Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Reexamination Certificate
1994-03-15
2002-10-29
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C435S320100
Reexamination Certificate
active
06472196
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to L-phenylalanine ammonia-lyase (hereinafter abbreviated as PAL) having a novel amino acid sequence and useful in the production of L-phenylalanine (sometimes abbreviated as L-Phe).
This invention also relates to the structural gene for PAL (sometimes also referred to as PAL gene) derived from
Rhodosporidium toruloides
, DNA sequences comprising the structural gene for PAL having joined thereto one or more necessary elements which permit the gene to be expressed in other prokaryotic or eukaryotic host microorganisms, and recombinant DNA plasmids for the expression of PAL containing such a DNA sequence.
This invention further relates to microorganisms transformed with such a recombinant DNA plasmid for the expression of PAL, a process for the production of PAL by using such a transformant, the PAL produced by such a transformant, and a process for the production of L-Phe by using the PAL so prepared.
2. Description of the Prior Art
L-Phe is one of the essential amino acids. It is an aromatic amino acid that is not only necessary for nutrition but also useful as the raw material for the manufacture of Aspartame recently in use as an artificial sweetener.
It is known that PALs derived from
Rhodosporidium toruloides
and other microorganisms of the genus Rhodotorula can be utilized for the production of L-Phe.
Processes for the production of PAL are disclosed, for example, in Japanese Patent Publication No. 10753/'69 and Japanese Patent Laid-Open No. 86082/'83.
In addition, Japanese Patent Publication No. 10753/'69 also teaches a method for inducing the production of the enzyme by contacting a microorganism of the genus Rhodotorula with L-phenylalanine, and Japanese Patent Laid-Open No. 86082/'83 also teaches a method for inducing the production of the enzyme by contacting a microorganism of the genus Rhodotorula with an amino acid such as L-isoleucine, L-valine, L-leucine or the like.
In the prior art including the aforesaid processes and methods, the production of PAL requires the step of bringing an amino acid suitable for the induction of PAL production into contact with a microorganism having the ability to produce PAL.
However, the industrial production of PAL by using the aforesaid processes and methods is disadvantageous from an economic or technical point of view, because an expensive amino acid must be used in large amounts, cultivation of the microorganism cannot be managed easily, and a satisfactorily high induction-efficiency cannot always be achieved.
Meanwhile, recent progress in molecular biology has made it possible to isolate a DNA strand coding for a protein originating from a certain microorganism and transform a microorganism of different species by introducing the DNA strand thereinto.
By utilizing such genetic engineering techniques, it may be expected that the necessity of inducing PAL production by contacting an expensive amino acid with a microorganism having the ability to produce PAL is eliminated and, therefore, cultivation of the microorganism can be managed easily.
However, no report has yet been published on the analysis of the structural gene for PAL, isolation and cloning thereof, and the amino acid sequence of PAL. Thus, the technical information necessary for the production of PAL by utilizing genetic engineering techniques has not been available in the existing state of the art.
SUMMARY OF THE INVENTION
The present invention has been made in view of the above-described existing state of the art concerning the production of PAL.
It is an object of the present invention to provide a novel structural gene for PAL which is useful in the production of PAL by utilizing genetic engineering techniques, a recombinant plasmid for the expression of PAL containing the gene, and a transformant having the recombinant plasmid.
It is another object of the present invention to provide a process for the production of PAL in which PAL can be efficiently produced at low cost, cultivation of the microorganism can be managed easily, and the production of PAL can be carried out on an industrial scale.
It is still another object of the present invention to provide PAL having a novel amino acid sequence and useful for the production of L-Phe, and a process for the production of L-Phe by using the PAL.
In order to accomplish these objects, the present inventors have made intensive studies and have achieved the following results:
(1) It has been made clear that PAL derived from
Rhodosporidium toruloides
has an amino acid sequence which will be shown later.
(2) Novel recombinant DNA plasmids (e.g., pSW101, pYtrp6 and pKY201) have been created by inserting a DNA strand coding for the PAL gene between the 3′-terminus of the promoter region and the 5′-terminus of the terminator region.
(3) Transformants having such a novel recombinant DNA plasmid [i.e.,
Escherichia coli
MT-10410 (FERM BR1710) and MT-10414 (FERM BR1712), and bakers' yeast MT-40390 (FERM P-8875)] have been created.
(4) There has been established a novel process for the production of PAL by growing such a novel transformant so as to cause PAL to be produced and accumulated in the culture.
(5) There has been established a novel technique for the production of L-phenylalanine by reacting an ammonia donor with cinnamic acid in the presence of the PAL produced by the aforesaid novel process and having a novel amino acid sequence.
The present invention, which includes the new findings and techniques described in the above Paragraphs 1 to 5, has made it possible to produce PAL efficiently at low cost in a simplified cultivation process utilizing genetic engineering techniques.
More specifically, according to the present invention, a microorganism easy of mass cultivation can be transformed with a recombinant plasmid for the expression of PAL, and the resulting transformant can be easily cultivated to produce PAL. Moreover, this cultivation process does not require any highly elaborate techniques or any expensive induction reagents. Thus, the production of PAL in accordance with the present invention does not require any conventional hard-to-manage cultivation process including the step of including PAL by contacting an expensive amino acid with a microorganism having the ability to produce PAL.
Moreover, the PAL produced by the microorganism obtained in accordance with the present invention is stable and suitable for use in the industrial production of L-phenylalanine. Furthermore, the present invention has the additional advantage that, where the PAL is expressed in
Escherichia coli
or the like, there is no need for contacting the microorganisms with a surface active agent to enhance the permeability of the cell wall.
REFERENCES:
Gilbert et al Journal of Bacteriol, vol. 161 1985. 314-320.*
Gilbert et al Journal of Bacteriol vol. 150 1982. 498-505.*
Hodgins Journal of Biol. Chem. vol. 246 (1971) 2977-2985.
Fukuhara Nobuhiro
Makiguchi Nobuyoshi
Nakajima Yoshiyuki
Se Tomoyuki
Sone Satori
Ketter James
Mitsu Chemicals, Inc.
Nixon & Vanderhye P.C.
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