Abstract:
The present invention provides methods for modifying cells. In particular, methods are provided which may be used to express an uncoupled ATPase activity in cells. By implementing these methods, the conversion of ATP to ADP can be increased in a cell without primary effects, e.g., on the other cellular metabolites or functions. The methods may therefore be used to increased ADP dependent metabolism, e.g., of a substrate. Accordingly, the methods of the invention are useful for improving the production of a biomass or other product of the ADP dependent metabolism of a substrate.

Description:
This invention relates to a method of improving the production of biomass or a desired product from a cell by inducing conversion of ATP to ADP without primary effects on other cellular metabolites or functions. The invention also relates to a method of optimizing the production of biomass or a desired product from a cell utilizing this first method. The desired product may for example be lactic acid produced by lactic acid bacteria and ethanol or carbon dioxide produced by yeast. 
     BACKGROUND OF THE INVENTION 
     A wide range of microorganisms are used for the production of various organic compounds and heterologous proteins. One example hereof is the production of lactic acid and other organic compounds by the lactic acid group of bacteria, which results in the acidification and flavouring of dairy products, better known as cheese and yoghurt production. 
     From the microorganism&#39;s point of view, the organic compounds which are excreted from the cells are often merely the by-product of a process that is vital to the cells: the production of various forms of free energy (ATP, NAD(P)H, membrane potential, etc.). Therefore, although many of the microorganisms which are being employed in these processes are reasonably well suited for the purpose, there is still a great potential for optimizing the productivity of these organisms when looking from the bioreactor point of view. Likewise, the production of heterologous proteins by a microorganism is not what the organism was adapted for and also here there is a potential for optimization. 
     Often when microorganisms are engineered for the purpose of optimizing an industrial production process, the reactions leading to the desired product will affect the delicate balance of co-factors involved in the energy metabolism of the cell. For instance if the glycolytic reactions producing lactate from sugar were somehow to be enhanced (e.g. by overexpressing the glycolytic enzymes) this would automatically lead to the convertion of ADP to ATP. The ratio between the concentrations of ATP and ADP is usually quite high in the growing cell ([ATP]/[ADP]&gt;10), and when the ratio [ATP]/[ADP] changes, the sum of [ATP] and [ADP] still remains virtually constant. Therefore, if in the example above, the enhanced production of ATP changes the [ATP]/[ADP] ratio from 10 to say 30, this will only marginally affect the concentration of ATP. The ADP concentration however will change by a factor of three. The cells will then hardly feel the surplus of ATP but the ADP pool in the cells may be depleted to such an extent that reactions in which ADP is a co-factor or allosteric regulator will be suppressed by the lack of ADP. The result may be that the total flux through the pathway (here through glycolysis) is only marginally increased. In the future, this situation is likely to occur more frequently, as the productivity of bioreactors are optimized by other means, and in these cases, it will be even more important (compared to the normal cell) to regenerate the ADP from ATP, in order to further increase the productivity. 
     Previously, attempts have been made to decrease the intracellular ATP concentration in yeast, employing sets of reactions which together form futile cycles, see EP patent No. 245 481. Often, the first reaction of a futile cycle is part of the regular metabolic network of the cell, for instance the phosphorylation of a glycolytic intermediate, coupled to the utilisation of ATP. The second reaction, which may also sometimes be part of the metabolic network, then de-phosphorylates the glycolytic intermediate without regenerating the ATP that was consumed in the first process, the overall effect being that a high energy phosphate bond is consumed. The limited success that this strategy has had so far, is probably due to the fact that it is impossible to obtain a significant futile flux without decreasing the concentration of the phosphorylated intermediate, thereby disturbing the cellular function and ultimately the growth. In addition, when the approach is to decrease the concentration of a glycolytic intermediate, this will effectively remove the substrate for the remaining part of the glycolysis, which will often result in a decreased flux through this pathway, rather than the desired increased flux. 
     Other strategies have been to use chemicals such as dinitrophosphate to stimulate the activity of the plasma membrane H + -ATPase by the addition of uncouplers of the membrane potential, or to genetically express the enzyme acid phosphatase in the cytoplasm, an enzyme that will remove phosphate groups from organic metabolites and proteins. However, both of these approaches suffer from the same inherent problem: they are unspecific and a range of cellular reactions/concentrations may be affected. For instance, the acid phosphatase will remove phosphate groups from essential metabolites and proteins, thus disturbing various metabolic fluxes and metabolic regulation. The uncoupling of the plasma membrane H + -ATPase will disturb the intracellular pH in addition to the gradient of numerous ions across the cytoplasmic membrane. Besides, the addition of chemicals such as dinitrophosphate is undesirable for most purposes. 
     SUMMARY OF THE INVENTION 
     The idea of the invention is to use a highly specific and clean way to increase the intracellular level of ADP, which does not suffer from the limitations described above: to express in a well-controlled manner an enzyme that has ATP-hydrolytic activity in the living cell without producing other products and without coupling this activity to energy conservation. Such an enzymatic activity is of course not likely to be found in a normal cell, because the cell would then loose some of its vital energy reservoir. 
     Accordingly the present invention provides a method of improving the production of biomass or a desired product from a cell, the method being 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. 
     One of the normal enzymes that comes closest to the ideal ATP-hydrolyzing enzyme, is the membrane bound H + -ATPase. This huge enzyme complex consists of two parts, the membrane integral part (F 0 ) and the cytoplasmic part (F 1 ). Together the two parts couples the hydrolysis of ATP to ADP and inorganic phosphate (P i ), to translocation of protons accross the cytoplasmic membrane, or vice versa, using the proton gradient to drive ATP synthesis from ADP and P i . 
     The method of the invention is conveniently carried out 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 the F 1  exhibiting ATPase activity. 
     The membrane bound H + -ATPase complex is found in similar form in prokaryotic as well as eukaryotic organisms, and thus F 1  and portions thereof expressing ATPase activity can be expressed in both prokaryotic and eukaryotic cells. 
     The organism from which the F1 ATPase or portions thereof is derived, or in which the F1 ATPase or portions thereof is expressed, may be selected from prokaryotes and eukaryotes, in particular from bacteria and eukaryotic microorganisms such as yeasts, other fungi and cell lines of higher organisms, in particular baker&#39;s and brewer&#39;s yeast. 
     A particularly interesting group of prokaryotes to which the method according to the invention can be implemented, i.a. in the dairy industry, are lactic acid bacteria of the genera Lactococcus, Streptococcus, Enterococcus, Lactobacillus and Leuconostoc, in particular strains of the species  Lactococcus lactis  and  Streptococcus thermophilus . Other interesting prokaryotes are bacteria belonging to the genera Escherichia, Zymomonas, Bacillus and Pseudomonas, in particular the species  Escherichia coli, Zymomonas mobilis, Bacillus subtilis  and  Pseudomonas putida.    
     In an expedient manner of carrying out the method according to the invention the 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 expressed in the cell. Said DNA encoding F 1  or a portion thereof may be derived from a prokaryotic or a eukaryotic organism, and it may be either homologous or heterologous to said cell. 
     The F 1  part of the bacterial H + -ATPase complex consists of several subunits that together are responsible for catalyzing ATP hydrolysis: the β-subunit is thought to carry the actual hydrolytic site for ATP hydrolysis, but in vitro ATPase activity requires that the β-subunit forms a complex together with the α- and γ-subunit (α 3 γβ 3 ). The activity of this complex is modulated by the ε-subunit, so that the in vitro activity of the α 3 γβ 3 ε complex is five fold less than the α 3 γβ 3  complex. 
     In a specific embodiment of the method according to the invention said DNA encoding F 1  or a catalytically active portion thereof, is derived from  Escherichia coli, Streptococcus thermophilus  or  Lactococcus lactis  and is selected from the group consisting of the gene encoding the F 1  subunit β or a catalytically active portion thereof and various combinations of said gene or portion with the genes encoding the F 1  subunits δ, α, γ and ε or catalytically active portions thereof. 
     In particular said DNA encoding F 1  or a portion thereof may be 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), atpDC (coding for subunits β, ε) and atpD (coding for subunit β alone). 
     Particularly interesting eukaryotes are the yeasts  Saccharomyces cerevisiae, Phaffia rhodozyma  or  Trichoderma reesei , and the DNA encoding F 1  or a portion thereof may be derived from such organisms 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. 
     Vectors 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, derived from the lactic acid bacteria  Lactococcus lactis  and  Streptococcus thermophilus  and from the yeasts  Saccharomyces cerevisiae, Phaffia rhodozyma  or  Trichoderma reesei  are also comprised by the invention as well as expression vectors including such DNA under the control of a promoter capable of directing the expression of said DNA in a prokaryotic or eukaryotic cell. 
     Specific vectors according to the invention are plasmids 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  (SEQ ID No. 1),  Lactococcus lactis  subsp.  lactis  (SEQ ID No. 6),  Streptococcus thermophilus  (SEQ ID No. 10),  Phaffia rhodozyma  (SEQ ID No. 14), and  Trichoderma reesei  (SEQ ID No. 16). 
     Further, the invention provides a method of optimizing the formation of biomass or a desired product by a cell, the method being 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. 
     Often, but not always, the optimization of a given product flux produced by a cell will entail the attainment of either maximum or minimum conversion rate of a substrate. 
     In an expedient manner of practicing this method of the invention 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 in each specimen, and choosing a specimen yielding an optimal conversion rate. In a particular embodiment of this manner, which is especially suited for scientific studies, the promoter in each expression vector is an inducible promoter, and each cell specimen is grown at different concentrations of inducer in order to fine-tune the optimal conversion rate. 
     In a preferred manner of practicing the above method of optimizing the performance of a cell 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 optimal conversion rate. In a more preferred embodiment of this manner, which is well suited to establish an optimal production strain, 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. 
     Also in this method of the invention the DNA encoding a portion of F 1  up to and including the entire F 1  may be derived from a prokaryotic or a eukaryotic organism, and it may be either homologous or heterologous to said organism. The specific DNAs mentioned above may also conveniently be employed in this method. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1. A linear representation of the plasmids constructed for modulating the cellular [ATP]/[ADP] ratio in  E. coli  (not drawn to scale). 
     FIG.  2 . Effect of induction of F 1 -ATPase activity on the growth of  E. coli  in batch culture. Cells were grown for more than 10 generations in minimal medium supplemented with glucose (0.4 g/l), ampicillin (0.1 g/l) and the indicated concentration of inducer, IPTG. 
     FIG.  3 . Effect of ATPase expression on the intracellular concentration of ATP and ADP (concentration in arbitrary units), and on the ratio [ATP]/[ADP]. 
     FIG. 4 Effect of increased ATPase expression on the glycolytic flux. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Many biosynthetic reactions in the living cell (anabolism), require an input of free energy (ATP), which is generated through a series of degrading reactions (catabolism). In the aerobic cell, there are two routes for ATP synthesis: 1) substrate level phosphorylation, where an energy rich phosphoryl group is transferred directly from a high energy intermediate metabolite to ADP, and 2) oxidative phosphorylation, where the free energy is first transformed into redox free energy by oxidizing the energy source, then into a proton gradient by respiration and finally the proton gradient is used by the H + -ATPase to drive ATP synthesis from ADP and inorganic phosphate. In other cases, e.g. anaerobic growth, there is only the first route, substrate level phosphorylation, that can be used for ATP synthesis. An example hereof is the homolactic LAB, where lactose is converted through the glycolytic pathway to lactic acid, which is excreted from the cells and thereby lowers the pH of the growth medium (usually milk products). With respect to ATP generation, homolactic fermentation is a very inefficient process, and only four moles of ATP are produced from 1 mole of lactose through substrate level phosphorylation. 
     The anabolic (ATP consuming) and catabolic (ATP producing) fluxes are normally well balanced in the living cell, and therefore, in the wild-type cell under normal growth conditions, the catabolic fluxes will be proportional to the anabolic fluxes. If a reaction is introduced that for instance hydrolyzes ATP in the cell and thereby lowers the cellular energy state (i.e. the [ATP]/[ADP] ratio), then either catabolism should increase or anabolism (growth) should decrease in order to make the consumption rate equal the production rate again. Which of these two scenarios will take place depends on whether, initially, the growth rate of the cell is limited through anabolism or through catabolism, i.e. whether there is a surplus or a shortage of energy in the cell to begin with. If there is a shortage of energy, then the rate of the anabolic reactions is limited by catabolism and these reactions will be sensitive to changes in the cellular energy state. Introduction of an ATP-hydrolyzing reaction is then most likely to affect the growth rate of the cells. On the other hand, if there is a surplus of energy, then the growth rate will be limited mainly by the anabolic reactions; the rate of anabolism will be insensitive to a decrease in the energy state, but the catabolic rate may increase due to a decrease in product inhibition at lower [ATP]/[ADP] ratio. 
     In vitro, the F 1  part of the H + -ATPase complex has been shown to have ATPase activity, see above. But so far nobody has managed to use the F 1  complex to stimulate the glycolytic flux, or even to show that the F 1  complex can hydrolyze ATP in intact cells. Indeed, when we first tried to overexpress the F1 complex, consisting of the genes for the subunits α, γ, β and ε, this had virtually no effect on the growth of  E. coli , even when the genes were transcribed from the maximally induced tac promoter and on a very high copy number vector (derived from pUC18). One skilled in the art of gene expression in  E. coli  will appreciate that this combination is one of the most efficient expression systems that exists for this organism. 
     We then decided to try to express different combinations of subunits of the F1 complex, in order to see if other combinations of subunits would be more powerful. Plasmids were constructed containing various combinations of the genes encoding the F 1  part of the bacterial F 1 F 0 -ATPase complex from  E. coli . The genes were expressed, either from an inducible (lac-type) promoter at various concentrations of inducer or from a series of constitutive promoters of varying promoter activity. These plasmids should express various levels of ATPase activity when introduced into the bacterial cell. Depending on which F 1  genes are present on the plasmid and the strength of the promoter which is used to drive the expression, we observed various degrees of inhibition of the growth of the cells harbouring these plasmids. Surprisingly, the beta subunit alone and in combination with the epsilon subunit turned out to be far more active in vivo than the entire F1 complex. 
     The objective of this work was to affect the energy state of the cells, as reflected in the ratio [ATP]/[ADP]. We therefore measured the intracellular concentration of ATP and ADP in growing cells expressing various activities of F 1 -ATPase. Indeed the ATP concentration decreased slightly with increasing ATPase activity and the ADP concentration increased, and therefore the [ATP]/[ADP] ratio decreased (the effect on the ATP concentration was less than the effect on the ADP concentration as expected, see above). We also calculated the glycolytic flux through the cells with various levels of ATPase activity. We found that the flux through the glycolytic pathway was first stimulated with increasing expression of ATPase activity, until a certain (optimal) ATPase activity which gave maximal glycolytic flux. Further increase of ATPase expression resulted in a lower glycolytic flux, due to a secondary effect of the ATPase activity on the growth of the cells. This emphasizes the need for optimization of gene expression rather than merely overexpressing the genes. 
     EXAMPLE 1 
     ATP Hydrolysis and Enhanced Glycolytic Flux in  Escherichia coli , Using an Inducible Promoter 
     Restriction enzymes, T4 DNA polymerase, calf intestine phosphatase (CIP) were obtained from Pharmacia. 
     Procedures for DNA isolation, cutting with restriction enzymes, filling in sticky DNA ends with T4 DNA polymerase in the presence of dATP, dCTP, dGTP and dTTP, treatment with calf intestine phophatase to remove phosphate groups from 5′ DNA ends and ligation of DNA fragments are carried out by standard methods as described by Maniatis et al., 1982. 
     Extraction and Measurement of ATP and ADP 
     0.9 ml of cell culture was mixed with 0.9 ml of (80° C.) phenol (equilibrated with 10 mM Tris, 1 mM EDTA pH=8) and immediately vortexed vigorously for 10 seconds. After 1 hour at room temperature the sample was vortexed again for 10 seconds and the two phases were separated by centrifugation at 14,000 rpm for 15 minutes, and then residual phenol in the water phase was removed by extraction with 1 volume of chloroform. ATP and ADP concentrations were then measured, using a luciferin-luciferase ATP monitoring kit (obtained from and used as recommended by LKB, except that 3 mM of phosphoenol-pyruvate was added). [ATP] was measured first. Subsequently the ADP in the same sample was converted to ATP by adding pyruvate kinase, and [ADP] was recorded as the concomitant increase in luminescence. 
     Construction of Plasmids Carrying Combinations of the  E. coil  atp Genes 
     The following combinations of  E. coli  genes coding for F 1  subunits were chosen for expressing ATPase activity in  E. coli : 1. atpAGDC (subunits α, γ, β, ε), 2. atpAGD (subunits α, γ, β) , 3. atpDC (subunits β, ε), and 4. atpD (subunit β alone). 
     Cloning of Fragments Carrying atp Genes onto pUC19 
     The plasmid pBJC917 (von Meyenburg, K., et al., 1984) which carries the entire atp operon was cut with 
     1) the restriction enzyme DraIII, and a 5009 bp DNA fragment containing the atpAGDC genes was isolated; 
     2) the restriction enzymes DraIII and Tth111I, and a 4106 bp DNA fragment containing the atpAGD genes was isolated; 
     3) the restriction enzymes DraIII and SacII, and a 2364 bp DNA fragment containing the atpDC genes was isolated; 
     4) the restriction enzymes AvaI and Tth111I, and a 1472 bp DNA fragment containing the atpD gene was isolated. 
     In all four cases the fragments were then treated with T4 DNA polymerase to create blunt ends, and subsequently the fragments were ligated into the cloning vector pUC19 (Yanisch-Perron et al.,1985) which had been cut with SmaI and treated with CIP. 
     The four ligation mixtures were transformed into the  E. coli  strain JM105 (Yanisch-Perron et al. ,1985), and the transformation mixtures were plated on LB (Luria-Bertani broth; Maniatis et al., 1982) plates containing 100 μg/ml ampicillin and 75 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal). In this strain background (JM105), plasmids formed by religation of pUC19 will give blue colonies, whereas plasmids that carry foreign DNA fragments inserted into the SmaI site of pUC19, will give white colonies. A number of white colonies from the four transformations were therefore picked for further analysis: plasmid DNA was isolated and analysed by cutting with various restriction enzymes. Clones were identified from each of the four series which had the desired fragment inserted into the SmaI site of pUC19, and in the proper orientation. These four plasmids were named, respectively: pATP-AGDC, pATP-AGD, pATP-DC and pATP-D, with reference to the specific atp genes carried by the plasmid. 
     Cloning Combinations of the atp Genes Under the Control of an Inducible (tac) Promoter 
     In order to be able to control the expression of the ATP-ase activity, we selected the expression vector pTTQ18 (Starck, 1987). This vector is a derivative of pUC18 (Yanisch-Perron et al. 1985), which carries a tac promoter and the lactose repressor gene, lacI. Immediately downstream of the tac promoter is a multiple cloning site (MCS; the polylinker from pUC18) in which genes can be inserted to be expressed from the tac promoter. The tac promoter is of the lac-type, i.e. repressed by the lactose repressor and inducible with isopropyl-β-D-thiogalactoside (IPTG). 
     The four plasmids, pATP-AGDC, pATP-AGD, pATP-DC and pATP-D were cut with KpnI and XbaI, which gave the four DNA fragments, 5023, 4120, 2378 and 1486 respectively. After purification, the fragments were ligated into the cloning vector, pTTQ18, which had also been cut with KpnI and XbaI (see FIG.  1 ). The ligation mixtures were transformed into  E. coil  K-12 MC1000 (Casabadan and Cohen, 1980), and the transformation mixtures were plated on LB plates containing 100 μg/ml ampicillin. A number of colonies from the four transformations were therefore picked for further analysis: plasmid DNA was isolated and analysed by cutting with various restriction enzymes. Clones were identified from each of the four series which had the desired fragment inserted into the MCS of pTTQ18 in the proper orientation. These four plasmids were named, respectively: pTAC-AGDC, pTAC-AGD, pTAC-DC and pTAC-D, with reference to the specific atp genes carried by these plasmids and the tac promoter used for their expression. For the purpose of subsequent physiological studies, the plasmids were transformed into the  E. coli  K-12 strain LM3118, which is used routinely for physiological experiments in this laboratory. The corresponding names for the LM3118 strain carrying these four plasmids are PJ4332, PJ4333, PJ4335 and PJ4334, respectively. 
     Effect of Induction of ATPase Activity on the Growth of  E. coli  on Plates 
     The strains containing the four plasmids were streaked on LB plates containing ampicillin (100 μg/ml) and 1 mM of IPTG which should give maximum expression from the tac promoter. Table I shows how the four strains responded: the strain carrying plasmid pATP-AGDC, which contains the genes for the four subunits, α, γ, β and ε, was only very slightly affected in growth, even in the presence of 1 mM IPTG. The other three plasmids, pTAC-AGD, pTAC-DC and pTAC-D caused severe growth inhibition in the presence of 1 mM IPTG, where colonies were no longer visible. With intermediate concentrations of IPTG, 0.01 mM and 0.1 mM, the plasmids affected the growth of their host cells to different extents: pTAC-AGD was the most active, giving rise to a strong inhibition of growth already with 0.01 mM IPTG, a concentration which gave only a slight inhibition with the plasmid pTAC-DC and no inhibition of the strain with pTAC-D. With 0.1 mM IPTG, colonies were hardly visible for the strain that carried the pTAC-AGD, the plasmid pTAC-DC caused strong growth inhibition, whereas the effect of pTAC-D was significant but small. 
     
