JP2003159073A - New saccharide-producing gene group - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for improving the productivity of a saccharide from a C1 compound and for inhibiting the production of an unnecessary saccharide to improve the production yield of a target substance, by using a C1 compound-assimilating microorganism. <P>SOLUTION: A gene which reduces the production of the saccharide, when a sequence for one or more amino acids is deleted, or a gene which increases the production of the saccharide, when the gene is amplified, are discovered from a microorganism capable of assimilating the C1 compound to produce the saccharide, and then used. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the microbial industry, and relates to genes involved in microbial sugar production and their use. The use of the gene provides, on the one hand, a means for improving the productivity of useful saccharides by the microorganism, or, on the other hand, it can suppress the unnecessary synthesis of saccharides produced by the microorganism, thereby increasing the productivity of the target substance produced by the microorganism. It is intended to provide a means for improving and a means for facilitating a method of obtaining a target substance.

In particular, it is effective to use a C1 compound, that is, a microorganism which assimilates a compound having one carbon atom such as methanol, methane or carbon dioxide.

[0003]

2. Description of the Related Art A method for producing a polysaccharide by culturing a microorganism belonging to the genus Pseudomonas having the ability to assimilate a C1 compound (for example, JP-A-51-151392 and JP-A-5-151392).
6-48891, JP-A-56-61996, JP-A-56-9979
No. 5, JP-A-56-140896) and CO2 assimilating Pseudomonas hydrogeno
vora) to produce polysaccharides (Agric. Bio
l. Chem. 44 (12), pp2925-2930 (1980)) are known.

U. Breuer et al., Can. J. Microbiol. 41 (Sup
pl.1); pp.55-59 (1995), Methylobaterium rhodecianum (Methylobaterium rhodesianum)
It is described that sianum) secretes polysaccharides outside the cells. B. Southgate et al., J. Gen. Microbiol. 135, pp.285.
9-2867 (1989) contains the methanol-assimilating bacterium Methylophilus
It is described that Methylophilus methylotrophus produces polysaccharides extracellularly. In addition, a mutant of Methylobacillus, a methanol-assimilating bacterium,
It is also known to produce monosaccharides from methanol (JP-A-10-113196). However, those C1
Nothing is known about the structure of genes involved in saccharide production of assimilating microorganisms.

The microorganisms that utilize C1 compounds are, for example, yeasts of the genus Methylobacillus, Methylobacterium, Methylophilus, Methylomonas, Methylobacter, Methylococcus, Methylocinas, Methylocistis and methanol. , For example, Candida, Pichia, Hansenula.

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to a gene involved in saccharide production of a C1 compound-assimilating microorganism, and utilization of the gene. The C1 compound utilizing a C1 compound-assimilating microorganism is used. To improve sugar productivity,
Alternatively, it is intended to provide a means for suppressing unnecessary saccharide production in some cases and improving the production yield of the target substance. Here, the target substance means useful substances such as amino acids, nucleic acids, vitamins, enzymes and proteins.

[0007]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies in order to solve the above-mentioned problems, and as a result, microorganisms capable of assimilating C1 compounds to produce sugars have We have discovered genes that reduce the amount produced, and conversely, genes that increase the amount of saccharide produced by gene amplification,
The present invention has been completed. Therefore, the present invention, when culturing a microorganism capable of assimilating a C1 compound and secreting and producing a saccharide using a C1 compound as a carbon source, a gene group necessary for improving the saccharide production amount, and a saccharide Providing genes necessary to suppress unwanted saccharide formation when producing target substances other than saccharides (eg, amino acids, nucleic acids, vitamins, enzymes, proteins, etc.) from secreted and produced microorganisms To do. It was unexpectedly difficult to clone some genes involved in sugar production. The reason for this is that by gene cloning, enzymes related to saccharide synthesis derived from heterologous microorganisms and proteins derived from DNA around the target gene are actively expressed in Escherichia coli, which is the host. It was thought to be disturbing and deteriorating the growth. Therefore, the DNA fragment carrying the target gene could not be obtained by the usual method. In such a case, the DNA region could be cloned by dividing it, or by devising such that the gene was not expressed, the cloning was successful and the DNA could be analyzed.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The gene group of the present invention utilizes the C1 compound to secrete sugars and
It is convenient to mutate a microorganism that can be produced and obtain a gene that restores saccharide productivity from a mutant strain that can no longer secrete and produce saccharides or a mutant strain that has reduced productivity. A variety of microorganisms capable of assimilating C1 compounds and capable of producing and secreting sugars are known, and they can be used. Alternatively, a newly isolated microorganism having the above properties can be used.

As a mutation method, a method commonly used for obtaining a mutant strain of a microorganism, for example, a physical method using ultraviolet rays or radiation, a chemical method using nitrite or the like, a biological method using a transposon or the like is used. Etc. can be used.
In the present invention, the method using a transposon is particularly convenient. In the present invention, a large number of methanol-assimilating bacteria were isolated from soil, and the 12S strain was selected as a strain that accumulates saccharides in the culture supernatant. This is a transposon (Tn)
By subjecting it to insertion mutation, a mutant strain Ma1 with reduced saccharide production was obtained in the culture supernatant. By analyzing the position of Tn inserted in the genome of this Ma1 strain, a gene involved in the production of saccharides was discovered and could be obtained.

The above 12S strain is Appl. Microbiol. Biotec
It is a bacterium belonging to the genus Methylobacillus described in hnol., 54, pp.341-347 (2000).

To cultivate this microorganism, a nitrogen source which can be assimilated by this microorganism, preferably an inorganic nitrogen source, such as ammonium sulfate, ammonium chloride, etc. is contained, and a C1 compound such as methanol is contained as a carbon source, More preferably inorganic salts such as phosphates, magnesium salts,
It may be cultured in a medium containing calcium chloride, iron sulfate and the like. The concentration of nitrogen source is, for example, 0.01 for inorganic substances.
.About.0.3 g / 100 ml medium, and as a carbon source, for example, methanol is 1.2 to 3.5 g / 100 ml, preferably 2 g / 100 ml.
The culture is carried out under aerobic conditions, and the culture temperature is preferably 25
C. to 35.degree. C., preferably 30.degree.

Various genes and methods of the present invention can be used. For example, in the production of sugars from C1 compounds,
By using the gene of the present invention, the production amount of the target sugar,
Productivity can be improved. What is a saccharide?
Oligos, monosaccharides, and nucleotide sugars. On the other hand, when the target substance is a substance other than saccharides, but the microorganism for its production produces saccharides as a by-product, by using the gene of the present invention, synthesis, secretion, production of unnecessary saccharides Is suppressed, and as a result, the productivity of the target substance can be improved.

[0013]

EXAMPLES The present invention will be described in more detail below with reference to examples.

