Process for producing non-proteinogenic L-amino acids by...

Details Process for producing non-proteinogenic L-amino acids by... Process for producing non-proteinogenic L-amino acids by...

2001-09-21

2004-06-29

Tate, Christopher R. (Department: 1654)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing alpha or beta amino acid or substituted amino acid...

C435S113000, C435S849000

Reexamination Certificate

active

06756216

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for producing non-proteinogenic L-amino acids by direct fermentation of microorganisms, and to L-amino acids obtained by the process.
2. The Prior Art
Non-proteinogenic amino acids are amino acids which are not used in nature as building blocks for protein biosynthesis and as a result may be clearly differentiated from the 20 proteinogenic amino acids. They are preferably &bgr;-substituted L-alanine derivatives.
Non-proteinogenic amino acids are compounds of interest, for example, for the preparation of pharmaceuticals and agricultural active compounds. They can, as active compound or as a part of an active compound imitate, in a type of molecular mimicry, the structure of natural amino acids and as a result, for example, in receptor interactions cause a modulation of the natural reaction. In addition, they can serve quite generally as synthesis building blocks as chiral compounds in the context of the “chiral pool”.
Previous production processes for non-proteinogenic amino acids in enantiomerically pure form are generally based on complex syntheses which generally only permit access to a defined compound. Only a few processes enable different compounds to be produced by simple replacement of a starting material.
In most cases chemical syntheses are involved which themselves mostly start from the beginning from chiral building blocks or are followed by a racemate resolution.
Alternatively, some enzymatic processes are described. Thus, using transaminases, various non-proteinogenic amino acids can be prepared from &agr;-keto acids using L-glutamic acid as amino donor. A different process utilizes hydantoinases in combination with carbamoylases. However, enzymatic processes are also cost-intensive, since the corresponding enzymes must be provided and these have only a limited life as catalysts (Rehm et al., Biotechnology 1996; Vol. 6, pp. 505-560).
In contrast, processes for producing non-proteinogenic amino acids by direct fermentation of microorganisms would be particularly simple and expedient. However, such processes have the risk that the non-proteinogenic amino acid produced interferes with the metabolism of the natural amino acids and thus growth inhibition occurs. Previously, within this subject area, a process for the direct fermentation of D-amino acids has been disclosed (WO98/14602). This application describes the production of D-amino acids by recombinant microorganisms into which a D-amino transferase gene and an L-amino deaminase gene have been introduced. Furthermore, Saito et al. (Biol. Pharm. Bull. 1997, 20: 47-53) described the production of the plant non-proteinogenic amino acid L-pyrazolylalanine by expressing plant genes in
Escherichia coli
. The yields, however, are too low for commercial production, at <1 g/l, and the costs, with the described use of L-serine as starting material, are very high.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an efficient process for producing a series of non-proteinogenic L-amino acids by direct fermentation.
This object is achieved according to the invention by a microorganism strain known per se having a deregulated cysteine metabolism being fermented in a manner known per se which comprises, during the fermentation, adding a nucleophilic compound to the fermentation batch in amounts such that this leads to the production of non-proteinogenic L-amino acids by the microorganism strain.
Preferably, at the end of the fermentation, the non-proteinogenic L-amino acids are separated off from the respective fermentation batch by means of methods known per se.
