Dihydrodipicolinate synthase from Bacillus methanolicus

1. Field of the Invention

The present invention relates to dihydrodipicolinate synthase and dihydrodipicolinate reductase derived thermophilic Bacillus bacteria and genes coding for them.

2. Related Art

In the production of L-lysine by fermentation, strains isolated from nature or artificial mutants thereof have been used in order to improve the productivity. Many artificial mutant strains that produce L-lysine are known, and many of them are aminoethylcysteine (AEC) resistant strains and belong to the genus Brevibacterium, Corynebacterium, Bacillus or Escherichia. Further, various techniques have been disclosed for increasing the amino acid production, for example, use of a transformant obtained by using recombinant DNA (U.S. Pat. No. 4,278,765).

Dihydrodipicolinate synthase (abbreviated as “DDPS” hereinafter) is an enzyme that synthesizes dihydrodipicolinate through dehydration condensation of aspartic acid semialdehyde and pyruvic acid, and this reaction serves as an entrance of the branching into the L-lysine biosynthesis system in the biosynthesis of amino acids of aspartic acid type. Further, dihydrodipicolinate reductase (abbreviated as “DDPR” hereinafter) is known as one of important enzymes of the L-lysine biosynthesis system, which catalyzes the reaction in which the dihydrodipicoliniate generated in the aforementioned reaction is reduced to generate piperidinedicarboxylic acid.

As for microorganisms belonging to the genus Escherichia or Corynebacterium, the gene (dapA) which codes for DDPS has been cloned, and the nucleotide sequence thereof has also be determined. As for the genus Escherichia, methods for producing L-lysine by enhancing DDPS have been disclosed in Japanese Patent Laid-open Publication (Kokai) No. 56-18596/1981, U.S. Pat. No. 4,346,170 and Applied Microbiology and Biotechnology, 15, pp.227-331 (1982). Furthermore, a method for producing L-lysine using an Escherichia bacterium introduced with DDPS derived form Corynebacterium bacteria, which is known not to suffer feedback inhibition by L-lysine, has been disclosed in Korean Patent Publication No. 92-8382.

The gene coding for DDPR (dapB) has also been already obtained from the genus Escherichia (Bouvier, J. et al., J. Biol. Chem., 259, 14829 (1984)) and the genus Corynebacterium ( Journal of Bacteriology, 175 (9), 2743-2749 (1993)). Furthermore, there has also been disclosed a method for improving production rate and productivity of L-lysine by enhancing the dapB gene derived from Corynebacterium bacterium together with the aspartokinase gene (WO96/40934).

The current mainstream of the L-lysine production is the fermentative production by using a coryneform bacterium or an Escherichia bacterium. In this production, however, enzymes required for the fermentation may be inactivated or the production bacteria may be killed due to temperature increase in the medium during the fermentation, and thus it is necessary to cool the medium during the fermentation.

By the way, enzymes and proteins produced by thermophilic bacteria are generally stable at elevated temperatures, and also stable against pH variation or organic solvents. Therefore, applications thereof as diagnostic regents, industrial catalysts and so forth have been highly developed. If it becomes possible to produce L-lysine by fermentation at elevated temperatures by utilizing such stability and durability of enzymes derived from thermophilic bacteria, the cooling of the medium becomes unnecessary, and therefore the cost for cooling during the fermentation can be reduced. Moreover, if fermentation at elevated temperatures is realized, it is expected that the reaction rate may also be improved

The present invention is accomplished in view of the aforementioned technical aspect, and its object is to obtain genes of the L-lysine biosynthesis system of thermophilic bacteria and thereby provide novel methods for producing L-lysine.

The inventors of the present invention assiduously studied in order to achieve the aforementioned object. As a result, they successfully isolated genes that coded for DDPS and DDPR from Bacillus methanolicus, which is one of the thermophilic Bacillus bacteria, and determined the nucleotide sequences of these genes. Thus, they accomplished the present invention.

That is, the present invention provides the followings.