[0014]

Example 1 Acquisition of Mutant Ma1 Strain of Methylobacillus Saccharide with Suppressed Extracellular Polysaccharide Production The 12S strain of Methylobacillus bacterium was subjected to transposon insertion mutation treatment as follows. Apply the transposon donor E. coli S17-1 (pSUP5011) to the LB medium agar plate. 3
Pre-incubate at 7 ℃ overnight, remove 1 platinum loop from it, and
Was inoculated into the LB liquid medium of and cultured at 37 ° C. overnight with shaking.
Recipient cells of 12S strain are methanol (2.26% (v / v))
Agar medium containing only as a carbon source, MMe agar medium (composition in 1 L is 1 g of (NH4) 2SO4, 5.5 g of KH2PO4, 10 g
Na2HPO4 / 12H2O, 0.1g MgSO4 / 7H2O, 0.01g FeSO4
・ 7H2O, 0.01g CaCl2 ・ 2H2O, and 22.6ml methanol, 15g agar containing medium), after culturing at 30 ℃, remove one platinum loop from it and plant in 5ml of the same liquid medium. The cells were cultivated and shake-cultured at 30 ° C. for several days.

After culturing, the transposon donor and recipient bacteria were
Mix 1: 1 and collect using a syringe on a filter with a pore size of 0.45 micrometer (μm), and filter the filter in LB solid medium (composition: tryptone 10 g, yeast extract 5 g, NaCl 10 g / L. The cells were placed face up on a solid medium containing agar at a final concentration of 1.5% (w / v), and statically cultured at 30 ° C for 6 hours for conjugal transfer.

After culturing, the cells on the filter were treated with 50 mM.
Suspend in phosphate buffer (KH2PO4-NaOH, pH 7.0), dilute the suspension appropriately, and apply to an agar medium containing methanol (2.26% (v / v)) and kanamycin (50 mg / L). Then
After culturing at 30 ° C. for several days, the emerged kanamycin-resistant colonies were regarded as transposon-inserted strains and used for the subsequent experiments. The number of strains obtained was 11,200.

Of these, the surface of the colony is mucoid
From (before mutation), 50 mutant strains having a non-mucoid phenotype were selected. Next, these mutant strains were shake-cultured in a liquid medium containing methanol as a carbon source, and mutant strains in which the viscosity of the culture solution was lower than that of the parent strain were selected. Among them, in particular, a strain in which the secretion and production of saccharides into the medium was reduced was isolated and named as Ma1 strain (AJ13934 strain). This strain was deposited at the Patent Microorganism Depositary Center, National Institute of Advanced Industrial Science and Technology as FERM P-18592 on November 7, 2001.

[0018]

[Example 2] Identification of mutation point of Ma1 strain and analysis of gene region involved in saccharide production The position of transposon Tn5 inserted in the genome of Ma1 strain was examined as follows. 12S with Tn5 inserted
Genomic DNA was prepared from the strain, namely Ma1 strain, and the restriction enzyme EcoR
The product digested with I was separated by 0.9% (w / v) agarose gel electrophoresis. A DNA having a length of 7 kbp to 10 kbp was purified from the gel, ligated to pUC19 as a vector, and transformed into Escherichia coli DH5α strain. By selecting a kanamycin resistant strain from among these, a plasmid having a genomic DNA in which Tn5 was inserted was obtained.

Genomic DN of 12S strain possessed by this plasmid DNA
The base sequence of the A portion was determined by a conventional method. And
DNA primers (SEQ ID NOs: 8 and 9) having sequences corresponding to both ends of the inserted DNA were synthesized by a conventional method. Using this pair of DNA primers, P
I did a CR. Conditions are 95 ℃, 95 ℃ after denaturing treatment for 5 minutes
-30 seconds, 57 ° C-30 seconds, 72 ° C-3 minutes treatment was repeated 30 times. The DNA fragment thus amplified is pT7
The resulting plasmid cloned into blue (R) (Novagen) was named pTMS3.

On the other hand, the wild type 12S strain genomic library was prepared by digesting the 12S strain genomic DNA with the restriction enzyme SalI, and
It was prepared by cloning a DNA fragment with A chain length of 4 kbp to 8 kbp into pUC19. Then, in order to examine the whole image of the Tn5 insertion site of the Ma1 strain, colony hybridization was performed on the above genomic library using the inserted DNA fragment of pTMS3 as a probe to obtain a positive clone. The probe is [α-32P] dCTP and Megaprime DNA.
labeling system (Amersham Pharmacia Biotech)
Was created using.

The positive clone obtained above was named pUMS1-X. Then, according to a conventional method, D of this inserted DNA fragment region is
The NA base sequence was determined. 4330 bp of this is SEQ ID NO: 7
Shown in. As a result of sequence analysis, it was found that all three open reading frames (orf) were in the same direction. Orf1 (epsA), orf2 (epsB), or
It was set to f3 (epsC). The base sequence of each orf and the encoded amino acid sequence are shown in SEQ ID NOs: 1 to 6. The sequence of orf1 is shown in Sequence Listings 1 and 2, the sequence of orf2 is shown in Sequence Listings 3 and 4, and the sequence of orf3 is shown in Sequence Listings 5 and 6, respectively. Tn5 was also inserted in epsB.

Orf1 (epsA) consists of 798 base pairs and can encode 265 amino acids. The amino acid sequence had some homology with those of transcriptional regulators belonging to the LuxR superfamily known in some microorganisms. orf2
(epsB) consists of 1371 base pairs and can encode 456 amino acids. Amino acid sequence homology was found with that of several glycosyltransferases. orf3
(epsC) consists of 1011 base pairs and can encode 336 amino acids. This amino acid sequence is Streptococcus
It was homologous to the amino acid sequence encoded by a gene that is said to be involved in CPS (capsular) synthesis in Streptococcus pneumoniae.

[0023]

[Example 3] A kanamycin resistance gene was inserted into the cloned coding regions of epsA, epsB, and epsC on the deletion effect vector pUC19 of Orf1 (epsA), orf2 (epsB), and orf3 (epsC), respectively. , PMm △ A and pMm respectively
ΔB and pMmΔC were prepared. These plasmids are
The strain was introduced by electroporation, and a kanamycin-resistant but ampicillin-sensitive strain was selected. Strains exhibiting this resistance phenotype are, for example, when pMmΔA is used, at the position of epsA in the genome of the 12S strain, epsA :: K of pMmΔA
A strain in which the m part has undergone homologous recombination twice (named MmΔA strain)
Is. In this way, gene-deficient strains of the other two genes were also prepared (named MmΔB strain and MmΔC strain, respectively).

