DNA fragment comprising a gene encoding ethylene forming enzyme

Details DNA fragment comprising a gene encoding ethylene forming enzyme DNA fragment comprising a gene encoding ethylene forming enzyme

1994-03-01

1996-07-16

Wax, Robert A.

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

4353201, 536 232, C12N 120, C12N 1563, C07H 2104

Patent

active

055366597

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

This invention relates to a DNA fragment comprising a gene encoding an ethylene-forming enzyme of bacteria, a vector including the DNA fragment, transformants transformed with the vector, and a method for producing ethylene using the transformants.


BACKGROUND ART

While ethylene is produced from crude petroleum or natural gas, it has long been known that ethylene could also be formed by plants and microorganisms.
Chou et al. reported that .alpha.-ketoglutaric acid(.alpha.-KG) or L-glutamic acid(Glu) is a precursor of ethylene in Penicillium digitatum, a species of fungi (Arch. Biochem. Biophys., 157, 73, 1973), and Goto et al. reported that .alpha.-KG serves as a substrate in the ethylene forming system in cell-free extracts of Pseudomonas syringae which is one of the species of pathogenic bacteria for plants (Plant Cell Physiol., 28, 405, 1987).
Primrose et al. reported that, in the ethylene-forming system in cell-free extracts of Escherichia coli, 2-keto-4-methylthiobutyric acid(KMBA) which is a metabolic intermediate of L-methionine(Met) is a precursor of ethylene biosynthesis (J. Gen. Microbiol.,98,519, 1977). In all these reports, however, the true substrates and biosynthetic pathway of ethylene-forming reaction were not established because materials such as cultured cells of bacteria or their cell free extracts, which are supposed to contain a lot of impurities, were used in the experiments.
To make clear the pathway of the ethylene biosynthesis caused by the bacteria and the ethylene-forming enzyme reaction, the present inventors purified the enzyme catalyzing the ethylene formation to electrophoretically homogeneous state. The results revealed that the ethylene-forming reaction via KMBA is really radical reactions in which active oxygen is concerned (Fukuda et al., FEMS Microbiol. Lett., 60, 107, 1989). We have also investigated the ethylene-forming enzyme of Penicillium digitatum IFO 9372 via a .alpha.-KG and its enzymatic reaction (Fukuda et al., FEMS Microbiol.lett., 59, 1989), and the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2 via .alpha.-KG and its characteristics (Nagahama, Fukuda et al., J. Gen. Microbiol., 137, 2228, 1991).
Although the enzyme catalyzing the ethylene biosynthesis by bacteria was identified as well as the characteristics of the enzyme, by the present inventors as described above, the ability of ethylene formation in these ethylene-forming bacteria was not adequately sufficient.
For the purpose of fundamental breeding improvement of the ethylene-forming bacteria through a gene manipulation technique, Pseudomonas syringae pv. phaseolicola PK2 was selected for the object and its DNA sequence encoding the ethylene-forming enzyme was analyzed, thereby completing this invention.


DISCLOSURE OF INVENTION

To achieve the above described object, this invention is characterized in that a gene encoding an amino acid sequence represented by: Cys Ala Ala Asp Ile Ser Leu Gly Arg Ala Leu Ile Gln Ala Trp Gln Lys Asp Gly Ile Phe Gln Ile Lys Thr Asp Ser Glu Gln Asp Arg Lys Thr Gln Glu Ala Met Ala Ala Ser Lys Gln Phe Cys Lys Glu Pro Leu Thr Phe Lys Ser Ser Cys Val Ser Asp Leu Thr Tyr Ser Gly Tyr Val Ala Ser Gly Glu Glu Val Thr Ala Gly Lys Pro Asp Phe Pro Glu Ile Phe Thr Val Cys Lys Asp Leu Ser Val Gly Asp Gln Arg Val Lys Ala Gly Trp Pro Cys His Gly Pro Val Pro Trp Pro Asn Asn Thr Tyr Gln Lys Ser Met Lys Thr Phe Met Glu Glu Leu Gly Leu Ala Gly Glu Arg Leu Leu Lys Leu Thr Ala Leu Gly Phe Glu Leu Pro Ile Asn Thr Phe Thr Asp Leu Thr Arg Asp Gly Trp His His Met Arg Val Leu Arg Phe Pro Pro Gln Thr Ser Thr Leu Ser Arg Gly Ile Gly Ala His Thr Asp Tyr Gly Leu Leu Val Ile Ala Ala Gln Asp Asp Val Gly Gly Leu Tyr Ile Arg Pro Pro Val Glu Gly Glu Lys Arg Asn Arg Asn Trp Leu Pro Gly Glu Ser Ser Ala Gly Met Phe Glu His Asp Glu Pro Trp Thr Phe Val Thr Pro Tbr Pro Gly Val Trp Tbr Val Phe Pro Gly Asp Ile Leu Gln Phe Met Thr Gly Gly Gln Leu Leu Ser Thr Pro His Lys Val Lys Leu Asn Thr Arg Glu Arg Phe Ala Cys Ala Tyr Phe His Glu Pro Asn Phe Glu Ala Ser Ala Tyr Pro Leu P

REFERENCES:
patent: 4877730 (1989-10-01), Urushizaki et al.
Nagahama, K. et al. (1992) "Classification of ethylene--producing bacteria in terms of biosynthetic pathways to ethylene" J. Ferment. Bioeng. 73(1):1-5.
Fukuda et al., Biochemical and Biophysical Research Communications, vol. 188, No. 2, Oct. 30, 1992, 826-832.
Nagahama et al., Journal of General Microbiology, vol. 137, 1991, 2281-2286.

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