DNA molecules encoding bacterial lysine 2,3-aminomutase

Details DNA molecules encoding bacterial lysine 2,3-aminomutase DNA molecules encoding bacterial lysine 2,3-aminomutase

1999-06-11

2001-06-19

Prouty, Rebecca E. (Department: 1653)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S183000, C435S233000, C435S069100, C435S252300, C435S252330, C536S023100

Reexamination Certificate

active

06248874

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to DNA molecules that encode lysine 2,3-aminomutase. More particularly, this invention relates to the use of recombinant host cells comprising such DNA molecules to produce pure L-&bgr;-lysine.
2. Related Art
Although less abundant than the corresponding &agr;-amino acids, &bgr;-amino acids occur in nature in both free forms and in peptides. Cardillo and Tomasini,
Chem. Soc. Rev.
25:77 (1996); Sewald,
Amino Acids
11:397 (1996). Since &bgr;-amino acids are stronger bases and weaker acids than &agr;-amino acid counterparts, peptides that contain a &bgr;-amino acid in place of an &agr;-amino acid, have a different skeleton atom pattern, resulting in new properties. For example, various peptides are protease inhibitors because the presence of the &bgr;-amino-&agr;-hydroxy acid motif acts as a transition state mimic of peptide hydrolysis.
&bgr;-Amino acids are of particular interest in the preparation of medicaments, such as &bgr;-lactams. Well-known &bgr;-lactam antimicrobial agents include penicillins, cephalosporins, carbapenems, and monobactams. Other examples of medically useful molecules that contain &bgr;-amino-&agr;-hydroxy acids include the anti-tumor agent taxol, the anti-bacterial agent, dideoxykanamicin A, bestatin, an immunological response modifier, the kynostatins, which are highly potent human immunodeficiency virus-1 protease inhibitors, and microginin, a tetrapeptide which has anti-hypertensive properties. Accordingly, enantiomerically pure &bgr;-amino-&agr;-hydroxy acids are of considerable importance as crucial components of pharmacologically active compounds.
In the 1950's, L-&bgr;-lysine was identified in several strongly basic peptide antibiotics produced by Streptomyces. Antibiotics that yield L-&bgr;-lysine upon hydrolysis include viomycin, streptolin A, streptothricin, roseothricin and geomycin. Stadtman,
Adv. Enzymol. Relat. Areas Molec. Biol.
38:413 (1973). &bgr;-Lysine is also a constituent of antibiotics produced by the fungi Nocardia, such as mycomycin, and &bgr;-lysine may be used to prepare other biologically active compounds. However, the chemical synthesis of &bgr;-lysine is time consuming, requires expensive starting materials, and results in a racemic mixture.
Therefore, a need exists for an improved method of preparing enantiomerically pure &bgr;-amino acids, such as &bgr;-lysine.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides an isolated DNA molecule comprising a nucleotide sequence that encodes lysine 2,3-aminomutase.
In another aspect, the present invention provides an expression vector comprising an isolated DNA molecule having a nucleotide sequence that encodes lysine 2,3-aminomutase.
The present invention additionally provides a method of producing lysine 2,3-aminomutase comprising the steps of culturing a host cell containing an expression vector having a nucleotide sequence that encodes lysine 2,3-aminomutase and isolating lysine 2,3-aminomutase from the cultured host cells.
The present invention provides, in a further aspect, a method of producing L-&bgr;-lysine from L-lysine comprising incubating L-lysine in a solution containing purified lysine 2,3-aminomutase and isolating the L-&bgr;-lysine from the solution.
Still another aspect of the present invention is a method of producing L-&bgr;-lysine from L-lysine comprising the steps of incubating culturing a host cell in the presence of L-lysine, wherein the cultured host cell expresses lysine 2,3-aminomutase and isolating the L-&bgr;-lysine from the cultured host cell.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
In the description that follows, a number of terms are utilized extensively. Definitions are herein provided to facilitate understanding of the invention.
Structural gene. A DNA sequence that is transcribed into messenger RNA (mRNA) which is then translated into a sequence of amino acids characteristic of a specific polypeptide (protein).
Promoter. A DNA sequence which directs the transcription of a structural gene to produce mRNA. Typically, a promoter is located in the 5′ region of a gene, proximal to the start codon of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
Enhancer. A promoter element. An enhancer can increase the efficiency with which a particular gene is transcribed into mRNA irrespective of the distance or orientation of the enhancer relative to the start site of transcription.
Complementary DNA (cDNA). Complementary DNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the art also use the term “cDNA” to refer to a double-stranded DNA molecule derived from a single mRNA molecule.
Expression. Expression is the process by which a polypeptide is produced from a structural gene. The process involves transcription of the gene into mRNA and the translation of such mRNA into polypeptide(s).
Cloning vector. A DNA molecule, such as a plasmid, cosmid, phagemid, or bacteriophage, which has the capability of replicating autonomously in a host cell and which is used to transform cells for gene manipulation. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences may be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene which is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
Expression vector. A DNA molecule comprising a cloned structural gene encoding a foreign protein which provides the expression of the foreign protein in a recombinant host. Typically, the expression of the cloned gene is placed under the control of (i.e., operably linked to) certain regulatory sequences such as promoter and enhancer sequences. Promoter sequences may be either constitutive or inducible.
Recombinant host. A recombinant host may be any prokaryotic or eukaryotic cell which contains either a cloning vector or expression vector. This term is also meant to include those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell. For examples of suitable hosts, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) [“Sambrook”].
As used herein, a substantially pure protein means that the desired purified protein is essentially free from contaminating cellular components, as evidenced by a single band following polyacrylamide-sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The term “substantially pure” is further meant to describe a molecule which is homogeneous by one or more purity or homogeneity characteristics used by those of skill in the art. For example, a substantially pure lysine 2,3-aminomutase will show constant and reproducible characteristics within standard experimental deviations for parameters such as the following: molecular weight, chromatographic migration, amino acid composition, amino acid sequence, blocked or unblocked N-terminus, HPLC elution profile, biological activity, and other such parameters. The term, however, is not meant to exclude artificial or synthetic mixtures of lysine 2,3-aminomutase with other compounds. In addition, the term is not meant to exclude lysine 2,3-aminomutase fusion proteins isolated from a recombinant host.
2. Isolation of a DNA Molecule That Encodes the Clostridium Lysine 2,3-Aminomutase
Lysine 2,3-aminomutase catalyzes the reversible isomerizat

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