CN114806982A - The modified 1,3-propanediol production strain and its application - Google Patents

Abstract

The invention discloses a modified 1, 3-propanediol production strain and application thereof. The modified strain is an engineering bacterium with improved 1, 3-propanediol production performance obtained by improving the content of the pdu microchamber in strain cells through manual operation. According to the invention, by increasing the level of the pdu microchamber in the 1, 3-propanediol production strain, 3-hydroxypropionaldehyde generated in cells can be quickly converted into 1, 3-propanediol in the process of producing the 1, 3-propanediol by using carbon sources such as glycerol or glucose, the toxic effect of the 3-hydroxypropionaldehyde on the cells is reduced, the reaction rate of glycerol to the 1, 3-propanediol is increased, and the conversion rate of the glycerol to the 1, 3-propanediol is increased.

Description

The modified 1,3-propanediol production strain and its application

technical field

The invention belongs to the technical field of bioengineering, in particular to a modified 1,3-propanediol production strain and its application.

Background technique

1,3-Propanediol is an important chemical that is mainly used as a raw material to synthesize polytrimethylene terephthalate with terephthalic acid. The polymer has excellent properties such as high elasticity and easy dyeing, and is a high-grade textile material. The biological production of 1,3-propanediol has been researched and developed for many years. Natural microorganisms such as genus Klebsiella, Citrobacter freundii, Clostridium butyricu and Lactobacillus reuteri can use glycerol as raw material to produce 1, 3-Propanediol. However, it has not been found that natural microorganisms can produce 1,3-propanediol using glucose and other saccharide raw materials. By artificially constructing metabolic pathways, microorganisms such as Escherichia coli and Corynebacterium glutamate can utilize carbon sources such as glucose to produce 1,3-propanediol.

The metabolic reaction for the conversion of glycerol to 1,3-propanediol consists of two steps, the dehydration of glycerol to form 3-hydroxypropanal, and the reduction of 3-hydroxypropanal to form 1,3-propanediol. In the artificially constructed Escherichia coli and Corynebacterium glutamicum for producing 1,3-propanediol from glucose raw materials, glucose is synthesized through multi-step reactions, and the subsequent glycerol forms 3-hydroxypropanal, which finally reacts to form 1,3-propanediol. 3-Hydroxypropionaldehyde has a toxic effect, and high concentration of 3-hydroxypropionaldehyde in the production process of 1,3-propanediol will inhibit the activity of the production strain and have a serious impact on the fermentation process.

There is an organelle called microcompartment or metabo losome in various bacteria such as citric acid freundii, Salmonella (Salmonella), and cyanobacteria. The microchamber is a protein organelle composed of a variety of structural proteins and catalytic proteins, the shape is icosahedron or irregular polyhedron, and the size is about 100-200nm. The microchamber separates its interior from the cytoplasm, making the catalytic reaction inside the microchamber a special environment. Microchambers in cyanobacteria are responsible for carbon dioxide fixation in cells. In citric acid freundii, Salmonella typhimurium and other bacteria, there is a microchamber responsible for the metabolism of 1,2-propanediol called pdu microchamber. The identified genes encoding pdu microchambers include pduA, pduB, pduC, pduD, pduE, pduG, pduH, pduJ, pduK, pduL, pduM, pduN, pduO, pduP, pduQ, pduS, pduT, pduU, pduV, pduW, pduX et al. Among them, pduA, pduB, pduJ, pduK, pduM, pduN, pduT, pduU, pduV, etc. are structural proteins. In the pdu microchamber, 1,2-propanediol is dehydrated to form propionaldehyde, which is reduced to form propanol, or oxidized to form propionyl CoA, which then enters the cytoplasm to form propionic acid.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a modified 1,3-propanediol production strain and its application.

The technical scheme that the present invention adopts for realizing the above-mentioned purpose is as follows:

Provided is a modified 1,3-propanediol production strain, which refers to an engineering strain with improved 1,3-propanediol production performance by increasing the content of pdu microchambers in strain cells by manual operation.