       
         
               
               
               
               
               
               
             
           
               
                 TABLE I 
               
               
                   
               
               
                   
                   
                   
                 0.01 mM 
                 0.1 mM 
                   
               
               
                 Strain 
                 Plasmid 
                 - IPTG 
                 IPTG 
                 IPTG 
                 1 mM IPTG 
               
               
                   
               
             
             
               
                 PJ4332 
                 pTAC-AGDC 
                 ++++ 
                 ++++ 
                 ++++ 
                 +++ 
               
               
                 PJ4333 
                 pTAC-AGD 
                 ++++ 
                 ++ 
                 + 
                 − 
               
               
                 PJ4335 
                 pTAC-DC 
                 ++++ 
                 +++ 
                 + 
                 − 
               
               
                 PJ4334 
                 pTAC-D 
                 ++++ 
                 ++++ 
                 ++ 
                 − 
               
               
                   
               
               
                 ++++ = normal colony size; +++ = slight inhibition; ++ = ½ normal size; + = {fraction (1/10)} normal size; − = no growth  
               
             
          
         
       
     
     The effect of ATPase expression from the four plasmids above was also studied in the  E. coli  mutant LM3115, in which the entire atp operon on the chromosome is deleted, but which grows with almost wild-type growth rate on LB medium. With this strain transformed with the four plasmids we observed a similar pattern of growth inhibition on LB plates as a function of IPTG concentration. This shows that the effect of ATPase expression was independent of the presence of the normal atp operon. 
     Effect of Induction of ATPase Activity on the Growth of  E. coli  in Liquid Cultures 
     The effect of induction of ATPase was also studied with cells grown in liquid cultures. For this purpose we chose the strain PJ4333, carrying the plasmid pTAC-AGD, because this plasmid appears to be the most active with respect to the inhibitory effect on the growth of  E. coli . FIG. 2 shows the growth of PJ4333 in minimal medium supplemented with a limiting concentration of glucose (0.4 g/l) and ampicillin (0.1 g/l), without IPTG and in the presence of increasing concentrations of IPTG. We observed that the growth rate of the strain was practically constant (within some 10%) with increasing amounts of IPTG up to about 30 μM. At higher than 40 μM IPTG, the growth of the cells were slightly inhibited, in accordance with the experiments on plates, see above. 
     However, what was affected was the final density of cells that one obtains from the limited amount of glucose that was included in each culture: The more ATPase that is expressed in the cells, the lower the yield of cell mass. Apparently, the cells become less economic with respect to converting the glucose into biomass, or in other words they consume more glucose per cell synthesized. If this is due to the expression of ATPase activity, then we would expect to see an effect hereof on the energy state of the cells. We therefore measured the concentrations of ATP and ADP in the cells growing with different expression levels of ATPase activity. 
     Indeed, the intracellular ATP concentration decreased gradually and the ADP concentration increased, with increased expression of ATPase; therefore the [ATP]/[ADP] ratio decreased with increased expression of ATPase, which implies that the increased glucose consumption is the result of increased ATP convertion to ADP, see FIG.  3 . The actual flux of glucose through the cells (J gluc , mmol glucose/g cell dry weight/hour) is also interesting, because this value tells us whether the performance of the cell increased as the ATPase activity increased. J gluc  can be calculated from the yield, Y (g cell dry weight/mol glucose) and the specific growth rate of the culture, μ (1/hours): 
     
       
         
           J 
           gluc 
           =μ/Y 
         
       
     
     FIG. 4 shows how the flux of glucose changed as the activity of ATPase increased: the glycolytic flux increased gradually as the ATPase expression increased, until a maximum was reached (at 30 μM IPTG). Further increase of ATPase expression had a slightly negative effect on the glucose flux. This was probably because the energy state of the cells became so low that this had a negative effect on some anabolic reactions, since the growth rate was lower for the culture that was grown in the presence of 40 μM IPTG. 
     The expression of subunits of the F 1  part of the bacterial H + -ATPase lowers the energy state of the bacterial cell. This is due to hydrolysis of ATP into ADP and P i . The expression of ATPase activity does not affect the growth rate of  E. coli  much at low levels of expression, but the efficiency by which the substrate is converted into biomass was strongly reduced. Under the set of conditions used here, the expression of ATPase activity has a stimulatory effect on the rate by which the cells consumes the exogenous glucose. 
     EXAMPLE 2 
     Expression of F 1 -ATPase Activity from Constitutive Promoters in  E. coli    
     In example 1 we used a lac-type promoter system to modulate the expression of the F 1  ATPase subunits in  E. coli . However, for the optimization of gene expression for instance in industrial bioreactors or for the use in fermented food products, the use of lac type promoters is not always feasible. In this example we illustrate the optimization of F 1 -ATPase expression in  E. coli , using a series of constitutive promoters of different strength, to control the expression of the atpAGD genes which here originates from  E. coli . The constitutive promoters (CP promoters) were selected from a library of artificial promoters which had previously been cloned onto a shuttle vector for  E. coli  and  L. lactis , pAK80 (Israelsen et al., 1995) as described in our co-pending PCT patent application PCT/DK97/00342. The selected plasmid derivatives of pAK80 were pCP34, pCP41 and CP44 (CPX cloning vectors). The atpAGD fragment from pTAC-AGD (from example 1) was first subcloned in a polylinker in order to have the atpAGD fragment flanked by two BamHI sites. Subsequently, this BamHI fragment was cloned into the unique BamHI site downstream of the CP promoters on the plasmids pCP34, pCP41 and CP44, resulting in the plasmids, pCP34::atpAGD, pCP34::2atpAGD, pCP41::atpAGD and CP44::atpAGD, where pCP34::2atpAGD contains two atpAGD fragments in tandem. 
     Subsequently, the strains were characterized with respect to growth rate, growth yield and glycolytic flux in glucose minimal medium supplemented with 200 μg/ml erythromycin, essentially as described in example 1, see table 2. 
     The expression of the F 1 -ATPase subunits had a slightly negative effect on the growth rate as the expression level increased. The effect on growth yield was much stronger and at the highest expression level the growth yield had dropped to 40% of the initial value. The glycolytic flux was stimulated 70% at the highest expression level of ATPase, and at this expression level the growth rate was lowered by 30%. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Effect of expression of uncoupled F 1 -ATPase activity 
               
               
                 ( E. coli  α, γ, β subunits) in  E. coli   
               
             
          
           
               
                   
                 Biomass 
                   
                 Glucose 
                   
                   
                   
               
               
                   
                 yield 
                 Growth 
                 flux 
                 Biomass 
                 Growth 
                 Glucose 
               
               
                   
                 gdw/mmol 
                 rate, μ 
                 mmol gluc- 
                 yield 
                 rate 
                 flux 
               
               
                 Plasmid 
                 glucose 
                 h-1 
                 cose/h/gdw 
                 % 
                 % 
                 % 
               
               
                   
               
             
          
           
               
                 pCP41 
                 0.067 
                 0.47 
                 6.9 
                 100 
                 100 
                 100 
               
               
                 pCP41::atpAGD 
                 0.047 
                 0.42 
                 9.1 
                  69 
                  90 
                 131 
               
               
                 pCP34 
                 0.063 
                 0.41 
                 6.6 
                 100 
                 100 
                 100 
               
               
                 pCP34::atpAGD 
                 0.034 
                 0.34 
                 9.9 
                  54 
                  81 
                 149 
               
               
                 pCP44 
                 0.067 
                 0.44 
                 6.5 
                 100 
                 100 
                 100 
               
               
                 pCP44::atpAGD 
                 0.027 
                 0.30 
                 11.2 
                  40 
                  69 
                 172 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 3 
     Expression of  E. coli  F 1 -ATPase Activity from Constitutive Promoters in  L. lactis    
     The plasmids from example 2 which express the  E. coli  F 1 -ATPase subunits to various extent are also capable of replicating in  L. lactis , and could therefore be used to test whether the  E. coli  F 1 -ATPase subunits can be used to hydrolyse ATP in  L. lactis.    
     The plasmids pCP34::atpAGD, pCP34::2atpAGD and pCP41::atpAGD, were transformed into the  L. lactis  subspecies cremoris strain, MG1363, which is used routinely for physiological experiments in this laboratory. In addition we transformed the respective vectors, pCP34 and pCP41 in order to have proper control strains. Subsequently, the resulting transformants were characterized with respect to growth rate, growth yield and glycolytic flux, in comparison to the respective vectors, pCP34 and pCP41, by growing the various cultures in defined medium (SA medium) supplemented with a limiting concentration of glucose (0.1%), see table 3. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Expression of  E. coli  F 1 -ATPase in  L. lactis   
               
             
          
           
               
                   
                 Biomass 
                   
                 Glucose 
                   
                   
                   
               
               
                   
                 yield 
                 Growth 
                 flux 
                 Biomass 
                 Growth 
                 Glucose 
               
               
                   
                 gdw/mmol 
                 rate, μ 
                 mmol gluc- 
                 yield 
                 rate 
                 flux 
               
               
                 Plasmid 
                 glucose 
                 h-1 
                 cose/h/gdw 
                 % 
                 % 
                 % 
               
               
                   
               
             
          
           
               
                 pCP34 
                 0.073 
                 0.664 
                 9.161 
                 100 
                 100 
                 100 
               
               
                 pCP34::atpAGD 
                 0.071 
                 0.653 
                 9.230 
                 97.5 
                 98.3 
                 100.8 
               
               
                 pCP34::2atpAGD 
                 0.069 
                 0.655 
                 9.560 
                 94.6 
                 98.7 
                 104.4 
               
               
                 pCP41 
                 0.072 
                 0.645 
                 8.925 
                 100 
                 100 
                 100 
               
               
                 pCP41::atpAGD 
                 0.070 
                 0.590 
                 8.461 
                 96.5 
                 91.5 
                 94.8 
               
               
                   
               
             
          
         
       
     
     The results show that the plasmids pCP34::atpAGD and pCP34::2atpAGD did affect the growth yield and the glycolytic flux to some extent, but the plasmids were far less efficient in  L. lactis , compared to  E. coli . This was probably a consequence of a lower expression of the  E. coli  ATPase subunits, or some of these, in  L. lactis , due to a lower copy number of the pAK80 vector in  L. lactis  (5-10), and due to differences in the translational efficiency of the three individual atp genes which originates from  E. coli . The plasmid pCP41::atpAGD also resulted in a lower growth yield, indicating that also in this case uncoupled ATP hydrolysis was taking place. However, the pCP41::atpAGD plasmid had a relatively strong inhibitory effect on the growth rate and therefore the glycolytic flux was not increased by this plasmid. It is possible that the heterologous expression of  E. coli  ATPase subunits resulted in growth inhibition due to effects other than ATP hydrolysis, e.g. by interfering with the function of the  L. lactis  F 1 F 0  H + -ATPase complex. 
     EXAMPLE 4 
     Expression of  L. lactis  F 1 -ATPase Subunits β and ε, in  L. lactis    
     In the example above we showed that the expression of F 1 -ATPase subunits from  E. coli  in  L. lactis , resulted in only a small stimulation of the glycolytic flux. It is possible that the heterologous expression of  E. coli  ATPase subunits resulted in growth inhibition due to effects other than ATP hydrolysis, e.g. by interfering with the function of the  L. lactis  F 1 F 0  H + -ATPase complex. In the present example we have expressed the  L. lactis  F 1 -ATPase subunits, β and ε, in  L. lactis , as this appeared to be an effective combination of subunits when expressed in  E. coli , see example 1. The atpDC Llc  genes from  L. lactis  subspecies  cremoris  (SEQ ID No. 1) was cloned on a 2.5 kb HindIII fragment into the HindIII restriction site on the standard cloning vector, pBluescript, into  E. coli  K-12, strain BOE270. Subsequently, the atpDC Llc  genes were cut out on a 2.5 kb BamHI-SalI fragment and cloned into 5 expression vectors, pCP32, pCP34, pCP37, pCP41 and pCP44 which had been digested with BamHI and SalI, resulting in the plasmids pCP32::atpDC Llc , pCP34::atpDC Llc , pCP37::atpDC Llc , pCP41::atpDC Llc  and pCP44::atpDC Llc , respectively, where the lacLM genes downstream of the CP promoters, have been replaced with the atpDC Llc  genes. These plasmids should express the  L. lactis  F 1 -ATPase subunits, β and ε, to different extent. The plasmids were then transformed into MG1363 with selection for the erythromycin resistance carried by these vectors. Experiments were then performed to test whether the constructs resulted in convertion of ATP into ADP in  L. lactis . The strains carrying the different constructs was then grown in GM17 medium supplemented with 5 μg/ml erythromycin. The plasmids did not have a strong effect on the growth rate of the cultures, which remained close to the growth rate of the respective vector control plasmids. The yield of biomass, however, decreases for all the cultures by up to 17%, which shows that the cultures did indeed express uncoupled ATPase activity, see table 4. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Effect of expression of  L. lactis  β and ε 
               
               
                 subunits on acid production by  L. lactis,  at 30° C. and 
               
               
                 with initial pH 6.7. 
               