The degree of saccharide production of the strain obtained as described above was examined in comparison with the wild strain. Each strain was inoculated into a medium containing methanol as a carbon source and cultured at 30 ° C. for 7 days. Then, the amount of saccharides in the medium supernatant was quantified by the phenol / sulfuric acid method (Anal. Chem. 28, pp.350-356, (1956)). As a result, the amount of saccharides produced by the MmΔB and MmΔC strains was reduced to less than 17% of the wild strain. This means that epsB
It was found that epsC is an important gene for the production of extracellular sugars in the 12S strain. In addition, the MmΔA strain was reduced to about half that of the wild strain.

[0025]

[Example 4] Gene expression enhancing effect of Orf1 (epsA) In order to examine the function of EpsA, the effect of enhancing the gene expression on saccharide synthesis was examined. First, using pUMS1 described above as a template for PCR amplification and using SEQ ID NOs: 10 and 11 as DNA primers, the epsA gene portion was increased. The reaction conditions of PCR were such that after denaturing treatment at 95 ° C for 5 minutes, treatments at 95 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 1 minute were repeated 30 times. 0.9 kbp DN amplified in this way
A fragment was inserted into pT7Blue (R) vector (Novagen),
got epsA. Furthermore, the restriction enzyme Hinc
Approximately 0.9 kbp including epsA gene excised by II and SmaI
Was inserted into a wide host range vector pBBR122 (manufactured by MobiTec) treated with a restriction enzyme DraI to prepare pBBA. By introducing this into 12S strain by electroporation and obtaining a kanamycin resistant strain, a 12S strain having pBBA, that is, 12SA strain was obtained.

The saccharide synthesizing ability of this 12SA strain was examined in the same manner as above. As a result, this strain was found to synthesize about 1.4 times more saccharide than the wild strain. Therefore, it was found that this gene is a gene that helps improve the ability to synthesize sugars.

[0027]

[Example 5] Evaluation of activity of epsB gene product EpsB was possibly a glycosyltransferase due to homology of amino acid sequences. Therefore, whether or not EpsB actually had the activity was examined as follows. Wild strain
The 12S strain and the epsB deficient strain were inoculated into 5 ml of MMe medium and cultured at 30 ° C. overnight. After that, 1 ml of each culture
Was transferred to 100 ml of the same medium and the culture was continued. After culturing the cells to a mid-logarithmic growth phase (OD560 = 0.6 to 1.0), each cell was collected and 10 ml of buffer A (composition:
1.2M sucrose, 50mM Tris-HCl (pH8.0), 1mM EDT
A, 20 mM lysozyme). Then, it incubated at 25 degreeC for 5 minutes.

Next, the protoplasts were 8000 at 4 ° C.
g, collected by centrifugation for 20 minutes, buffer B (composition: 20 mM Tris
-HCl (pH8.0), 1 mM EDTA, 200 μM phenylmethylsulfonyl fluoride), and this was subjected to ultrasonic treatment. After crushing the microbial cells, the debris of the microbial cells is at 4 ° C
The cells were removed by centrifugation at 8,000 g for 20 minutes, and then the membrane fraction of the bacterial cells was collected by ultracentrifugation at 30,000 g for 1 hour at 0 ° C. This fraction is buffer C (composition: 50 mM Tris-acetate (pH 8.3), 1 mM
After washing with EDTA and 200 μM phenylmethylsulfonyl fluoride), the cells were collected again by the above ultracentrifugation and finally suspended in 0.5 ml of buffer C.

The glycosyltransferase activity is basically measured by the method of Kolkman et al. (J. Bacteriol., 178, p.
p.3736-3741, (1996)). However, uridine diphosphate D- [U- ¹⁴ C] glucose (300 mCi / mmol; NE
N Life Science), uridine diphosphate D- [U-
¹⁴ C] Galactose (300 mCi / mmol; Amersham PharmaciaB
iotech) and guanosine diphosphate D- [U-
¹⁴ C] mannose (300 mCi / mmol; Amersham Pharmacia Bi
otech) was used as the reaction substrate.

As a result, when the membrane fraction prepared from the 12S strain was used, glucose uptake into the lipid carrier in the membrane was observed. However, in the case of the epsB-deficient strain, uptake of glucose, galactose and mannose was not observed. Thus, EpsB was shown to exhibit glucosyl-1-phosphate transferase activity.

[0031]

[Example 6] Improvement of saccharide production by amplification of epsB and epsC genes pUMS1-X prepared in Example 2 contained epsA, epsB, and e.
Although there are three orfs of psC, the kanamycin resistance gene was inserted only within the coding region of epsA to construct pUMSΔ0. When this plasmid was introduced into a Methylobacillus bacterium 12S strain and a kanamycin resistant strain was obtained, a strain that was also ampicillin resistant was obtained at the same time. Analysis of the chromosome structure revealed that this is a strain in which homologous recombination occurred once between the chromosome portion of the host and the plasmid DNA portion. So this strain Mm0-2
It was named a strain.

Then, when this Mm0-2 strain and the original wild strain 12S strain were cultured and assayed for the amount of saccharides produced in the culture solution, the Mm0-2 strain was about 1.3 times as much as the control strain. It was found to produce a lot of sugars. From this, it was revealed that the increase of the genes of epsB and epsC had an effect on saccharide production.

EFFECTS OF THE INVENTION According to the present invention, it is possible to optionally overproduce or suppress the production of saccharides, particularly polysaccharides, in microorganisms.