As a preferred embodiment, the starting strain of the modified strain is a wild strain with 1,3-propanediol production capacity or an engineered strain that is genetically modified for 1,3-propanediol production.

As a preferred embodiment, the 1,3-propanediol producing strains include: Klebsiella, Citrobacter freundii, Clostridium butyricum, Lactobacillus roceii and other wild strains with the ability to produce 1,3-propanediol, and other strains with these strains It is the genetically modified strain of the starting strain, including the artificially constructed Escherichia coli and Corynebacterium glutamicum and other engineered strains with the ability to produce 1,3-propanediol.

As a preferred embodiment, the method for increasing the content of pdu microchambers in cells of a strain adopts at least one of the following methods:

(1) The pdu microchamber encoding gene is connected to the expression plasmid through molecular biological operations, and the expression plasmid is transferred into the 1,3-propanediol production strain;

(2) Recombining the pdu microchamber-encoding gene into the chromosome of the 1,3-propanediol-producing strain by molecular manipulation;

(3) by replacing the promoter of the pdu microchamber-encoding gene on the chromosome of the 1,3-propanediol-producing strain with a high-level promoter;

(4) PocR, a regulatory gene that expresses pdu microchamber-encoding genes at a high level.

The pdu microchamber is a microchamber that metabolizes 1,2-propanediol in bacteria.

The coding genes of pdu microchambers include pduA, pduB, pduC, pduD, pduE, pduG, pduH, pduJ, pduK, pduL, pduM, pduN, pduO, pduP, pduQ, pduS, pduT, pduU, pduV, pduW, pduX, etc. Among them, pduA, pduB, pduJ, pduK, pduM, pduN, pduT, pduU, pduV, etc. are structural proteins.

In the genome (NC_003197.2) of Salmonella enterica subsp.enterica serovar Typhimuriumstr.LT2, the pdu microchamber-encoding gene pduA-pduX is located between 2116843-2133611 bases (the sequence is shown in SEQ ID No.1), and each Gene transcription direction is the same and arranged in order. The pdu microchamber regulatory protein pocR in the genome is located between 2114396-2115307 bases (the sequence is shown in SEQ ID No. 2), which is opposite to the transcription direction of pduA-pduX.

In the genome (NC_011283.1) of Klebsiella variicola 342 (NC_011283.1), the pdu microchamber encoding gene pduA-pduX is located between 906824-923600 bases, (the sequence is shown in SEQ ID No.3) . The pdu microchamber regulatory protein pocR in the genome is located between 925138-926049 bases (the sequence is shown in SEQ ID No. 4).

In the genome (NZ_CACD01000056.1) of Citrobacter freundii str. ballerup 7851, the pdu microchamber encoding gene pduA-pduX is located between bases 5309-22069 (the sequence is shown in SEQ ID No. 5). In the genome, the pdu microchamber regulatory protein pocR is located between 2861-3772 bases (the sequence is shown in SEQ ID No. 6).

Salmonella LT2, Klebsiella mutans 342 and Citrobacter freundii 7851 have high homology in the pdu microchamber-encoding gene sequences, all of which contain 21 genes.

The present invention also provides the application of the modified 1,3-propanediol producing strain in the production of 1,3-propanediol and the method for producing 1,3-propanediol. The method for producing 1,3-propanediol is as follows: the modified 1,3-propanediol production strain is inoculated into a medium containing a carbon source for fermentation culture, and the bacterium transforms the carbon source in the medium into 1,3-propanediol.

As a preferred embodiment, the medium uses glycerol or glucose as the main carbon source.

Compared with the prior art, the beneficial effects of the present invention are:

By increasing the level of the pdu microchamber in the 1,3-propanediol producing strain, the invention enables the strain to produce 3-hydroxypropanal in the cell in the process of using glycerol or glucose and other carbon sources to produce 1,3-propanediol. The conversion to 1,3-propanediol reduces the toxic effect of 3-hydroxypropionaldehyde on cells, increases the reaction rate of glycerol to 1,3-propanediol, and improves the conversion rate of glycerol to 1,3-propanediol. Using the strain transformed by the invention to produce 1,3-propanediol makes the overall process control of the 1,3-propanediol fermentation process simple, the production intensity is increased, and the conversion rate of raw materials is improved.