             
          
           
               
                   
                   
                   
                 Acid formation, 
               
               
                   
                   
                   
                 relative to biomass* 
               
               
                 Plasmid 
                 Biomass* 
                 Final pH* 
                 % of vector 
               
               
                   
               
             
          
           
               
                 pCP34 
                 5.08 
                 4.27 
                 100 
               
               
                 pCP34::atpDC11c 
                 4.72 
                 4.31 
                 98.0 
               
               
                 pCP41 
                 4.66 
                 4.34 
                 100 
               
               
                 pCP41::atpDC11c 
                 5.21 
                 4.24 
                 113.5 
               
               
                 pCP37 
                 4.89 
                 4.28 
                 100 
               
               
                 pCP37::atpDC11c 
                 4.63 
                 4.24 
                 116.1 
               
               
                 pCP32 
                 4.86 
                 4.34 
                 100 
               
               
                 pCP32::atpDC11c 
                 3.95 
                 4.36 
                 116.9 
               
               
                   
               
               
                 *Each value is the average of 3-4 independent cultures. The acid production was calculated from the pH change, and normalized by the biomass produced.  
               
             
          
         
       
     
     The GM17 growth medium used in these experiments contains a surplus of glucose (1%), and growth only stops when the pH of the growth medium becomes lower than approximately pH 4.3. To some extent, this mimics the situation that the lactic acid bacteria experience during cheese and yoghurt production. In this medium, the growth yield, in terms of the final cell mass of the cultures, reflects the acid production by these cultures. 
     In these cultures, the expression of F 1 -ATPase subunits will increase three fold at approximately OD600 equal to 1.5. This is a consequence of the three fold amplification of the plasmid copy number that has been shown to take place at this point of the growth curve. In reality, the effect of expressing the F 1 -ATPase subunits may therefore be larger. 
     To test this hypothesis, we grew some of the strains which expressed the  L. lactis  F 1 -ATPase subunits β and ε in batch cultures of GM17 medium which had been adjusted to pH 5.9, see Table 5. In addition, the temperature of the growth medium may also affect the plasmid copy number and thus the expression of the F 1 -ATPase subunits. The experiments were therefore performed at 37° C. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Effect of expression of  L. lactis  β and ε 
               
               
                 subunits on acid production by  L. lactis,  at 37° C. and 
               
               
                 with initial pH 5.9. 
               
             
          
           
               
                   
                   
                   
                 Acid formation, 
               
               
                   
                 Biomass* 
                   
                 relative to biomass* 
               
               
                 Plasmid 
                 OD 450   
                 Ffinal pH* 
                 % of vector 
               
               
                   
               
             
          
           
               
                 pCP34 
                 1.24 
                 4.95 
                 100 
               
               
                 pCP34::atpDC 11c   
                 1.06 
                 4.87 
                 141.4 
               
               
                 pCP37 
                 1.00 
                 4.96 
                 100 
               
               
                 pCP37::atpDC 11c   
                 0.58 
                 4.92 
                 188.4 
               
               
                   
               
             
          
         
       
     
     Clearly, the effect of the F 1 -ATPase activity was much stronger under these growth conditions: the amount of acid produced was almost doubled for the strain carrying the plasmid pCP37::atpDC llc . 
     EXAMPLE 5 
     Expression of the F 1 -ATPase Subunits, α, γ, and β, from  L. lactis  Subspecies  cremoris  in  L. lactis  Subspecies  cremoris    
     In example 4, only the  L. lactis  F 1 -ATPase β and ε subunits were expressed in  L. lactis . However, from the experiments with  E. coli  (example 1), we know that the simultaneous expression of subunits α, γ, and β, is a more powerful combination, which could also be the case for  L. lactis . In order to obtain the same strong stimulation of the glycolytic flux and acid production in  L. lactis , a set of vectors similar to the vectors described in example 4 was constructed, in which the atpAGD Llc  genes derived from  L. lactis , encoding the subunits α, γ, and β (SEQ ID No. 1) was expressed from CP promoters with different activities. The atpAGD Llc  genes from  L. lactis  was cloned on a 2.5 kb BamHI-SalI fragment into the 5 vectors, pCP32, pCP34, pCP37, pCP41 and pCP44, resulting in the plasmids, pCP32::atpAGD Llc , pCP34::atpAGD Llc , pCP37::atpAGD Llc , pCP41::atpAGD Llc , pCP44::atpAGD Llc , respectively, where the lacLM genes downstream of the CP promoters, has been replaced with the atpAGD Llc  genes. These plasmids will express the  L. lactis  F 1 -ATPase subunits α, γ, and β, to different extent. The plasmids were transformed into MG1363 with selection for the Erythromycin resistance carried by these vectors. Experiments were then performed to show that the constructs were effective in ATP hydrolysis in  L. lactis  and to what extent the glycolytic flux was enhanced, by growing the five different constructs in GM17 medium supplemented with erythromycin, and measuring the growth rate, ATP and ADP concentrations, the yield of biomass and the rate of acid production. 
     EXAMPLE 6 
     Expression of F 1 -ATPase Subunits from  L. lactis  Subsp  lactis,  in  L. lactis  Subspecies  lactis    
     In the examples 3-5 above, we used the strain  L. lactis  subsp.  cremoris , MG1363. This strain is plasmid-free and is used routinely in our laboratory as a simple model organism for our physiological studies. But strains belonging to the subspecies  lactis  are also important in cheese production. We therefore cloned and sequenced the atpAGD Lll  genes from  L. lactis  subsp.  lactis,  (SEQ ID No. 6). Subsequently, a 4.2 kb fragment habouring the atpAGD Lll  genes was cloned into 5 vectors, pCP32, pCP34, pCP37, pCP41 and pCP44, resulting in the plasmids, pCP32::atpAGD Lll , pCP34::atpAGD Lll , pCP37::atpAGD Lll , pCP41::atpAGD Lll , pCP44::atpAGD Lll , respectively. These plasmids were then transformed into  L. lactis  subsp.  lactis  as described in example 3. The resulting strains with different expression levels of the F 1 -ATPase subunits α, γ and β were then used to characterize the effect hereof on the growth yield, growth rate, glycolytic flux, and the cellular energy state of  L. lactis  subsp.  lactis , as described in the examples 1-5. 
     EXAMPLE 7 
     Expression of F 1 -ATPase Subunits from  S. thermophilus , ST3, in  S. thermophilus , ST3 
     In the examples 3-6 above, we used strains of the genus Lactococcus. These strains are important in cheese production. As starter cultures for yoghurt production, the dairy industry often uses strains of  S. thermophilus . We therefore cloned and sequenced the atpAGD St  genes from  S. thermophilus , strain ST3 (SEQ ID No. 10). Subsequently, a 4.2 kb fragment habouring the atpAGD St  genes was cloned into the 5 vectors, pCP32, pCP34, pCP37, pCP41 and pCP44, resulting in the plasmids, pCP32::atpAGD St , pCP34::atpAGD St , pCP37::atpAGD St , pCP41::atpAGD St , pCP44::atpAGD St , respectively. These plasmids were then transformed into  S. thermophilus  strain ST3. The resulting strains have different expression levels of the F 1 -ATPase subunits α, γ, and β, and were then used to characterize the effect hereof on the growth yield, growth rate, glycolytic flux, and the cellular energy state of  S. thermophilus , as described in the previous examples. 
     EXAMPLE 8 
     Expression of a Truncated F 1 -ATPase β Subunit from  Phaffia rhodozyma  in  Saccharomyces cerevisiae    
     In this example we show that uncoupled F 1 -ATPase expression can also be used to hydrolyze ATP in yeast cells of  Saccharomyces cerevisiae.    
     A cDNA gene library was prepared from total RNA, isolated from  Phaffia rhodozyma , by cloning the cDNA fragments into the expression vector, pYES2.0. One of the resulting plasmids, pATPbeta, gave rise to an ade +  phenotype in the  Saccharomyces cerevisiae  strain, W301 , which carries a mutation in the ADE2 gene. Sequencing of the clone revealed a 0.9 kb insert, which encoded a protein of 254 amino acids (SEQ ID No. 14). The encoded protein had a very high homology to the C-terminal part of F 1 -ATPase β subunits from other organisms, prokaryotic as well as eukaryotic, including the β subunit from  S. cerevisiae  (86% identity). 
     The ADE2 mutation results in starvation for an intermediate further down in the purine metabolism, AICAR (which under normal conditions is produced by ADE3, two steps further down in this pathway). AICAR is essential for de novo biosynthesis of AMP and GMP, and ADE2 mutants therefore need an alternative purine source in the growth medium. However, there is an alternative route for synthesis of AICAR which involves some of the genes involved in histidine biosynthesis. These genes are normally repressed under the conditions used for the complementation, but when the HIS3 gene is introduced on a plasmid, this complements the ADE2 mutation because the cells start to produce AICAR. Since AICAR is a precursor for ATP, it is likely that a lack of ATP (or increased levels of ADP and AMP) provides a signal to derepress the HIS3 gene and generates AICAR (which will subsequently end up as ATP). Indeed, cross-pathway regulation between purine and histidine biosynthesis has been found in yeast and involves the transcription factors BAS1 and BAS2. A reasonable explanation for the ade +  phenotype conferred by the plasmid, is therefore that the plasmid gives rise to ATP hydrolysis in the cytoplasm, thereby effecting the concentrations of adenine nucleotides in the cytoplasm. 
     Importantly, this truncated β subunit from  Phaffia rhodozyma  that was encoded on pATPbeta, included the region of the β subunit which is thought to encode the catalytic site for ATP hydrolysis. The truncation of the N-terminal part of the β subunit probably means that the protein will no longer be exported into the mitochondrion, but should stay within the yeast cytoplasm. 
     The truncated β subunit pATPbeta is expressed from a gal promoter, i.e. it can be induced with galactose. If the truncated β subunit encoded by the clone is active in ATP hydrolysis it should result in a decrease in the growth yield, and at sufficiently high expression level, we should also observe inhibition of growth. The strain which expressed the truncated β subunit and a control strain (which contained a plasmid pHIS3 containing a HIS3 gene from  Phaffia rhodozyma ), were streaked on plates containing galactose as the energy source, which will give maximal expression of the truncated β subunit. Indeed, the growth of the strain which expressed the truncated β subunit was strongly inhibited by the presence of galactose, whereas the control strain grew normally. As a control, the growth of the two strains were also compared on a plate containing glucose as the energy source, conditions under which the expression of the β subunit should be repressed, and indeed we observed little difference in growth of the two strains on these plates, see table 6. 
     Subsequently, for the purpose of the physiological investigations, the two strains were converted into Rho −  strains (petit mutants, defective in oxidative phosphorylation) by standard treatment with ethidium bromide. The induction with galactose caused even stronger inhibition of growth in the Rho −  background, which further indicates that the cause of the growth inhibition is uncoupled ATP hydrolysis in the cytoplasm. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Effect of expression of a truncated F 1 -ATPase β 
               
               
                 subunit from  Phaffia rhodozyma  in  S. cerevisiae  on SC 
               
               
                 plates 
               
             
          
           
               
                   
                 Strain/plasmid 
                 SC-ura + glucose 
                 SC-ura + galactose 
               
               
                   
                   
               
               
                   
                 Rho + /pATPbeta 
                 +++++ 
                 + 
               
               
                   
                 Rho + /pHIS3 
                 ++++ 
                 +++ 
               
               
                   
                 Rho − /pATPbeta 
                 +++++ 
                 − 
               
               
                   
                 Rho − /pHIS3 
                 ++++ 
                 +++ 
               
               
                   
                   
               
             
          
         
       
     
     Growth experiments were performed to measure the resulting changes in the ATP/ADP ratio and the degree of stimulation of the glycolytic flux and ethanol formation, essentially as described in the examples above, and to show that the truncated 1 subunit from  Phaffia rhodozyma  is active with respect to converting ATP into ADP in the yeast cell. 
     EXAMPLE 9 
     Expression of F 1 -ATPase β subunit from  Trichoderma reesei  in  Saccharomyces cerevisiae    
     In this example we show that the expression of the F 1 -ATPase β subunit from the filamentous fungus,  Trichoderma reesei  can be used to improve the product formation of  Saccharomyces cerevisiae.    
     The gene encoding the F 1 -ATPase β subunit homologue from  Trichoderma reesei  was isolated from a cDNA library, inserted into a multicopy expression vector, pAJ401. DNA sequencing (SEQ ID 16) revealed that the cloned gene had very high homology to the β subunits from  Neurospora crassa  (91% identity),  Kluyveromyces lactis  (68%) and  Saccharomyces cerevisiae  (68%). Importantly, the first 43 amino acids in this β subunit, which encodes the signal for exporting the protein into the mitochondria, was homologous to the N-terminal part of the β subunit from  Neurospora crassa  (58% identity), but not to that of  Saccharomyces cerevisiae . It is therefore likely that the β subunit from  Trichoderma reesei  will stay within the cytoplasm when expressed in  Saccharomyces cerevisiae . This is important for the many cases where the fermentation is carried out anaerobically, because in these cases it is probably most efficient if the ATP hydrolysis takes place in the cytoplasm. Alternatively, in those cases where the β subunit is transported into the mitochondrion, it may be useful to genetically modify the β subunit so that is stays within the cytoplasm. 
     The gene encoding the F 1 -ATPase β subunit homologue from  Trichoderma reesei  was expressed in  S.cerevisiae  strain VW1b (MAT alpha, leu2-3/112, ura3-52, trp1-289, his3D1MAL2-8c, SUC2). To test whether the presence of the  T. reesei  β subunit resulted in ATP hydrolysis in the cytoplasm of the  Saccharomyces cerevisiae  host cells, we measured the intracellular concentrations of ATP, ADP and AMP, under various growth conditions in cultures of two strains expressing the β subunit (pATPβ34 and pATPβ44) and a strain carrying the vector plasmid, pFL60, see table 7. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Effect of expression of  T. reesei  β subunit on 
               
               
                 ATP, ADP and AMP concentrations  S. cerevisiae   
               
             
          
           
               
                   
                 ATP 
                 ADP 
                 AMP 
                   
               
               
                 Strain 
                 μmol/gdw 
                 μmol/gdw 
                 μmol/gdw 
                 ATP/ADP ratio 
               
               
                   
               
             
          
           
               
                 Aerobic/exp.phase 
                   
                   
                   
                   
               
               
                 pATPβ34 
                 19.3 
                 5.58 
                 3.31 
                 3.5 
               
               
                 pATPβ44 
                 13.9 
                 5.15 
                 3.25 
                 2.7 
               
               
                 pVECTOR 
                 16.6 
                 5.47 
                 3.43 
                 3.0 
               
               
                 Aerobic/stat.phase 
               
               
                 pATPβ34 
                 9.30 
                 4.03 
                 2.89 
                 2.3 
               
               
                 pATPβ44 
                 8.99 
                 3.90 
                 2.42 
                 2.3 
               
               
                 pVECTOR 
                 19.5 
                 4.62 
                 2.87 
                 4.2 
               
               
                 anaerobic/stat.phase 
               
               
                 pATPβ34 
                 4.39 
                 11.6 
                 6.72 
                 0.4 
               
               
                 pATPβ44 
                 3.14 
                 10.5 
                 6.65 
                 0.3 
               
               
                 pVECTOR 
                 8.84 
                 10.2 
                 6.37 
                 0.9 
               
               
                   
               
               
                 *according tc Bergmeyer (1985)  
               
             
          
         
       
     
     The β subunit did not appear to have a significant effect on the concentrations of ATP, ADP and AMP in cells growing on glucose in the exponential growth phase. The reason is probably that the ATP concentration that the homeostatic control of ATP synthesis can here keep up with the extra drain on ATP conferred by the β subunit F 1 -ATPase activity. Indeed, the growth rate of these cultures was unaffected by the presence of the F 1 -ATPase activity, see table 7. But in the stationary cultures the concentration of ATP decreased significantly in the cultures expressing the β subunit, compared to the control. The effect was strongest in the anaerobically grown cultures where the ATP was lowered by a factor of 2-3. In these cultures, ATP must be generated through oxidative phosphorylation, (which is not even an option for the anaerobic cultures), and any effect of uncoupled ATP hydrolysis should therefore indeed be stronger in these cells. 
     Shake Flask Cultivations of Cultures Expressing the F 1 -ATPase β Subunit Homologue in  Saccharomyces cerevisiae    
     Shake flask cultivations were performed under microaerobic/anaerobic conditions with volume ratio 1/1.25 and no agitation; with 400 ml growth media in 500 ml Erlenmeyers on magnetic stirring. The growth media contained 5 g/l of glucose and amino acids and bases according to synthetic complete medium (SC−ura+0.5%G). OD 600 ) was monitored during the cultivation (OD 600 =1.0 is equal to 0.3 g/l dry weight). Ethanol and glucose were measured with HPLC (Waters, Sugar-Pak or IC-Pak columns). Production of ethanol (grams of ethanol per grams of cell dry weight) is shown in Table 8. 
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Effect of expression of  T. reesei  β subunit, 
               
               
                 on fluxes of ethanol and glucose in  S. cerevisiae   
               
             
          
           
               
                   
                   
                   
                   
                 J gluc   
                 J etch   
               
               
                   
                   
                   
                   
                 relative 
                 relative 
               
               
                   
                 μ 
                 J gluc   
                 J etch   
                 to 
                 to 
               
               
                 Strain 
                 h −1   
                 g/h/gdw 
                 g/h/gdw 
                 control 
                 control 
               
               
                   
               
             
          
           
               
                 pATPβ34 
                 0.40 
                 2.811 
                 1.190 
                 107.7 
                 105.6 
               
               
                 pATPβ44 
                 0.40 
                 2.750 
                 1.187 
                 105.3 
                 105.3 
               
               
                 pVECTOR 
                 0.39 
                 2.611 
                 1.127 
                 100 
                 100 
               
               
                 control 
               
               
                   
               
             
          
         
       
     
     These data show that the presence of the  T. reesei  F 1 -ATPase β subunit resulted in an increased flux of glucose, as well as ethanol, in the  Saccharomyces cerevisiae  host cells. 
     REFERENCES 
     Casabadan, M. J., and Cohen, S. N. (1980).  J. Mol. Biol.,  138, 179-207. 
     Israelsen, H. (1995). Cloning and partial characterization of regulated promoters from  Lactococcus lactis  Tn917-lacZ integrants with the new promoter probe vector, pAK80.  Appl. Environ. Microbiol.,  61, 2540-2547. 
     Maniatis, T., Fritsch, E. F., and Sambrook, J., (1982).  Molecular cloning . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 
     Miller, J. H., (1972).  Experiments in molecular genetics , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 
     Starck, M. J. R. (1987). Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in  Escherichia coli. Gene  51, 255-267. 
     von Meyenburg, K., Joergensen, B. B., and van Deurs, B. (1984). Physiological and morphological effects of overproduction of membrane-bound ATP synthase in  Escherichia coli  K-12.  EMBO J.  3, 1791-1797. 
     Yanisch-Perron, C., Vieira, J., and Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.  Gene  33, 103-109. 
     
