Pseudouridine synthetase mutant, mutant gene and application thereof in preparation of vitamin B2In (1)

文档序号：1916778 发布日期：2021-12-03 浏览：18次中文

阅读说明：本技术 假尿苷合成酶突变体、突变基因及其在制备维生素b2中的应用 (Pseudouridine synthetase mutant, mutant gene and application thereof in preparation of vitamin B2In (1) ) 是由解永梅马成兵尤淑芬李铭越刘川于 2021-08-18 设计创作，主要内容包括：本发明公开了一种假尿苷合成酶突变体、突变基因及其在制备维生素B2中的应用。其中所述突变体的多肽氨基酸序列相对于SEQ ID No.3所示序列仅存在下述突变：第239位组氨酸突变为亮氨酸。本发明以假尿苷合成酶ylyB为基础,选择第239位的氨基酸进行定点突变,将枯草芽孢杆菌BS-1中假尿苷合成酶编码基因ylyB变为假尿苷合成酶突变体(H239L)与枯草芽孢杆菌BS-1相比,含有假尿苷合成酶突变体(H239L)的菌株可以提高产维生素B-(2)的能力达10.8％,因而在制备维生素B-(2)中具有很大的应用价值。(The invention discloses a pseudouridine synthetase mutant, a mutant gene and application thereof in preparing vitamin B2. Wherein the polypeptide amino acid sequence of the mutant only has the following mutations relative to the sequence shown in SEQ ID No. 3: histidine at 239 was mutated to leucine. The invention selects the 239 th amino acid to perform site-directed mutagenesis on the basis of pseudouridine synthetase yB, and carries out site-directed mutagenesis on the pseudouridine synthetase coding gene yly in bacillus subtilis BS-1B-to-Pseudouridine synthetase mutant (H239L) the strain containing Pseudouridine synthetase mutant (H239L) had increased vitamin B production compared to Bacillus subtilis BS-1 2 The content of vitamin B in the product can reach 10.8%, and the vitamin B can be used for preparing vitamin B 2 Has great application value.)

1. A pseudouridine synthase mutant, characterized in that the polypeptide amino acid sequence thereof has only the following mutations relative to the sequence shown in SEQ ID No. 3: histidine at 239 was mutated to leucine.

2. The mutant pseudouridine synthase according to claim 1, wherein the amino acid sequence is represented by SEQ ID No. 4.

3. The gene encoding a pseudouridine synthase mutant according to claim 1.

4. The coding gene of claim 3, wherein the nucleotide sequence is as shown in SEQ ID No. 2.

5. A recombinant vector comprising a gene encoding the pseudouridine synthase mutant according to claim 3.

6. A recombinant host cell comprising a gene encoding the pseudouridine synthase mutant according to claim 3.

7. Use of the pseudouridine synthase mutant according to claim 1 or the pseudouridine synthase mutant according to claim 3 in production of vitamin B₂The use of (1).

8. Enhancing vitamin B production of bacillus subtilis₂The method is characterized in that the RNA helicase coding gene of the amino acid sequence shown as SEQ ID No.3 on the chromosome is subjected to site-directed mutagenesis to obtain the vitamin B₂Wherein the site-directed mutation is site-directed mutation of a nucleotide encoding histidine at 239 th position of the encoded gene to encode leucine.

9. The method of claim 8, wherein the bacillus subtilis is bacillus subtilis BS-1 as the starting strain.

10. A method for preparing vitamin B by using Bacillus subtilis obtained by the method of claim 8 or 9₂The method of (1), comprising culturing said Bacillus subtilis, and collecting vitamin B₂The step (2).

Technical Field

The invention relates to pseudouridine synthetase mutant, mutant gene and application thereof in preparing vitamin B₂Belonging to the field of biotechnology.

