Fermenco: The Technology

ATPase activity uncouples the production of biochemicals from biomass production
The production of chemical compounds such as organic acids (ex. lactate) and alcohols (ex. ethanol) by a variety of micro-organisms are by-products of naturally occurring processes which, from the micro-organisms point of view, serve to supplement the organisms with energy (ATP) for biomass production (Fig. 2). The production of chemicals is therefore coupled to the production of biomass, i.e. a certain amount of biomass must be produced for each amount of biochemical produced. The coupling of product formation to biomass production has important implications for the industrial application of these bioprocesses as follows:

A fraction of the starting material (various sugars) in these processes ends up in biomass rather than in the desired product, with a resulting loss in yield depending on the actual process.

Often, a major part of the production process takes place under conditions where the micro-organisms grow slowly or not at all. Since the rate of product formation is coupled to growth, the process time is extended significantly, depending on the actual bioprocess and production phase.

Figure 2. Simplified illustration of energy metabolism in a cell. Sugar is converted to product with concomitant generation of ATP, which is subsequently exploited mainly for growth. Introduction of an uncoupled ATPase regenerates the ADP consumed in the sugar metabolism.

It is therefore highly desirable to uncouple chemical production process from the biomass production process, since less biomass would then be produced as by-product and the full capacity of the bioprocess could be exploited. Fermenco has achieved this by introducing an uncoupled ATPase in the microbial cell. The ATPase converts the ATP produced by the bioprocess back into ADP and P_i which has two important implications for the bioprocess: 1) the bioprocess is uncoupled from biomass production, i.e. more substrate is converted to the desired product, and 2) the bioprocess is allowed to proceed at maximal rate, even under conditions where the cells do not grow at all.

Bioprocesses that will benefit from the ATPase technology
The biochemical processes which are the objects of the ATPase technology are primarily bulk chemicals such as ethanol and lactate, but expected results are also likely to have a spill off on the production of various amino acids, sweeteners and other chemicals produced by micro-organisms. In general, compounds whose synthesis is accompanied by a net ATP synthesis would be a potential target for ATPase optimisation.

The F₁-ATPase is highly superior to other ATPases
In any living cell there are numerous reactions which require ATP. All the corresponding ATP consuming enzymes are ATPases in the sense that they hydrolyse ATP to ADP and P_i as part of the reaction that they carry out and the coupling between the ATPase reaction and another biochemical reaction is necessary in order for the overall process to be beneficial to the cell.

The ATPase reaction of these enzymes can in theory be uncoupled from the other reaction that they normally catalyses. Therefore, all enzymes, which carry catalytic sites for ATP hydrolysis, are potential candidates for use as an uncoupled ATPase. However, the F₁-ATPase part of the universal enzyme complex F₁F₀-ATPase is by far the most important enzyme for this purpose: the catalytic activity of the enzyme is 100-1000 fold faster than other ATPases (Fig. 3). Moreover, ATP hydrolysis with overexpression of the F₁-domain is retained, which may not be the case for uncoupling of other ATPases from their native function.

Figure 3. The F₁F₀ ATPase from bacteria and the separate F₁-ATPase consisting of the a, b and g subunits. The figure also shows the atp operon en-coding the F₁F₀ ATPase in Escherichia coli.

A combination of three different proteins forms the active F₁-ATPase complex
In vitro experiments have demonstrated that F₁-ATPase subunits a, b and g forms a highly active ATPase complex and we have demonstrated with the bacteria Escherichia coli and Lactococcus lactis that this is also the combination of subunits that is most active in the living cell (Koebmann et al., 2002a, b). In bacteria, the atp genes encoding the F₁-ATPase is conveniently located in a so-called operon structure (Fig. 2), which greatly facilitates the construction of plasmids for over-expression of three subunits in concert. Examples on development of the ATPase technology for the two prokaryotes Escherichia coli and Lactococcus lactis are in the following described in details.

The ATPase technology in Escherichia coli
The ATPase technology was originally developed in Escherichia coli. The three genes atpA, atpG, and atpD coding for the a-, g-, and b-subunits, respectively, from the functional F₁F₀-ATPase were overexpressed from a set of synthetic promoters with different strengths, which enabled us to modulate the uncoupled ATPase activity to different extents (Fig. 4).

Figure 4. Plasmids for introduction of F₁-ATPase in E. coli and L. lactis. A DNA fragment encoding atpAGD is placed after synthetic promoters (CP) in transcriptional fusion with the reporter gene lacLM, coding for b-galactosidase.