       
         
           
             17 
           
           
             
               4815 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Lactococcus lactis subsp. cremoris 
               MG1363 
             
             
               CDS 
               26..550 
               /codon_start= 26
               /product= “ATPase subunit”
               /gene= “atpH”
               /standard_name= “delta subunit of the F1 portion
               of the F0F1 ATPase”
               /label= delta-subunit
 
             
             
               CDS 
               742..2241 
               /codon_start= 742
               /product= “ATPase subunit”
               /gene= “atpA”
               /standard_name= “alpha subunit of the F1 portion
               of the F0F1 ATPase”
               /label= alpha-subunit
 
             
             
               CDS 
               2260..3126 
               /codon_start= 2260
               /product= “ATPase subunit”
               /gene= “atpG”
               /standard_name= “gamma subunit of the F1 portion
               of the F0F1 ATPase”
               /label= gamma-subunit
 
             
             
               CDS 
               3301..4707 
               /codon_start= 3301
               /product= “ATPase subunit”
               /gene= “atpD”
               /standard_name= “beta subunit of the F1 portion of
               the F0F1 ATPase”
               /label= beta-subunit
 
             
              1
TATCTCGCTA AGTTAGGAGA ATAAG ATG ACA AAA GTA AAT TCA CAA AAA TAC       52
                            Met Thr Lys Val Asn Ser Gln Lys Tyr
                              1               5
AGT AAA GCT TTA CTT GAG GTC GCC CGA GAA AAA GGA CAA CTT GAA GCA      100
Ser Lys Ala Leu Leu Glu Val Ala Arg Glu Lys Gly Gln Leu Glu Ala
 10                  15                  20                  25
ATT CTT ACT GAA GTT AGC GAA ATG ATT CAG CTT TTC AAA GAA AAT AAC      148
Ile Leu Thr Glu Val Ser Glu Met Ile Gln Leu Phe Lys Glu Asn Asn
                 30                  35                  40
TTA GGT GCT TTT TTA GCA AAT GAA GTT TAT TCA TTC TCT GCT AAA TCT      196
Leu Gly Ala Phe Leu Ala Asn Glu Val Tyr Ser Phe Ser Ala Lys Ser
             45                  50                  55
GAA TTG ATT GAT ACT TTG CTT CAA ACT TCA TCA GAA GTG ATG TCA AAT      244
Glu Leu Ile Asp Thr Leu Leu Gln Thr Ser Ser Glu Val Met Ser Asn
         60                  65                  70
TTC CTG AAT ACT ATT CGT TCT AAT GGA CGT CTA GCT GAC CTC GGA GAA      292
Phe Leu Asn Thr Ile Arg Ser Asn Gly Arg Leu Ala Asp Leu Gly Glu
     75                  80                  85
ATA CTT GAA GAA ACT AAA AAT GCA GCA GAT GAC ATG TTC AAA ATT GCT      340
Ile Leu Glu Glu Thr Lys Asn Ala Ala Asp Asp Met Phe Lys Ile Ala
 90                  95                 100                 105
GAC GTT GAA GTT GTT TCA AGT ATT GCA TTG TCA GAA GCT CAA ATT GAA      388
Asp Val Glu Val Val Ser Ser Ile Ala Leu Ser Glu Ala Gln Ile Glu
                110                 115                 120
AAA TTT AAA GCA ATG GCT AAA TCA AAA TTT GAT TTA AAC GAA GTA ACA      436
Lys Phe Lys Ala Met Ala Lys Ser Lys Phe Asp Leu Asn Glu Val Thr
            125                 130                 135
GTA ATT AAT ACA GTC AAT GAA AAA ATT CTC GGA GGA TTC ATT GTG AAC      484
Val Ile Asn Thr Val Asn Glu Lys Ile Leu Gly Gly Phe Ile Val Asn
        140                 145                 150
TCT CGT GGA AAA ATT ATT GAC GCC TCA TTA AAA ACA CAA TTG GCT AAA      532
Ser Arg Gly Lys Ile Ile Asp Ala Ser Leu Lys Thr Gln Leu Ala Lys
    155                 160                 165
ATC GCC GCT GAA ATC CTC TAATCAGGAT AGAAAAATTT TCTTCCTTTG             580
Ile Ala Ala Glu Ile Leu
170                 175
TTAAAAACTT AGTGGAGAAT TTTTCAAACT CAAACTGTTA AACTTTTGAA AACATGCAAA    640
GGTAATTTTA AAACTTGCTT ATTCATGCTC AAAAAGTATA ACTGCAGTTT AAAGCTAAAT    700
AGCCTTGAAC TAGTAAAAAA TTTCTAGAAG GGAGCATATT T TTG GCA ATT AAA        753
                                              Leu Ala Ile Lys
                                                1
GCT AAT GAA ATC AGC TCA CTG ATT AAA AAA CAA ATT GAA AAT TTC ACA      801
Ala Asn Glu Ile Ser Ser Leu Ile Lys Lys Gln Ile Glu Asn Phe Thr
  5                  10                  15                  20
CCA GAT TTT GAA GTT GCT GAA ACT GGT GTC GTT ACC TAT GTT GGT GAT      849
Pro Asp Phe Glu Val Ala Glu Thr Gly Val Val Thr Tyr Val Gly Asp
                 25                  30                  35
GGT ATC GCG CGT GCC TAT GGC CTT GAA AAT GCG ATG AGC GGT GAG CTT      897
Gly Ile Ala Arg Ala Tyr Gly Leu Glu Asn Ala Met Ser Gly Glu Leu
             40                  45                  50
GTT GAG TTT TCA AAT GGT ATA CTT GGT ATG GCG CAA AAC TTG GAT GCT      945
Val Glu Phe Ser Asn Gly Ile Leu Gly Met Ala Gln Asn Leu Asp Ala
         55                  60                  65
ACA GAC GTT GGT ATT ATC GTA CTT GGT GAT TTC CTC TCA ATT CGT GAA      993
Thr Asp Val Gly Ile Ile Val Leu Gly Asp Phe Leu Ser Ile Arg Glu
     70                  75                  80
GGT GAC ACT GTT AAA CGT ACA GGT AAA ATC ATG GAA ATC CAA GTT GGT     1041
Gly Asp Thr Val Lys Arg Thr Gly Lys Ile Met Glu Ile Gln Val Gly
 85                  90                  95                 100
GAA GAA CTC ATC GGA CGT GTT GTA AAC CCA CTT GGA CAA CCC GTT GAT     1089
Glu Glu Leu Ile Gly Arg Val Val Asn Pro Leu Gly Gln Pro Val Asp
                105                 110                 115
GGA CTT GGA GAA CTT AAT ACA GGT AAA ACT CGT CCA GTT GAA GCA AAA     1137
Gly Leu Gly Glu Leu Asn Thr Gly Lys Thr Arg Pro Val Glu Ala Lys
            120                 125                 130
GCT CCT GGT GTT ATG CAA CGT AAA TCA GTC TCT GAG CCA TTA CAA ACT     1185
Ala Pro Gly Val Met Gln Arg Lys Ser Val Ser Glu Pro Leu Gln Thr
        135                 140                 145
GGT CTT AAA GCG ATT GAT GCC CTC GTT CCA ATT GGA CGT GGA CAA CGT     1233
Gly Leu Lys Ala Ile Asp Ala Leu Val Pro Ile Gly Arg Gly Gln Arg
    150                 155                 160
GAA TTA ATT ATC GGA GAC CGT CAA ACT GGT AAA ACT TCA GTC GCT ATT     1281
Glu Leu Ile Ile Gly Asp Arg Gln Thr Gly Lys Thr Ser Val Ala Ile
165                 170                 175                 180
GAT GCA ATC TTG AAC CAA AAA GGT CAA GAT ATG ATT TGT ATC TAT GTT     1329
Asp Ala Ile Leu Asn Gln Lys Gly Gln Asp Met Ile Cys Ile Tyr Val
                185                 190                 195
GCG ATT GGA CAA AAA GAG TCA ACA GTT CGT ACA CAA GTT GAA ACG CTC     1377
Ala Ile Gly Gln Lys Glu Ser Thr Val Arg Thr Gln Val Glu Thr Leu
            200                 205                 210
CGT AAA CTC GGT GCG ATG GAT TAT ACA ATC GTC GTA ACT GCG TCA GCT     1425
Arg Lys Leu Gly Ala Met Asp Tyr Thr Ile Val Val Thr Ala Ser Ala
        215                 220                 225
TCT CAA CCT TCT CCA CTC CTT TAC ATC GCT CCT TAC GCT GGA GCT GCA     1473
Ser Gln Pro Ser Pro Leu Leu Tyr Ile Ala Pro Tyr Ala Gly Ala Ala
    230                 235                 240
ATG GGT GAA GAA TTT ATG TAT AAC GGT AAA CAT GTC TTG GTT GTT TAT     1521
Met Gly Glu Glu Phe Met Tyr Asn Gly Lys His Val Leu Val Val Tyr
245                 250                 255                 260
GAT GAT TTA TCT AAA CAA GCG GTC GCT TAC CGT GAA CTT TCT CTC TTG     1569
Asp Asp Leu Ser Lys Gln Ala Val Ala Tyr Arg Glu Leu Ser Leu Leu
                265                 270                 275
CTC CGT CGT CCA CCA GGT CGT GAA GCA TAC CCA GGT GAC GTT TTC TAC     1617
Leu Arg Arg Pro Pro Gly Arg Glu Ala Tyr Pro Gly Asp Val Phe Tyr
            280                 285                 290
TTG CAC TCA CGT CTT TTG GAA CGT GCT GCT AAA TTG TCT GAT GAT CTT     1665
Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys Leu Ser Asp Asp Leu
        295                 300                 305
GGT GGT GGA TCA ATG ACG GCT TTG CCA TTC ATT GAA ACA CAA GCA GGT     1713
Gly Gly Gly Ser Met Thr Ala Leu Pro Phe Ile Glu Thr Gln Ala Gly
    310                 315                 320
GAT ATC TCA GCT TAT ATT CCA ACA AAC GTT ATC TCT ATT ACC GAC GGT     1761
Asp Ile Ser Ala Tyr Ile Pro Thr Asn Val Ile Ser Ile Thr Asp Gly
325                 330                 335                 340
CAA ATT TTC CTT GAA AAT GAC TTG TTC TAT TCA GGT GTA CGT CCT GCC     1809
Gln Ile Phe Leu Glu Asn Asp Leu Phe Tyr Ser Gly Val Arg Pro Ala
                345                 350                 355
ATT GAT GCT GGT TCA TCA GTA TCA CGT GTT GGT GGT GCC GCA CAA ATC     1857
Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly Ala Ala Gln Ile
            360                 365                 370
AAA GCC ATG AAG AAA GTA GCT GGT ACT TTG CGT CTT GAC CTT GCG TCG     1905
Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu Asp Leu Ala Ser
        375                 380                 385
TTC CGT GAA CTT GAA GCC TTT ACA CAA TTT GGT TCT GAC CTT GAT GAA     1953
Phe Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser Asp Leu Asp Glu
    390                 395                 400
GCG ACT CAA GCA AAA TTG AAT CGT GGT CGT CGT ACC GTT GAA GTC TTG     2001
Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr Val Glu Val Leu
405                 410                 415                 420
AAA CAA CCA TTG CAC AAA CCA TTG GCT GTT GAA AAA CAA GTT TTG ATT     2049
Lys Gln Pro Leu His Lys Pro Leu Ala Val Glu Lys Gln Val Leu Ile
                425                 430                 435
CTC TAT GCA TTG ACT CAT GGT CAT CTT GAT AAT GTT CCA GTT GAT GAT     2097
Leu Tyr Ala Leu Thr His Gly His Leu Asp Asn Val Pro Val Asp Asp
            440                 445                 450
GTT CTT GAT TTT GAA ACT AAA ATG TTC GAT TTC TTC GAT GCA AAT TAT     2145
Val Leu Asp Phe Glu Thr Lys Met Phe Asp Phe Phe Asp Ala Asn Tyr
        455                 460                 465
GCA GAT CTC TTG AAC GTA ATT ACT GAC ACT AAA GAT TTG CCA GAA GAA     2193
Ala Asp Leu Leu Asn Val Ile Thr Asp Thr Lys Asp Leu Pro Glu Glu
    470                 475                 480
GCA AAA CTT GAC GAA GCA ATT AAA GCA TTC AAA AAT ACA ACG AAT TAT     2241
Ala Lys Leu Asp Glu Ala Ile Lys Ala Phe Lys Asn Thr Thr Asn Tyr
485                 490                 495                 500
TAATAAGGAG GCTAACTA ATG GGA GCT TCA CTT AAC GAA ATA AAA ACT AAG     2292
                    Met Gly Ala Ser Leu Asn Glu Ile Lys Thr Lys
                      1               5                  10
ATT GCG TCA ACA AAG AAA ACA AGT CAA ATC ACA GGT GCC ATG CAA ATG     2340
Ile Ala Ser Thr Lys Lys Thr Ser Gln Ile Thr Gly Ala Met Gln Met
             15                  20                  25
GTT TCT GCT GCT AAA CTT CAA AAA GCA GAA TCT CAC GCT AAA GCT TTT     2388
Val Ser Ala Ala Lys Leu Gln Lys Ala Glu Ser His Ala Lys Ala Phe
         30                  35                  40
CAG ACT TAT GCT GAA AAA GTA CGT AAG ATT ACG ACT GAC TTA GTT TCA     2436
Gln Thr Tyr Ala Glu Lys Val Arg Lys Ile Thr Thr Asp Leu Val Ser
     45                  50                  55
AGC GAT AAT GAG CCG GCC AAA AAT CCG ATG ATG ATT AAA CGT GAA GTC     2484
Ser Asp Asn Glu Pro Ala Lys Asn Pro Met Met Ile Lys Arg Glu Val
 60                  65                  70                  75
AAG AAA ACT GGC TAT CTC GTT ATC ACA TCA GAT CGT GGG CTT GTT GGC     2532
Lys Lys Thr Gly Tyr Leu Val Ile Thr Ser Asp Arg Gly Leu Val Gly
                 80                  85                  90
AGT TAT AAT TCA AAT ATT TTG AAG TCT GTT ATA AGT AAT ATA CGT AAA     2580
Ser Tyr Asn Ser Asn Ile Leu Lys Ser Val Ile Ser Asn Ile Arg Lys
             95                 100                 105
CGC CAC ACA AAT GAG AGT GAG TAT ACA ATA CTT GCC CTT GGT GGT ACG     2628
Arg His Thr Asn Glu Ser Glu Tyr Thr Ile Leu Ala Leu Gly Gly Thr
        110                 115                 120
GGA GCG GAC TTT TTC AAA GCC CGT AAC GTC AAA GTT TCT TAT GTT CTT     2676
Gly Ala Asp Phe Phe Lys Ala Arg Asn Val Lys Val Ser Tyr Val Leu
    125                 130                 135
CGC GGA CTT TCA GAT CAA CCG ACC TTT GAA GAG GTT CGG GCA ATT GTT     2724
Arg Gly Leu Ser Asp Gln Pro Thr Phe Glu Glu Val Arg Ala Ile Val
140                 145                 150                 155
ACA GAA GCC GTA GAA GAA TAT CAA GCA GAA GAA TTC GAT GAA CTC TAT     2772
Thr Glu Ala Val Glu Glu Tyr Gln Ala Glu Glu Phe Asp Glu Leu Tyr
                160                 165                 170
GTT TGT TAC AAC CAC CAT GTG AAC TCA TTG GTA AGT GAG GCA CGG ATG     2820
Val Cys Tyr Asn His His Val Asn Ser Leu Val Ser Glu Ala Arg Met
            175                 180                 185
GAA AAA ATG TTA CCT ATT TCT TTT GAT GAA AAA GGT GAC GAA AAA GCA     2868
Glu Lys Met Leu Pro Ile Ser Phe Asp Glu Lys Gly Asp Glu Lys Ala
        190                 195                 200
TCT CTT GTT ACA TTT GAA TTA GAA CCA GAT CGT GAA ACA ATC TTA AAT     2916
Ser Leu Val Thr Phe Glu Leu Glu Pro Asp Arg Glu Thr Ile Leu Asn
    205                 210                 215
CAG TTG TTG CCG CAA TAT GCT GAA AGT ATG ATT TAT GGC TCA ATT GTT     2964
Gln Leu Leu Pro Gln Tyr Ala Glu Ser Met Ile Tyr Gly Ser Ile Val
220                 225                 230                 235
GAT GCA AAA ACA GCA GAA CAT GCT GCA GGT ATG ACC GCA ATG CGT ACT     3012
Asp Ala Lys Thr Ala Glu His Ala Ala Gly Met Thr Ala Met Arg Thr
                240                 245                 250
GCA ACA GAT AAT GCA CAT TCT GTC ATT AAT GAT TTA ACC ATT CAA TAT     3060
Ala Thr Asp Asn Ala His Ser Val Ile Asn Asp Leu Thr Ile Gln Tyr
            255                 260                 265
AAC CGT GCT CGT CAA GCT TCA ATT ACG CAA GAA ATT ACG GAA ATT GTT     3108
Asn Arg Ala Arg Gln Ala Ser Ile Thr Gln Glu Ile Thr Glu Ile Val
        270                 275                 280
GCG GGT GCT TCA GCG CTA TAATTACTGT CAAACATTAT TCTCAATGTT            3156
Ala Gly Ala Ser Ala Leu
    285
ACGATTTATC AACTTGAGGA ATAAATGTTC TGTCAGTAAA GGCTTTGAAT TTTAAATACG   3216
TTTGTCAGTA AATTTTTACT GATTAGCTTA AAAATGAATA GAAATTCTGT TGTTAGACAG   3276
AAAATAAAAA CAGGAGGAAA AACA TTG AGT TCT GGT AAA ATT ACT CAG GTT      3327
                           Leu Ser Ser Gly Lys Ile Thr Gln Val
                             1               5
ATC GGT CCC GTC GTT GAC GTG GAA TTT GGT TCT GAT GCC AAA CTG CCT     3375
Ile Gly Pro Val Val Asp Val Glu Phe Gly Ser Asp Ala Lys Leu Pro
 10                  15                  20                  25
GAG ATT AAC AAT GCC TTG ATT GTC TAC AAA GAT GTC AAT GGT TTA AAA     3423
Glu Ile Asn Asn Ala Leu Ile Val Tyr Lys Asp Val Asn Gly Leu Lys
                 30                  35                  40
ACA AAA ATT ACT CTT GAA GTT GCT TTG GAA CTT GGT GAT GGT GCA GTT     3471
Thr Lys Ile Thr Leu Glu Val Ala Leu Glu Leu Gly Asp Gly Ala Val
             45                  50                  55
CGT ACG ATC GCT ATG GAA TCT ACT GAT GGA TTG ACT CGT GGA CTT GAA     3519
Arg Thr Ile Ala Met Glu Ser Thr Asp Gly Leu Thr Arg Gly Leu Glu
         60                  65                  70
GTC CTT GAT ACA GGT AAA GCG GTC AGC GTT CCT GTT GGT GAA TCT ACT     3567
Val Leu Asp Thr Gly Lys Ala Val Ser Val Pro Val Gly Glu Ser Thr
     75                  80                  85
CTT GGT CGT GTT TTT AAT GTC CTT GGT GAC GTT ATT GAT GGT GGA GAA     3615
Leu Gly Arg Val Phe Asn Val Leu Gly Asp Val Ile Asp Gly Gly Glu
 90                  95                 100                 105
GAT TTC CCT GCT GAT GCA GAA CGT AAT CCT ATC CAC AAG AAA GCT CCA     3663
Asp Phe Pro Ala Asp Ala Glu Arg Asn Pro Ile His Lys Lys Ala Pro
                110                 115                 120
ACT TTT GAC GAA TTG TCA ACT GCA AAT GAA GTT CTT GTA ACA GGG ATT     3711
Thr Phe Asp Glu Leu Ser Thr Ala Asn Glu Val Leu Val Thr Gly Ile
            125                 130                 135
AAA GTT GTC GAT TTA CTT GCC CCT TAT CTT AAA GGT GGG AAA GTC GGA     3759
Lys Val Val Asp Leu Leu Ala Pro Tyr Leu Lys Gly Gly Lys Val Gly
        140                 145                 150
CTC TTC GGT GGT GCC GGT GTT GGT AAA ACC GTC CTT ATC CAA GAA TTG     3807
Leu Phe Gly Gly Ala Gly Val Gly Lys Thr Val Leu Ile Gln Glu Leu
    155                 160                 165
ATT CAC AAT ATT GCC CAA GAA CAC GGT GGT ATT TCT GTA TTT ACA GGT     3855
Ile His Asn Ile Ala Gln Glu His Gly Gly Ile Ser Val Phe Thr Gly
170                 175                 180                 185
GTT GGC GAT CGT ACT CGT GAC GGG AAT GAC CTT TAC TGG GAA ATG AAA     3903
Val Gly Asp Arg Thr Arg Asp Gly Asn Asp Leu Tyr Trp Glu Met Lys
                190                 195                 200
GAA TCA GGC GTT ATT GAA AAA ACA GCC ATG GTC TTT GGT CAA ATG AAT     3951
Glu Ser Gly Val Ile Glu Lys Thr Ala Met Val Phe Gly Gln Met Asn
            205                 210                 215
GAA CCA CCT GGA GCA CGT ATG CGT GTT GCC CTT ACT GGT TTA ACA ATT     3999
Glu Pro Pro Gly Ala Arg Met Arg Val Ala Leu Thr Gly Leu Thr Ile
        220                 225                 230
GCG GAA TAT TTC CGT GAT GTT CAA GGA CAA GAC GTA TTG CTT TTC ATC     4047
Ala Glu Tyr Phe Arg Asp Val Gln Gly Gln Asp Val Leu Leu Phe Ile
    235                 240                 245
GAT AAC ATC TTC CGT TTC ACT CAA GCT GGT TCA GAA GTT TCT GCC CTT     4095
Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly Ser Glu Val Ser Ala Leu
250                 255                 260                 265
TGG GGA CGT ATG CCT TCT GCC GTT GGT TAC CAA CCA ACT CTT GCA ACT     4143
Trp Gly Arg Met Pro Ser Ala Val Gly Tyr Gln Pro Thr Leu Ala Thr
                270                 275                 280
GAA ATG GTT CAA TTA CAG GAA CGT ATC ACT TCT ACT AAG AAG GGT TCT     4191
Glu Met Val Gln Leu Gln Glu Arg Ile Thr Ser Thr Lys Lys Gly Ser
            285                 290                 295
GTT ACA TCT ATC CCA GCG ATT TAT GTC CCT GCC GAT GAC TAT ACT GAC     4239
Val Thr Ser Ile Pro Ala Ile Tyr Val Pro Ala Asp Asp Tyr Thr Asp
        300                 305                 310
CCA GCG CCA GCT ACA GCC TTC GCT CAC TTG GAC GCA ACA ACT AAC TTG     4287
Pro Ala Pro Ala Thr Ala Phe Ala His Leu Asp Ala Thr Thr Asn Leu
    315                 320                 325
GAA CGT CGT TTG ACA CAA ATG GGT ATC TAT CCA GCC GTT GAC CCA CTT     4335
Glu Arg Arg Leu Thr Gln Met Gly Ile Tyr Pro Ala Val Asp Pro Leu
330                 335                 340                 345
GCT TCA TCA TCA CGT GCG CTT ACA CCT GAA ATT GTT GGT GAA GAA CAC     4383
Ala Ser Ser Ser Arg Ala Leu Thr Pro Glu Ile Val Gly Glu Glu His
                350                 355                 360
TAT GAA GTT GCA ATG GAA GTT CAA CGT GTC CTT CAA CGC TAC AAA GAA     4431
Tyr Glu Val Ala Met Glu Val Gln Arg Val Leu Gln Arg Tyr Lys Glu
            365                 370                 375
TTG CAA GAT ATC ATT GCC ATT CTT GGT ATG GAT GAA TTG TCA GAT GAT     4479
Leu Gln Asp Ile Ile Ala Ile Leu Gly Met Asp Glu Leu Ser Asp Asp
        380                 385                 390
GAA AAA ATT CTC GTT GGA CGT GCA CGT CGT ATC CAA TTC TTC CTT TCA     4527
Glu Lys Ile Leu Val Gly Arg Ala Arg Arg Ile Gln Phe Phe Leu Ser
    395                 400                 405
CAA AAC TTC CAC GTT GCT GAA CAG TTT ACT GGT CAA CCT GGT TCA TAT     4575
Gln Asn Phe His Val Ala Glu Gln Phe Thr Gly Gln Pro Gly Ser Tyr
410                 415                 420                 425
GTA CCA ATT GAC AAA ACA GTT CAT GAC TTC AAG GAA ATT TTG GAA GGT     4623
Val Pro Ile Asp Lys Thr Val His Asp Phe Lys Glu Ile Leu Glu Gly
                430                 435                 440
AAA TAT GAC GAA GTC CCT GAA GAT GCT TTC CGT GGA GTA GGT CCA ATT     4671
Lys Tyr Asp Glu Val Pro Glu Asp Ala Phe Arg Gly Val Gly Pro Ile
            445                 450                 455
GAA GAC GTA CTT GCA AAA GCA AAA TCA ATG GGT TAT TAATTCGATT          4717
Glu Asp Val Leu Ala Lys Ala Lys Ser Met Gly Tyr
        460                 465
TCTTATGAAA TGACAAAGTG AAAATACATT ATTGAATCGC AAAATTTACT GACAATAATT   4777
CTGTCGTAAG TGCTCACTTT TAAGTTGTTC CGATCGTT                           4815
 
