Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2000-11-30
2003-09-30
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
active
06627420
ABSTRACT:
The present invention relates to a process for preparing alanine, especially L-alanine, by culturing a genetically modified microorganism that is able to produce alanine from a carbon source.
A process of this type is already known from Uhlenbusch et at., Appl. Environ. Microbiol. 57: 1360-1366 (1991). In this process, L-alanine is produced by culturing a microorganism (
Zymomonas mobilis
) that has been genetically modified in such a manner that it expresses an alanine dehydrogenase gene. The expressed alanine dehydrogenase converts the glucose in the medium, via conversion into pyruvate, into a mixture of alanine and ethanol, with a maximum alanine yield of 16%, calculated on the amount of glucose converted.
However,
Zymomonas mobilis
is not a food-grade organism. Nor is
Zymomonas mobilis
a homofermentative organism, since it forms one molecule of CO
2
for each molecule of ethanol produced so that it forms 66% of ethanol and 33% of CO
2
, calculated on the carbon balance.
L-alanine is used in pharmaceutical and veterinary applications. For example, it is included, together with other amino acids, in preparations for parenteral administration as clinical preoperative and postoperative foods, as well as an animal feed supplement. Furthermore, alanine is used as a food additive on account of its sweet taste.
L-alanine is produced industrially by the decarboxylation of L-aspartate by means of immobilized cells or cell suspensions of
Pseudomonas dacunhae.
The L-alanine yield may, in this case, exceed 90%, starting from aspartic acid, but this is an expensive substrate.
D/L-alanine can also be prepared by chemical synthesis or by the direct fermentation of sugars. The microorganisms involved in this fermentation, such as
Corynebacterium gelatinosium, Arthrobacter oxydans, Brevibacterium lactofermentum,
Clostridium sp. and
Pyrococcus furiosus,
produce D/L-alanine with a maximum conversion rate of 50 to 60%, probably via catalysis by an endogenous alanine dehydrogenase.
EP 0 603 865 describes a process for the production of alanine, in which a microorganism of the genus Escherichia, Cornyebacterium or Brevibacterium, provided with a heterologous L-alanine dehydrogenase activity (for example from Arthrobacter), is used.
None of these microorganisms belongs to the lactic acid bacteria. However, a number of above-mentioned Brevibacterium-strains—such as
B.lactofermentum
and
B.flavum
—are classified as among the “food grade” microorganisms. However, this is because they are isolated from food products; they are not applied in the (fermentative) preparation of foodstuffs.
Finally—as far as can be derived from the data given- according to Example 7 of EP 0 603 865 only a maximum conversion of 30% is reached, calculated on the amount of glucose in the medium.
EP 0 567 644 describes a process for the preparation of L-alanine by fermentation using an Arthrobacter-strain. In doing so—as far as can be derived from the data given—according to Example 5 a maximal conversion of 30% is reached, calculated on the amount of glucose in the medium.
However, the Arthrobacter-strains used are not homo-fermentative and do not belong to the food grade or lactic acid bacteria. Furthermore, according to EP 0 603 865 page 5, lines 1-4 the use of Arthrobacter leads to contamination with “odor producing materials”.
J. Oerlgysson et al.,
Anthonie van Leeuwenhoek,
68 no.4, p. 273-280 (1995) describe the production of alanine using a Clostridium-strain. As far as can be derived from the data given the maximal conversion reached in this case is always less than 50%, calculated on the amount of glucose in the medium. Furthermore, Clostridium is homo-fermentative and does not belong to the food grade- or lactic acid bacteria. Also, on p. 279 it is mentioned that “Clostridium P2 produced almost no alanine from pyruvate, and, in fact, could not grow on pyruvate”.
The use of
E.coli
-strains in the production of alanine is described in A. Galkin et al., Appl. Environ. Microbiology, 1997, p.4651-4656, and in Patent Abstracts of Japan, vol. 11, no.222 (C-435), corresponding to JP-A-62936196. However,
E.coli
is a hetero-fermentative organism (especially when cultured under minimum O
2
conditions) and not a food grade- or lactic acid bacterium.
T. Ferain et al., J. of Bacteriology 1996, p. 5431-5437, describe a
L.plantarum
-strain (LDH), of which the two genes encoding the native lactate dehydrogenase are disrupted.
However, this reference does not describe the re-routing of the carbon/pyruvate metabolism in this organism towards the formation of alanine; therefore the LDH
−
-mutants can not be applied in the production of alanine.
The aim of the invention is to provide an improved process for the production of alanine, especially L-alanine.
It has now been found that a process of this type can be provided by replacing, in a suitable microorganism, the native metabolic conversion or conversions of sugars into their derivatives (such as lactate) by the formation of alanine, especially L-alanine.
The invention therefore relates to a process for preparing alanine, especially L-alanine, by culturing a genetically modified microorganism that is able to produce alanine from a suitable carbon source in the medium, characterized in that the microorganism used is one whose native sugar metabolism has been diverted to alanine formation, especially L-alanine formation, in such a manner that essentially more than 20%, preferably more than 40%, more preferably more than 60% and most preferably more than 75% of the carbon consumed from the medium is converted into alanine.
According to the invention preferably food grade- and/or lactic acid bacteria are applied and more preferably food grade strains of lactic acid bacteria. However, the invention is not limited to this:
when in the process of the invention a lactic acid bacterium is used (including the preferred food grade lactic acid bacteria), according to the invention essentially more than 20%, preferably more than 40%, more preferably more than 60% and most preferably more than 75% of the carbon consumed from the medium is converted into alanine;
when a food grade microorganism is used that does not belong to the lactic acid bacteria, according to the invention essentially more than 40%, preferably more than 60% and more preferably more than 75% of the carbon consumed from the medium is converted into alanine;
when a microorganism is used that is neither a food grade microorganism nor a lactic acid bacterium, according to the invention essentially more than 60%, preferably more than 75% of the carbon consumed from the medium is converted into alanine.
Furthermore, the microorganism used is preferably essentially homo fermentative.
In particular, according to the invention the native metabolic conversion or conversions of pyruvate (derived from the conversion of glucose or another suitable source of sugar in the medium) is/are diverted to alanine formation, in such a manner that essentially more than 20%, preferably more than 40%, more preferably more than 60% and most preferably more than 75% of the pyruvate formed being converted into alanine, on the basis of carbon in the pyruvate formed:
when in the process of the invention a lactic acid bacterium is used (including the preferably applied food grade lactic acid bacteria), according to the invention essentially more than 20%, preferably more than 40%, more preferably more than 60% and most preferably more than 75% of the pyruvate formed is converted into alanine;
when a food grade microorganism is used that does not belong to the lactic acid bacteria, according to the invention essentially more than 40%, preferably more than 60% and more preferably more than 75% of the pyruvate formed is converted into alanine;
when a microorganism is used that is neither a food grade microorganism nor a lactic acid bacterium, according to the invention essentially more than 60%, preferably more than 75% of the pyruvate formed is converted into alanine.
The process provides alanine to a high degree of purity and in a hi
De Vos Willem Meindert
Delcour Jean Marcelin Alain Marie Ghislain
Ferain Thierry
Hols Pascal
Kleerebezem Michiel
Achutamurthy Ponnathapu
Browdy and Neimark
Kerr Kathleen
Nederlands Instituut voor Zuivelonderzoek
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