[Sequence list]                                SEQUENCE LISTING         <110> Ajinomoto Co., Inc. <120> <130> <160> 11 <170> PatentIn Ver. 2.1 <210> 1 <211> 798 <212> DNA <213> Methylobacillus sp. <220> <221> CDS <222> (1) .. (798) <400> 1 atg ttt att acc aaa gaa cta acc gat att gag aag gca cag ctt att 48 Met Phe Ile Thr Lys Glu Leu Thr Asp Ile Glu Lys Ala Gln Leu Ile   1 5 10 15 gac ttg atg cac gag tct ttg cga atc cgc tcg cac ttc gag ttc ttc 96 Asp Leu Met His Glu Ser Leu Arg Ile Arg Ser His Phe Glu Phe Phe              20 25 30 ctt tgg atg caa ggc aag ctt cag cag ttt tta ccg cat gaa atc atg 144 Leu Trp Met Gln Gly Lys Leu Gln Gln Phe Leu Pro His Glu Ile Met          35 40 45 atc acc gcc tgg ggc gat ttc tcc atg ggc gtc atc tat ttc aat att 192 Ile Thr Ala Trp Gly Asp Phe Ser Met Gly Val Ile Tyr Phe Asn Ile      50 55 60 gtg tcg ccg ctc ccc gga gta cgt acc gag aag ata tca agt ggc gat 240 Val Ser Pro Leu Pro Gly Val Arg Thr Glu Lys Ile Ser Ser Gly Asp  65 70 75 80 ctg aat cca ttg ctc aaa cgg ctt ttc aat tac tgg ctt agt cat acc 288 Leu Asn Pro Leu Leu Lys Arg Leu Phe Asn Tyr Trp Leu Ser His Thr                  85 90 95 aaa gcc ccg ttt acc tta tcg gcg gag aat ggc gta ttc cag gac tgc 336 Lys Ala Pro Phe Thr Leu Ser Ala Glu Asn Gly Val Phe Gln Asp Cys             100 105 110 gat gta ctg cca gcg gaa gtg aac agc cat ctg aaa aac atg aag tca 384 Asp Val Leu Pro Ala Glu Val Asn Ser His Leu Lys Asn Met Lys Ser         115 120 125 gcg ctg gtg cac ggc atc aag gat ttc cgt ggg cgc cat gac tgc ctt 432 Ala Leu Val His Gly Ile Lys Asp Phe Arg Gly Arg His Asp Cys Leu     130 135 140 tac ata ttg ctg aat tca acc ccc acc atg ccg aat acc tca cgg tac 480 Tyr Ile Leu Leu Asn Ser Thr Pro Thr Met Pro Asn Thr Ser Arg Tyr 145 150 155 160 atg ctg gag tca ttg cta cct tac att gac agc gcg ctc agg caa ctg 528 Met Leu Glu Ser Leu Leu Pro Tyr Ile Asp Ser Ala Leu Arg Gln Leu                 165 170 175 gag cat ttg cct gtg cag cac ccg gca gat aaa gaa gcc agt gaa gat 576 Glu His Leu Pro Val Gln His Pro Ala Asp Lys Glu Ala Ser Glu Asp             180 185 190 ctg cac gaa gaa gaa aac gaa gcg ctg gag cag ctt tct tcc cgt gag 624 Leu His Glu Glu Glu Asn Glu Ala Leu Glu Gln Leu Ser Ser Arg Glu         195 200 205 atc gaa atc atg gaa tgg gtg cgc aac ggc aaa acc aat cag gaa atc 672 Ile Glu Ile Met Glu Trp Val Arg Asn Gly Lys Thr Asn Gln Glu Ile     210 215 220 ggc atg atc ctg gac att agt tca ttt acc gtc aaa aac cat ttg caa 720 Gly Met Ile Leu Asp Ile Ser Ser Phe Thr Val Lys Asn His Leu Gln 225 230 235 240 cgc ata ttt aaa aag ctt gat gtt ctg aat aga gcg caa gcc gtt tcc 768 Arg Ile Phe Lys Lys Leu Asp Val Leu Asn Arg Ala Gln Ala Val Ser                 245 250 255 aaa ttc aag cag act cca cgg gcc tca tga 798 Lys Phe Lys Gln Thr Pro Arg Ala Ser             260 265 <210> 2 <211> 265 <212> PRT <213> Methylobacillus sp. <400> 2 Met Phe Ile Thr Lys Glu Leu Thr Asp Ile Glu Lys Ala Gln Leu Ile   1 5 10 15 Asp Leu Met His Glu Ser Leu Arg Ile Arg Ser His Phe Glu Phe Phe              20 25 30 Leu Trp Met Gln Gly Lys Leu Gln Gln Phe Leu Pro His Glu Ile Met          35 40 45 Ile Thr Ala Trp Gly Asp Phe Ser Met Gly Val Ile Tyr Phe Asn Ile      50 55 60 Val Ser Pro Leu Pro Gly Val Arg Thr Glu Lys Ile Ser Ser Gly Asp  65 70 75 80 Leu Asn Pro Leu Leu Lys Arg Leu Phe Asn Tyr Trp Leu Ser His Thr                  85 90 95 Lys Ala Pro Phe Thr Leu Ser Ala Glu Asn Gly Val Phe Gln Asp Cys             100 105 110 Asp Val Leu Pro Ala Glu Val Asn Ser His Leu Lys Asn Met Lys Ser         115 120 125 Ala Leu Val His Gly Ile Lys Asp Phe Arg Gly Arg His Asp Cys Leu     130 135 140 Tyr Ile Leu Leu Asn Ser Thr Pro Thr Met Pro Asn Thr Ser Arg Tyr 145 150 155 160 Met Leu Glu Ser Leu Leu Pro Tyr Ile Asp Ser Ala Leu Arg Gln Leu                 165 170 175 Glu His Leu Pro Val Gln His Pro Ala Asp Lys Glu Ala Ser Glu Asp             180 185 190 Leu His Glu Glu Glu Asn Glu Ala Leu Glu Gln Leu Ser Ser Arg Glu         195 200 205 Ile Glu Ile Met Glu Trp Val Arg Asn Gly Lys Thr Asn Gln Glu Ile     210 215 220 Gly Met Ile Leu Asp Ile Ser Ser Phe Thr Val Lys Asn His Leu Gln 225 230 235 240 Arg Ile Phe Lys Lys Leu Asp Val Leu Asn Arg Ala Gln Ala Val Ser                 245 250 255 Lys Phe Lys Gln Thr Pro Arg Ala Ser             260 265 <210> 3 <211> 1374 <212> DNA <213> Methylobacillus sp. <220> <221> CDS <222> (1) .. (1374) <400> 3 ctg gca tta cct ttg cac gtg aca ata ttt tgc acg gtg ttg aag ccc 48 Met Ala Leu Pro Leu His Val Thr Ile Phe Cys Thr Val Leu Lys Pro   1 5 10 15 tgc ttg atc cgc ttg tcg tcg tca ttt cac tgt gga tcg tgg cgt ttt 96 Cys Leu Ile Arg Leu Ser Ser Ser Phe His Cys Gly Ser Trp Arg Phe              20 25 30 gtt tat gaa aac gat ctg cca cct cac tac ctg atc atg tcg ctg att 144 Val Tyr Glu Asn Asp Leu Pro Pro His Tyr Leu Ile Met Ser Leu Ile          35 40 45 ctg ttt tcg ctg atg ttc ccc agc acc acc aaa atc agc cag ccc atc 192 Leu Phe Ser Leu Met Phe Pro Ser Thr Thr Lys Ile Ser Gln Pro Ile      50 55 60 agt tca gtt att caa aac acg ttg ctg gca tgg ttt gtt ctg gcc ggg 240 Ser Ser Val Ile Gln Asn Thr Leu Leu Ala Trp Phe Val Leu Ala Gly  65 70 75 80 cta ttg atg gcg ttt gga tat gct tct gaa tac atc tat atc ttc ccc 288 Leu Leu Met Ala Phe Gly Tyr Ala Ser Glu Tyr Ile Tyr Ile Phe Pro                  85 90 95 aaa gcc tcg att aca tta tgg ctc tgg