           
           
             
               175 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              2
Met Thr Lys Val Asn Ser Gln Lys Tyr Ser Lys Ala Leu Leu Glu Val
  1               5                  10                  15
Ala Arg Glu Lys Gly Gln Leu Glu Ala Ile Leu Thr Glu Val Ser Glu
             20                  25                  30
Met Ile Gln Leu Phe Lys Glu Asn Asn Leu Gly Ala Phe Leu Ala Asn
         35                  40                  45
Glu Val Tyr Ser Phe Ser Ala Lys Ser Glu Leu Ile Asp Thr Leu Leu
     50                  55                  60
Gln Thr Ser Ser Glu Val Met Ser Asn Phe Leu Asn Thr Ile Arg Ser
 65                  70                  75                  80
Asn Gly Arg Leu Ala Asp Leu Gly Glu Ile Leu Glu Glu Thr Lys Asn
                 85                  90                  95
Ala Ala Asp Asp Met Phe Lys Ile Ala Asp Val Glu Val Val Ser Ser
            100                 105                 110
Ile Ala Leu Ser Glu Ala Gln Ile Glu Lys Phe Lys Ala Met Ala Lys
        115                 120                 125
Ser Lys Phe Asp Leu Asn Glu Val Thr Val Ile Asn Thr Val Asn Glu
    130                 135                 140
Lys Ile Leu Gly Gly Phe Ile Val Asn Ser Arg Gly Lys Ile Ile Asp
145                 150                 155                 160
Ala Ser Leu Lys Thr Gln Leu Ala Lys Ile Ala Ala Glu Ile Leu
                165                 170                 175
 
           
           
             
               500 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              3
Leu Ala Ile Lys Ala Asn Glu Ile Ser Ser Leu Ile Lys Lys Gln Ile
  1               5                  10                  15
Glu Asn Phe Thr Pro Asp Phe Glu Val Ala Glu Thr Gly Val Val Thr
             20                  25                  30
Tyr Val Gly Asp Gly Ile Ala Arg Ala Tyr Gly Leu Glu Asn Ala Met
         35                  40                  45
Ser Gly Glu Leu Val Glu Phe Ser Asn Gly Ile Leu Gly Met Ala Gln
     50                  55                  60
Asn Leu Asp Ala Thr Asp Val Gly Ile Ile Val Leu Gly Asp Phe Leu
 65                  70                  75                  80
Ser Ile Arg Glu Gly Asp Thr Val Lys Arg Thr Gly Lys Ile Met Glu
                 85                  90                  95
Ile Gln Val Gly Glu Glu Leu Ile Gly Arg Val Val Asn Pro Leu Gly
            100                 105                 110
Gln Pro Val Asp Gly Leu Gly Glu Leu Asn Thr Gly Lys Thr Arg Pro
        115                 120                 125
Val Glu Ala Lys Ala Pro Gly Val Met Gln Arg Lys Ser Val Ser Glu
    130                 135                 140
Pro Leu Gln Thr Gly Leu Lys Ala Ile Asp Ala Leu Val Pro Ile Gly
145                 150                 155                 160
Arg Gly Gln Arg Glu Leu Ile Ile Gly Asp Arg Gln Thr Gly Lys Thr
                165                 170                 175
Ser Val Ala Ile Asp Ala Ile Leu Asn Gln Lys Gly Gln Asp Met Ile
            180                 185                 190
Cys Ile Tyr Val Ala Ile Gly Gln Lys Glu Ser Thr Val Arg Thr Gln
        195                 200                 205
Val Glu Thr Leu Arg Lys Leu Gly Ala Met Asp Tyr Thr Ile Val Val
    210                 215                 220
Thr Ala Ser Ala Ser Gln Pro Ser Pro Leu Leu Tyr Ile Ala Pro Tyr
225                 230                 235                 240
Ala Gly Ala Ala Met Gly Glu Glu Phe Met Tyr Asn Gly Lys His Val
                245                 250                 255
Leu Val Val Tyr Asp Asp Leu Ser Lys Gln Ala Val Ala Tyr Arg Glu
            260                 265                 270
Leu Ser Leu Leu Leu Arg Arg Pro Pro Gly Arg Glu Ala Tyr Pro Gly
        275                 280                 285
Asp Val Phe Tyr Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys Leu
    290                 295                 300
Ser Asp Asp Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Phe Ile Glu
305                 310                 315                 320
Thr Gln Ala Gly Asp Ile Ser Ala Tyr Ile Pro Thr Asn Val Ile Ser
                325                 330                 335
Ile Thr Asp Gly Gln Ile Phe Leu Glu Asn Asp Leu Phe Tyr Ser Gly
            340                 345                 350
Val Arg Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly
        355                 360                 365
Ala Ala Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu
    370                 375                 380
Asp Leu Ala Ser Phe Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser
385                 390                 395                 400
Asp Leu Asp Glu Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr
                405                 410                 415
Val Glu Val Leu Lys Gln Pro Leu His Lys Pro Leu Ala Val Glu Lys
            420                 425                 430
Gln Val Leu Ile Leu Tyr Ala Leu Thr His Gly His Leu Asp Asn Val
        435                 440                 445
Pro Val Asp Asp Val Leu Asp Phe Glu Thr Lys Met Phe Asp Phe Phe
    450                 455                 460
Asp Ala Asn Tyr Ala Asp Leu Leu Asn Val Ile Thr Asp Thr Lys Asp
465                 470                 475                 480
Leu Pro Glu Glu Ala Lys Leu Asp Glu Ala Ile Lys Ala Phe Lys Asn
                485                 490                 495
Thr Thr Asn Tyr
            500
 
           
           