The introduction of uncoupled ATPase activity in steadily growing E. coli cells resulted in a gradual increase in the glycolytic flux to 170% of the wildtype level, while the biomass yield was significantly reduced to 45% of the wildtype level (Fig. 5a). The growth rate was also affected, although to a lesser extent. These results demonstrate that glycolysis was uncoupled from growth in the presence of an uncoupled ATPase. The results also demonstrate why earlier approach with overexpression of glycolytic enzymes has been futile: the majority of control resides not inside but outside the glycolytic pathway, i.e. with the enzymes that hydrolyse ATP. The control of the ATP consuming processes on the glycolytic flux was quantified and found to have full control at the wild-type level (Koebmann et al., 2002a; See also commentary in Nature by Oliver, 2002)

The uncoupling of glycolysis from growth consequently resulted in a lower amount of sugar converted into biomass. Instead, the sugar was to a higher extent converted into byproduct, in this case acetate, resulting in an increase in the acetate flux to 220% of the wildtype level (Fig. 5b). Consequently, the sugar recovered as acetate was found to increase by 40% relative to wildtype (Fig. 5b). This shows that the efficiency of fermentation processes can be improved significantly by uncoupling of the product formation from biomass production by introducing an additional ATP consuming process in E. coli.

Figure 5. The effect of uncoupled F₁-ATPase activity in exponentially growing E. coli with respect to (a) growth rate, biomass yield and glycolytic flux, and (b) the flux and yield of acetate.

The ATPase technology in Lactococcus lactis
The effect of uncoupled ATPase activity was next studied in the lactic acid bacterium Lactococcus lactis. Also here the F₁-genes atpA, atpG, and atpD were expressed from a library of synthetic promoters with different strengths (Fig. 4). This resulted in a gradual increase in uncoupled F₁-ATPase activity and a reduction in biomass yield to 69% of the wildtype level in exponentially growing cells (Fig. 6) (Koebmann et al., 2002b). Here the growth rate was also reduced to 69%. Thus, in contrast to what was found for E. coli, no net change in the glycolytic flux was obtained when ATPase activity was introduced in exponentially growing cells (Fig. 6). However, the finding of a reduction in growth rate, while the glycolytic flux remained essentially unchanged showed that glycolysis was also here uncoupled from biomass production, resulting in less sugar incorporated into cell biomass. Consequently, the yield of fermentation product (lactate) increased compared to the wildtype strain, resulting in a higher yield of lactate.

Figure 6. The effect of uncoupled F₁-ATPase in exponentially growing L. lactis with respect to biomass yield, growth rate and glycolytic flux.

Industrial fermentations are often performed with slow- or non-growing cultures. An example is fermentation processes with immobilised cells which has slow or no growth but should still sustain a high fermentation rate. This can be achieved by an uncoupling of glycolysis from growth. Thus, the ATPase technology was tested under conditions with non-growing cells. In one experiment L. lactis cells were resuspended in buffer containing glucose but without essential amino acids and vitamins. The lack of essential building blocks abolished growth, and resulted in a significant reduction in fermentation rate. But a gradual increase in the production of uncoupled F₁-ATPase resulted in a gradual increase in the glycolytic flux until the flux found in exponentially grown cells was approached (Fig. 7a). Further increase in ATPase activity resulted in a decrease in the glycolytic flux from the optimal level.

The approach to increase the ATP demand was also applied to increase acidification of a L. lactis mutant unable to replicate in milk (Pedersen et al., 2002). A similar response in the cells containing an uncoupled ATPase was observed. In the non-growing L. lactis cells the glycolytic flux was almost doubled by the ATPase activity (Fig. 7b).

Figure 7. The effect of uncoupled ATPase in (a) slow-growing L. lactis cells resuspended in buffer supplemented with glucose, but without essential amino acids and vitamins. (b) Non-replicating thyA mutants. In both cases the rate of lactate production was accelerated with increasing levels of ATPase activity.

References

Koebmann, B. J., Westerhoff, H.V., Snoep, J. L., Nilsson, D., and Jensen, P. R. 2002a. The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J. Bacteriol. 184:3909-3916.

Koebmann, B. J., Solem, C., Pedersen, M. B., Nilsson, D., and Jensen, P. R. 2002b. Expression of genes encoding F₁-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis. Appl. Environ. Microbiol. 68:4274-4282.

Oliver, S. 2002. Metabolism: demand management in cells. Nature. July 4, 418 :33-34.

Pedersen, M. B., Koebmann, B. J., Jensen, P. R., and Nilsson, D. 2002. Increasing acidification of nonreplicating Lactococcus lactis D thyA mutants by incorporating ATPase activity. Appl. Environ. Microbiol. 68:5249-5257.