Specific implementation method

The technical solutions of the present invention will be described in detail below with reference to the embodiments.

The reagents and biological materials used below are all commercial products unless otherwise specified. Various molecular biology manipulations are general manipulation methods.

Example 1: Increasing the level of pdu microchambers of Citrobacter freundii

Citrobacter freundii is a laboratory preserved wild strain, abbreviated as Cf. According to the genome information of Citrobacter freundii str.ballerup 7851, primers were designed, and the pduA-pduX gene sequence of Citrobacter freundii was cloned and ligated. The pSARI-pdu plasmid was formed from the pSARI (Genbank MH037013) plasmid, and the pSARI-pdu plasmid was transferred back to CF to obtain Cf-pdu. The specific operations are as follows:

1) Linearization of the expression plasmid. Using pSARI-s: ATCAGTACTCCTAGGGTACCAGCTG (shown in SEQ ID No. 7) and pSARI-a: CACCTTCATGGTGGTCAGTGC (shown in SEQ ID No. 8) as specific primers and pSARI plasmid as a template, a linearized pSARI vector was obtained.

2) Amplify the overexpressed target gene. Taking the genome of Citrobacter freundii as a template, using pdu-s: GGATCGCCATCAGATCTGATATGCAACAAGAAGCGTTAGGAAT (shown in SEQ ID No. 9) and pdu-a: GGTACCCTAGGAGTACTGATTTATTGCAGTTCGACCCCGC (shown in SEQ ID No. 10) as specific primers for PCR amplification, Obtain the target gene fragment pduA-pduX.

3) The target gene is connected with the linearized vector. According to the operation steps in the ClonExpress Ultra One Step Cloning Kit (commercial product), the amplified target gene fragment pduA-pduX is ligated with the linear pSARI vector to form a circle. The recombinant product was transformed into DH5α competent cells, and the positive clone pSARI-pdu plasmid was screened by Kan ^r -resistant plate.

4) Construction of overexpression strains. After the pSARI-pdu plasmid was constructed, the target plasmid was extracted from DH5α according to the method of the plasmid extraction kit and electrotransferred into the competent cells of Citrobacter freundii to obtain Cf-pdu.

Shake flask fermentation of Cf and Cf-pdu was used to produce 1,3-propanediol. The strains were inoculated into 250ml Erlenmeyer flasks containing 50ml of fermentation medium. The rotating speed of the shaker cabinet was 150 rpm, and the fermentation was carried out at a constant temperature of 37 °C.

Fermentation medium formula: ammonium sulfate 4g/L; dipotassium hydrogen phosphate 0.69g/L; potassium dihydrogen phosphate 0.25g/L; magnesium sulfate 0.2g/L; yeast powder 1.5g/L; glycerol 30g/L; trace Element 1.0ml/L; iron solution 1.0ml/L.

The trace elements are: manganese sulfate 100mg/L; zinc chloride 70mg/L; sodium molybdate 35mg/L; boric acid 60mg/L; cobalt chloride 200mg/L; copper sulfate 29.28mg/L; nickel chloride 25mg/L; Concentrated hydrochloric acid (37%) 0.9ml/L.

The iron solution is as follows: 5.0 g of ferrous sulfate and 4 ml of 37% concentrated hydrochloric acid are added to 1 liter of water.

Samples were taken at 12 hours of culture, and cell microchambers were extracted according to the method in the literature (Journal of Bacteriology 2012: 194, 1912-1918), and the samples were subjected to SDS electrophoresis. Electrophoresis results showed that the Cf strain samples had vague multiple protein bands; the Cf-pdu strain samples had obvious multiple protein bands. It indicated that the expression level of pdu microchamber of Cf-pdu strain was improved.

