produce said biomass or product. This is conveniently done by expressing in said cell the soluble part (F 1 ) of the membrane bound (F 0 F 1 type) H + -ATPase or a portion of F 1 exhibiting ATPase activity. The organism from which the F 1 ATPase or portions thereof is derived, or in which the F 1 ATPase or portions thereof is expressed, may be selected from prokaryotes and eukaryotes. In particular, the DNA encoding F 1 or a portion thereof may be derived from bacteria and eukaryotic microorganisms such as yeast, other fungi and cell lines of higher organisms and be selected from the group consisting of the gene encoding the F 1 subunit beta or a portion thereof and various combinations of said gene or portion with the genes encoding the other F 1 subunits or portions thereof. The method can be used i.a. for optimizing the formation of biomass or a desired product by a cell by expressing different levels of uncoupled ATPase activity in the cell, incubating the cell on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product at each level of ATPase expression, and choosing a level of ATPase expression at which the conversion rate is optimized.

What is claimed is:  
     
         1 . A method of improving the production of biomass or a desired product from a cell, characterized by expressing an uncoupled ATPase activity in said cell to induce conversion of ATP to ADP without primary effects on other cellular metabolites or functions, and incubating the cell with a suitable substrate to produce said biomass or product.  
     
     
         2 . A method according to  claim 1 , characterized by expressing in said cell the soluble part (F 1 ) of the membrane bound (F 0 F 1  type) H + -ATPase or a portion of F 1  exhibiting ATPase activity.  
     
     
         3 . A method according to  claim 1 , wherein said cell is a prokaryotic cell.  
     
     
         4 . A method according to  claim 3 , wherein said cell is selected from the group consisting of bacteria belonging to the genera Lactococcus, Streptococcus, Enterococcus, Lactobacillus, Leuconostoc, Escherichia, Zymomonas, Bacillus and Pseudamonas.  
     
     
         5 . A method according to  claim 1 , wherein said cell is a eukaryotic cell.  
     
     
         6 . A method according to  claim 5 , wherein said cell is a yeast cell.  
     
     
         7 . A method according to  claim 6 , wherein said cell belongs to  Saccharomyces cerevisiae  or  TriChoderma reesei.    
     
     
         8 . A method according to  claim 1 , wherein said cell is transformed or transfected with an expression vector including DNA encoding F 1  or a portion thereof exhibiting ATPase activity under the control of a promoter functioning in said cell, and said DNA is expresses in the cell.  
     
     
         9 . A method according to  claim 8 , wherein said DNA encoding F 1  or a portion thereof is homologous to said cell.  
     
     
         10 . A method according to  claim 8 , wherein said DNA encoding F 1  or a portion thereof is heterologous to said cell.  
     
     
         11 . A method according to  claim 8 , wherein said DNA encoding F 1  or a portion thereof is derived from a prokaryotic organism.  
     
     
         12 . A method according to  claim 11 , wherein said DNA encoding F 1 or a portion thereof is derived from  Escherichia coli, Lactococcus lactis  or  Streptococcus thermophilus  and is selected from the group consisting of the gene encoding the F 1  subunit β or a portion thereof and various combinations of said gene or portion with the genes encoding the F 1  subunits δ, α, γ and ε or portions thereof.  
     
     
         13 . A method according to  claim 12 , wherein said DNA encoding F 1  or a portion thereof is selected from the group consisting of the  Escherichia coli, Streptococcus thermophilus  and  Lactococcus lactis  genes atpHAGDC (coding for subunits δ, α, γ, β, ε), atpAGDC (coding for subunits α, γ, β, ε), atpAGD (coding for subunits α, γ, β, ε), atpDC (coding for subunits β, ε) and atpD (coding for subunit β alone).  
     
     
         14 . A method according to  claim 8 , wherein said DNA encoding F 1  or a portion thereof is derived from a eukaryotic organism.  
     
     
         15 . A method according to  claim 14 , wherein said DNA encoding F 1  or a portion thereof is derived from  Saccharomyces cerevisiae, Phaffia rhodozyma  or  Trichoderma reesei  and is selected from the group consisting of the gene encoding the F 1  subunit β or a portion thereof and various combinations of said gene or portion with the genes encoding the other F 1  subunits or portions thereof.  
     
     
         16 . A vector including DNA encoding the soluble part (F 1 ) of the membrane bound (F 0 F 1  type) H + -ATPase or a portion of F 1  exhibiting ATPase activity, said DNA being derived from  Lactococcus lactis  subsp. cremoris and having the sequence stated in SEQ ID No. 1.  
     
     
         17 . A vector including DNA encoding the soluble part (F 1 ) of the membrane bound (F 0 F 1  type) H + -ATPase or a portion of F 1  exhibiting ATPase activity, said DNA being derived from  Lactococcus lactis  subsp. lactis and having the sequence stated in SEQ ID No. 6.  
     