Background

Riboflavin (Riboflavin) also called vitamin B₂Molecular formula is C₁₇H₂₀O₆N₄The vitamin B is a water-soluble vitamin in a vitamin B group, exists in two forms of Flavin Mononucleotide (FMN) and Flavin Adenine Dinucleotide (FAD) in organisms, participates in redox reaction as a coenzyme of some important oxidoreductases in organisms, and plays a role in delivering hydrogen. The lack of riboflavin causes the metabolic disorder of organisms, but neither humans nor animals can synthesize riboflavin by themselves and only ingest riboflavin from food, so that the production of riboflavin has a very wide market in the food, feed and pharmaceutical industries.

Currently, the industrial production methods of riboflavin include plant extraction methods, chemical synthesis methods, semi-synthesis methods, and microbial synthesis methods. Among them, the microbial synthesis method has advantages of low cost, environmental friendliness, renewable energy and the like, and gradually takes a leading position, and becomes a main method for most industrial production. Among a plurality of microorganisms capable of producing riboflavin, bacillus subtilis is taken as a non-pathogenic microorganism, has clear physiological metabolism and genetic background, is convenient to determine a metabolic target point and modify genetic engineering, has reliable safety, has long-term application of fermentation products in food and feed industries, and is very important for environmental, medical and industrial fermentation production. Secondly, the bacillus subtilis genetic engineering strain can excessively synthesize folic acid, inosine or guanosine and has the potential of providing enough precursors for excessive synthesis of riboflavin, so the bacillus subtilis genetic engineering strain gradually shows strong vitality in the microbial fermentation production of riboflavin and becomes a main production strain.

Riboflavin synthesis in bacillus subtilis (b.subtilis) requires two precursor substances, ribulose-5-phosphate (Ru5P) and guanine-5' -triphosphate (GTP), where Ru5P is derived from the pentose phosphate pathway and GTP is derived from the de novo purine synthesis pathway. Both of them finally synthesize riboflavin from the riboflavin synthesis pathway through 7-step reactions under the action of a number of riboflavin operons.

Pseudouridine synthase, encoded by the yB gene, catalyzes the production of pseudouridine from uracil. Among pyrimidine metabolic pathways, there are de novo and salvage synthetic pathways. Uracil reacts with phosphoribosylpyrophosphate (PRPP) in a salvage synthesis pathway to form uracil nucleotides. Thereby consuming free uracil and PRPP. While PRPP can be accumulated after uracil forms pseudouridine. PRPP, in turn, can be used as a precursor for de novo and salvage purine synthesis pathways to produce GTP, a precursor of riboflavin. Thus, it is thought that the pseudouridine synthase affects the production of riboflavin, i.e., the expression level of the pseudouridine synthase-encoding gene has a certain potential relationship with the production of riboflavin. At present, no pseudouridine synthase mutant gene has been used for vitamin B₂And (5) reporting the synthesis.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a pseudouridine synthetase mutant, a mutant gene and application thereof in preparing vitamin B₂The use of (1).

Firstly, the invention provides a pseudouridine synthetase mutant, which is characterized in that the polypeptide amino acid sequence only has the following mutations relative to the sequence shown in SEQ ID No. 3: histidine at 239 was mutated to leucine.

Preferably, according to the invention, the amino acid sequence is shown in SEQ ID No. 4.

Secondly, the present invention provides the coding gene of the pseudouridine synthetase mutant.

Preferably, according to the invention, the nucleotide sequence is shown as SEQ ID No. 2.

Accordingly, the third aspect of the present invention also provides an expression cassette, a recombinant vector containing a gene encoding the pseudouridine synthetase mutant. The recombinant vector is not particularly limited to the starting vector, and may be any vector known in the art as long as it can replicate in a host. For example, the vector includes, but is not limited to, a plasmid, a phage. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, integrate into the genome itself.

More preferably a recombinant expression vector, more preferably a prokaryotic re-expression vector. Most preferred are expression vectors suitable for expression in Bacillus subtilis.

In a fourth aspect, the present invention provides a recombinant host cell containing a gene encoding said pseudouridine synthase mutant. Wherein said "host cell" is a cell having a meaning generally understood in the art, which is capable of introducing a gene encoding a mutant of the invention, the introduction being hereinafter referred to as recombinant host cell. The host cell of the invention may be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, more preferably Bacillus subtilis.