             
               289 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              4
Met Gly Ala Ser Leu Asn Glu Ile Lys Thr Lys Ile Ala Ser Thr Lys
  1               5                  10                  15
Lys Thr Ser Gln Ile Thr Gly Ala Met Gln Met Val Ser Ala Ala Lys
             20                  25                  30
Leu Gln Lys Ala Glu Ser His Ala Lys Ala Phe Gln Thr Tyr Ala Glu
         35                  40                  45
Lys Val Arg Lys Ile Thr Thr Asp Leu Val Ser Ser Asp Asn Glu Pro
     50                  55                  60
Ala Lys Asn Pro Met Met Ile Lys Arg Glu Val Lys Lys Thr Gly Tyr
 65                  70                  75                  80
Leu Val Ile Thr Ser Asp Arg Gly Leu Val Gly Ser Tyr Asn Ser Asn
                 85                  90                  95
Ile Leu Lys Ser Val Ile Ser Asn Ile Arg Lys Arg His Thr Asn Glu
            100                 105                 110
Ser Glu Tyr Thr Ile Leu Ala Leu Gly Gly Thr Gly Ala Asp Phe Phe
        115                 120                 125
Lys Ala Arg Asn Val Lys Val Ser Tyr Val Leu Arg Gly Leu Ser Asp
    130                 135                 140
Gln Pro Thr Phe Glu Glu Val Arg Ala Ile Val Thr Glu Ala Val Glu
145                 150                 155                 160
Glu Tyr Gln Ala Glu Glu Phe Asp Glu Leu Tyr Val Cys Tyr Asn His
                165                 170                 175
His Val Asn Ser Leu Val Ser Glu Ala Arg Met Glu Lys Met Leu Pro
            180                 185                 190
Ile Ser Phe Asp Glu Lys Gly Asp Glu Lys Ala Ser Leu Val Thr Phe
        195                 200                 205
Glu Leu Glu Pro Asp Arg Glu Thr Ile Leu Asn Gln Leu Leu Pro Gln
    210                 215                 220
Tyr Ala Glu Ser Met Ile Tyr Gly Ser Ile Val Asp Ala Lys Thr Ala
225                 230                 235                 240
Glu His Ala Ala Gly Met Thr Ala Met Arg Thr Ala Thr Asp Asn Ala
                245                 250                 255
His Ser Val Ile Asn Asp Leu Thr Ile Gln Tyr Asn Arg Ala Arg Gln
            260                 265                 270
Ala Ser Ile Thr Gln Glu Ile Thr Glu Ile Val Ala Gly Ala Ser Ala
        275                 280                 285
Leu
 
           
           
             
               469 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              5
Leu Ser Ser Gly Lys Ile Thr Gln Val Ile Gly Pro Val Val Asp Val
  1               5                  10                  15
Glu Phe Gly Ser Asp Ala Lys Leu Pro Glu Ile Asn Asn Ala Leu Ile
             20                  25                  30
Val Tyr Lys Asp Val Asn Gly Leu Lys Thr Lys Ile Thr Leu Glu Val
         35                  40                  45
Ala Leu Glu Leu Gly Asp Gly Ala Val Arg Thr Ile Ala Met Glu Ser
     50                  55                  60
Thr Asp Gly Leu Thr Arg Gly Leu Glu Val Leu Asp Thr Gly Lys Ala
 65                  70                  75                  80
Val Ser Val Pro Val Gly Glu Ser Thr Leu Gly Arg Val Phe Asn Val
                 85                  90                  95
Leu Gly Asp Val Ile Asp Gly Gly Glu Asp Phe Pro Ala Asp Ala Glu
            100                 105                 110
Arg Asn Pro Ile His Lys Lys Ala Pro Thr Phe Asp Glu Leu Ser Thr
        115                 120                 125
Ala Asn Glu Val Leu Val Thr Gly Ile Lys Val Val Asp Leu Leu Ala
    130                 135                 140
Pro Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe Gly Gly Ala Gly Val
145                 150                 155                 160
Gly Lys Thr Val Leu Ile Gln Glu Leu Ile His Asn Ile Ala Gln Glu
                165                 170                 175
His Gly Gly Ile Ser Val Phe Thr Gly Val Gly Asp Arg Thr Arg Asp
            180                 185                 190
Gly Asn Asp Leu Tyr Trp Glu Met Lys Glu Ser Gly Val Ile Glu Lys
        195                 200                 205
Thr Ala Met Val Phe Gly Gln Met Asn Glu Pro Pro Gly Ala Arg Met
    210                 215                 220
Arg Val Ala Leu Thr Gly Leu Thr Ile Ala Glu Tyr Phe Arg Asp Val
225                 230                 235                 240
Gln Gly Gln Asp Val Leu Leu Phe Ile Asp Asn Ile Phe Arg Phe Thr
                245                 250                 255
Gln Ala Gly Ser Glu Val Ser Ala Leu Trp Gly Arg Met Pro Ser Ala
            260                 265                 270
Val Gly Tyr Gln Pro Thr Leu Ala Thr Glu Met Val Gln Leu Gln Glu
        275                 280                 285
Arg Ile Thr Ser Thr Lys Lys Gly Ser Val Thr Ser Ile Pro Ala Ile
    290                 295                 300
Tyr Val Pro Ala Asp Asp Tyr Thr Asp Pro Ala Pro Ala Thr Ala Phe
305                 310                 315                 320
Ala His Leu Asp Ala Thr Thr Asn Leu Glu Arg Arg Leu Thr Gln Met
                325                 330                 335
Gly Ile Tyr Pro Ala Val Asp Pro Leu Ala Ser Ser Ser Arg Ala Leu
            340                 345                 350
Thr Pro Glu Ile Val Gly Glu Glu His Tyr Glu Val Ala Met Glu Val
        355                 360                 365
Gln Arg Val Leu Gln Arg Tyr Lys Glu Leu Gln Asp Ile Ile Ala Ile
    370                 375                 380
Leu Gly Met Asp Glu Leu Ser Asp Asp Glu Lys Ile Leu Val Gly Arg
385                 390                 395                 400
Ala Arg Arg Ile Gln Phe Phe Leu Ser Gln Asn Phe His Val Ala Glu
                405                 410                 415
Gln Phe Thr Gly Gln Pro Gly Ser Tyr Val Pro Ile Asp Lys Thr Val
            420                 425                 430
His Asp Phe Lys Glu Ile Leu Glu Gly Lys Tyr Asp Glu Val Pro Glu
        435                 440                 445
Asp Ala Phe Arg Gly Val Gly Pro Ile Glu Asp Val Leu Ala Lys Ala
    450                 455                 460
Lys Ser Met Gly Tyr
465
 
           
           
             
               2207 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Lactococcus lactis subsp. lactis 
             
             
               CDS 
               4..633 
               /partial
               /codon_start= 4
               /product= “ATPase subunit, partial sequence”
               /gene= “atpA”
               /standard_name= “alpha subunit of the F1 portion
               of the F0F1 ATPase”
               /label= alpha-subunit
 
             
             
               CDS 
               652..1518 
               /codon_start= 652
               /product= “ATPase subunit”
               /gene= “atpG”
               /standard_name= “gamma subunit of the F1 portion
               of the F0F1 ATPase”
               /label= gamma-subunit
 
             
             
               CDS 
               1654..2205 
               /partial
               /codon_start= 1654
               /product= “ATPase subunit, partial sequence”
               /gene= “atpD”
               /standard_name= “beta subunit of the F1 portion of
               the F0F1 ATPase”
               /label= beta-subunit
 
             
              6
TGA TTC TAC TTA CAT TCA CGT CTT TTG GAA CGT GCT GCC AAA TTA TCT       48
    Phe Tyr Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys Leu Ser
    470                 475                 480
GAC TAT CTT GGT GGT GGT TCA ATG ACT GCA CTG CCA TTC ATT GAA ACA       96
Asp Tyr Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Phe Ile Glu Thr
485                 490                 495                 500
CAA GCC GGA GAT ATC TCA GCT TAT ATT GCA ACA AAC GTT ATC TCT ATT      144
Gln Ala Gly Asp Ile Ser Ala Tyr Ile Ala Thr Asn Val Ile Ser Ile
                505                 510                 515
ACT GAC GGT CAA ATT TTC CTT GAA AAT GAC TTA TTC TAT TCA GGT GTA      192
Thr Asp Gly Gln Ile Phe Leu Glu Asn Asp Leu Phe Tyr Ser Gly Val
            520                 525                 530
CGT CCT GCC ATC GAT GCT GGT TCT TCA GTT TCT CGG GTT GGT GGT GCT      240
Arg Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly Ala
        535                 540                 545
GCA CAG ATC AAA GCC ATG AAG AAA GTT GCT GGT ACT TTG CGT CTT GAC      288
Ala Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu Asp
    550                 555                 560
CTT GCG TCA TTC CGT GAA CTT GAA GCC TTT ACT CAA TTT GGT TCT GAT      336
Leu Ala Ser Phe Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser Asp
565                 570                 575                 580
CTT GAT GAA GCG ACT CAA GCA AAA TTG AAT CGT GGT CGT CGT ACC GTT      384
Leu Asp Glu Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr Val
                585                 590                 595
GAA GTT TTG AAG CAA CCA TTG CAC AAA CCA TTG GCT GTT GAA AAA CAA      432
Glu Val Leu Lys Gln Pro Leu His Lys Pro Leu Ala Val Glu Lys Gln
            600                 605                 610
GTT TTA ATT CTT TAT GCA TTG ACT CAT GGT CAC TTG GAT GAT GTT CCA      480
Val Leu Ile Leu Tyr Ala Leu Thr His Gly His Leu Asp Asp Val Pro
        615                 620                 625
GTT GAT GAC GTC CTT GAT TTT GAA ACA AAC AAT GTC CGA TTC TTC GAT      528
Val Asp Asp Val Leu Asp Phe Glu Thr Asn Asn Val Arg Phe Phe Asp
    630                 635                 640
GCA AAT TAT GCA AAA CTC TTG AAC GTG ATT ACT GAA ACT AAA GAT TGC      576
Ala Asn Tyr Ala Lys Leu Leu Asn Val Ile Thr Glu Thr Lys Asp Cys
645                 650                 655                 660
CAG AAG AAG CAA AAC TCG ACG AAG CAA TTA AAG CAT TCT AAA ATA CAA      624
Gln Lys Lys Gln Asn Ser Thr Lys Gln Leu Lys His Ser Lys Ile Gln
                665                 670                 675
CGA ATT ATT AATAAGGAGG CTAATCTA ATG GGA GCT TCA CTT AAT GAA ATA      675
Arg Ile Ile                     Met Gly Ala Ser Leu Asn Glu Ile
                                  1               5
AAA ACT AAG ATT GCC TCA ACG AAG AAA ACA AGT CAA ATA ACT GGA GCC      723
Lys Thr Lys Ile Ala Ser Thr Lys Lys Thr Ser Gln Ile Thr Gly Ala
     10                  15                  20
ATG CAA ATG GTT TCC GCT GCG AAA CTT CAA AAA GCT GAA TCT CAT GCC      771
Met Gln Met Val Ser Ala Ala Lys Leu Gln Lys Ala Glu Ser His Ala
 25                  30                  35                  40
AAA GCA TTT CAA ATT TAT GCT GAA AAA GTT CGT AAA ATT ACA ACT GAT      819
Lys Ala Phe Gln Ile Tyr Ala Glu Lys Val Arg Lys Ile Thr Thr Asp
                 45                  50                  55
TTA GTT TCC TCT GAC AAA GAG CCA GCT AAG AAT CCA ATG ATG ATA GGA      867
Leu Val Ser Ser Asp Lys Glu Pro Ala Lys Asn Pro Met Met Ile Gly
             60                  65                  70
AGA GAA GTC AAA AAA ACT GGC TAT CTT GTA ATT ACT TCG GAT CGT GGA      915
Arg Glu Val Lys Lys Thr Gly Tyr Leu Val Ile Thr Ser Asp Arg Gly
         75                  80                  85
CTT GTC GGT GGC TAT AAT TCA TAT ATT TTG AAA TCT GTC ATG AAT ACT      963
Leu Val Gly Gly Tyr Asn Ser Tyr Ile Leu Lys Ser Val Met Asn Thr
     90                  95                 100
ATC CGT AAA CGT CCT GCT AAT GAA AGT GAA TAT ACT ATT CTT GCA CTT     1011
Ile Arg Lys Arg Pro Ala Asn Glu Ser Glu Tyr Thr Ile Leu Ala Leu
105                 110                 115                 120
GGC GGT ACT GGA GCA GAT TTC TTC GGA GCA AGC AAT GTT AAA AGT TTC     1059
Gly Gly Thr Gly Ala Asp Phe Phe Gly Ala Ser Asn Val Lys Ser Phe
                125                 130                 135
TTA GTC CTT TGT GGT TTT TCA GAC CAA CCA AAT TTT GAA GAA GTT AGA     1107
Leu Val Leu Cys Gly Phe Ser Asp Gln Pro Asn Phe Glu Glu Val Arg
            140                 145                 150
GCG ATT GTT ACA GAA GCG GTA ACT GAA TAT CAA GCA GAA GAA TTT GAT     1155
Ala Ile Val Thr Glu Ala Val Thr Glu Tyr Gln Ala Glu Glu Phe Asp
        155                 160                 165
GAA CTT TAT GTT TGC TAT AAT CAC CAT GTG AAC TCA TTG GTA AGT GAA     1203
Glu Leu Tyr Val Cys Tyr Asn His His Val Asn Ser Leu Val Ser Glu
    170                 175                 180
GCA AGT ATG GAA AAA ATG TTG CCT ATT TTT TTT GAA GCA TCA GGT CAA     1251
Ala Ser Met Glu Lys Met Leu Pro Ile Phe Phe Glu Ala Ser Gly Gln
185                 190                 195                 200
CAA AAA CCA TTT TTT GAA ACA TTT GAA TTA GAA CCA GAT TGT GAA ACA     1299
Gln Lys Pro Phe Phe Glu Thr Phe Glu Leu Glu Pro Asp Cys Glu Thr
                205                 210                 215
ATT TTA AAC CAA TTG TTG CCA CCA TAC GCT GAA AGT ATG ATT TAT GGT     1347
Ile Leu Asn Gln Leu Leu Pro Pro Tyr Ala Glu Ser Met Ile Tyr Gly
            220                 225                 230
TCA ATC GTT GAT GCT AAG ACA GCA GAA CAT GCT GCA GGT ATG ACA GCA     1395
Ser Ile Val Asp Ala Lys Thr Ala Glu His Ala Ala Gly Met Thr Ala
        235                 240                 245
ATG CGT ACT GCA ACT GAT AAT GCT CAC TCT GTT ATC AAT GAT TTG ACT     1443
Met Arg Thr Ala Thr Asp Asn Ala His Ser Val Ile Asn Asp Leu Thr
    250                 255                 260
ATT CAA TAC AAC CGT GCT CGT CAA GCA TCG ATT ACG CAA GAA ATT ACG     1491
Ile Gln Tyr Asn Arg Ala Arg Gln Ala Ser Ile Thr Gln Glu Ile Thr
265                 270                 275                 280
GAA ATC GTT GCA GGA GCC TCA GCG CTT TAATTTACTG ATAGGAATTC           1538
Glu Ile Val Ala Gly Ala Ser Ala Leu
                285
TGTCAGTGAT GGCTTTGAAT CTTAATTGTT TTTGTCAGTA AAATTTTTAC TGACAAACAT   1598
AAAAATGAAT AGAAATTCTG TTCTTTGACA GAAAATAAAA ACAGGAGGAA AAACA TTG    1656
                                                             Leu
                                                               1
AGT TCT GGT AAA ATT ACT CAG ATT ATC GGT CCC GTC GTT GAC GTG GAA     1704
Ser Ser Gly Lys Ile Thr Gln Ile Ile Gly Pro Val Val Asp Val Glu
              5                  10                  15
TTT GGT TCT GAT GCC AAA TTG CCT GAG ATT AAC AAT GCC TTG ATT GTC     1752
Phe Gly Ser Asp Ala Lys Leu Pro Glu Ile Asn Asn Ala Leu Ile Val
         20                  25                  30
TAC AAA GAT GTC AAT GGC CTA AAA ACA AAA ATT ACT CTT GAA GTT GCT     1800
Tyr Lys Asp Val Asn Gly Leu Lys Thr Lys Ile Thr Leu Glu Val Ala
     35                  40                  45
TTG GAA CTT GGT GAT GGT GCA GTT CGT ACA ATC GCT ATG GAA TCT ACT     1848
Leu Glu Leu Gly Asp Gly Ala Val Arg Thr Ile Ala Met Glu Ser Thr
 50                  55                  60                  65
GAT GGC TTG ACT CGT GGA CTT GAA GTC CTT GAT ACA GGT AAA GCA GTC     1896
Asp Gly Leu Thr Arg Gly Leu Glu Val Leu Asp Thr Gly Lys Ala Val
                 70                  75                  80
AGC GTT CCT GTT GGG GAA GCC ACT CTT GGT CGT GTT TTT AAC GTC CTT     1944
Ser Val Pro Val Gly Glu Ala Thr Leu Gly Arg Val Phe Asn Val Leu
             85                  90                  95
GGT GAT GTT ATT GAC GGT GGG GAA GAA TTT GCT GCT GAT GCA GAA CGT     1992
Gly Asp Val Ile Asp Gly Gly Glu Glu Phe Ala Ala Asp Ala Glu Arg
        100                 105                 110
AAT CCT ATC CAT AAA AAA GCT CCA ACA TTT GAC GAA TTG TCA ACT GCA     2040
Asn Pro Ile His Lys Lys Ala Pro Thr Phe Asp Glu Leu Ser Thr Ala
    115                 120                 125
AAC GAA GTT CTC GTA ACT GGG ATT AAA GTT GTC GAT TTG CTT GCA CCT     2088
Asn Glu Val Leu Val Thr Gly Ile Lys Val Val Asp Leu Leu Ala Pro
130                 135                 140                 145
TAC CTT AAA GGT GGT AAA GTT GGA CTT TTC GGT GGT GCC GGA GTT GGT     2136
Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe Gly Gly Ala Gly Val Gly
                150                 155                 160
AAA GCC GTC CTT ATT CAA GAA TTG AAA CAC AAC ATC GCC CAA GAA CAC     2184
Lys Ala Val Leu Ile Gln Glu Leu Lys His Asn Ile Ala Gln Glu His
            165                 170                 175
GGA GGT ATT TCT GTG TTT ACC GG                                      2207
Gly Gly Ile Ser Val Phe Thr
        180
 