(1) A protein defined in the following (A) or (B):

(A) a protein which has the amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing, or (B) a protein which has an amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate synthase activity.

(2) A DNA which codes for a protein defined in the following (A) or (B):

(A) a protein which has the amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing, or

(B) a protein which has an amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate synthase activity.

(3) The DNA according to (2), which is a DNA defined in the following (a) or (b):

(a) a DNA which has a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 924 in SEQ ID NO: 1 shown in Sequence Listing; or

(b) a DNA which is hybridizable with a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 924 in SEQ ID NO: 1 shown in Sequence Listing under a stringent condition, and codes for a protein having dihydrodipicolinate synthase activity.

(4) The DNA according to (3), wherein the stringent condition is a condition in which washing is performed at 60° C., 1×SSC and 0.1% SDS.

(5) A protein defined in the following (C) or (D):

(C) a protein which has the amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing, or

(D) a protein which has an amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate reductase activity.

(6) A DNA which codes for a protein defined in the following (C) or (D):

(C) a protein which has the amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing, or

(D) a protein which has an amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate reductase activity.

(7) The DNA according to (6), which is a DNA defined in the following (c) or (d):

(c) a DNA which has a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 798 in SEQ ID NO: 3 shown in Sequence Listing; or

(d) a DNA which is hybridizable with a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 798 in SEQ ID NO: 3 shown in Sequence Listing under a stringent condition, and codes for a protein having dihydrodipicolinate reductase activity.

(8) The DNA according to (7), wherein the stringent condition is a condition in which washing is performed at 60° C., 1×SSC and 0.1% SDS.

(9) A microorganism which is introduced with the DNA according to (2) or the DNA according to (6) or the both in a form that allows expression of a protein encoded by each DNA.

(10) A method for producing L-lysine, which comprises culturing the microorganism according to (9) in a medium to produce and accumulate L-lysine in the medium, and collecting the L-lysine from the medium.

In the present invention, the expression of “to have dihydrodipicolinate synthase activity” is used to mean to have an activity for catalyzing the reaction of dehydration condensation of aspartic acid semialdehyde and pyruvic acid to generate dihydrodipicolinate. The expression of “to have dihydrodipicolinate reductase activity” is used to mean to have an activity for catalyzing the reaction of reduction of dihydrodipicolinate to generate piperidinedicarboxylic acid, and to mean that the activity is higher at 50° C. than at 37° C.

According to the present invention, there are provided DDPS and DDPR, which are involved in the L-lysine biosynthesis, with excellent heat resistance, and genes coding for them.

Hereafter, the present invention will be explained in detail.

The DNA of the present invention can be obtained through selection of clones containing a DDPS gene or a DDPR gene based on recovery of auxotrophy in mutant strains of microorganisms deficient in DDPS or DDPR used as an index from a gene library of thermophilic Bacillus bacteria, for example, Bacillus methanolicus.

The method for obtaining the DNA of the present invention will be explained below.

<1> Production of Gene Library of Bacillus methanolicus

A gene library of Bacillus methanolicus can be produced, for example, as follows. First, the total chromosome DNA is prepared by the method of Saito et al. (Saito, H. and Miura, K., Biochem. Biophys. Acta, 72, 619-629, (1963)) or the like from a wild-type strain of Bacillus methanolicus, for example, Bacillus methanolicus PB1 (NCIMB13113) strain, and partially digested with a suitable restriction enzyme, for example, Sau3AI and so forth to obtain a mixture of various fragments. If the degree of the digestion is controlled by adjusting digestion reaction time and so forth, restriction enzymes of a wide range can be used.

Subsequently, the digested chromosome DNA fragments are ligated to vector DNA autonomously replicable within Escherichia coli cells to produce recombinant DNA. More specifically, a restriction enzyme producing the same end nucleotide sequence as the restriction enzyme used for the digestion of the chromosome DNA is allowed to act on the vector DNA to fully digest the vector and cleave it. Then, the mixture of the chromosome DNA fragments and the cleaved vector DNA obtained as described above are mixed, and a DNA ligase, preferably T4 DNA ligase, is allowed to act on the mixture to obtain recombinant DNA.

