Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-08-04
2003-01-28
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S004000, C435S015000, C435S252300, C435S252330, C435S252500, C435S255100, C435S254100, C435S320100, C536S023200, C536S023700
Reexamination Certificate
active
06511836
ABSTRACT:
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'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]>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's and brewer'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 e
Jensen Peter Ruhdal
Snoep Jacky Leendert
Westerhoff Hans Victor
Darby & Darby
Jensen Peter Ruhdal
Saidha Tekchand
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