After culturing for 24 hours, the components in the fermentation broth were determined by liquid chromatography. The liquid chromatography method was used to separate the components of the fermentation broth by HPX-87H chromatographic column, and detected by parallax and ultraviolet detector. The fermentation results of the strains are shown in Table 1.

Table 1, Citrobacter freundii and its modified strain shake flask fermentation experiment results

strain Glycerin (g/L) Acetic acid (g/L) 1,3-Propanediol (g/L) Cf twenty three 1.7 1.2 Cf-pdu 18 2.1 3.6

It can be seen from Table 1 that after increasing the pdu microchamber level of Citrobacter freundii, compared with the wild-type strain, the glycerol consumption rate of the engineered strain increased, the concentration of 1,3-propanediol increased, and the glycerol was converted into 1,3-propanediol. Conversion rate increases.

Example 2: Increasing the level of Klebsiella pdu microchambers

Klebsiella CGMCC 1.6366 strain (this strain is also called TUAC01, AC01) has been disclosed in the literature (World Journal of Microbiology Biotechnology 2008, 24: 1731-1740), referred to as Kp, and this strain is used as the starting strain for inactivation The strains for lactate dehydrogenase and alcohol dehydrogenase were simply constructed as Kp-ΔldhA-ΔadhE (Biotechnol Lett 2016, 38:975-982).

According to the genome information of Klebsiella mutans 342, primers were designed to construct DNA fragments, and the promoter of the 2,3-butanediol operon in the cell was recombined to the upstream of the pduA gene on the chromosome to obtain the Kp-pdu1 strain. The operation steps are as follows:

1) Amplify the gene sequence of the long homology arm at the upstream and downstream ends of the pduA gene, the promoter sequence of the 2,3-butanediol operon and the gene sequence of the resistance fragment. With pduA-Q600-s: CCCGGGGATCCTCTAGAGATCCGACGTCAGATAGACGGCA (shown in SEQ ID No. 11), pduA-Q600-a: CTCCAGCCTACAGGATAAGATAAAATTGTCGTTTATTTATTTAA (shown in SEQ ID No. 12) and pdu-H600-s: GAGGAAGTGGTATATGCAACAAGAAGCACTAGGAATG (shown in SEQ ID No. 13) , pdu-H600-a: CATGCCTGCAGGTCGACGATCAGTGTTGGTGGCTTTCACCG (shown in SEQ ID No. 14) is a specific primer, and the Klebsiella pneumoniae CGMCC 1.6366 genome is used as a template, and PCR amplification is carried out to obtain 600bp of long homology arms at both ends respectively. DNA sequence. PCR amplification was performed using pduA-FRT-s: TCTTATCCTGTAGGCTGGAGCTGCTTCG (shown in SEQ ID No. 15), pduA-FRT-a: AAGGAATTCCGGGGATCCGTCGA (shown in SEQ ID No. 16) as specific primers, and PIJ773 plasmid (commercial product) as a template. to obtain the aac(3)IV gene fragment containing FRT sites in the middle with Apr ^r resistance at both ends. Using Pbud-s: ACGGATCCCCGGAATTCCTTGGCGGCCTGGGTG (shown in SEQ ID No. 17), Pbud-a: GTTGCATATACCACTTCCTCGTTCAACAAATAT (shown in SEQ ID No. 18) as specific primers, using Klebsiella pneumoniae CGMCC 1.6366 genome as a template, PCR Amplify the promoter sequence on the 2,3-butanediol operon. PCR amplification was performed using pMD18-Ts: ATCGTCGACCTGCAGGCA (SEQ ID No. 19) and pMD18-Ta: ATCTCTAGAGGATCCCCGGGT (SEQ ID No. 20) as specific primers, and plasmid pMD-18T simple (commercial product) as a template. increased to obtain a linearized pMD18-T gene fragment.

2) The recombination ligation of gene fragments was performed using the operation method in the ClonExpress Ultra One Step Cloning Kit. The gene fragments amplified in step 1 were recombined and ligated, and then placed on ice immediately after reacting at 50°C for 15 minutes. Afterwards, it was transformed into DH5α competent cells and screened with apramycin-resistant plates.