By using the obtained recombinant DNA, Escherichia coli, for example, the Escherichia coli JM109 strain and so forth, is transformed, and a gene library solution can be prepared from culture of obtained transformants. The transformation can be performed by, for example, the method of D. M. Morrison (Methods in Enzymology, 68, 326 (1979)) and the method in which recipient cells are treated with calcium chloride so as to increase the permeability of the cells for DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)). Electroporation was employed in the examples mentioned below.

Examples of the vector include, for example, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF100, pMW119, pMW118, pMW219, pMW218, pSTV28 and so forth. In addition, phage vectors can also be used. For example, since a chloramphenicol resistance gene is contained in pSTV28, only transformants harboring the vector or the recombinant DNA can be grown by using a medium containing chloramphenicol when that vector is used.

Examples of the method for collecting the recombinant DNA from the cells after the transformants are cultured include the alkali SDS method and so forth.

<2> Screening of Clones Containing DDPS Gene or DDPR Gene

By using a gene library solution of Bacillus methanolicus obtained as described above, a mutant strain of microorganism deficient in DDPS or DDPR is transformed, and clones showing recovery of auxotrophy are selected. Examples of such a mutant strain of microorganism deficient in DDPS include the Escherichia coli AT998 (CGSC4548) strain, which is deficient in DDPS. Since the Escherichia coli AT998 strain is deficient in the DDPS gene, it cannot grow in a minimal medium that does not contain diaminopimelate. On the other hand, a transformant strain thereof which harbors the DDPS gene derived from Bacillus methanolicus can grow in the minimal medium, because the gene functions. Therefore, a DNA fragment containing the DDPS gene can be obtained by selecting a transformant strain that can grow in the minimal medium and collecting recombinant DNA from the strain.

Examples of the mutant strain of microorganism deficient in DDPR include the Escherichia coli AT999 strain (CGSC 4549), which is deficient in the DDPR gene. Since the Escherichia coil AT999 strain is deficient in the DDPR gene, it shows only a slow growth rate even in a complete medium such as L medium if it does not contain diaminopimelate. On the other hand, a transformant strain thereof which harbors the DDPR gene derived from Bacillus methanolicus shows normal growth, because the gene functions. Further, the Escherichia coli AT999 cannot grow in a minimal medium, whereas a transformant strain thereof which harbors the DDPR gene derived from Bacillus methanolicus can grow in the minimal medium, because the gene functions. Therefore, a DNA fragment containing the DDPR gene can be obtained by selecting a transformant strain which can grow in the minimal medium and collecting recombinant DNA from the strain.

By extracting an inserted DNA fragment from the obtained recombinant DNA and determining nucleotide sequence of the fragment, the nucleotide sequence and the amino acid sequence of the DDPS gene or the DDPR gene and DDPS or DDPR can be determined.

Determination of nucleotide sequences, digestion and ligation of DNA and so forth may be attained by those methods commonly used for gene cloning (detailed in, for example, Sambrook J., Fritsch, E. F. and Maniatis, T., 1989, Molecular Cloning: A Laboratory Manual, Second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. etc.). They can also be conducted according to instructions attached to reagents such as restriction enzymes and kits.

The DDPS gene of the present invention codes for DDPS that has the amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing. Specific examples of the DDPS gene of the present invention include DNA that has the nucleotide sequence of SEQ ID NO: 1. Further, the DDPS gene of the present invention may have a nucleotide sequence including replacement of codons for each amino acid with equivalent codons, so long as the sequence codes for the same amino acid sequence as the amino acid sequence shown as SEQ ID NO: 2.