           
           
             
               210 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              7
Phe Tyr Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys Leu Ser Asp
  1               5                  10                  15
Tyr Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Phe Ile Glu Thr Gln
             20                  25                  30
Ala Gly Asp Ile Ser Ala Tyr Ile Ala Thr Asn Val Ile Ser Ile Thr
         35                  40                  45
Asp Gly Gln Ile Phe Leu Glu Asn Asp Leu Phe Tyr Ser Gly Val Arg
     50                  55                  60
Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly Ala Ala
 65                  70                  75                  80
Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu Asp Leu
                 85                  90                  95
Ala Ser Phe Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser Asp Leu
            100                 105                 110
Asp Glu Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr Val Glu
        115                 120                 125
Val Leu Lys Gln Pro Leu His Lys Pro Leu Ala Val Glu Lys Gln Val
    130                 135                 140
Leu Ile Leu Tyr Ala Leu Thr His Gly His Leu Asp Asp Val Pro Val
145                 150                 155                 160
Asp Asp Val Leu Asp Phe Glu Thr Asn Asn Val Arg Phe Phe Asp Ala
                165                 170                 175
Asn Tyr Ala Lys Leu Leu Asn Val Ile Thr Glu Thr Lys Asp Cys Gln
            180                 185                 190
Lys Lys Gln Asn Ser Thr Lys Gln Leu Lys His Ser Lys Ile Gln Arg
        195                 200                 205
Ile Ile
    210
 
           
           
             
               289 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              8
Met Gly Ala Ser Leu Asn Glu Ile Lys Thr Lys Ile Ala Ser Thr Lys
  1               5                  10                  15
Lys Thr Ser Gln Ile Thr Gly Ala Met Gln Met Val Ser Ala Ala Lys
             20                  25                  30
Leu Gln Lys Ala Glu Ser His Ala Lys Ala Phe Gln Ile Tyr Ala Glu
         35                  40                  45
Lys Val Arg Lys Ile Thr Thr Asp Leu Val Ser Ser Asp Lys Glu Pro
     50                  55                  60
Ala Lys Asn Pro Met Met Ile Gly Arg Glu Val Lys Lys Thr Gly Tyr
 65                  70                  75                  80
Leu Val Ile Thr Ser Asp Arg Gly Leu Val Gly Gly Tyr Asn Ser Tyr
                 85                  90                  95
Ile Leu Lys Ser Val Met Asn Thr Ile Arg Lys Arg Pro Ala Asn Glu
            100                 105                 110
Ser Glu Tyr Thr Ile Leu Ala Leu Gly Gly Thr Gly Ala Asp Phe Phe
        115                 120                 125
Gly Ala Ser Asn Val Lys Ser Phe Leu Val Leu Cys Gly Phe Ser Asp
    130                 135                 140
Gln Pro Asn Phe Glu Glu Val Arg Ala Ile Val Thr Glu Ala Val Thr
145                 150                 155                 160
Glu Tyr Gln Ala Glu Glu Phe Asp Glu Leu Tyr Val Cys Tyr Asn His
                165                 170                 175
His Val Asn Ser Leu Val Ser Glu Ala Ser Met Glu Lys Met Leu Pro
            180                 185                 190
Ile Phe Phe Glu Ala Ser Gly Gln Gln Lys Pro Phe Phe Glu Thr Phe
        195                 200                 205
Glu Leu Glu Pro Asp Cys Glu Thr Ile Leu Asn Gln Leu Leu Pro Pro
    210                 215                 220
Tyr Ala Glu Ser Met Ile Tyr Gly Ser Ile Val Asp Ala Lys Thr Ala
225                 230                 235                 240
Glu His Ala Ala Gly Met Thr Ala Met Arg Thr Ala Thr Asp Asn Ala
                245                 250                 255
His Ser Val Ile Asn Asp Leu Thr Ile Gln Tyr Asn Arg Ala Arg Gln
            260                 265                 270
Ala Ser Ile Thr Gln Glu Ile Thr Glu Ile Val Ala Gly Ala Ser Ala
        275                 280                 285
Leu
 
           
           
             
               184 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              9
Leu Ser Ser Gly Lys Ile Thr Gln Ile Ile Gly Pro Val Val Asp Val
  1               5                  10                  15
Glu Phe Gly Ser Asp Ala Lys Leu Pro Glu Ile Asn Asn Ala Leu Ile
             20                  25                  30
Val Tyr Lys Asp Val Asn Gly Leu Lys Thr Lys Ile Thr Leu Glu Val
         35                  40                  45
Ala Leu Glu Leu Gly Asp Gly Ala Val Arg Thr Ile Ala Met Glu Ser
     50                  55                  60
Thr Asp Gly Leu Thr Arg Gly Leu Glu Val Leu Asp Thr Gly Lys Ala
 65                  70                  75                  80
Val Ser Val Pro Val Gly Glu Ala Thr Leu Gly Arg Val Phe Asn Val
                 85                  90                  95
Leu Gly Asp Val Ile Asp Gly Gly Glu Glu Phe Ala Ala Asp Ala Glu
            100                 105                 110
Arg Asn Pro Ile His Lys Lys Ala Pro Thr Phe Asp Glu Leu Ser Thr
        115                 120                 125
Ala Asn Glu Val Leu Val Thr Gly Ile Lys Val Val Asp Leu Leu Ala
    130                 135                 140
Pro Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe Gly Gly Ala Gly Val
145                 150                 155                 160
Gly Lys Ala Val Leu Ile Gln Glu Leu Lys His Asn Ile Ala Gln Glu
                165                 170                 175
His Gly Gly Ile Ser Val Phe Thr
            180
 
           
           
             
               2161 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               Streptococcus thermophilus 
               ST3 
             
             
               CDS 
               2..637 
               /partial
               /codon_start= 2
               /product= “ATPase subunit, partial sequence”
               /gene= “atpA”
               /standard_name= “alpha subunit of the F1 portion
               of the F0F1 ATPase”
               /label= alpha-subunit
 
             
             
               CDS 
               659..1537 
               /codon_start= 659
               /product= “ATPase subunit”
               /gene= “atpG”
               /standard_name= “gamma subunit of the F1 portion
               of the F0F1 ATPase”
               /label= gamma-subunit
 
             
             
               CDS 
               1616..2161 
               /partial
               /codon_start= 1616
               /product= “ATPase subunit, partial sequence”
               /gene= “atpD”
               /standard_name= “beta subunit of the F1 portion of
               the F0F1 ATPase”
               /label= beta-subunit
 
             
              10
T GAT TCT CAT CTC CAC TCA CGT CTT TTG GAA CGT TCA GCT AAG CTT         46
  Asp Ser His Leu His Ser Arg Leu Leu Glu Arg Ser Ala Lys Leu
  185                 190                 195
TCA GAT GAT CTT GGT GGT GGT TCA ATG ACT GCC TTG CCA ATC ATC CAA       94
Ser Asp Asp Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Ile Ile Gln
200                 205                 210                 215
ACA CAA GCA GGA GAT ATC TCA GCT TAT ATC GCG ACA AAC GTT ATT TCT      142
Thr Gln Ala Gly Asp Ile Ser Ala Tyr Ile Ala Thr Asn Val Ile Ser
                220                 225                 230
ATC ACA GAT GGA CAA ATC TTC TTG CAA GAA AAT CTT TTC AAC TCA GGT      190
Ile Thr Asp Gly Gln Ile Phe Leu Gln Glu Asn Leu Phe Asn Ser Gly
            235                 240                 245
ATT CGT CCT GCG ATT GAT GCT GGT TCT TCA GTA TCA CGT GTT GGT GGT      238
Ile Arg Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly
        250                 255                 260
TCA GCA CAA ATC AAA GCA ATG AAG AAA GTT GCT GGT ACC CTT CGT CTT      286
Ser Ala Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu
    265                 270                 275
GAC TTG GCT TCT CAC CGT GAA CTT GAA GCC TTT ACA CAA TTC GGT TCT      334
Asp Leu Ala Ser His Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser
280                 285                 290                 295
GAT TTG GAT GCC GCA ACA CAA GCT AAA CTT AAT CGT GGA CGT CGT ACA      382
Asp Leu Asp Ala Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr
                300                 305                 310
GTT GAA GTG CTT AAA CAA CCA CTT CAT AAC CCA CTT CCG GTT GAA AAA      430
Val Glu Val Leu Lys Gln Pro Leu His Asn Pro Leu Pro Val Glu Lys
            315                 320                 325
CAA GTT CTT ATT CTT TAC GCT TTG ACA CAT GGC TTC TTG GAC AGT GTT      478
Gln Val Leu Ile Leu Tyr Ala Leu Thr His Gly Phe Leu Asp Ser Val
        330                 335                 340
CCG GTT GAT CAA ATC TTG GAT TTT GAA GAA GCC CTC TAT GAC TAC TTT      526
Pro Val Asp Gln Ile Leu Asp Phe Glu Glu Ala Leu Tyr Asp Tyr Phe
    345                 350                 355
GAT AGC CAT CAT GAG GAT ATC TTT GAA ACA ATC CGT TCA ACT AAG GAT      574
Asp Ser His His Glu Asp Ile Phe Glu Thr Ile Arg Ser Thr Lys Asp
360                 365                 370                 375
CTT CCT GAA GAA GCT GTG CTT AAT GAA GCT ATC CAA GCT TTC AAA GAT      622
Leu Pro Glu Glu Ala Val Leu Asn Glu Ala Ile Gln Ala Phe Lys Asp
                380                 385                 390
CAA TCG GAA TAC AAA TAGAGATAGG GAGGACAGCA T ATG GCA GGC TCT CTA      673
Gln Ser Glu Tyr Lys                         Met Ala Gly Ser Leu
            395                               1               5
AGA GAA ATC AAA GCA AAA ATT GCT TCA ATT AAG CAA ACG AGT CAT ATT      721
Arg Glu Ile Lys Ala Lys Ile Ala Ser Ile Lys Gln Thr Ser His Ile
                 10                  15                  20
ACA GGA GCC ATG CAA ATG GTT TCT GCT TCT AAA TTG ACA CGT TCT GAG      769
Thr Gly Ala Met Gln Met Val Ser Ala Ser Lys Leu Thr Arg Ser Glu
             25                  30                  35
CAG GCT GCT AAA GAT TTC CAA ATC TAT GCC TCA AAA ATT AGA CAG ATC      817
Gln Ala Ala Lys Asp Phe Gln Ile Tyr Ala Ser Lys Ile Arg Gln Ile
         40                  45                  50
ACA ACA GAT CTT CTA CAT TCA GAA TTG GTT AAT GGT TCT TCA AAT CCG      865
Thr Thr Asp Leu Leu His Ser Glu Leu Val Asn Gly Ser Ser Asn Pro
     55                  60                  65
ATG TTG GAT GCA CGT CCA GTT CGT AAG TCA GGG TAT ATT GTC ATT ACT      913
Met Leu Asp Ala Arg Pro Val Arg Lys Ser Gly Tyr Ile Val Ile Thr
 70                  75                  80                  85
TCA GAT AAG GGA TTA GTT GGA GGA TAT AAT TCA ACC ATT CTT AAA GCT      961
Ser Asp Lys Gly Leu Val Gly Gly Tyr Asn Ser Thr Ile Leu Lys Ala
                 90                  95                 100
GTC TTG GAT ATG ATT AAA CGT GAC CAT GAT TCT GAA GAT GAA TAT GCT     1009
Val Leu Asp Met Ile Lys Arg Asp His Asp Ser Glu Asp Glu Tyr Ala
            105                 110                 115
ATC ATC TCT ATT GGT GGA ACA GGT TCA GAT TTC TTC AAA GCT CGT AAC     1057
Ile Ile Ser Ile Gly Gly Thr Gly Ser Asp Phe Phe Lys Ala Arg Asn
        120                 125                 130
ATG AAT GTT GCT TTT GAA CTT CGT GGC CTT GAA GAT CAA CCT AGT TTC     1105
Met Asn Val Ala Phe Glu Leu Arg Gly Leu Glu Asp Gln Pro Ser Phe
    135                 140                 145
GAT CAA GTC GGG GAA ATC ATT CTA AAA GCT GTA GGA ATG TAT CAA AAT     1153
Asp Gln Val Gly Glu Ile Ile Leu Lys Ala Val Gly Met Tyr Gln Asn
150                 155                 160                 165
GAG CTT TTT GAT GAA CTT TAT GTG TGT TAC AAT CAT CAT ATT AAT AGT     1201
Glu Leu Phe Asp Glu Leu Tyr Val Cys Tyr Asn His His Ile Asn Ser
                170                 175                 180
TTG TTT TGT GAA GTT TGT GTT GAA AAA ATG CTT CCA ATT GCT GAT TTT     1249
Leu Phe Cys Glu Val Cys Val Glu Lys Met Leu Pro Ile Ala Asp Phe
            185                 190                 195
GAT CCT AAT GAA TTT GAA GGC CAT GTA TTG ACC AAG TTT GAA TTG GAA     1297
Asp Pro Asn Glu Phe Glu Gly His Val Leu Thr Lys Phe Glu Leu Glu
        200                 205                 210
CCA AGT TGT GAT ACT ATT TTG GAT CAA CTT TTG CCC ACA ATA GTC GGT     1345
Pro Ser Cys Asp Thr Ile Leu Asp Gln Leu Leu Pro Thr Ile Val Gly
    215                 220                 225
GAG AGT TTT ATC TAC GGT GCT ATC GTA GAT GCC AAA ACA GCT GAG CAT     1393
Glu Ser Phe Ile Tyr Gly Ala Ile Val Asp Ala Lys Thr Ala Glu His
230                 235                 240                 245
GCT GCT GGT ATG ACC GCA ATG CAG ACT GCC ACT GAT AAT GCT AAG AAA     1441
Ala Ala Gly Met Thr Ala Met Gln Thr Ala Thr Asp Asn Ala Lys Lys
                250                 255                 260
ATA ATT AAC GAT TTA ACA ATT CAA TAC AAC CGT GCA CGT CAA GCA GCC     1489
Ile Ile Asn Asp Leu Thr Ile Gln Tyr Asn Arg Ala Arg Gln Ala Ala
            265                 270                 275
ATT ACT CAG GAA ATC ACT GAG ATT GTT GGC GGT GCT AGT GCA CTT GAA     1537
Ile Thr Gln Glu Ile Thr Glu Ile Val Gly Gly Ala Ser Ala Leu Glu
        280                 285                 290
TAGCTAGAGA TTTGTCTTGA TTTGACATAC AATAAAAAGG GATGATTGTC ATCCAGAAAA   1597
CTTCATAAGG AGAAAACA ATG AGC TCA GGC AAA ATT GCT CAG GTT GTT GGT     1648
                    Met Ser Ser Gly Lys Ile Ala Gln Val Val Gly
                      1               5                  10
CCT GTT GTA GAC GTA GCG TTT GCA ACT GGC GAT AAA CTT CCT GAG ATT     1696
Pro Val Val Asp Val Ala Phe Ala Thr Gly Asp Lys Leu Pro Glu Ile
             15                  20                  25
AAC AAT GCA TTG GTC GTT TAC ACT GAG AAG AAA AGT CTT AGA CGG ATG     1744
Asn Asn Ala Leu Val Val Tyr Thr Glu Lys Lys Ser Leu Arg Arg Met
         30                  35                  40
GTG CTC GAA GTA GCT TCG TTG AAA CTT GGA GAA GGT GTG GTT CGT ACC     1792
Val Leu Glu Val Ala Ser Leu Lys Leu Gly Glu Gly Val Val Arg Thr
     45                  50                  55
ATT GCC ATG GAA TCT ACT GAT GGA TTG ACT CGT GGG CTA GAA GTT CTG     1840
Ile Ala Met Glu Ser Thr Asp Gly Leu Thr Arg Gly Leu Glu Val Leu
 60                  65                  70                  75
GAC ACA GGT CGT CCA ATC AGT GTT CCT GTT GGT AAA GAA CTT CTT GGA     1888
Asp Thr Gly Arg Pro Ile Ser Val Pro Val Gly Lys Glu Leu Leu Gly
                 80                  85                  90
CGT GTC TTT AAC GTG CTT GGA GAT ACC ATT GAC ATG GAA GCA CCT TTT     1936
Arg Val Phe Asn Val Leu Gly Asp Thr Ile Asp Met Glu Ala Pro Phe
             95                 100                 105
GCA GAT GAT GCA GAG CGT GAA CCA ATT CAT AAA AAA GCA CCT ACC TTC     1984
Ala Asp Asp Ala Glu Arg Glu Pro Ile His Lys Lys Ala Pro Thr Phe
        110                 115                 120
GAT GAA TTG TCA ACA AGT ACT GAA ATC CTT GAA ACA GGG ATT AAA GTT     2032
Asp Glu Leu Ser Thr Ser Thr Glu Ile Leu Glu Thr Gly Ile Lys Val
    125                 130                 135
ATC GAC TTG CTT GCC CCT TAT CTT AAA GGT GGT AAA GTC GGA CTT TTC     2080
Ile Asp Leu Leu Ala Pro Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe
140                 145                 150                 155
GGT GGT GCC GGT GTT GGT AAG GCC GTT CTT ATT CAA GAG CTG AAT CAC     2128
Gly Gly Ala Gly Val Gly Lys Ala Val Leu Ile Gln Glu Leu Asn His
                160                 165                 170
AAC ATT GCT CAA GAA CAC GGT GGC ATT TCC GTG                         2161
Asn Ile Ala Gln Glu His Gly Gly Ile Ser Val
            175                 180
 