3) Extract the plasmid pMD18T-aac(3)IV in the positive clone DH5α-T-aac(3)IV in step 2, and amplify the plasmids with pduA-Q600-s and pduA-H600-a as templates. 600bp homology arm, resistance cassette fragment with FRT sequence in the middle and fragment A1 of 2,3-butanediol operon promoter, and this fragment A1 was electroporated into Krebs with plasmid pDK6-red Pneumoniae CGMCC 1.6366 competent cells. Spread it on a plate with Apr ^r resistance, and after it grows colonies, use pduA-Q600-s and pduA-H600-a as primers for PCR amplification, and the positive clones screened are 2,3- The promoter of the butanediol operon was recombined and replaced upstream of the pduA gene on the chromosome to obtain the Kp-pdu1 strain.

According to the genome information of Klebsiella mutans 342, primers were designed to clone the pocR gene, which was connected to the expression plasmid pDK6 plasmid to form pDK6-pocR, and the plasmid was transformed into Kp-ΔldhA-ΔadhE to obtain Kp-pdu2 strain. The specific operations are as follows :

1) Linearization of expression plasmids. Using pDK6-s: AAGCTTGGCTGTTTTGGCG (shown in SEQ ID No. 21) and pDK6-a: GAATTCTGTTTCCTGTGTGAAATTG (shown in SEQ ID No. 22) as specific primers, and using pDK6 plasmid (commercial product) as a template, linearized pDK6 was obtained vector.

2) Amplify the overexpressed target gene. PCR was performed using the genome of Klebsiella pneumoniae CGMCC 1.6366 as a template and pocR-s: TCACACAGGAAACAGAATTCATGATTTCTGCGAGCACACTGA (SEQ ID No. 23) and pocR-a: CCGCCAAAACAGCCAAGCTTTTAAACTGACTCAGTTTGTGATCTGG (SEQ ID No. 24) as specific primers Amplify to obtain the target gene fragment pocR.

3) The target gene is connected with the linearized vector. According to the operation steps in the ClonExpress Ultra One Step Cloning Kit (commercial product), the amplified target gene fragment pocR is ligated with the linear pDK6 vector to form a circle. The recombinant product was transformed into DH5α competent cells, and positive clones were screened by Kan ^r -resistant plates.

4) Construction of overexpression strains. After the pDK6-pocR plasmid was constructed, the target plasmid was extracted from DH5α according to the method of plasmid extraction kit and electroporated into Kp-ΔldhA-ΔadhE competent cells of Klebsiella pneumoniae to obtain Kp-pdu2 strain.

These strains were subjected to 5L fermentor fermentation experiments.

The Kp and Kp-pdu1 strains were inoculated into 250 mL conical flasks, which were filled with 50 mL of LB medium, and the rotating speed of the flask cabinet was 150 rpm, and the constant temperature was 37 °C for seed culture.

The seeds were cultivated for 12 hours, inoculated into a 5L fermentor, and filled with a 3L fermentation medium. The glycerol concentration in the fermentation medium was 50 g/L, and the other components were the same as the fermentation medium used in Example 1. Keep the ventilation rate of fermentation process at 2L/min, the stirring speed at 200 rpm, and the fermentation temperature at 37°C. The pH value of the fermentation broth is stabilized at 7.0 by using sodium hydroxide solution, and the fermentation is sampled for 9 hours and finished in 15 hours. The components in the fermentation broth were determined by the method of Example 1. The content of 3-hydroxypropanal in fermentation broth was determined by tryptophan display reaction.

The 9-hour samples were extracted from cell microchambers according to the method in the literature (Journal of Bacteriology 2012: 194, 1912-1918), and the samples were subjected to SDS electrophoresis. Electrophoresis results showed that the Kp strain samples had vague multiple protein bands; the Kp-pdu1 strain samples had obvious multiple protein bands. It indicated that the expression level of pdu microchamber was increased in Kp-pdu1 strain.

The fermentation results of each strain are shown in Table 2.