Further, the DDPS gene of the present invention may be one coding for a protein which has an amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, addition or inversion of one or several amino acids, or one coding for a protein which has DDPS activity. The term “several” amino acids used herein means preferably 1-50 amino acids, more preferably 1-10 amino acids. Homology between the DDPS gene of the present invention and a known DDPS gene of Bacillus subtilis ( B. subtilis ) is 65.9% on the basis of the nucleotide sequence, and 64.8% on the basis of the encoded amino acid sequence.

The DDPR gene of the present invention codes for DDPR that has the amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing. Specific examples of the DDPR gene of the present invention include DNA that has the nucleotide sequence of SEQ ID NO: 3. Further, the DDPR gene of the present invention may have a nucleotide sequence including replacement of codons for each amino acid with equivalent codons, so long as the sequence codes for the same amino acid sequence as the amino acid sequence shown as SEQ ID NO: 4.

Further, the DDPR gene of the present invention may be one coding for a protein which has an amino acid sequence of SEQ ID NO: 4 including substitution, deletion, insertion, addition or inversion of one or several amino acids, or one coding for a protein which has DDPR activity. The term “several” amino acids used herein means preferably 1-40 amino acids, more preferably 1-10 amino acids. Homology between the DDPR gene of the present invention and a known DDPR gene of Bacillus subtilis ( B. subtilis ) is 66.7% on the basis of the nucleotide sequence, and 67.5% on the basis of the encoded amino acid sequence.

DNA that codes for the substantially same protein as DDPS or DDPR as described above is obtained by modifying the nucleotide sequence, for example, by means of the site-directed mutagenesis method so that one or more amino acid residues at a specific site should contain substitution, deletion, insertion, addition or inversion. DNA modified as described above may also be obtained by conventionally known mutation treatments. Such mutation treatments includes a method for treating DNA coding for DDPS or DDPR in vitro, for example, with hydroxylamine or the like, and a method for treating a microorganism, for example, a bacterium belonging to the genus Escherichia, harboring DNA coding for DDPS or DDPR with ultraviolet irradiation or a mutagenizing agent usually used for the mutation treatment such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid.

The substitution, deletion, insertion, addition, or inversion of nucleotides as described above also includes mutation (mutant or variant) which naturally occurs, for example, due to the individual difference or the difference in species or genus of the microorganism that harbors DDPS or DDPR.

Such DNA coding for substantially the same protein as DDPS or DDPR is obtained by expressing DNA having mutation described above in an appropriate cell, and investigating the DDPS or DDPR activity of the expression product. DNA coding for substantially the same protein as DDPS or DDPR is also obtained by isolating DNA which is hybridizable with a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1 or 3 shown in Sequence Listing or a part thereof, for example, a probe which can be prepared from the nucleotide sequence of SEQ ID NO: 1 or 3 by PCR, under a stringent condition, and codes for a protein having DDPS or DDPR activity.

The “stringent condition” referred to herein is a condition under which a so-called specific hybrid is formed, and a non-specific hybrid is not formed. It is difficult to clearly define this condition by using numerical values. However, for example, the stringent condition includes a condition under which DNA's having high homology, for example, DNA's having homology of not less than 40% are hybridized with each other, and DNA's having homology lower than the above are not hybridized with each other. Alternatively, the stringent condition is exemplified by a condition under which DNA's are hybridized with each other at a salt concentration corresponding to an ordinary condition of washing in Southern hybridization, i.e., 60° C., 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS (see, for example, Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. etc.).

Such genes, which are hybridizable under the condition as described above, includes those having a stop codon generated in a coding region of the genes, and those having no activity due to mutation of active center. However, such mutants can be easily removed by ligating the genes with a commercially available activity expression vector, and measuring the DDPS or DDPR activity. The DDPS activity can be measured by, for example, the method of Yugari et al. (Yugari Y. and Gilvarg C., Journal of Biological Chemistry, 240, 4710 (1962)). Specifically, for example, the DDPS activity can be measured by allowing a reaction of 100 μl of 500 mM imidazole hydrochloride (pH 7.5), 100 μl of 20 mM aspartic acid semialdehyde (which can be synthesized by the method described in Black S. and Write N., Journal of Biological Chemistry, 213, 51 (1955)), 100 μl of 20 mM sodium pyruvate and 100 μl of an enzyme solution in a total volume of 1 ml and measuring increase of absorbance at 270 nm.

