Hydroperoxide lyases

Details Hydroperoxide lyases Hydroperoxide lyases

1999-10-14

2001-05-29

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Preparing oxygen-containing organic compound

C435S157000, C435S232000, C435S252330, C435S254110, C435S254210, C536S023200

Reexamination Certificate

active

06238898

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to hydroperoxide lyases (hereinafter also referred to as HPO lyase proteins or proteins with HPO lyase activity), their microbial production via recombinant DNA technology, and their use for the production of aliphatic aldehydes and alcohols, flavor molecules known as “green notes”.
BACKGROUND OF THE INVENTION
“Green notes” are volatile flavor and fragrance molecules present in a wide variety of plant leaves, vegetables and fruits characterized in organoleptic terms as fresh “green” and grassy. These compounds are produced by the plant from the degradation of unsaturated fatty acids (linoleic and linolenic acid). In
FIG. 1
the formation of a variety of linolenic acid degradation products is summarized.
Degradation of polyunsaturated fatty acids starts by the oxygenation at cis—cis double bonds of polyunsaturated fatty acids. This reaction is catalyzed by lipoxygenase (EC 1.13.11.12)-enzymes which are present in plants, animals and microorganisms. The oxygenated products, fatty acid hydroperoxides, are precursors for many important hormones (e.g. prostaglandins, lipoxins, jasmonic acid, traumatic acid) and flavor/fragrance molecules (e.g., cis-3-hexenol, 1-octen-3-ol). In plants, cleavage of the hydroperoxides occurs through the action of specific hydroperoxide lyases.
Commercial production of natural “green note” compounds is currently achieved by fractional distillation of essential oils such as mint oil or by the combined action of lipoxygenase and hydroperoxide lyase on unsaturated fatty acids using plant material from different sources.
However, these processes have the drawbacks that they provide low yields and/or depend on specific plant materials.
SUMMARY OF THE INVENTION
It has now been found that high reproducible yields of “green note” compounds (e.g., cis-3-hexenol) can be obtained independent of plant materials and in the absence of unwanted side reaction (e.g. isomerase activity) by transfer of the gene coding for HPO lyase from plant into host cells, subsequent expression of the gene, addition of linolenic acid hydroperoxide as substrate, and reduction of cleaved substrate by aldehyde dehydrogenase. In
FIG. 2
the formation of cis-3-hexenol from 13-(S)-hydroperoxy linolenic acid by recombinant HPO lyase is summarized.
Thus, in a first aspect of this invention, there are provided isolated DNA sequences encoding proteins with HPO lyase activity or fragments thereof. Specifically, the DNA sequences of this invention are defined to include the nucleotide sequence SEQ ID No:1 or a fragment thereof or any DNA sequence which is substantially homologous to the nucleotide sequence SEQ ID No:1 or a fragment thereof.
As used hereinbefore the term “substantially homologous”, means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes of the present invention, DNA sequences having greater than 95 percent homology, encoding equivalent biological properties, and showing equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the DNA sequence should be disregarded. Sequences having lesser degrees of homology, encoding comparable bioactivity, and showing equivalent expression characteristics, e.g., fragments of the nucleotide sequence SEQ ID No:1 are considered substantial equivalents. Generally, homologous DNA sequences can be identified by cross-hybridization under standard hybridization conditions of moderate stringency.
There are also provided vectors and expression vectors containing the DNA sequences of the present invention, hosts containing such vectors for the production of proteins with HPO lyase activity, and processes for the production of such DNA sequences, recombinant vectors and host cells.
There are further provided recombinant proteins with HPO lyase activity. Specifically a protein with HPO lyase activity is defined to include the amino acid sequence SEQ ID No:2 or any protein or polypeptide having an amino acid sequence which is substantially homologous to the amino acid sequence SEQ ID No:2 and further having the following biological activity: When the protein or polypeptide is incubated under suitable conditions and a suitable amount of substrate such as 13-(S)-linolenic acid hydroperoxide is added, the formation of cis-3-hexenal is observed.
As used hereinbefore the term “substantially homologous” means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes of the present invention, sequences having greater than 95 percent homology, equivalent biological activity and equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the sequence should be disregarded. Sequences having lesser degrees of homology, comparable bioactivity, and equivalent expression characteristics, e.g., fragments of the amino acid sequence SEQ ID No:2 are considered substantial equivalents.
As used herein the term recombinant proteins with HPO lyase activity includes proteins modified deliberately, as for example, by addition of specific sequences that preferably bind to an affinity carrier material. Examples of such sequences are sequences containing at least two adjacent histidine residues (see in this respect European Patent No. 282 042). Such sequences bind selectively to nitrilotriacetic acid nickel chelate resins (Hochuli and Döbeli, Biol. Chem. Hoope-Seyler 368, 748 (1987); European Patent No. 253 303). Proteins with HPO lyase activity which contain such a specific sequence can, therefore, be separated selectively from the remaining polypeptides. The specific sequence can be linked either to the C-terminus or the N-terminus of the proteins with HPO lyase activity.
Methods for the expression, isolation and purification of the proteins with HPO lyase activity are also provided.
The following steps outline the methods for recombinantly expressing the proteins with HPO lyase activity.
1) Cloning of DNA sequences encoding proteins with HPO lyase activity
DNA sequences encoding proteins with HPO lyase activity can be cloned using a variety of techniques. Using the methods described in this application cDNAs encoding proteins with HPO lyase activity or fragments thereof can be produced. These cDNAs can be isolated and amplified by PCR technique using oligodeoxynucleotide DNA primers by conventional techniques.
The cDNA (SEQ ID No:1) encoding the amino acid sequence SEQ ID No:2 is obtained using the DNA primers described in the examples. By using conventional technique, this cDNA has been isolated from a lambda phage cDNA library made from RNA derived from banana (Musa sp.) leaves.
The cDNA may be obtained not only from cDNA libraries, but by other conventional techniques, e.g., by cloning genomic DNA, or fragments thereof, purified from the desired cells. These procedures are described by Sambrook et al., in “DNA Cloning: A Practical Approach”, Vol. I and II, D. N. Glover, ed., 1985, MRL Press, Ltd., Oxford, U. K; Benton and Davis, Science 196, 180-182 (1977); and Grunstein and Hogness, Proc. Nat. Acad. Sci. 72, 3961-3965 (1975).
To obtain the cDNA encoding the proteins with HPO lyase activity cDNA libraries are screened by conventional DNA hybridization techniques by the methods of Benton and Davis, supra, or Grunstein and Hogness, supra, using radioactive HPO lyase gene fragments. Clones which hybridize to the radioactive gene fragments are analyzed, e.g., by restriction endonuclease cleavage or agarose gel electrophoresis. After isolating several positive clones the positive insert of one clone is subcloned, e.g.,

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