Table 2, Klebsiella strain fermentor fermentation results 1

strain Glycerin (g/L) 1,3-Propanediol (g/L) 3-Hydroxypropionaldehyde (mmol/L) Kp(9h) 28 7.1 14 Kp-pdu1(9h) 28 8.3 2 Kp(15h) 25 8.7 4 Kp-pdu1(14h) 0 19 0

From the data results in Table 2, it can be seen that the wild bacteria produced 14 mmol/L of 3-hydroxypropionaldehyde during the fermentation process, and inhibited the activity of the cells. The conversion rate of glycerol into 1,3-propanediol was 0.35 g/L. g, fermented for 15 hours to form 8.7 g/L of 1,3-propanediol. However, the Kp-pdu1 strain had low 3-hydroxypropionaldehyde content in the fermentation broth during the fermentation process, and the growth of the strain was not inhibited. The fermentation of 14 hours could consume all glycerol and form 19g/L of 1,3-propanediol. The conversion rate of glycerol into 1,3-propanediol was 0.38 g/g, and the performance of the strain was significantly improved after the level of pdu microchamber was increased.

The Kp-ΔldhA-ΔadhE and Kp-pdu2 strains were inoculated into 250 mL conical flasks, which were filled with 50 mL of LB medium, and the flask was shaken at 150 rpm at a constant temperature of 37 °C for seed culture.

The seeds were cultivated for 12 hours, inoculated into a 5L fermentor, and filled with 3L of fermentation medium. The components of the fermentation medium were the same as those of the fermentation medium used in Example 1. During the fermentation process, the ventilation rate was kept at 2L/min, the stirring speed was 200 rpm/min, the fermentation temperature was 37°C, and the pH value of the fermentation broth was stabilized at 7.0 by using sodium hydroxide solution. During the fermentation process, the concentration of each substance in the fermentation broth was detected. When the glycerol concentration in the fermentation broth of the Kp-ΔldhA-ΔadhE strain is lower than 2g/L (when the glycerol supplementation is higher than 2g/L, the activity of the bacterial cells will be inhibited due to the high level of 3-hydroxypropionaldehyde), the glycerol supplementation The glycerol concentration was controlled not to be higher than 5g/L 20 hours before the fermentation, and the fermentation was finished in 4 hours. When the glycerol concentration in the fermentation broth of Kp-pdu2 strain was lower than 5 g/L, glycerol feed was added. Samples were taken at 12 hours of fermentation to determine the cellular microchamber level.

The 12-hour samples were extracted from cell microchambers according to the method in the literature (Journal of Bacteriology 2012: 194, 1912-1918), and the samples were subjected to SDS electrophoresis. Electrophoresis results showed that the Kp-ΔldhA-ΔadhE strain samples had vague multiple protein bands; the Kp-pdu2 strain samples had obvious multiple protein bands. It indicated that the expression level of pdu microchamber of Kp-pdu2 strain was improved. Fermentation is over for 40 hours. The fermentation results of each strain are shown in Table 3.

Table 3, Klebsiella genus modified strains fed with fermentor fermentation results

strain Glycerol consumption (g/L) 1,3-Propanediol (g/L) Conversion rate (g/g) Kp-ΔldhA-ΔadhE 162 60 0.37 Kp-pdu2 180 70 0.39

From the data results in Table 3, it can be seen that after the level of pdu microchambers was increased in the strains after knocking out lactate dehydrogenase and alcohol dehydrogenase, the modified strains had higher levels of 1,3-propanediol in the fed-feed fermentation culture. Both final concentration and feedstock conversion are improved.

Example 3: Increasing the level of Klebsiella pdu microchambers

Klebsiella Kp2 is an engineering strain that can produce 1,3-propanediol from glucose, constructed with reference to the literature (Metabolic Engineering 2020: 62, 116–125), which expresses a strain derived from Saccharomyces cerevisiae Dihydroxyacetone phosphate dehydrogenase and 3-phosphoglycerol phosphorylase, overexpress its own glycerol dehydratase, glycerol dehydratase activating protein and 1,3-propanediol oxidoreductase.