ctg acc cct acc ctg ctt atc 336 Lys Ala Ser Ile Thr Leu Trp Leu Trp Leu Thr Pro Thr Leu Leu Ile             100 105 110 gca gca acc ctc ggt tta cgc atg gcg gcg ccg cta ctc atc aag atg 384 Ala Ala Thr Leu Gly Leu Arg Met Ala Ala Pro Leu Leu Ile Lys Met         115 120 125 caa ggg cct atg cga cgg gcg gtg att gcg ggc atg aac gaa cag ggc 432 Gln Gly Pro Met Arg Arg Ala Val Ile Ala Gly Met Asn Glu Gln Gly     130 135 140 ctg gcc ctg gca gaa cgc ttg cag cgt agc cac tac cat cca aca gag 480 Leu Ala Leu Ala Glu Arg Leu Gln Arg Ser His Tyr His Pro Thr Glu 145 150 155 160 ttg cag ggc ttc ttt gag gat cgc agt gtg gac cga ctc aat tca gaa 528 Leu Gln Gly Phe Phe Glu Asp Arg Ser Val Asp Arg Leu Asn Ser Glu                 165 170 175 ttg cat tac ccc att ctt ggc cgc atc gac agc atg gcg gag tat gtg 576 Leu His Tyr Pro Ile Leu Gly Arg Ile Asp Ser Met Ala Glu Tyr Val             180 185 190 aaa gag cat cac atc aac acc atc tac ctt tca ctg ccc atg gcg agc 624 Lys Glu His His Ile Asn Thr Ile Tyr Leu Ser Leu Pro Met Ala Ser         195 200 205 caa cca cgc atc atg aag ttg tta gat gat ttg aaa gac acc acg gcg 672 Gln Pro Arg Ile Met Lys Leu Leu Asp Asp Leu Lys Asp Thr Thr Ala     210 215 220 tcc att tac ttt gtc ccg gat att ttc atg aca gat ctg ata caa ggg 720 Ser Ile Tyr Phe Val Pro Asp Ile Phe Met Thr Asp Leu Ile Gln Gly 225 230 235 240 cgt gtc gat cag gta gaa aac atc ccg gtg gtc agc gta tgt gaa acc 768 Arg Val Asp Gln Val Glu Asn Ile Pro Val Val Ser Val Cys Glu Thr                 245 250 255 cca ttt acc ggc ttt gac ggt ctg ttg aaa cgc tca gct gat atc gct 816 Pro Phe Thr Gly Phe Asp Gly Leu Leu Lys Arg Ser Ala Asp Ile Ala             260 265 270 ttc tcg ttg ctg atc ctg atc ctt atc tcg ccg gta tta ttg gcg atc 864 Phe Ser Leu Leu Ile Leu Ile Leu Ile Ser Pro Val Leu Leu Ala Ile         275 280 285 gcg att ggc gtg aag atg agt tca cca ggc cca gtc att ttc aaa cag 912 Ala Ile Gly Val Lys Met Ser Ser Pro Gly Pro Val Ile Phe Lys Gln     290 295 300 cgc cgt tat ggg ctt gat ggc gaa gag atc ctt gtc tac aaa ttc cgc 960 Arg Arg Tyr Gly Leu Asp Gly Glu Glu Ile Leu Val Tyr Lys Phe Arg 305 310 315 320 tcc atg acg gta tgc gaa gat ggc gca aaa gtg acg caa gcg acg aaa 1008 Ser Met Thr Val Cys Glu Asp Gly Ala Lys Val Thr Gln Ala Thr Lys                 325 330 335 aat gac cag cgc acc acc aag ttt ggc gcc ttc ctg cgc cgc aac tcg 1056 Asn Asp Gln Arg Thr Thr Lys Phe Gly Ala Phe Leu Arg Arg Asn Ser             340 345 350 ctg gat gaa ctg ccg cag ttc atc aat gtg ctg caa ggg cgt atg agt 1104 Leu Asp Glu Leu Pro Gln Phe Ile Asn Val Leu Gln Gly Arg Met Ser         355 360 365 gtc gtg ggt ccc cgc cct cac gcc gtc tcc cac aac gag atg tac cgc 1152 Val Val Gly Pro Arg Pro His Ala Val Ser His Asn Glu Met Tyr Arg     370 375 380 aag ctg atc aaa ggc tac atg atc cgg cac aag gta aaa cct ggc att 1200 Lys Leu Ile Lys Gly Tyr Met Ile Arg His Lys Val Lys Pro Gly Ile 385 390 395 400 aca ggc tgg gcg caa gtg aat ggc ctg cgc ggg gaa act gaa acc ctg 1248 Thr Gly Trp Ala Gln Val Asn Gly Leu Arg Gly Glu Thr Glu Thr Leu                 405 410 415 gac aaa atg aag gca cgc atc gac tac gac atc gac tat ttg cgt aac 1296 Asp Lys Met Lys Ala Arg Ile Asp Tyr Asp Ile Asp Tyr Leu Arg Asn             420 425 430 tgg acg ccc aaa ctg gac atg tac atc att tat aaa aca gtc agc gta 1344 Trp Thr Pro Lys Leu Asp Met Tyr Ile Ile Tyr Lys Thr Val Ser Val         435 440 445 gtg ttt acc ggt gag aaa aac gct tac tag 1374 Val Phe Thr Gly Glu Lys Asn Ala Tyr     450 455 <210> 4 <211> 457 <212> PRT <213> Methylobacillus sp. <400> 4 Met Ala Leu Pro Leu His Val Thr Ile Phe Cys Thr Val Leu Lys Pro   1 5 10 15 Cys Leu Ile Arg Leu Ser Ser Ser Phe His Cys Gly Ser Trp Arg Phe              20 25 30 Val Tyr Glu Asn Asp Leu Pro Pro His Tyr Leu Ile Met Ser Leu Ile          35 40 45 Leu Phe Ser Leu Met Phe Pro Ser Thr Thr Lys Ile Ser Gln Pro Ile      50 55 60 Ser Ser Val Ile Gln Asn Thr Leu Leu Ala Trp Phe Val Leu Ala Gly  65 70 75 80 Leu Leu Met Ala Phe Gly Tyr Ala Ser Glu Tyr Ile Tyr Ile Phe Pro                  85 90 95 Lys Ala Ser Ile Thr Leu Trp Leu Trp Leu Thr Pro Thr Leu Leu Ile             100 105 110 Ala Ala Thr Leu Gly Leu Arg Met Ala Ala Pro Leu Leu Ile Lys Met         115 120 125 Gln Gly Pro Met Arg Arg Ala Val Ile Ala Gly Met Asn Glu Gln Gly     130 135 140 Leu Ala Leu Ala Glu Arg Leu Gln Arg Ser His Tyr His Pro Thr Glu 145 150 155 160 Leu Gln Gly Phe Phe Glu Asp Arg Ser Val Asp Arg Leu Asn Ser Glu                 165 170 175 Leu His Tyr Pro Ile Leu Gly Arg Ile Asp Ser Met Ala Glu Tyr Val             180 185 190 Lys Glu His His Ile Asn Thr Ile Tyr Leu Ser Leu Pro Met Ala Ser         195 200 205 Gln Pro Arg Ile Met Lys Leu Leu Asp Asp Leu Lys Asp Thr Thr Ala     210 215 220 Ser Ile Tyr Phe Val Pro Asp Ile Phe Met Thr Asp Leu Ile Gln Gly 225 230 235 240 Arg Val Asp Gln Val Glu Asn Ile Pro Val Val Ser Val Cys Glu Thr                 245 250 255 Pro Phe Thr Gly Phe Asp Gly Leu Leu Lys Arg Ser Ala Asp Ile Ala             260 265 270 Phe Ser Leu Leu Ile Leu Ile Leu Ile Ser Pro Val Leu Leu Ala Ile         275 280 285 Ala Ile Gly Val Lys Met Ser Ser Pro Gly Pro Val Ile Phe Lys Gln     290 295 300 Arg Arg Tyr Gly Leu Asp Gly Glu Glu Ile Leu Val Tyr Lys Phe Arg 305 310 315 320 Ser Met Thr Val Cys Glu Asp Gly Ala Lys Val Thr Gln Ala Thr Lys                 325 330 335 