DNA shuffling to produce nucleic acids for mycotoxin...

Details DNA shuffling to produce nucleic acids for mycotoxin... DNA shuffling to produce nucleic acids for mycotoxin...

1999-10-06

2002-12-31

Venkat, Jyothsna (Department: 1627)

Chemistry: molecular biology and microbiology

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

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

C435S455000, C435S468000, C435S471000, C435S091500, C435S091500, C800S279000

Reexamination Certificate

active

06500639

ABSTRACT:

COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
This invention pertains to the shuffling of nucleic acids to achieve or enhance mycotoxin detoxification, especially in plants.
BACKGROUND OF THE INVENTION
“Mycotoxins” generically refer to a number of toxic molecules produced by fungal species, such as polyketides (including aflatoxins, demethylsterigmatocystin, O-methylsterigmatocystin etc.), fumonisins, alperisins (e.g., A
1
, A
2
, B
1
, B
2
), sphingofungins (A, B, C and D), trichothecenes, fumifungins, and the like. Polyketides are a large structurally diverse class of secondary metabolites synthesized by bacteria, fungi, and plants and are formed by a polyketide synthase (PKS) through the sequential condensation of small carboxylic acids. Katz and Donandio (1993)
Annu Rev. Microbiool.
47:875-912; Brown et al. (1996)
PNAS
93:14873-14877; Silva et al. (1996)
J. Biol Chem.
271: 13600-608.
Aflatoxin B1, is the principal member of the aflatoxin (AF) family of polyketide mycotoxins produced by
Aspergillus parasiticus, Aspergillus flavus
and
Aspergillus nomius.
Aflatoxin B1 is the most potent mycotoxin known to man. For example, AF was characterized as the causative agent for the death of more than a hundred thousand poultry in England that had ingested AF-contaminated peanut meal. This discovery led to legislation regulating the trade of AF-contaminated agricultural commodities.
Sterigniatocystin (ST) is a related polyketide mycotoxin, which is produced by several members of the Aspergillus. ST is the second to last intermediate in the biosynthesis of AF. Kelkar et al. (1997)
J. Biol Chem.
272: 1589-94. Various Aspergillus species that produce AF and ST are known to be pathogenic to corn, grains and nuts and are known to produce these mycotoxins during the growth of the crops and during storage, leading to the introduction of AF and ST into primary food stuffs. AF and ST are acutely toxic and carcinogenic and are a serious concern from human and animal health perspective. Busby & Wogan (1985) in
Chemical Carcinogens
(Searle ed., 1985) pp 945-1136, American Chemical Society, Washington D.C.
Trichothecenes are another family of sesquiterpenoid mycotoxins produced by Fusarium species and other molds that are known plant pathogens. These compounds are potent inhibitors of protein synthesis in eukaryotes (Kimura et al. (1998)
J. Biol Chem.
273: 1654-1661) and reportedly bind to the 60S ribosomal subunits to prevent polypeptide chain initiation or elongation. Trichothecenes are also an important group of mycotoxins that cause serious problems of food pollution. They have been implicated in incidents of mycotoxicosis including vomiting, dermatitis and hemorrhagic septicemia in humans and livestock, resulting in loss of productivity and even death. Lastly, fumonisins (F) are another structurally distinct class of mycotoxins produced by several Fusarium species that is involved in food poisoning and toxic effects. Scott (1993)
International Journal of Food Microbiology
18:257-270 and the references therein provide a review of the Fuminosins.
Thus, the contamination of corn, grains and nuts with various types of mycotoxins produced by pathogenic species such as Aspergillus and Fusarium is a major health and food pollution problem, as well as causing reduction in crop yields by being toxic to infected plants. These mycotoxins survive food processing, which adds to the problem. It is well known that ST and AF induce liver cancer and are linked to a specific mutation in a tumor suppressor gene. Brown et al. (1996)
PNAS
93: 14873-14877. Natural aflatoxins and other mycotoxins like ST do not pose a major health threat per se; however, renal and hepatic oxidative detoxification of these compounds in contaminated foods by cytochrome P450 enzymes yields an epoxide which is cytotoxic.
For example, AFB1 is converted to its 15,16-exo-epoxide, which is a highly toxic mutagen. Silva et al. (1996), supra and references therein. It has been shown that this epoxide targets guanine residues and selectively alkylates the N-7 position of this purine in double-stranded DNA. Depurination of the alkylated base has been correlated to bladder cancer in laboratory mice, teratogenic effects in chicken embryos and liver cancer in humans. A direct correlation between DNA damage and human cancer has been established and is related to the mutational hot spots of p53, an important tumor suppressor gene. Approximately 50% of all cancers have associated altered p53 sequences.
Trading of AF-contaminated agricultural commodities is tightly regulated at both national and international levels. Compliance to these regulations causes the loss of millions of dollars in agricultural produce in US each year. Trade sanctions and health effects on mycotoxin contaminated grains add significantly to the losses (Brown et al. (1996)
PNAS
93: 14873-14877).
Accordingly, it is highly desirable to transform various mycotoxins produced by fungal pathogens in various crops into inactive compounds with respect to plant, human and animal toxicity. This would alleviate important food pollution problems, as well as cost associated with complying with detecting AF-contamination in various crop commodities and destroying them. Surprisingly, the present invention provides for the detoxification of mycotoxis by transformation of the mycotoxins into non-toxic compounds. This detoxification is particularly useful in crops, thereby solving each of the problems outlined above, as well as providing a variety of other features which will be apparent upon review.
SUMMARY OF THE INVENTION
In the present invention, DNA shuffling is used to generate new or improved mycotoxin detoxification genes. These mycotoxin detoxification genes are used to provide enzymes which degrade mycotoxins, in agricultural and industrial processes. These new and/or improved genes have surprisingly superior properties as compared to naturally occurring mycotoxin detoxification genes.
In the methods for obtaining mycotoxin resistant genes, a plurality of parental forms (homologs) of a selected nucleic acid are recombined. The selected nucleic acid is derived either from one or more parental nucleic acid(s) which encodes an enzyme which degrades or modifies a mycotoxin, or a fragment thereof, or from a parental nucleic acid which does not encode mycotoxin detoxification, but which is a substrate for DNA shuffling to develop monooxygenase activity. The plurality of forms of the selected nucleic acid differ from each other in at least one (and typically two or more) nucleotides, and, upon recombination, provide a library of recombinant mycotoxin detoxification nucleic acids. The library can be an in vitro set of molecules, or present in cells, phage or the like. The library is screened to identify at least one recombinant mycotoxin detoxification nucleic acid that exhibits distinct or improved mycotoxin detoxification activity (typically in an encoded polypeptide) compared to the parental nucleic acid or nucleic acids.
In selecting for mycotoxin detoxification activity, a candidate shuffled DNA can be tested for encoded mycotoxin detoxification activity in essentially any process. Common processes that can be screened include screening for inactivation or modification of an aflatoxin, inactivation or modification of a sterigmatocystin, inactivation or modification of a trichothecene, and inactivation or modification of a fumonisin. Similarly, instead of, or in addition to, testing for an increase in mycotoxin detoxification activity, it is also desirable to screen for shuffled nucleic acids which produce higher levels of a mycotoxin detoxification nucleic acid or enhanced or reduc

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