The pDK6-pocR constructed in Example 2 was transformed into Kp2 to obtain Kp2-pdu strain.

Kp2 and Kp2-pdu were subjected to shake flask fermentation. The strains were inoculated into 250ml Erlenmeyer flasks containing 50ml of fermentation medium. The rotating speed of the shaker cabinet was 150 rpm, and the fermentation was carried out at a constant temperature of 37 °C.

Fermentation medium: ammonium sulfate 4g/L; dipotassium hydrogen phosphate 0.69g/L; potassium dihydrogen phosphate 0.25g/L; magnesium sulfate 0.2g/L; yeast powder 1.5g/L; glucose 30g/L; trace elements 1.0ml/L; iron solution 1.0ml/L.

Samples were taken at 12 hours of fermentation to determine the cellular microchamber level.

The 12-hour samples were extracted from cell microchambers according to the method in the literature (Journal of Bacteriology 2012: 194, 1912-1918), and the samples were subjected to SDS electrophoresis. Electrophoresis results showed that only one vague protein band and several weak bands could be seen in the Kp2 strain samples; the Kp2-pdu strain samples had obvious multiple protein bands. It indicated that the expression level of pdu microchamber of Kp2-pdu strain was improved. The components in the fermentation broth were determined for 24 hours of fermentation, and the results are shown in Table 4

Table 4. Production of 1,3-propanediol by Escherichia coli and modified strains

strain Glucose (g/L) 1,3-Propanediol (g/L) Conversion rate (g/g) Kp2 5 8 0.32 Kp2-pdu 0 10 0.33

From the data results in Table 4, it can be seen that after the level of pdu microchamber was increased in the Klebsiella engineered strain, the modified strain produced 1,3-propanediol by fermentation of glucose. - Both propylene glycol concentration and feedstock conversion are improved.

Example 4: Increasing the level of E. coli pdu microchambers.

Escherichia coli Ec is an engineering strain constructed from Escherichia coli Dh5α with reference to literature (Current Opinion in Biotechnology. 2003; 14:454-9.) that can utilize glucose to produce 1,3-propanediol, which expresses a strain derived from Saccharomyces cerevisiae Dihydroxyacetone phosphate dehydrogenase and 3-phosphoglycerol phosphorylase, glycerol dehydratase and glycerol dehydratase activating protein from Klebsiella, and nonspecific glycol dehydrogenase from Escherichia coli. The Ec strain itself does not have pdu microchamber-encoding genes in its genome.

The pSARI-pdu plasmid constructed in Example 1 was transformed into Ec to form Ec-pdu. Ec and Ec-pdu were subjected to shake flask fermentation. The strains were inoculated into 250ml Erlenmeyer flasks containing 50ml of fermentation medium. The rotating speed of the shaker cabinet was 150 rpm, and the fermentation was carried out at a constant temperature of 37 °C.

Fermentation medium: ammonium sulfate 4g/L; dipotassium hydrogen phosphate 0.69g/L; potassium dihydrogen phosphate 0.25g/L; magnesium sulfate 0.2g/L; yeast powder 1.5g/L; glucose 50g/L; trace elements 1.0ml/L; iron solution 1.0ml/L, vitamin B12 2 mg/L. The components in the fermentation broth were determined for 24 hours of fermentation, and the results are shown in Table 5

Table 5. Production of 1,3-propanediol by Escherichia coli and modified strains

strain Glucose (g/L) 1,3-Propanediol (g/L) Conversion rate (g/g) Ec 20 9 0.3 Ec-pdu 10 13 0.325

It can be seen from the data results in Table 4 that after increasing the level of pdu microchambers in E. coli, the final concentration of 1,3-propanediol and the conversion rate of raw materials in the fermentation culture of the modified strains are improved.