     
         18 . A vector including DNA encoding the soluble part (F 1 ) of the membrane bound (F 0 F 1  type) H + -ATPase or a portion of F 1  exhibiting ATPase activity, said DNA being derived from  Streptococcus thermophilus  and having the sequence stated in SEQ ID No. 10.  
     
     
         19 . A vector including DNA encoding the soluble part (F 1 ) of the membrane bound (F 0 F 1  type) H + -ATPase or a portion of F 1  exhibiting ATPase activity, said DNA being derived from  Phaffia rhodozyma  and having the sequence stated in SEQ ID No. 14.  
     
     
         20 . A vector including DNA encoding the soluble part (F 1 ) of the membrane bound (F 0 F 1  type) H + -ATPase or a portion of F 1  exhibiting ATPase activity, said DNA being derived from  Trichoderma reesei  and having the sequence stated in SEQ ID No. 16.  
     
     
         21 . An expression vector including DNA as defined in any one of claims  16 - 20  under the control of a promoter capable of directing the expression of said DNA in a prokaryotic or eukaryotic cell.  
     
     
         22 . A method of optimizing the formation of biomass or a desired product by a cell, characterized by expressing different levels of uncoupled ATPase activity in the cell, incubating the cell on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product at each level of ATPase expression, and choosing a level of ATPase expression at which the conversion rate is optimized.  
     
     
         23 . A method according to  claim 22 , wherein a number of specimens of said cell are transformed or transfected with their respective expression vector each including DNA encoding a different portion of the cytoplasmic part (F 1 ) of the membrane bound (F 0 F 1  type) H + -ATPase up to and including the entire F 1 , each portion exhibiting ATPase activity, said DNA in each expression vector being under the control of a promoter functioning in said cell, incubating each cell specimen on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product by each specimen, and choosing a specimen yielding an optimized conversion rate.  
     
     
         24 . A method according to  claim 22 , wherein a number of specimens of said cell are transformed or transfected with their respective expression vector including DNA encoding a portion of the cytoplasmic part (F 1 ) of the membrane bound (F 0 F 1  type) H + -ATPase up to and including the entire F 1 , said portion exhibiting ATPase activity, said DNA in the respective expression vectors being under the control of each of a series of promoters covering a broad range of promoter activities and functioning in said cell, incubating each cell specimen on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product by each specimen, and choosing a specimen yielding an optimized conversion rate.  
     
     
         25 . A method according to  claim 24 , wherein the respective expression vectors include DNA encoding different such portions of F 1  up to and including the entire F 1 , each DNA in respective expression vectors being under the control of each of a series of promoters covering a broad range of promoter activities and functioning in said cell.  
     
     
         26 . A method according to any one of claims  23 - 25 , wherein the promoter in each expression vector is an inducible promoter, and each cell specimen is grown at different concentrations of inducer.  
     
     
         27 . A method according to any one of claims  23 - 25 , wherein said DNA encoding a portion of F 1  up to and including the entire F 1  is homologous to said cell.  
     
     
         28 . A method according to any one of claims  23 - 25 , wherein said DNA encoding a portion of F 1  up to and including the entire F 1  is heterologous to said cell.  
     
     
         29 . A method according to any one of claims  23 - 25  wherein said DNA encoding a portion of F 1  up to and including the entire F 1  is derived from a prokaryotic organism.  
     
     
         30 . A method according to  claim 29 , wherein said DNA encoding a portion of F 1  up to and including the entire F 1  is derived from  Escherichia coli, Lactococcus lactis  or  Streptococcus thermophilus  and is selected from the group consisting of the gene encoding the F 1  subunit β or a portion thereof and various combinations of said gene or to portion with the genes encoding the F 1  subunits δ, α, γ and ε r portions thereof.  
     
     
         31 . A method according to  claim 30 , wherein said DNA encoding a portion of F 1  up to and including the entire F 1  is selected from the group consisting of the  E. coli  genes atpAGDC (coding for subunits α, γ, β, ε), atpAGD (coding for subunits α, γ, β, atpDC (coding for subunits β, ε) and atpD (coding for subunit β alone).  
     
     
         32 . A method according to any one of claims  23 - 25 , wherein said DNA encoding a portion of F 1  up to and including the entire F 1  is derived from a eukaryotic organism.  
     
     
         33 . A method according to  claim 32 , wherein said DNA encoding F 1  or a portion thereof is derived from  Saccharomyces cerevisiae, Phaffia rhodozyma  or  Trichoderma reesei  and is selected from the group consisting of the gene encoding the F 1  subunit β or a portion thereof and various combinations of said gene or portion with the genes encoding the other F 1  subunits or portions thereof.