The DDPR activity can be measured by, for example, the method of Tamir et al. (Tamir H. and Gilvarg C., Journal of Biological Chemistry, 249, 3034 (1974)). Specifically, for example, the DDPR activity can be measured by allowing a reaction of 100 μl of 500 mM imidazole hydrochloride (pH 7.5), 100 μl of dihydrodipicolinic acid, 100 μl of NADPH and 100 μl of an enzyme solution in a total volume of 1 ml and measuring decrease of absorbance at 340 nm.

Because the nucleotide sequences of the genes which code for DDPS and DDPR derived from Bacillus methanolicus were elucidated by the present invention, the DNA sequence which codes for DDPS or DDPR can be obtained from Bacillus methanolicus gene library by hybridization using an oligonucleotide probe prepared based on each of the sequences. DNA sequences which code for the enzymes can also be obtained by amplification from Bacillus methanolicus chromosome DNA by PCR (polymerase chain reaction) using oligonucleotide primers prepared based on the aforementioned nucleotide sequences.

<3> Application of DDPS Gene and DDPR Gene

The DDPS gene and the DDPR gene of the present invention can be used for production of DDPS and DDPR. That is, DDPS can be produced by introducing DNA containing the DDPS gene into a suitable host cell, and culturing the obtained transformant to allow expression of the DNA. DDPR can be produced by introducing DNA containing the DDPR gene into a suitable host cell, and culturing the obtained transformant to allow expression of the DNA. The produced DDPS protein or DDPR protein can be collected from the culture and purified by techniques used for usual purification of proteins such as salting out, solvent precipitation, gel filtration chromatography and ion exchange chromatography.

The DDPS gene and the DDPR gene can also be utilized for breeding of L-lysine producing bacteria. By introducing the DDPS gene, the DDPR gene or the both into a microorganism, L-lysine biosynthesis is enhanced and thus L-lysine producing ability is improved.

Examples of the host cell into which the DDPS gene or the DDPR gene is introduced include Escherichia bacteria such as Escherichia coli, coryneform bacteria such as Brevibacterium lactofermentum, Bacillus bacteria such as Bacillus methanolicus and so forth. Examples of the vector used for introducing the DDPS gene or the DDPR gene into these hosts include, as for Escherichia bacteria, those mentioned above. As for coryneform bacteria, the following vectors can be mentioned. There are indicated microorganisms which harbors each vector, and accession numbers thereof at international depositories are shown in the parentheses, respectively.

pAJ655

Escherichia coli AJ11882 (FERM BP-136)

Corynebacterium glutamicum SR8201

(ATCC39135)

pAJ1844

Escherichia coli AJ11883(FERM BP-137)

Corynebacterium glutamicum SR8202

(ATCC39136)

pAJ611 Escherichia coli AJ11884 (FERM BP-138)

pAJ3148 Corynebacterium glutamicum SR8203

(ATCC39137)

pAJ440 Bacillus subtilis AJ11901 (FERM BP-140)

These vectors are obtained from deposited microorganisms as follows. Cells collected at the logarithmic growth phase are lysed with lysozyme and SDS to give a lysate, from which a supernatant solution is obtained by centrifugation at 30,000×g. Polyethylene glycol is added to the supernatant solution to perform fractional purification by means of cesium chloride-ethidium bromide equilibrium density gradient centrifugation.

In order to introduce a plasmid into E. coli to transform it, there may be used a method in which recipient cells are treated with calcium chloride so as to increase the permeability of the cells for DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and so forth.

Examples of the vector for Bacillus bacteria include, for example, pUB110, pHY300PLK, pHV1248, pE194, pC194, pBC16, pSA0501, pSA2100, pAM77, pT181, pBD6, pBD8, pBD64, pHV14 and so forth.