In a fifth aspect, the present invention provides a pseudouridine synthetase mutant encoding gene in the production of vitamin B₂The use of (1).

In a sixth aspect, the invention provides a method for enhancing the production of vitamin B by bacillus subtilis₂The method of (1), wherein the pseudouridine synthetase encoding gene on the chromosome is subjected to site-directed mutagenesis to obtain vitamin B₂Wherein the site-directed mutation is a substitution of the nucleotide encoding the 239 th histidine H of the coding gene with a nucleotide encoding leucine L, more particularly a mutation of the 716 th nucleotide a of the coding gene to T. The site-directed mutagenesis can be carried out by various methods known in the art.

Preferably, according to the invention, the original starting strain of Bacillus subtilis is Bacillus subtilis BS-1.

In a seventh aspect of the present invention, there is provided a method for preparing vitamin B using Bacillus subtilis obtained by the method of the above sixth aspect₂The method of (1), which comprises culturing the Bacillus subtilis, and collecting vitamin B₂More preferably further comprises purifying vitamin B₂The step (2).

Wherein the Bacillus subtilis is culturedThe cultivation can be carried out according to the conventional methods in the art, such as shake flask cultivation, batch cultivation, continuous cultivation, fed-batch cultivation, etc., and suitable cultivation conditions such as temperature, time, pH of the medium, etc., can be selected as the case may be. In addition, vitamin B can be recovered or purified from the cells or the culture medium₂The method of (a) can be carried out by a method conventional in the art, for example, filtration, anion exchange chromatography, crystallization, HPLC and the like.

The experimental procedures not described in detail in the present invention can be performed according to the routine experimental procedures in the technical field.

The invention has the technical characteristics that:

selecting pseudouridine synthetase gene yB in bacillus subtilis, and carrying out site-directed mutagenesis on the 239 th histidine of the pseudouridine synthetase gene yB to obtain a pseudouridine synthetase mutant (H239L). The activity of pseudouridine synthetase gene yB is obviously enhanced after mutation to drive uracil to generate pseudouridine, intracellular uracil is reduced after uracil generates pseudouridine, phosphoribosyl pyrophosphate (PRPP) can be accumulated, and PRPP can be used as a precursor of purine de novo synthesis pathway and salvage synthesis pathway, so that GTP synthesis can be enhanced, and vitamin B can be used as a precursor of purine de novo synthesis pathway and salvage synthesis pathway₂Providing sufficient precursor, and increasing vitamin B₂The yield of (2).

The invention has the beneficial effects that:

1. the invention takes pseudouridine synthetase yB as the basis, selects the 239 th amino acid to carry out site-directed mutagenesis, changes the pseudouridine synthetase encoding gene yB in bacillus subtilis BS-1 into a pseudouridine synthetase mutant (H239L), and compared with the bacillus subtilis BS-1, the strain containing the pseudouridine synthetase mutant (H239L) can improve the vitamin B production₂The content of vitamin B in the product can reach 10.8%, and the vitamin B can be used for preparing vitamin B₂Has great application value.

2. The research proves that the genetically engineered bacteria containing the mutant gene of the pseudouridine synthetase are biologically safe, and furthermore, the experiment shows that the gene mutation on the chromosome not only does not influence the growth of the bacteria, but also is beneficial to the growth of the bacteria, so that the content of the pseudouridine synthetase mutant gene can be further improvedStrain of pseudouridine synthetase mutant (H239L) produces vitamin B₂The ability of the cell to perform.

Drawings

FIG. 1 shows VB of different Bacillus subtilis strains after fermentation for 41h₂And (4) yield.

Detailed Description

The following examples and figures of the present invention are merely illustrative of specific embodiments for carrying out the invention and these should not be construed as limiting the invention and any changes which may be made without departing from the principles and spirit of the invention are within the scope of the invention.

