Process for producing S-hydroxynitrile lyase

Details Process for producing S-hydroxynitrile lyase Process for producing S-hydroxynitrile lyase

1999-12-27

2002-05-14

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

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

Recombinant dna technique included in method of making a...

C435S320100, C536S023100, C536S024100

Reexamination Certificate

active

06387659

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for producing S-hydroxynitrile lyase using a recombinant yeast cell into which S-hydroxynitrile lyase coding gene derived from cassava (
Manihot esculenta
) is introduced.
2. Description of the Prior Art
S-hydroxynitrile lyase derived from cassava (EC 4.1.2.37) is an enzyme useful for synthesis of optically active S-cyanohydrins from aromatic and/or aliphatic carbonyl compounds and hydrocyanic acid. Synthesis of optically active cyanohydrins using the enzyme is very useful for synthesis of various optically active intermediates.
However, it has been difficult to industrially utilize the present enzyme since cassava tissue contains an extremely low amount of the enzyme. One example of process for producing the enzyme is conventionally known, which comprises the steps of growing
E. coli
transformed with recombinant DNA into which S-hydroxynitrile lyase coding gene derived from cassava has been introduced. (Angew. Chem. Int. Ed. Engl. 35, 437-439, 1996; Biotechnol. Bioeng. 53, 332-338, 1997). However, such a process using
E. coli
as a host had several disadvantages including: a low productivity of the enzyme; and requirements to add expensive antibiotics and/or an inducer substrate to medium for producing a large amount of the enzyme. Because of these problems, it has been difficult to accomplish a process for efficient production of the enzyme with reduced cost.
OBJECTS AND SUMMARY OF THE INVENTION
The object of the present invention is to provide a process for producing a large amount of S-hydroxynitrile lyase by genetic-engineer technique.
After intensive studies, the present inventors finally succeeded in production of a large amount of S-hydroxynitrile lyase using yeast rather than using
E. coli
as a host for expression of S-hydroxynitrile lyase coding gene derived from cassava.
The present invention generally relates to a process for producing S-hydroxynitrile lyase, comprising the steps of culturing, in a medium, yeast cells transformed with recombinant DNA consisting of an expression vector into which S-hydroxynitrile lyase (EC 4. 1. 2. 37) coding gene derived from cassava (
Manihot esculenta
) has been incorporated, and collecting S-hydroxynitrile lyase from the culture.
Particularly, the present invention relates to the followings:
(1) a process for producing S-hydroxynitrile lyase, comprising the steps of culturing, in a medium, yeast cells transformed with recombinant DNA consisting of yeast episome expression vector into which S-hydroxynitrile lyase (EC 4. 1. 2. 37) coding gene derived from cassava (
Manihot esculenta
) has been incorporated, and collecting S-hydroxynitrile lyase from the culture;
(2) a process for producing S-hydroxynitrile lyase, comprising the steps of culturing, in a medium, the yeast Saccharomyces transformed with recombinant DNA consisting of yeast integrating expression vector into which S-hydroxynitrile lyase (EC 4. 1. 2. 37) coding gene derived from Cassava (
Manihot esculenta
) has been incorporated, and collecting S-hydroxynitrile lyase from the culture; and
(3) a process for producing S-hydroxynitrile lyase, comprising the steps of culturing, in a medium, the yeast Pichia transformed with recombinant DNA consisting of yeast integrating expression vector into which S-hydroxynitrile lyase (EC 4.1.2.37) coding gene derived from cassava (
Manihot esculenta
) has been incorporated, and collecting S-hydroxynitrile lyase from the culture.
This specification includes all or part of the contents as disclosed in the specification of Japanese Patent Applications Nos. 373246/1998, 373247/1998 and 373248/1998, which are priority documents of the present application.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be hereinafter described in detail.
In accordance with the present invention, S-hydroxynitrile lyase coding gene of interest derived from cassava is cloned at first. The DNA sequence of the enzyme is well known and has been already disclosed. (Arch. Biochem, Biophys. 311, 496-502, 1994). Total mRNA including mRNA of the enzyme gene is extracted from cassava leaves and cDNA thereof is synthesized according to any conventional method. The enzyme coding gene is amplified by PCR using primers designed based on the well-known sequence data of S-hydroxynitrile lyase cDNA.
Next, an expression cassette was constructed by inserting a transcription promoter at upstream site and a transcription-terminator at downstream site of the enzyme gene to allow the enzyme gene obtained as described above to express in the recombinant yeast cells, and the constructed expression cassette is then introduced into an expression vector. Alternatively, where transcription promoter and terminator are already present in an expression vector into which the enzyme gene is to be introduced, the transcription promoter and terminator may be used and only the enzyme gene may be introduced therebetween, i.e., there is no need to construct an expression cassette. In either cases, multiple expression cassettes can be present in one expression vector.
Since the expression level of the enzyme greatly depends on the selection of a promoter to be used in an expression cassette, appropriate promoter should be selected. Examples of yeast promoter for efficient expression of the introduced gene in a yeast cell include native promoters such as PGK, GAP, TPI, GAL1, GAL10, ADH2, PHO5, CUP1 and MF&agr;1, recombinant promoters such as PGK/&agr;2 operator, GAP/GAL, PGK/GAL, GAP/ADH2, CYC/GRE and PGK/ARE, and mutated promoters such as Leu2-d. Particularly, GAP promoter is preferred.
Each of the promoters described above may have DNA consisting of the nucleotide sequence of a native promoter, or DNA consisting of the native promoter sequence having deletion, substitution and/or addition of one or more bases but still retaining the promoter activity. Deletion, substitution or addition of base(s) may be generated by using any conventional techniques such as site-directed mutagenesis.
On the other hand, transcription-terminator may be present downstream of the enzyme gene to allow efficient transcription-termination to obtain maximum gene expression. Examples of such transcription-terminator include ADH1, TDH1, TFF and TRP5.
Where the yeast Pichia is used as a host to be transformed, promoter in such an expression cassette as described above may be one which promotes enzyme expression within a methanol-utilizing strain in the yeast Pichia in the presence of methanol carbon source. Terminator in such an expression cassette may be one which allows efficient transcription-termination to obtain maximum gene expression. Particularly, AOX1 promoter and AOX1 terminator are preferred.
According to one embodiment of the present invention, yeast episome expression vector (autonomously replicating plasmid) is used as expression vector.
Yeast episome plasmid vector contains 2&mgr; plasmid sequence, which is native to yeast. The vector can be replicated within a host yeast cell by utilizing the replication origin of the 2&mgr; plasmid sequence.
Yeast episome expression vector to be used in the present invention may not be limited to particular vectors as long as it comprises at least ORI sequence of yeast 2&mgr; plasmid sequence and can be autonomously replicated in a host yeast cell. Examples of such vector include YEp51, pYES2, YEp351 and YEp352 but are not limited thereto.
Preferably, the above-described yeast episome expression vector may be a shuttle vector which can replicate in a
E. coli
cell for subcloning in the recombinant
E. coli
. More preferably, such expression vectors may also contain a selective marker gene such as ampicillin-resistant gene. Also, such expression vectors contain a marker gene by which yeast clones can be selected depending on their auxotrophy and/or drug-resistance when recombinant yeast is prepared. Examples of marker gene include HIS3, TRP1, LEU2, URA3, LYS2, Tn903 kan
r
, Com
r
, Hyg
r
, CUP1 and DHFR though they are not limited thereto. Preferably, a ma

Affiliated with

Also associated with

Rating

Process for producing S-hydroxynitrile lyase does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Process for producing S-hydroxynitrile lyase, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for producing S-hydroxynitrile lyase will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2890451