DNA constructs and methods of producing cellulytic enzymes

Details DNA constructs and methods of producing cellulytic enzymes DNA constructs and methods of producing cellulytic enzymes

1999-01-07

2001-08-28

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Preparing oxygen-containing organic compound

C435S204000

Reexamination Certificate

active

06280984

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to isolated nucleic acid sequences and constructs encoding cellulytic enzymes derived from a strain of Bacillus, recombinant expression vectors and host cells comprising such constructs, and methods for obtaining cellulytic enzymes.
BACKGROUND OF THE INVENTION
PCT publication WO 94/01532 describes a new species of alkalophilic Bacillus, initially named Bacillus sp. AC13, as well as proteases, xylanases and cellulases obtainable therefrom. A sample of the strain was deposited as NCIMB 40482. WO 94/01532 also describes methods for the production of these enzymes by cultivation of a strain of Bacillus sp. AC13. However, WO 94/01532 does not describe nucleic acid constructs comprising a nucleic acid sequence encoding cellulytic enzymes derived from a strain Bacillus sp. AC13, or methods of producing these cellulytic enzymes by recombinant DNA technology.
The same new species as described in WO 94/01532 has been described by Nielsen et al. (1995) Microbiology 141:1745-1761, with the now established name,
Bacillus agaradhierens.
A sample of the strain has been deposited as DSM 8721. Nielsen et al. (1995) supra, however, do not describe nucleic acid sequences or constructs encoding cellulytic enzymes derived from a strain
Bacillus agaradhierens,
or methods of producing these cellulytic enzymes by recombinant DNA technology.
SUMMARY OF THE INVENTION
The invention features an isolated DNA sequence derived from Bacillus encoding a cellulytic enzyme, thereby making it possible to prepare a mono-component enzyme preparation.
Accordingly, in one aspect, the invention provides an isolated DNA sequence derived from
Bacillus agaradhierens
encoding a polypeptide having cellulytic enzyme activity. In a specific embodiment, the isolated DNA sequence is the sequence of SEQ ID NO:1. In another related embodiment, the isolated DNA sequence is a DNA sequence encoding a cellulytic enzyme having more than 98% homology to the cellulytic enzyme encoded by the DNA sequence of SEQ ID NO:1. Included in the invention is an isolated DNA sequence complementary to SEQ ID NO:1, and a fragment of the sequence of SEQ ID NO:1 (or its complementary sequence) that is at least 15 base pairs in length that selectively hybridizes under stringent conditions to DNA sequences encoding the cellulytic enzyme of SEQ ID NO:1. In still further embodiments, the DNA sequence is isolated from a Bacillus strain identified by the deposit accession number DSM 8721 or NCIMB 40482.
In further aspects, the invention provides a DNA construct having the DNA sequence of SEQ ID NO:1, an expression vector harboring the DNA construct of the invention, a cell having the DNA construct or expression vector of the invention, as well as a method of producing a cellulytic enzyme by culturing the cell of the invention under conditions permitting the production of the cellulytic enzyme, and recovering the cellulytic enzyme from the culture.
In another aspect, the invention features an isolated polypeptide encoded by SEQ ID NO:1 and having cellulytic activity. The invention includes an isolated polypeptide having the amino acid sequence of SEQ ID NO:2, or a polypeptide having an amino acid sequence with at least 80%, 90%, or 95% identity with the amino acid sequence of SEQ ID NO:2.
The invention further features an enzyme preparation comprising the cellulytic enzyme encoded by the DNA sequence of SEQ ID NO:1.
DETAILED DISCLOSURE OF THE INVENTION
Before the methods and compositions of the present invention are described and disclosed it is to be understood that this invention is not limited to the particular methods and compositions described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.
It must be noted that as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a DNA sequence” includes a plurality of DNA sequences and different types of DNA sequences.
Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials or methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the particular information for which the publication was cited. The publications discussed above are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventor is not entitled to antedate such disclosure by virtue of prior invention.
Isolated DNA Sequences and DNA Constructs
The present invention provides an isolated DNA sequence and a construct comprising the DNA sequence encoding a cellulytic enzyme. The DNA sequence of the invention includes (a) the DNA sequence of SEQ ID NO:1, (b) a DNA sequence encoding a polypeptide having more than 98% homology with the cellulytic enzyme encoded by SEQ ID NO:1, (c) a sequence complementary to SEQ ID NO:1, and (d) a fragment of the sequence of (a), (b), or (c) that is at least 15 base pairs in length that selectively hybridizes under stringent conditions to DNA sequences encoding the cellulytic enzyme of SEQ ID NO:1.
As defined herein the term “DNA construct” is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin. The term “construct” is intended to indicate a nucleic acid segment which may be single- or double-stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a cellulytic enzyme of interest. It is understood that such nucleotide sequences include intentionally manipulated nucleotide sequences, e.g., subjected to site-directed mutagenesis, and sequences that are degenerate as a result of the genetic code. All degenerate nucleotide sequences are included in the invention so long as the cellulytic enzyme encoded by the nucleotide sequence is functionally unchanged. The construct may optionally contain other nucleic acid segments.
The DNA construct of the invention preferably is of microbial origin, preferably derived from a strain of Bacillus. In its most preferred embodiments, the DNA construct of the invention is derived from a strain of the new alkalophilic species
Bacillus agaradhierens,
formerly referred to as Bacillus AC13.
The DNA construct of the invention encoding the cellulytic enzyme may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the cellulytic enzyme by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. e.g. Sambrook et al. (1989)
Molecular Cloning. A Laboratory Manual
, Cold Spring Harbor, N.Y.).
The nucleic acid construct of the invention encoding the cellulytic enzyme may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers (1981) Tetrahedron Letters 22:1859-1869, or the method described by Matthes et al. (1984) EMBO J. 3:801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
The nucleic acid construct may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or by Saiki et al. (1988) Science 239:487-491.
Furthermore, the nucleic acid construct may be of mixed synthetic and genomic DNA, mixed synthetic and

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