Transformation of coryneform bacteria may be performed by the electric pulse method (Sugimoto et al., Japanese Pat. Publication Laid-Open No. 2-207791/1990). Transformation of Bacillus bacteria may be performed by a method of making host sells into the protoplast or spheroplast followed by introducing recombinant DNA into the DNA-recipient cells (Chang, S. and Choen, S. N., Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G. R., Proc. Natl. Acad. Sci., USA, 75, 1929 (1978)).

The DDPS gene or the DDPR gene to be introduced may be introduced into a host with a promoter proper to the gene, or the structural gene ligated to another promoter may be introduced. Examples of such a promoter include lac promoter, trp promoter, trc promoter, tac promoter, P R promoter and P L promoter of λ phage, tet promoter, amyE promoter, spac promoter and so forth.

L-lysine can be produced by culturing a microorganism introduced with the DDPS gene or the DDPR gene or the both genes and having L-lysine producing ability in a medium to produce and accumulate L-lysine in the medium, and collecting the L-lysine from the medium.

Although medium and culture conditions can suitably be selected according to kind of the host microorganism to be used, usual media can be used, which contain a nitrogen source, inorganic ions and other organic trace amount nutrients as required.

As the carbon source, there can be used saccharides such as glucose, lactose, galactose, fructose and hydrolysate of starch, alcohols such as glycerol and sorbitol, organic acids such as fumaric acid, citric acid and succinic acid and so forth.

When a methanol assimilating bacterium such as Bacillus methanolicus is used as the microorganism of the present invention, methanol can be preferably used as the carbon source.

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and so forth can be used.

As the inorganic ions or sources thereof, a small amount of potassium phosphate, magnesium sulfate, iron ions, manganese ions and so forth may be added. As a trace amount organic nutrient, it is desirable to add a suitable amount of required substances such as L-homoserine and vitamin B 1 , yeast extract and so forth as required.

The culture is performed under conditions suitable for growth of a microorganism to be used. In general, it is preferably performed for 16 to 72 hours under an aerobic condition, and the culture temperature is controlled to be 20 to 45° C., and pH to be 5-8.5 during the culture. For adjusting pH, inorganic or organic acidic or alkaline substances, ammonia gas and so forth can be used. Further, when a thermophilic bacterium is used as a host, it can be cultured at a culture temperature of 42 to 60° C.

Collection of L-lysine from the culture can usually be carried out by using a combination of known techniques such as techniques using ion exchange resins, precipitation methods and so forth.

Hereafter, the present invention will be further specifically explained with reference to the following examples.

The reagents used were obtained from Wako Pure Chemicals or Nakarai Tesque unless otherwise indicated. The compositions of the media used in each example are shown below. All the media were subjected to steam sterilization at 120° C. for 20 minutes after the components were dissolved.

[The components except for magnesium solution and glucose were steam-sterilized, then added with 2 M magnesium stock solution (1 M MgSO 4 , 1 M MgCl 2 ) and 2 M glucose solution, which solutions had been passed through a 0.22 μm filter beforehand, and passed through a 0.22 μm filter again.]

[MgSO 4 and glucose were separately sterilized and added. A suitable amount of amino acids and vitamins were added as required.]