The experimental techniques and experimental methods used in this example are conventional techniques unless otherwise specified. The materials, reagents and the like used in the present examples are all available from normal commercial sources unless otherwise specified.

High vitamin B production as described in this example₂The preservation number of the bacillus subtilis is CGMCC NO. 4018. The Bacillus subtilis BS-1 has a preservation number of CGMCC NO. 4019. The Bacillus subtilis 168 is a Bacillus subtilis model strain.

The formula of the culture medium is as follows:

LB medium (g/L): 10 parts of sodium chloride, 10 parts of tryptone, 5 parts of yeast extract and 18 parts of agar powder added into a solid culture medium.

Fermentation medium (g/L): corn steep liquor dry powder 2, glucose 50, yeast extract 5, magnesium sulfate 0.05, ammonium sulfate 0.5 and the balance of distilled water.

Detection of OD 600:

and (3) measuring the turbidity of the fermentation liquor under 600nm by using a spectrophotometer, and quickly and accurately obtaining the biomass in the fermentation liquor. Accurately transferring a certain volume of fermentation liquor into a brown volumetric flask, fixing the volume with distilled water, uniformly mixing, placing into a cuvette, measuring the biomass at a wavelength of 600nm by taking the distilled water as a reference blank, and determining the OD600 as the reading multiplied by the dilution.

Vitamin B₂The detection method comprises the following steps:

mixing the fermentation liquor uniformly, diluting the fermentation liquor to a proper multiple by using 0.01mol/L NaOH, mixing uniformly, carrying out light-shielding alkali dissolution for 20min, centrifuging at 12000rpm for 2min, taking supernate, taking 0.01mol/L NaOH as a blank, measuring absorbance (the display value is controlled between 0.2 and 0.8) at 444nm, and calculating the content of riboflavin according to the following formula: FB (mg/L) ═ (dilution times absorbance)/0.0321.

Example 1: confirmation and verification of yBgene mutation site

Since the yB gene is responsible for encoding pseudouridine synthase in Bacillus subtilis, driving uracil to produce pseudouridine, site-directed mutagenesis was chosen for the yB gene.

The inventors analyzed the highly conserved regions of the amino acid sequence of the pseudouridine synthetase encoded by the yyB gene using the Jalview software. Meanwhile, the protein simulation modeling of the pseudo uridine synthetase of the yyB gene is carried out by using a Swiss-Model tool, and the active center of the pseudo uridine synthetase is predicted by using a HotSpot Wizard 2.0. It was found that the 239 th position of the amino acid sequence of pseudouridine synthetase encoded by the yBgene is located near the active center and the highly conserved region, and it is highly probable that site-directed mutagenesis thereof results in an increase in the pseudouridine synthetase activity.

The inventor selects a high-yield vitamin B strain in the earlier stage₂The bacillus subtilis (CGMCC NO.4018) can be fermented to produce vitamin B₂. To further verify the role of the 239 th position of the pseudo uridine synthetase amino acid sequence encoded by the yyB gene, the genome of Bacillus subtilis 168 is taken as a reference genome, and vitamin B is highly produced₂The Bacillus subtilis is sent to Jinzhi Biotechnology limited for sequencing to find vitamin B₂Genes with an effect on competence. Through sequence comparison analysis, high-yield vitamin B is found₂The bacillus subtilis has a large number of mutations, the total number of the mutations comprises 203 mutation sites, and through mutation statistics, the mutations relate to 50 genes, including the yyB gene.

Further, vitamin B is produced at a high yield₂The genome of the bacillus subtilis is taken as a template, UPyB-F and DNyB-R are taken as primers, yB gene and upstream and downstream sequences are cloned to obtain a PCR product, and then the PCR product is sent to Jinzhi Biotech limited to carry out sequencing.Through sequence comparison analysis, high-yield vitamin B is found₂Compared with Bacillus subtilis 168, the yBgene has mutation, and contains 4 mutation sites including 239 th site. Wherein, the nucleotide sequence of the yB gene of the Bacillus subtilis 168 is shown as SEQ ID No.1, and the amino acid sequence of the coded RNA helicase is shown as SEQ ID No. 3.