Asn Asp Gln Arg Thr Thr Lys Phe Gly Ala Phe Leu Arg Arg Asn Ser             340 345 350 Leu Asp Glu Leu Pro Gln Phe Ile Asn Val Leu Gln Gly Arg Met Ser         355 360 365 Val Val Gly Pro Arg Pro His Ala Val Ser His Asn Glu Met Tyr Arg     370 375 380 Lys Leu Ile Lys Gly Tyr Met Ile Arg His Lys Val Lys Pro Gly Ile 385 390 395 400 Thr Gly Trp Ala Gln Val Asn Gly Leu Arg Gly Glu Thr Glu Thr Leu                 405 410 415 Asp Lys Met Lys Ala Arg Ile Asp Tyr Asp Ile Asp Tyr Leu Arg Asn             420 425 430 Trp Thr Pro Lys Leu Asp Met Tyr Ile Ile Tyr Lys Thr Val Ser Val         435 440 445 Val Phe Thr Gly Glu Lys Asn Ala Tyr     450 455 <210> 5 <211> 1011 <212> DNA <213> Methylobacillus sp. <220> <221> CDS <222> (1) .. (1011) <400> 5 atg tat cct cag cag gga aaa acc agc gtc cct cgt cga cgg ctt cgg 48 Met Tyr Pro Gln Gln Gly Lys Thr Ser Val Pro Arg Arg Arg Leu Arg   1 5 10 15 ccg ccg gga tgt cat gaa cac caa gga att ttc gac atg aac ttc act 96 Pro Pro Gly Cys His Glu His Gln Gly Ile Phe Asp Met Asn Phe Thr              20 25 30 tcg gcc cat ttc tgc aat cga gta tta ggc aag gcc agc aag ctg att 144 Ser Ala His Phe Cys Asn Arg Val Leu Gly Lys Ala Ser Lys Leu Ile          35 40 45 caa cgt tcg ttc atc cct gtg ggc atc cag tct gct gtt cat cgc gcc 192 Gln Arg Ser Phe Ile Pro Val Gly Ile Gln Ser Ala Val His Arg Ala      50 55 60 agg att ggc att gtc cag cat tac ctc acc caa caa tac ggc tcg ctt 240 Arg Ile Gly Ile Val Gln His Tyr Leu Thr Gln Gln Tyr Gly Ser Leu  65 70 75 80 atc aac gca tac cag cct ctg gtg aaa gac ccc gca gaa cta aac ctg 288 Ile Asn Ala Tyr Gln Pro Leu Val Lys Asp Pro Ala Glu Leu Asn Leu                  85 90 95 cct gct gac aag atc atc tgg gta tgc tgg cta cag ggc ctg gac cac 336 Pro Ala Asp Lys Ile Ile Trp Val Cys Trp Leu Gln Gly Leu Asp His             100 105 110 gct cca gag ata gtg aaa aaa tgc att gcc aac ctc cag ttg ttt cac 384 Ala Pro Glu Ile Val Lys Lys Cys Ile Ala Asn Leu Gln Leu Phe His         115 120 125 ggt gag gac tgc act att cat ctg ata acg ctg gag aat tat aaa aac 432 Gly Glu Asp Cys Thr Ile His Leu Ile Thr Leu Glu Asn Tyr Lys Asn     130 135 140 ttc gtg acg ctc cca aga cat att gag gag aag ttc gaa aag aaa ctc 480 Phe Val Thr Leu Pro Arg His Ile Glu Glu Lys Phe Glu Lys Lys Leu 145 150 155 160 atg aag cca gcg cat ttt tcc gat gtg cta cgg cta tgt tta ttg gca 528 Met Lys Pro Ala His Phe Ser Asp Val Leu Arg Leu Cys Leu Leu Ala                 165 170 175 aaa cac ggc ggt gcg tgg ata gac tcc acc gta ctc atc ctg aag aaa 576 Lys His Gly Gly Ala Trp Ile Asp Ser Thr Val Leu Ile Leu Lys Lys             180 185 190 ctg cct gat gac gtt ttc aat gaa caa ttt ttt aca ctg aaa agt cac 624 Leu Pro Asp Asp Val Phe Asn Glu Gln Phe Phe Thr Leu Lys Ser His         195 200 205 tcc tac tca acc ata tca agc att tcc ctg ggt aag tgg acc act ttt 672 Ser Tyr Ser Thr Ile Ser Ser Ile Ser Leu Gly Lys Trp Thr Thr Phe     210 215 220 ttt ctc cag gcc agg cgc ggg tac acc cct gtt tta ttc ttg cgg gac 720 Phe Leu Gln Ala Arg Arg Gly Tyr Thr Pro Val Leu Phe Leu Arg Asp 225 230 235 240 ctc ctt att cag tat tgg caa ggc cat gat aaa gag ttg gat tat ttt 768 Leu Leu Ile Gln Tyr Trp Gln Gly His Asp Lys Glu Leu Asp Tyr Phe                 245 250 255 ctt ttc gac cat gcc atc agc atc gcc tat caa cac ttc cca gag ttt 816 Leu Phe Asp His Ala Ile Ser Ile Ala Tyr Gln His Phe Pro Glu Phe             260 265 270 aaa tac cag gta gat cac ctg ggt tat ttt ggc aat cat cgc cac cta 864 Lys Tyr Gln Val Asp His Leu Gly Tyr Phe Gly Asn His Arg His Leu         275 280 285 tta atg cca tta cta ttg acc gaa tat agc gca gaa gcc gcc agc ccc 912 Leu Met Pro Leu Leu Leu Thr Glu Tyr Ser Ala Glu Ala Ala Ser Pro     290 295 300 atc aac gaa gat ccg gtg cag ata tat aaa ttg acc tac aag atc gac 960 Ile Asn Glu Asp Pro Val Gln Ile Tyr Lys Leu Thr Tyr Lys Ile Asp 305 310 315 320 tca acg cac act gtt ccg cag cat tcg ttt tat cag cat tac att aag 1008 Ser Thr His Thr Val Pro Gln His Ser Phe Tyr Gln His Tyr Ile Lys                 325 330 335 tag 1011 <210> 6 <211> 336 <212> PRT <213> Methylobacillus sp. <400> 6 Met Tyr Pro Gln Gln Gly Lys Thr Ser Val Pro Arg Arg Arg Leu Arg   1 5 10 15 Pro Pro Gly Cys His Glu His Gln Gly Ile Phe Asp Met Asn Phe Thr              20 25 30 Ser Ala His Phe Cys Asn Arg Val Leu Gly Lys Ala Ser Lys Leu Ile          35 40 45 Gln Arg Ser Phe Ile Pro Val Gly Ile Gln Ser Ala Val His Arg Ala      50 55 60 Arg Ile Gly Ile Val Gln His Tyr Leu Thr Gln Gln Tyr Gly Ser Leu  65 70 75 80 Ile Asn Ala Tyr Gln Pro Leu Val Lys Asp Pro Ala Glu Leu Asn Leu                  85 90 95 Pro Ala Asp Lys Ile Ile Trp Val Cys Trp Leu Gln Gly Leu Asp His             100 105 110 Ala Pro Glu Ile Val Lys Lys Cys Ile Ala Asn Leu Gln Leu Phe His         115 120 125 Gly Glu Asp Cys Thr Ile His Leu Ile Thr Leu Glu Asn Tyr Lys Asn     130 135 140 Phe Val Thr Leu Pro Arg His Ile Glu Glu Lys Phe Glu Lys Lys Leu 145 150 155 160 Met Lys Pro Ala His Phe Ser Asp Val Leu Arg Leu Cys Leu Leu Ala                 165 170 175 Lys His Gly Gly Ala Trp Ile Asp Ser Thr Val Leu Ile Leu Lys Lys             180 185 190 Leu Pro Asp Asp Val Phe Asn Glu Gln Phe Phe Thr Leu Lys Ser His         195 200 205 Ser Tyr Ser Thr Ile Ser Ser Ile Ser Leu Gly Lys Trp Thr Thr Phe     210 215 220 Phe Leu Gln Ala Arg Arg Gly Tyr Thr Pro Val Leu Phe Leu Arg Asp 225 230 235 240 