Bacillus methanolicus

2

Met Val Ser Phe Gly Arg Ile Ser Thr Ala Met Val Thr Pro Phe Asp

1 5 10 15

Asn Lys Gly His Ile Asp Phe Ala Lys Thr Thr Gln Leu Val Asn His

20 25 30

Leu Ile Asn Asn Gly Ser Asp Ser Leu Val Val Val Gly Thr Thr Gly

35 40 45

Glu Ser Ala Thr Leu Thr Lys Glu Glu Lys Leu Ala Leu Phe Gln His

50 55 60

Val Val Lys Val Val Glu Lys Arg Val Pro Val Ile Ala Gly Thr Gly

65 70 75 80

Ser Asn Asn Thr Tyr Asp Ser Ile Glu Met Thr Lys Lys Ala Glu Lys

85 90 95

Met Gly Val Asp Ala Ile Leu Ala Val Ala Pro Tyr Tyr Asn Lys Pro

100 105 110

Asn Gln Glu Gly Leu Tyr Gln His Phe Lys Ala Ile Ala Glu Ser Thr

115 120 125

Ser Leu Pro Val Ile Ile Tyr Asn Ile Pro Gly Arg Ser Val Val Asn

130 135 140

Ile Glu Pro Glu Thr Val Ile Arg Leu Ser Lys Ile Pro Asn Ile Val

145 150 155 160

Gly Ile Lys Glu Ala Gly Gly Asn Leu Ser Ala Met Thr Gln Ile Ile

165 170 175

Ala Asn Thr Asp Asp Asp Phe Leu Leu Tyr Ser Gly Asp Asp Gly Leu

180 185 190

Thr Leu Pro Val Leu Ser Ile Gly Gly Thr Gly Val Ile Ser Val Ala

195 200 205

Ser His Val Ile Gly Asn Glu Met Gln Glu Met Ile Ser Ala Phe Leu

210 215 220

Asn Gly Asp Tyr Glu Arg Ala Ala Lys Ile His Gln Lys Leu Leu Pro

225 230 235 240

Leu Met Asp Gly Leu Phe Ile Ala Pro Asn Pro Val Pro Val Lys Thr

245 250 255

Ala Leu Gln Ile Lys Gly Met Asp Val Gly Ser Val Arg Leu Pro Leu

260 265 270

Val Pro Leu Thr Glu Gln Glu Arg Asn Thr Val Ala Ala Leu Leu Asn

275 280 285

Ala Leu

290

3

atg gaa att gta aaa att gtt gta gca ggc ccg cgc gga cga atg ggg 48

Met Glu Ile Val Lys Ile Val Val Ala Gly Pro Arg Gly Arg Met Gly

1 5 10 15

cgg gaa gca gtc cat ctt gtc ggg aga aca gaa aat ttc gag ttg gca 96

Arg Glu Ala Val His Leu Val Gly Arg Thr Glu Asn Phe Glu Leu Ala

20 25 30

gca gtg ctg gat aat aag aat gac gga aaa aat ctt tcc gaa ttg gaa 144

Ala Val Leu Asp Asn Lys Asn Asp Gly Lys Asn Leu Ser Glu Leu Glu

35 40 45

ggt ttt caa gga ttt gat gcc cct gtg tat aca aat att gaa aaa tgt 192

Gly Phe Gln Gly Phe Asp Ala Pro Val Tyr Thr Asn Ile Glu Lys Cys

50 55 60

ttt caa gat acc ggc gca gat gtc tta atc gat ttg acg act cct gaa 240

Phe Gln Asp Thr Gly Ala Asp Val Leu Ile Asp Leu Thr Thr Pro Glu

65 70 75 80

gta ggc tac tat cat aca aaa acg gct ctc gaa tat gga gtg cgg cct 288

Val Gly Tyr Tyr His Thr Lys Thr Ala Leu Glu Tyr Gly Val Arg Pro

85 90 95

gta gtt ggg acg acg ggt ttt acg aaa gat caa tta aaa gaa att gaa 336

Val Val Gly Thr Thr Gly Phe Thr Lys Asp Gln Leu Lys Glu Ile Glu

100 105 110

gaa att tgc gaa gaa aag aaa ctt ggc tgc att ata gcg cca aat ttt 384

Glu Ile Cys Glu Glu Lys Lys Leu Gly Cys Ile Ile Ala Pro Asn Phe

115 120 125

