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
1995-06-07
2001-04-03
Prouty, Rebecca E. (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...
C435S193000, C435S128000, C435S071200, C435S252300, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06210934
ABSTRACT:
German Offenlegungsschrift 38 18 851 (which has not been prior-published and corresponds to EP-A2 0 344 683 published on Dec. 6, 1989) has already proposed an aminotransferase (transaminase) which was isolated from
E. coli
DH-1.
The gene which codes for this new transaminase has now been found. It is possible thereby, according to the invention, to prepare the enzyme in larger amounts than in accordance with the earlier proposal, but also to carry out the specific transamination reactions with a microorganism transformed according to the invention. Thus, the isolation and characterization of the gene permits very much more efficient transaminations than are possible with the enzyme isolated according to the earlier proposal.
German Offenlegungsschrift 38 18 851 characterizes the new enzyme, inter alia, by the amino acid sequence of the N terminus. The first 30 of these amino acids are shown below:
1 5 10 15
NH
2
-MET ASN SER ASN LYS GLU LEU MET GLN ARG ARG SER GLN ALA ILE
16 20 25 30
PRO ARG GLY VAL GLY GLN ILE HIS PRO ILE PHE ALA ASP ARG ALA --
In the region of amino acids 4 to 10 there are methionine, which is coded for by only one triplet, as well as four amino acids which are encoded by only two triplets. Only leucine is six-fold “degenerate” in the genetic code. This sequence is thus particularly well suited for the construction of a probe of 20 nucleotides (20mer):
5 -AAC AAA GAA TTA ATG CAA CG-3
T G G C G G A
C
T
This probe was synthesized by the phosphoramidite method in a manner known per se. Additionally synthesized was a 38mer of the non-coding strand for amino acids 15 to 27.
3-TAG GGC GCG CCG CAA CCG GTC TAG GTG GGC TAG AAG CG-5
T T T T
This oligonucleotide was also synthesized by the phosphoramidite method.
These probes were employed to screen a cosmid gene bank of
E. coli
DH 1. Hybridization-positive clones were initially assayed for elevated L-PPT transaminase activity and then characterized in detail by restriction mapping. It was possible by subcloning and activity assays of subfragments to localize the position of the gene in the genome and subsequently to define it even further by exonuclease degradation. Thus, initially a 15 kb SalI fragment on which the gene according to the invention is located was identified, as was a 3.8 kb SalI/BamHI fragment which allowed the orientation of the gene to be established (FIG.
1
). The latter fragment also contains the gene's own promoter. It was thus possible, merely by cloning restriction fragments into suitable vectors, to increase the transaminase activity by about fifty times compared with the starting strain.
The yield of enzyme or enzyme activity can also be influenced by choosing suitable culture conditions. Thus, for example, the glucose content in the medium plays a considerable role, depending on the choice of the expression system: at concentrations above 0.05% there may be a drastic fall in the enzyme activity. This dependence is evident even with control strains which express only the copy of the transaminase gene in the bacterial chromosome.
In a further development of this concept of the invention it was then possible to localize the gene coding for the aminotransferase more accurately: the gene is located on a 1.6 kb DraI/BamHI fragment (
FIG. 2
) which contains an open reading frame which is 1281 nucleotides long (including the stop codon) and codes for a protein of 426 amino acids.
The DNA sequence is depicted in Table 1. The ATG start codon starts with nucleotide no. 275, and the TAG stop codon starts with nucleotide no. 1553.
Table 2 shows the coding strand of the gene as well as the amino acid sequence of the transaminase according to the invention. The latter shows only a few homologies of sequence with the other known transaminases from
E. coli
(aspC, tyrB, hisC, ilvE, avtA and serC).
Because of the substrate specificity of the L-PPT transaminase for 4-aminobutyric acid (GABA) and comparison of the restriction map of the 15 kb SalI fragment (see
FIG. 1
) with the physical map of the
E. coli
K-12 genome [Kohara et al. (1987), Cell 50: 495-508], it was possible to identify the cloned transaminase gene as gabT, a locus from the
E. coli
K-12 gab cluster at 57.5 min [Metzer et al. (1979), J. Bacteriol. 137; 1111-1118].
Knowledge of the gene allows the structural gene to be provided with strong promoters in a directed manner. The gene constructs obtained in this way not only show higher expression rates than the previously mentioned expression plasmids but also permit their activity to be controlled by inducers. It is furthermore possible to choose expression systems which exhibit no catabolite repression, such as the tac system, so that bacteria transformed with such gene constructs can also be fermented in the presence of glucose in the nutrient medium. This makes high cell densities possible and thus achieves high yields relative to the fermenter volume.
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patent: 73794/87 (1987-12-01), None
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H. Shiraishi et al., “A rapid and efficient method for targeted random mutagenesis”, Gene 64:313-319 (1988).
Wilkinson et al., “A Large increase in enzyme-substrate affinity by protein engineering”, Nature 307:187-188. (1984).
Smith, “In Vitro Mutagenesis”, Ann. Rev. Genet. 19:423-462 (1985).
Zoller and Smith, “Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA, Nucleic Acids Research”, 10(20):6487-6500 (1982).
E. Metzer et al. “Isolation and Properties ofEscherichia coliK-12 Mutants Impaired in the Utilization of Gamma-Aminobutyrate”, J. Bacteriol. 137(3): 1111-1118, Mar. 1979.
Bartsch Klaus
Schulz Arno
Uhlmann Eugen
Aventis Pharma Deutschland GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Prouty Rebecca E.
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