Leu Leu Ile Gln Tyr Trp Gln Gly His Asp Lys Glu Leu Asp Tyr Phe                 245 250 255 Leu Phe Asp His Ala Ile Ser Ile Ala Tyr Gln His Phe Pro Glu Phe             260 265 270 Lys Tyr Gln Val Asp His Leu Gly Tyr Phe Gly Asn His Arg His Leu         275 280 285 Leu Met Pro Leu Leu Leu Thr Glu Tyr Ser Ala Glu Ala Ala Ser Pro     290 295 300 Ile Asn Glu Asp Pro Val Gln Ile Tyr Lys Leu Thr Tyr Lys Ile Asp 305 310 315 320 Ser Thr His Thr Val Pro Gln His Ser Phe Tyr Gln His Tyr Ile Lys                 325 330 335 <210> 7 <211> 3690 <212> DNA <213> Methylobacillus sp. <400> 7 tgtatggact gtagagggtg acaacaatag tccaaaagaa ctaccactga tgtaggcaaa 60 agttttatca tagtgacagc cgtcacatac aagaatacgc taggattcca cgcttgatcc 120 aaatataaaa tggccaccta caaacacatg agcaaatatc cttttcagca tgtcccacaa 180 tttgagtgga ggatctactg ctggacagca ctggcgccct tgcagctggc ccaataattg 240 ctctcaaaga caagtgtttg caacgtaaat gattttttat atttgaacgg ctatgtttat 300 taccaaagaa ctaaccgata ttgagaaggc acagcttatt gacttgatgc acgagtcttt 360 gcgaatccgc tcgcacttcg agttcttcct ttggatgcaa ggcaagcttc agcagttttt 420 accgcatgaa atcatgatca ccgcctgggg cgatttctcc atgggcgtca tctatttcaa 480 tattgtgtcg ccgctccccg gagtacgtac cgagaagata tcaagtggcg atctgaatcc 540 attgctcaaa cggcttttca attactggct tagtcatacc aaagccccgt ttaccttatc 600 ggcggagaat ggcgtattcc aggactgcga tgtactgcca gcggaagtga acagccatct 660 gaaaaacatg aagtcagcgc tggtgcacgg catcaaggat ttccgtgggc gccatgactg 720 cctttacata ttgctgaatt caacccccac catgccgaat acctcacggt acatgctgga 780 gtcattgcta ccttacattg acagcgcgct caggcaactg gagcatttgc ctgtgcagca 840 cccggcagat aaagaagcca gtgaagatct gcacgaagaa gaaaacgaag cgctggagca 900 gctttcttcc cgtgagatcg aaatcatgga atgggtgcgc aacggcaaaa ccaatcagga 960 aatcggcatg atcctggaca ttagttcatt taccgtcaaa aaccatttgc aacgcatatt 1020 taaaaagctt gatgttctga atagagcgca agccgtttcc aaattcaagc agactccacg 1080 ggcctcatga aaccaatatt agccgccacc aagccgactt cacgaaaaac cttcgctggc 1140 attacctttg cacgtgacaa tattttgcac ggtgttgaag ccctgcttga tccgcttgtc 1200 gtcgtcattt cactgtggat cgtggcgttt tgtttatgaa aacgatctgc cacctcacta 1260 cctgatcatg tcgctgattc tgttttcgct gatgttcccc agcaccacca aaatcagcca 1320 gcccatcagt tcagttattc aaaacacgtt gctggcatgg tttgttctgg ccgggctatt 1380 gatggcgttt ggatatgctt ctgaatacat ctatatcttc cccaaagcct cgattacatt 1440 atggctctgg ctgaccccta ccctgcttat cgcagcaacc ctcggtttac gcatggcggc 1500 gccgctactc atcaagatgc aagggcctat gcgacgggcg gtgattgcgg gcatgaacga 1560 acagggcctg gccctggcag aacgcttgca gcgtagccac taccatccaa cagagttgca 1620 gggcttcttt gaggatcgca gtgtggaccg actcaattca gaattgcatt accccattct 1680 tggccgcatc gacagcatgg cggagtatgt gaaagagcat cacatcaaca ccatctacct 1740 ttcactgccc atggcgagcc aaccacgcat catgaagttg ttagatgatt tgaaagacac 1800 cacggcgtcc atttactttg tcccggatat tttcatgaca gatctgatac aagggcgtgt 1860 cgatcaggta gaaaacatcc cggtggtcag cgtatgtgaa accccattta ccggctttga 1920 cggtctgttg aaacgctcag ctgatatcgc tttctcgttg ctgatcctga tccttatctc 1980 gccggtatta ttggcgatcg cgattggcgt gaagatgagt tcaccaggcc cagtcatttt 2040 caaacagcgc cgttatgggc ttgatggcga agagatcctt gtctacaaat tccgctccat 2100 gacggtatgc gaagatggcg caaaagtgac gcaagcgacg aaaaatgacc agcgcaccac 2160 caagtttggc gccttcctgc gccgcaactc gctggatgaa ctgccgcagt tcatcaatgt 2220 gctgcaaggg cgtatgagtg tcgtgggtcc ccgccctcac gccgtctccc acaacgagat 2280 gtaccgcaag ctgatcaaag gctacatgat ccggcacaag gtaaaacctg gcattacagg 2340 ctgggcgcaa gtgaatggcc tgcgcgggga aactgaaacc ctggacaaaa tgaaggcacg 2400 catcgactac gacatcgact atttgcgtaa ctggacgccc aaactggaca tgtacatcat 2460 ttataaaaca gtcagcgtag tgtttaccgg tgagaaaaac gcttactaga tagtcttgtg 2520 cattcgtgca tatcagattg agcgagctgg acgccattct agaaccaagg aatgtatcct 2580 cagcagggaa aaaccagcgt ccctcgtcga cggcttcggc cgccgggatg tcatgaacac 2640 caaggaattt tcgacatgaa cttcacttcg gcccatttct gcaatcgagt attaggcaag 2700 gccagcaagc tgattcaacg ttcgttcatc cctgtgggca tccagtctgc tgttcatcgc 2760 gccaggattg gcattgtcca gcattacctc acccaacaat acggctcgct tatcaacgca 2820 taccagcctc tggtgaaaga ccccgcagaa ctaaacctgc ctgctgacaa gatcatctgg 2880 gtatgctggc tacagggcct ggaccacgct ccagagatag tgaaaaaatg cattgccaac 2940 ctccagttgt ttcacggtga ggactgcact attcatctga taacgctgga gaattataaa 3000 aacttcgtga cgctcccaag acatattgag gagaagttcg aaaagaaact catgaagcca 3060 gcgcattttt ccgatgtgct acggctatgt ttattggcaa aacacggcgg tgcgtggata 3120 gactccaccg tactcatcct gaagaaactg cctgatgacg ttttcaatga acaatttttt 3180 acactgaaaa gtcactccta ctcaaccata tcaagcattt ccctgggtaa gtggaccact 3240 ttttttctcc aggccaggcg cgggtacacc cctgttttat tcttgcggga cctccttatt 3300 cagtattggc aaggccatga taaagagttg gattattttc ttttcgacca tgccatcagc 3360 atcgcctatc aacacttccc agagtttaaa taccaggtag atcacctggg ttattttggc 3420 aatcatcgcc acctattaat gccattacta ttgaccgaat atagcgcaga agccgccagc 3480 cccatcaacg aagatccggt gcagatatat aaattgacct acaagatcga ctcaacgcac 3540 actgttccgc agcattcgtt ttatcagcat tacattaagt agctcacttt aatatccgga 3600 atatccgcga caccattttc aaatcactcc gcagaattca aggcgaaaaa aaaccccgca 3660 tttgcggggt tttttattac ttaagcagct 3690 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 8 aacgaatgct gcggaacagt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 9 accttacatt gacagcgcgc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 10 gcccaataat tgctctcaaa g 21 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 11 ttggtggcgg ctaatattg 19