gcg gtt ggg gct gta tta atg atg aaa ttt tca caa atg gca gcc aag 432

Ala Val Gly Ala Val Leu Met Met Lys Phe Ser Gln Met Ala Ala Lys

130 135 140

tat ttt caa gat att gaa att att gaa ctg cat cat gat caa aaa ttg 480

Tyr Phe Gln Asp Ile Glu Ile Ile Glu Leu His His Asp Gln Lys Leu

145 150 155 160

gat gca ccg tcc gga aca gct gtc aaa aca gct gag atg att gcg gaa 528

Asp Ala Pro Ser Gly Thr Ala Val Lys Thr Ala Glu Met Ile Ala Glu

165 170 175

gtg aga gaa gca aag aag cag ggt cat cca aat gaa aaa gaa acg atc 576

Val Arg Glu Ala Lys Lys Gln Gly His Pro Asn Glu Lys Glu Thr Ile

180 185 190

atc ggt gca agg ggt gcg gat tat gaa gga atg cat att cat tct gtt 624

Ile Gly Ala Arg Gly Ala Asp Tyr Glu Gly Met His Ile His Ser Val

195 200 205

cgt ttg ccg gga tta att gcc cat cag cag gtg atg ttt gga tca gac 672

Arg Leu Pro Gly Leu Ile Ala His Gln Gln Val Met Phe Gly Ser Asp

210 215 220

ggg caa aca ttg acg atc cgc cac gat tcg tat aac cgg gca tct ttc 720

Gly Gln Thr Leu Thr Ile Arg His Asp Ser Tyr Asn Arg Ala Ser Phe

225 230 235 240

atg tct ggc gta aag cat gcc gtt gag acg gtt tta aaa att gat acg 768

Met Ser Gly Val Lys His Ala Val Glu Thr Val Leu Lys Ile Asp Thr

245 250 255

ttt gtt tac gga tta gaa aat att att gaa tag 801

Phe Val Tyr Gly Leu Glu Asn Ile Ile Glu

260 265

4

266

Bacillus methanolicus

4

Met Glu Ile Val Lys Ile Val Val Ala Gly Pro Arg Gly Arg Met Gly

1 5 10 15

Arg Glu Ala Val His Leu Val Gly Arg Thr Glu Asn Phe Glu Leu Ala

20 25 30

Ala Val Leu Asp Asn Lys Asn Asp Gly Lys Asn Leu Ser Glu Leu Glu

35 40 45

Gly Phe Gln Gly Phe Asp Ala Pro Val Tyr Thr Asn Ile Glu Lys Cys

50 55 60

Phe Gln Asp Thr Gly Ala Asp Val Leu Ile Asp Leu Thr Thr Pro Glu

65 70 75 80

Val Gly Tyr Tyr His Thr Lys Thr Ala Leu Glu Tyr Gly Val Arg Pro

85 90 95

Val Val Gly Thr Thr Gly Phe Thr Lys Asp Gln Leu Lys Glu Ile Glu

100 105 110

Glu Ile Cys Glu Glu Lys Lys Leu Gly Cys Ile Ile Ala Pro Asn Phe

115 120 125

Ala Val Gly Ala Val Leu Met Met Lys Phe Ser Gln Met Ala Ala Lys

130 135 140

Tyr Phe Gln Asp Ile Glu Ile Ile Glu Leu His His Asp Gln Lys Leu

145 150 155 160

Asp Ala Pro Ser Gly Thr Ala Val Lys Thr Ala Glu Met Ile Ala Glu

165 170 175

Val Arg Glu Ala Lys Lys Gln Gly His Pro Asn Glu Lys Glu Thr Ile

180 185 190

Ile Gly Ala Arg Gly Ala Asp Tyr Glu Gly Met His Ile His Ser Val

195 200 205

Arg Leu Pro Gly Leu Ile Ala His Gln Gln Val Met Phe Gly Ser Asp

210 215 220

Gly Gln Thr Leu Thr Ile Arg His Asp Ser Tyr Asn Arg Ala Ser Phe

225 230 235 240

Met Ser Gly Val Lys His Ala Val Glu Thr Val Leu Lys Ile Asp Thr

245 250 255

Phe Val Tyr Gly Leu Glu Asn Ile Ile Glu

260 265