           
           
             
               212 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              11
Asp Ser His Leu His Ser Arg Leu Leu Glu Arg Ser Ala Lys Leu Ser
  1               5                  10                  15
Asp Asp Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Ile Ile Gln Thr
             20                  25                  30
Gln Ala Gly Asp Ile Ser Ala Tyr Ile Ala Thr Asn Val Ile Ser Ile
         35                  40                  45
Thr Asp Gly Gln Ile Phe Leu Gln Glu Asn Leu Phe Asn Ser Gly Ile
     50                  55                  60
Arg Pro Ala Ile Asp Ala Gly Ser Ser Val Ser Arg Val Gly Gly Ser
 65                  70                  75                  80
Ala Gln Ile Lys Ala Met Lys Lys Val Ala Gly Thr Leu Arg Leu Asp
                 85                  90                  95
Leu Ala Ser His Arg Glu Leu Glu Ala Phe Thr Gln Phe Gly Ser Asp
            100                 105                 110
Leu Asp Ala Ala Thr Gln Ala Lys Leu Asn Arg Gly Arg Arg Thr Val
        115                 120                 125
Glu Val Leu Lys Gln Pro Leu His Asn Pro Leu Pro Val Glu Lys Gln
    130                 135                 140
Val Leu Ile Leu Tyr Ala Leu Thr His Gly Phe Leu Asp Ser Val Pro
145                 150                 155                 160
Val Asp Gln Ile Leu Asp Phe Glu Glu Ala Leu Tyr Asp Tyr Phe Asp
                165                 170                 175
Ser His His Glu Asp Ile Phe Glu Thr Ile Arg Ser Thr Lys Asp Leu
            180                 185                 190
Pro Glu Glu Ala Val Leu Asn Glu Ala Ile Gln Ala Phe Lys Asp Gln
        195                 200                 205
Ser Glu Tyr Lys
    210
 
           
           
             
               293 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              12
Met Ala Gly Ser Leu Arg Glu Ile Lys Ala Lys Ile Ala Ser Ile Lys
  1               5                  10                  15
Gln Thr Ser His Ile Thr Gly Ala Met Gln Met Val Ser Ala Ser Lys
             20                  25                  30
Leu Thr Arg Ser Glu Gln Ala Ala Lys Asp Phe Gln Ile Tyr Ala Ser
         35                  40                  45
Lys Ile Arg Gln Ile Thr Thr Asp Leu Leu His Ser Glu Leu Val Asn
     50                  55                  60
Gly Ser Ser Asn Pro Met Leu Asp Ala Arg Pro Val Arg Lys Ser Gly
 65                  70                  75                  80
Tyr Ile Val Ile Thr Ser Asp Lys Gly Leu Val Gly Gly Tyr Asn Ser
                 85                  90                  95
Thr Ile Leu Lys Ala Val Leu Asp Met Ile Lys Arg Asp His Asp Ser
            100                 105                 110
Glu Asp Glu Tyr Ala Ile Ile Ser Ile Gly Gly Thr Gly Ser Asp Phe
        115                 120                 125
Phe Lys Ala Arg Asn Met Asn Val Ala Phe Glu Leu Arg Gly Leu Glu
    130                 135                 140
Asp Gln Pro Ser Phe Asp Gln Val Gly Glu Ile Ile Leu Lys Ala Val
145                 150                 155                 160
Gly Met Tyr Gln Asn Glu Leu Phe Asp Glu Leu Tyr Val Cys Tyr Asn
                165                 170                 175
His His Ile Asn Ser Leu Phe Cys Glu Val Cys Val Glu Lys Met Leu
            180                 185                 190
Pro Ile Ala Asp Phe Asp Pro Asn Glu Phe Glu Gly His Val Leu Thr
        195                 200                 205
Lys Phe Glu Leu Glu Pro Ser Cys Asp Thr Ile Leu Asp Gln Leu Leu
    210                 215                 220
Pro Thr Ile Val Gly Glu Ser Phe Ile Tyr Gly Ala Ile Val Asp Ala
225                 230                 235                 240
Lys Thr Ala Glu His Ala Ala Gly Met Thr Ala Met Gln Thr Ala Thr
                245                 250                 255
Asp Asn Ala Lys Lys Ile Ile Asn Asp Leu Thr Ile Gln Tyr Asn Arg
            260                 265                 270
Ala Arg Gln Ala Ala Ile Thr Gln Glu Ile Thr Glu Ile Val Gly Gly
        275                 280                 285
Ala Ser Ala Leu Glu
    290
 
           
           
             
               182 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              13
Met Ser Ser Gly Lys Ile Ala Gln Val Val Gly Pro Val Val Asp Val
  1               5                  10                  15
Ala Phe Ala Thr Gly Asp Lys Leu Pro Glu Ile Asn Asn Ala Leu Val
             20                  25                  30
Val Tyr Thr Glu Lys Lys Ser Leu Arg Arg Met Val Leu Glu Val Ala
         35                  40                  45
Ser Leu Lys Leu Gly Glu Gly Val Val Arg Thr Ile Ala Met Glu Ser
     50                  55                  60
Thr Asp Gly Leu Thr Arg Gly Leu Glu Val Leu Asp Thr Gly Arg Pro
 65                  70                  75                  80
Ile Ser Val Pro Val Gly Lys Glu Leu Leu Gly Arg Val Phe Asn Val
                 85                  90                  95
Leu Gly Asp Thr Ile Asp Met Glu Ala Pro Phe Ala Asp Asp Ala Glu
            100                 105                 110
Arg Glu Pro Ile His Lys Lys Ala Pro Thr Phe Asp Glu Leu Ser Thr
        115                 120                 125
Ser Thr Glu Ile Leu Glu Thr Gly Ile Lys Val Ile Asp Leu Leu Ala
    130                 135                 140
Pro Tyr Leu Lys Gly Gly Lys Val Gly Leu Phe Gly Gly Ala Gly Val
145                 150                 155                 160
Gly Lys Ala Val Leu Ile Gln Glu Leu Asn His Asn Ile Ala Gln Glu
                165                 170                 175
His Gly Gly Ile Ser Val
            180
 
           
           
             
               914 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             C-terminal 
             
               Phaffia rhodozyma 
             
             
               CDS 
               51..824 
               /partial
               /codon_start= 51
               /product= “ATPase subunit, partial sequence”
               /gene= “ATP2”
               /standard_name= “beta subunit of the F1 portion of
               the F0F1 ATPase”
               /label= beta-subunit
 
             
              14
GAATTCTCAA CCTTGAGGGT GACTCCAAGG TCGCTCTTGT CTTCGGACAG ATG AAC        56
                                                       Met Asn
GAG CCC CCG GGT GCT CGA GCC CGA GTC GCT TTG ACT GGT TTG ACC ATC      104
Glu Pro Pro Gly Ala Arg Ala Arg Val Ala Leu Thr Gly Leu Thr Ile
185                 190                 195                 200
GCC GAG TAC TTC CGA GAC GAG GAA GGA CAG GAT GTC TTG CTT TTC ATC      152
Ala Glu Tyr Phe Arg Asp Glu Glu Gly Gln Asp Val Leu Leu Phe Ile
                205                 210                 215
GAC AAC ATT TTC CGA TTC ACC CAG GCC GGT TCT GAG GTG TCT GCC TTG      200
Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly Ser Glu Val Ser Ala Leu
            220                 225                 230
CTT GGT CGA ATT CCC TCC GCC GTC GGA TAC CAG CCC ACT CTT TCC ACC      248
Leu Gly Arg Ile Pro Ser Ala Val Gly Tyr Gln Pro Thr Leu Ser Thr
        235                 240                 245
GAT ATG GGA GGT ATG CAG GAG CGA ATT ACC ACC ACC AAG AAG GGA TCC      296
Asp Met Gly Gly Met Gln Glu Arg Ile Thr Thr Thr Lys Lys Gly Ser
    250                 255                 260
ATC ACT TCC GTC CAG GCC GTC TAC GTG CCT GCT GAT GAT TTG ACC GAT      344
Ile Thr Ser Val Gln Ala Val Tyr Val Pro Ala Asp Asp Leu Thr Asp
265                 270                 275                 280
CCT GCC CCC GCC ACC ACC TTC GCC CAC TTG GAC GCC ACC ACT GTG TTG      392
Pro Ala Pro Ala Thr Thr Phe Ala His Leu Asp Ala Thr Thr Val Leu
                285                 290                 295
TCT CGA GGT ATC GCT GAG TTG GGT ATC TAC CCC GCT GTC GAT CCC CTT      440
Ser Arg Gly Ile Ala Glu Leu Gly Ile Tyr Pro Ala Val Asp Pro Leu
            300                 305                 310
GAT TCT AAG TCC CGA ATG CTC GAC CCC CGA ATT GTC GGA CAG GAG CAC      488
Asp Ser Lys Ser Arg Met Leu Asp Pro Arg Ile Val Gly Gln Glu His
        315                 320                 325
TAC GAC ATC GCC ACC AAG ACC CAG AAG ATC CTC CAG GAC TAC AAG TCC      536
Tyr Asp Ile Ala Thr Lys Thr Gln Lys Ile Leu Gln Asp Tyr Lys Ser
    330                 335                 340
CTC CAG GAT ATC ATT GCC ATT CTT GGT ATG GAT GAG TTG TCT GAG GAG      584
Leu Gln Asp Ile Ile Ala Ile Leu Gly Met Asp Glu Leu Ser Glu Glu
345                 350                 355                 360
GAC AAG TTG ACC GTC GAG CGA GCC CGA AAG ATC CAG CGA TTC ATG TCG      632
Asp Lys Leu Thr Val Glu Arg Ala Arg Lys Ile Gln Arg Phe Met Ser
                365                 370                 375
CAG CCT TTC GCT GTC GCT CAG GTC TTC ACT GGT ATC GAG GGA AAG CTT      680
Gln Pro Phe Ala Val Ala Gln Val Phe Thr Gly Ile Glu Gly Lys Leu
            380                 385                 390
GTT CCC TTG AAG ACT ACT TTG GAG TCC TTT AAG GAG CTT CTT TCC GGA      728
Val Pro Leu Lys Thr Thr Leu Glu Ser Phe Lys Glu Leu Leu Ser Gly
        395                 400                 405
GCC TGC GAC CAC CTC CCT GAG TCT GCT TTC TAC ATG GTT GGT GAC ATC      776
Ala Cys Asp His Leu Pro Glu Ser Ala Phe Tyr Met Val Gly Asp Ile
    410                 415                 420
GCT GAT GTC AAG GCC AAG GCT GCT GCC CAG GCT AAG GAG TTG GCT GCT      824
Ala Asp Val Lys Ala Lys Ala Ala Ala Gln Ala Lys Glu Leu Ala Ala
425                 430                 435                 440
TAAGAGAAGA GTTGTCGAAT GTGTTTCGAG GTGTCAGAGT TGTCTTTTAT GAATGTTTCT    884
ATCTCCTTAA AAAAAAAAAA AAAAAAAAAA                                     914
 
           
           
             
               258 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              15
Met Asn Glu Pro Pro Gly Ala Arg Ala Arg Val Ala Leu Thr Gly Leu
  1               5                  10                  15
Thr Ile Ala Glu Tyr Phe Arg Asp Glu Glu Gly Gln Asp Val Leu Leu
             20                  25                  30
Phe Ile Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly Ser Glu Val Ser
         35                  40                  45
Ala Leu Leu Gly Arg Ile Pro Ser Ala Val Gly Tyr Gln Pro Thr Leu
     50                  55                  60
Ser Thr Asp Met Gly Gly Met Gln Glu Arg Ile Thr Thr Thr Lys Lys
 65                  70                  75                  80
Gly Ser Ile Thr Ser Val Gln Ala Val Tyr Val Pro Ala Asp Asp Leu
                 85                  90                  95
Thr Asp Pro Ala Pro Ala Thr Thr Phe Ala His Leu Asp Ala Thr Thr
            100                 105                 110
Val Leu Ser Arg Gly Ile Ala Glu Leu Gly Ile Tyr Pro Ala Val Asp
        115                 120                 125
Pro Leu Asp Ser Lys Ser Arg Met Leu Asp Pro Arg Ile Val Gly Gln
    130                 135                 140
Glu His Tyr Asp Ile Ala Thr Lys Thr Gln Lys Ile Leu Gln Asp Tyr
145                 150                 155                 160
Lys Ser Leu Gln Asp Ile Ile Ala Ile Leu Gly Met Asp Glu Leu Ser
                165                 170                 175
Glu Glu Asp Lys Leu Thr Val Glu Arg Ala Arg Lys Ile Gln Arg Phe
            180                 185                 190
Met Ser Gln Pro Phe Ala Val Ala Gln Val Phe Thr Gly Ile Glu Gly
        195                 200                 205
Lys Leu Val Pro Leu Lys Thr Thr Leu Glu Ser Phe Lys Glu Leu Leu
    210                 215                 220
Ser Gly Ala Cys Asp His Leu Pro Glu Ser Ala Phe Tyr Met Val Gly
225                 230                 235                 240
Asp Ile Ala Asp Val Lys Ala Lys Ala Ala Ala Gln Ala Lys Glu Leu
                245                 250                 255
Ala Ala
 
           
           
             
               375 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               Trichoderma reesei 
             
             
               CDS 
               50..361 
               /partial
               /codon_start= 50
               /product= “ATPase subunit, partial sequence”
               /gene= “ATP2”
               /standard_name= “beta subunit of F1 portion of the
               F0F1 ATPase”
               /label= beta-subunit
 
             
              16
TACTCGAAGA ATTCGGCACG AGGCTGATTG CTCTCGGTCA TCTGCCAAG ATG TTC         55
                                                      Met Phe
                                                          260
AAG AGC GGC GTT TCG TCC CTC GCC AGG GCT GCC CGC CCA TCA ATT ACC      103
Lys Ser Gly Val Ser Ser Leu Ala Arg Ala Ala Arg Pro Ser Ile Thr
                265                 270                 275
GCT CGA CGA GCT ATC CGA CCA GCC TTC CCT CGA ACC CCC CTC GCG AGG      151
Ala Arg Arg Ala Ile Arg Pro Ala Phe Pro Arg Thr Pro Leu Ala Arg
            280                 285                 290
CTT GCC AGC ACC CAG AGC GTC GGA GAT GGC AAG ATC CAC CAG GTC ATT      199
Leu Ala Ser Thr Gln Ser Val Gly Asp Gly Lys Ile His Gln Val Ile
        295                 300                 305
GGT GCC GTC GTC GAC GTC AAG TTC GAC ACC GCC AAG CTG CCT CCT ATC      247
Gly Ala Val Val Asp Val Lys Phe Asp Thr Ala Lys Leu Pro Pro Ile
    310                 315                 320
CTG AAC GCC CTG GAG ACC ACC AAC AAC AAC CAG AAG CTG GTC CTC GAG      295
Leu Asn Ala Leu Glu Thr Thr Asn Asn Asn Gln Lys Leu Val Leu Glu
325                 330                 335                 340
GTG GCT CAA CAC TTG GGC GAG AAT GTC GTT CGC TGC ATT GCC ATG GAC      343
Val Ala Gln His Leu Gly Glu Asn Val Val Arg Cys Ile Ala Met Asp
                345                 350                 355
GGA TCC GAG GGT CTC GTC GTGGTTCCAA GGCA                              375
Gly Ser Glu Gly Leu Val
            360
 
           
           
             
               104 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
              17
Met Phe Lys Ser Gly Val Ser Ser Leu Ala Arg Ala Ala Arg Pro Ser
  1               5                  10                  15
Ile Thr Ala Arg Arg Ala Ile Arg Pro Ala Phe Pro Arg Thr Pro Leu
             20                  25                  30
Ala Arg Leu Ala Ser Thr Gln Ser Val Gly Asp Gly Lys Ile His Gln
         35                  40                  45
Val Ile Gly Ala Val Val Asp Val Lys Phe Asp Thr Ala Lys Leu Pro
     50                  55                  60
Pro Ile Leu Asn Ala Leu Glu Thr Thr Asn Asn Asn Gln Lys Leu Val
 65                  70                  75                  80
Leu Glu Val Ala Gln His Leu Gly Glu Asn Val Val Arg Cys Ile Ala
                 85                  90                  95
Met Asp Gly Ser Glu Gly Leu Val
            100