Except that the mutation at the 239 th site is non-synonymous mutation, the mutations at the other three sites are synonymous mutations, and the protein structure is not influenced. From the above results, it was found that the 239 th position in the amino acid sequence of pseudouridine synthase encoded by the yyB gene affects vitamin B₂A key site of yield is designed according to the site, a pseudouridine synthetase mutant (H239L) is designed, the amino acid sequence of the pseudouridine synthetase mutant is shown as SEQ ID No.4, and the nucleotide sequence of the pseudouridine synthetase mutant is shown as SEQ ID No. 2.

Example 2: construction of engineering Strain 18-yB with yB Back-mutation

Amplifying a wild type UPylyB (containing DR) fragment with a joint by using a primer UPylyB-F, UPylyB-R (containing DR) and amplifying an araR (containing DR) fragment with a joint by using a primer araR-F, araR-R (containing DR) by using a Bacillus subtilis 168 chromosome as a template; using pC194 plasmid as a template, and amplifying a cat fragment with a joint by using a primer cat-F, cat-R; a chromosome of Bacillus subtilis 168 is taken as a template, and a downstream homology arm segment DNyB is amplified by using a primer DNyB-F, DNylyB-R, wherein the nucleotide sequence of the yB gene is shown as SEQ ID No.1, and the coded amino acid sequence is shown as SEQ ID No. 3.

And (3) performing fusion PCR by using a primer UPylyB-F, DNylyB-R by using the UPylyB fragment, the cat fragment, the araR fragment and the DNylyB fragment as templates to obtain an assembled fragment UCR-yB, detecting the assembled fragment UCR-yB correctly by nucleic acid electrophoresis, and recovering the gel to obtain a purified UCR-yB fragment.

Transformation of UCR-yB fragment Spizizen into high-yield vitamin B₂The bacillus subtilis (CGMCC NO.4018) is coated on an LB solid plate containing 8mg/L chloramphenicol, colony PCR verification is carried out after 24h culture, intellectual sequencing is carried out when nucleic acid electrophoresis is correct, and the intermediate strain 4018-UCR-And (2) yB, wherein the nucleotide sequence of the yB gene is shown as SEQ ID No.1, and the coded amino acid sequence is shown as SEQ ID No. 3.

Selecting a single colony of an intermediate strain 4018-UCR-yB in a test tube containing 5mL LB, carrying out shake culture at 37 ℃ for 8h, then taking 200uL bacterial liquid to coat on an LB solid plate containing 40mg/L neomycin, carrying out colony PCR verification after culturing for 24h, carrying out accurate nucleic acid electrophoresis and carrying out intelligent sequencing, and obtaining an engineering strain 18-yB which removes a screening marker cat-araR through DR and contains the back mutation of the yB gene after the sequencing is accurate, wherein the nucleotide sequence of the yB gene is shown as SEQ ID No.1, and the coded amino acid sequence is shown as SEQ ID No. 3.

The primers used in this section were as follows:

the strains and plasmids used in this section were as follows:

example 3: construction of engineering Strain 19-yB containing yB mutant

Amplifying a UPylyB (containing DR) fragment with a joint and a point mutation by using a primer UPylyB-F, UPylyB-R (containing DR) by using a coding gene of an artificially synthesized pseudouridine synthetase mutant (H239L) as a template, and amplifying an araR (containing DR) fragment with a joint by using a primer araR-F, araR-R (containing DR); using pC194 plasmid as a template, and amplifying a cat fragment with a joint by using a primer cat-F, cat-R; a downstream homology arm fragment DNyB was amplified by using a primer DNyB-F, DNylyB-R, using the chromosome of B.subtilis 168 as a template. Wherein, the nucleotide sequence of the pseudouridine synthetase mutant (H239L) is shown as SEQ ID No.2, and the coded amino acid sequence is shown as SEQ ID No. 4.