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] December 10, 2001 (2001.12.
10)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Title of invention

[Correction method] Change

[Correction content]

[Title of Invention] Novel saccharide-producing gene group

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // (C12N 1/20 C12N 15/00 ZNAA C12R 1:01) (72) Inventor Hisashi Yasuda Kawasaki, Kanagawa Prefecture 1-1, Suzuki-cho, Kawasaki-ku, Ajinomoto Co., Inc. F-Technology Research Institute F-term (reference) 4B024 AA20 BA80 CA04 DA05 DA12 EA04 FA02 GA11 HA01 4B064 AF01 AG01 CA02 CA06 CA19 CC24 4B065 AA01X AA01Y AA72X AB01 AC14 AC20 BA01 BB19 BB29 BD27 CA19 CA24

Claims (8)

[Claims] 1. A DNA encoding a protein shown in (A) to (F) below. (A) A protein having the amino acid sequence of SEQ ID NO: 2. (B) A protein consisting of the amino acid sequence of SEQ ID NO: 2, which comprises an amino acid sequence including substitution, deletion, insertion or addition of one or more amino acids. (C) A protein having the amino acid sequence of SEQ ID NO: 4. (D) A protein consisting of the amino acid sequence of SEQ ID NO: 4, which has one or more amino acid substitutions, deletions, insertions or additions. (E) A protein having the amino acid sequence of SEQ ID NO: 6. (F) A protein consisting of the amino acid sequence of SEQ ID NO: 6, which has one or more amino acid substitutions, deletions, insertions or additions. 2. The DNA according to claim 1, which is the DNA shown in the following (a) to (f). (A) The nucleotide sequence set forth in SEQ ID NO: 1. (B) A DNA that hybridizes under stringent conditions with the base sequence shown in SEQ ID NO: 1 or a probe that can be prepared from the base sequence. (C) The nucleotide sequence set forth in SEQ ID NO: 3. (D) A DNA that hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 3 or a probe that can be prepared from the nucleotide sequence. (E) The nucleotide sequence set forth in SEQ ID NO: 5. (F) A DNA that hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 5 or a probe that can be prepared from the nucleotide sequence. 3. The gene according to claim 1, wherein the gene involved in sugar production is derived from the chromosome of a microorganism capable of assimilating methane and / or methanol. 4. The gene according to claim 3, wherein the microorganism capable of assimilating methanol is a microorganism belonging to the genus Methylobacillus. 5. A microorganism having improved saccharide-producing ability by using the gene according to claim 1. 6. A method for producing a saccharide using the microorganism according to claim 5. 7. A microorganism having a reduced saccharide-producing ability by using the gene according to claim 1. 8. A method for producing a target substance other than saccharides using the microorganism according to claim 7 as a host.