Surprisingly, it has been found that in the fermentation of microorganism strains having deregulated cysteine metabolism, instead of sulfide, a series of other nucleophilic compounds enter very efficiently into amino acid metabolism and the corresponding reaction products are excreted into the culture medium. Advantageously, glucose can be used here as an inexpensive source of carbon.
By means of the inventive addition of nucleophilic compounds during the fermentation, non-proteinogenic L-amino acids are accordingly formed. Preferably, therefore, a nucleophilic compound which enters into amino acid metabolism is added during the fermentation.
Preferably, nucleophilic compounds are added which comprise a radical selected from the group consisting of
Particularly preferably, a nucleophilic compound selected from the following group is added to the fermentation batch:
Thiol of the general formula (1):
H—S—R
1
  (1)
where R
1
is monovalent substituted or unsubstituted alkyl, alkoxy, aryl or heteroaryl radical having a maximum of 15 carbon atoms;
azole of the general formula (2) or (3):
 and their esters, ethers or salts,
where X and Y are identical or different and denote CR
4
or N, and R
4
is —H, —COOH, —OH, —NH
2
, —NO
2
, —SH, —SO
3
, —F, —Cl, —Br, —I, C
1
-C
5
-alkylcarbonyl or R
1
, and R
1
has the meaning specified under formula (1) and
where R
2
and R
3
are identical or different and are R
4
or where C
1
and C
2
in formula (3), instead of the substituents R
2
and R
3
, are linked by means of a bridge [—CR
5
R
6
—]
a
, where a is 1, 2, 3 or 4, to form a ring, where R
5
and R
6
are identical or different and are R
4
and one or more non-adjacent groups [—CR
5
R
6
—] can be replaced by oxygen, sulfur, or an imino radical, which may be unsubstituted or substituted by C
1
-C
5
-alkyl, and two adjacent groups [—CR
5
R
6
—] can be replaced by a group [—CR
5
═CR
6
—] or by a group [—CR
5
═N—].
Isoxazolinone of the general formula (4) or (5):
 and their esters, ethers or salts,
where X, R
1
, R
2
, R
3
have the meaning specified above and where C
1
and C
2
in formula (5), instead of the substituents R
2
and R
3
, can be linked by means of a bridge defined as for formula (3) to form a ring.
Examples of thiols are compounds selected from the group consisting of 2-mercaptoethanol, 3-mercaptopropanol, 3-mercaptopropionic acid, 3-mercapto-1-propanesulfonic acid, mercaptoethanesulfonic acid, 2-mercaptoethylamine, thioglycollc acid, thiolactic acid, thioacetic acid, mercaptosuccinic acid, mercaptopyruvic acid, dithiothreitol, dithioerythritol, 1-thioglycerol, thiophenol, 4-fluorothiophenol, 4-mercaptophenol, p-thiocresol, 5-thio-2-nitrobenzoic acid, 2-mercaptothiazole, 2-mercaptothiazoline, 2-mercaptoimidazole, 3-mercapto-1,2,4,-triazole, 2-thiophenethiol, 2-mercaptopyridine, 2-mercaptopyrimidine, 2-thiocytosine, 2-mercaptonicotinic acid, 2-mercapto-1-methylimidazole, 2-mercaptobenzothiazole, 2-mercaptobenzoxazole, 6-mercaptopurine.
Examples of azoles are compounds selected from the group consisting of 1,2-pyrazole, 3-methylpyrazole, 4-methylpyrazole, 3,5-dimethylpyrazole, 3-aminopyrazole, 4-aminopyrazole, pyrazole-4-carboxylic acid, pyrazole-3,5-dicarboxylic acid, 1,2,3-triazole, 1,2,4-triazole, 3-amino-1,2,4-triazole, 1,2,3,4-tetrazole, indazole, indazole-3-carboxylic acid, indazole-5-carboxylic acid, 5-aminoindazole, benzotriazole, benzotriazole-5-carboxylic acid, 5-aminobenzotriazole, aminopyrazolopyrimidine, 8-azaguanine, 8-azaadenine.
Examples of isoxazolinones are compounds selected from the group consisting of isoxazolin-2-one, 4-methylisoxazolin-2-one, 5-methylisoxazolin-2-one, 4,5-dimethylisoxazolin-2-one, 1,2,4-oxadiazolidin-3,5-dione.
Microorganism strains having deregulated cysteine metabolism that can be used in the inventive process are known from the prior art. They are distinguished by endogenic production of O-acetyl-L-serine, the immediate biosynthetic precursor of L-cysteine, which is increased in comparison with the wild type-strain. In a microorganism, it is known that in the last step of cysteine biosynthesis, due to the activity of O-acetyl-serine-sulfhydrylases, the acetyl function of the O-acetyl-L-serine is replaced by a thiol function and L-cysteine is thus formed. This reaction type is termed &bgr;-substitution, since at the &bgr

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