Transforming the UCR-yB fragment Spizizen into Bacillus subtilis BS-1(CGMCC NO.4019), coating the Bacillus subtilis BS-1 on an LB solid plate containing 8mg/L chloramphenicol, culturing for 24H, then carrying out colony PCR verification, carrying out intelligent sequencing on nucleic acid electrophoresis correctly, and obtaining the intermediate strain 4019-UCR-yB containing the pseudouridine synthetase mutant (H239L) after the sequencing is correct. Wherein, the nucleotide sequence of the pseudouridine synthetase mutant (H239L) is shown as SEQ ID No.2, and the coded amino acid sequence is shown as SEQ ID No. 4.

Selecting a single colony of an intermediate strain 4019-UCR-yB, placing the single colony in a test tube containing 5mL LB, carrying out shake culture at 37 ℃ for 8H, then taking 200uL bacterial liquid, coating the bacterial liquid on an LB solid plate containing 40mg/L neomycin, carrying out colony PCR verification after culturing for 24H, carrying out intelligent sequencing on nucleic acid electrophoresis correctly, and obtaining an engineering strain 19-yB which has the internal chromosome removed the screening marker cat-araR through DR homologous recombination and contains the pseudouridine synthetase mutant (H239L). Wherein. The nucleotide sequence of the pseudouridine synthetase mutant (H239L) is shown as SEQ ID No.2, and the coded amino acid sequence is shown as SEQ ID No. 4.

The strains and plasmids used in this section were as follows:

example 4: evaluation of vitamin B in different strains₂Capacity of production

Engineering strains 19-yB and Bacillus subtilis BS-1(CGMCC NO.4019) are respectively used as an experimental group and a control group for engineeringStrain 18-yB and high-yield vitamin B₂Respectively taking Bacillus subtilis (CGMCC NO.4018) as experimental group and control group, and performing strain vitamin B₂And (5) evaluating the production capacity.

1. Conditions for culturing the strains

LB solid plates containing 20mg/L neomycin were streaked with an inoculating needle under aseptic conditions, and inverted in an incubator at 37 ℃ for 24-48 hours to obtain freshly activated single colonies. A single colony was picked with an inoculating needle, streaked on an LB solid slant containing 20mg/L neomycin, and cultured in an incubator at 37 ℃ for 48 hours. Scraping 1/3 thallus Porphyrae on the inclined plane, inoculating into 500mL baffle triangular flask containing 70mL fermentation medium (3 parallel strains), culturing at 37 deg.C under shaking at 200rpm for 41 hr, and measuring OD600 and vitamin B in the fermentation broth₂And (4) yield.

2. Different strains OD600 and vitamin B₂Comparison of yields

Engineered Strain 19-yB and Bacillus subtilis BS-1, Strain 19-yB vitamin B containing Pseudouridine synthetase mutant (H239L)₂The yield is improved by 10.8 percent, the engineering strain is 18-yB and the high-yield vitamin B₂Return mutant Strain 18-yB vitamin B compared to Bacillus subtilis₂The yield decreased by 9.8% (see table 4 below and figure 1).

The experimental result shows that the invention takes pseudouridine synthetase yB as the basis, selects the 239 th amino acid for site-directed mutagenesis to change the pseudouridine synthetase yB in bacillus subtilis BS-1 into a pseudouridine synthetase mutant (H239L), and compared with the original strain, the strain containing the pseudouridine synthetase mutant (H239L) can improve the vitamin B production₂The content of vitamin B in the product can reach 10.8%, and the vitamin B can be used for preparing vitamin B₂Has great application value.

In addition, the genetically engineered bacteria containing the mutant gene of the pseudouridine synthase are biologically safe, and furthermore, the biological quantity of the engineered strain 19-yB containing the mutant gene of the pseudouridine synthase is higher than that of the original strain of the bacillus subtilisBacillus BS-1, which shows that point mutation of yyB gene is beneficial to growth of thallus, can further improve the production of vitamin B by the strain containing pseudouridine synthetase mutant (H239L)₂The ability of the cell to perform.

SEQUENCE LISTING

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