The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

sequence listing

<110> Shanghai Institutes for Advanced Study, Chinese Academy of Sciences

<120> Modified 1,3-propanediol production strain and its application

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cactggcgaa acgccctgtg aatcaggacg gctttgttaa ggagtggatc gaagaaggct 1200

ttatcgcgat ggaaagcccg aacgacccaa aaccgtcgat aaaaatcgtt aacggcgcgg 1260

taaccgagct ggacggaaaa ccggttagcg aattcgacct gatcgaccac tttatcgccc 1320

gctacggcat caacctgaac cgcgccgaag aagtgatggc gatggattcg gtcaagctgg 1380

ctaacatgct gtgcgatccg aacgtcaagc gcagcgaaat cgttccgcta accaccgcga 1440

tgaccccagc gaaaattgtc gaagtggttt cgcatatgaa cgtggttgag atgatgatgg 1500

cgatgcagaa aatgcgcgcc cgccgtactc catctcaaca ggcgcacgtc accaacgtta 1560

aagacaaccc ggtgcaaatt gccgccgatg ccgccgaagg cgcatggcgc gggtttgacg 1620

aacaagagac gacggttgcg gtagcgcgct atgcgccgtt caacgccatc gcgctgctgg 1680

ttggttctca ggtaggtcgt ccgggggtac tgactcaatg ctcgctggaa gaagccaccg 1740

agctgaagct cggcatgctg ggccacacct gctacgccga aaccatctcc gtttacggca 1800

ccgagccggt cttcaccgac ggtgacgata ccccatggtc gaagggcttc ttagcctctt 1860

cctacgcctc tcgcggcctg aaaatgcgct tcacctccgg ctccggctcc gaagtgcaga 1920

tgggctacgc cgaaggcaaa tccatgctgt atctggaagc gcgctgcatc tatatcacca 1980

aagccgcggg cgttcagggg ctgcaaaacg gctccgtaag ctgcatcggc gtaccgtctg 2040

ccgtgccgtc aggcattcgt gccgtgctgg cggaaaacct gatctgctct tcgctggatc 2100

tggaatgcgc ctccagtaac gaccagacct tcacccactc cgatatgcgt cgtaccgctc 2160

gcctgctgat gcagttcctg ccgggtaccg actttatctc ctccggttat tccgcggtgc 2220

cgaactacga caacatgttc gccggttcca acgaagatgc ggaagacttt gacgactaca 2280

acgttatcca gcgtgacctg aaagtggacg gcggtctgcg cccggttcgc gaagaggacg 2340

ttatcgccat ccgtaacaaa gccgcccgcg cgctgcaggc cgtgtttgcc ggaatgggac 2400

tgccgccgat taccgatgaa gaagttgaag ccgcgaccta tgcccacggt tcgaaagata 2460

tgccggagcg caacatcgtc gaagacatca agttcgccca ggaaatcatc aataaaaacc 2520

gcaacggtct ggaagttgtg aaagcgctgg ctcagggcgg gtttaccgac gtggcccagg 2580

acatgctcaa catccagaaa gccaagctaa ccggcgacta tttgcacacc tccgccatta 2640

tcgtcggcga cggacaagtg ctctctgcgg ttaatgacgt caatgactat gccggtccgg 2700

caacaggtta tcgcctgcag ggagaacgct gggaagagat taaaaacatc cctggcgctc 2760

ttgatcccaa cgagattgat taaggggtga gaaatggaaa ttaatgaaaa attgctgcgc 2820

cagataattg aagacgtact ccgcgatatg aagggcagcg ataaacccgt ctcgtttaat 2880

gcgcctgcgg catccacagc accacagacc gctgcgcctg cgggcgacgg ctttctgacc 2940

gaagtgggcg aagcgcgcca gggcactcag caggacgaag tcattatcgc cgtcggcccg 3000

gcatttggcc tggcgcaaac cgtcaatatc gtcggcttac cgcataagag cattctgcgc 3060

gaagtcattg ccggtattga agaagaaggc atcaaggcgc gcgtgattcg ctgctttaaa 3120

tcttccgacg tggcgttcgt cgccgttgaa ggtaaccgcc tgagcggatc cggcatctcc 3180

atcggcatcc agtcgaaagg tactacggtt atccaccagc aggggctacc gccgctctcc 3240

aacctggagc tgttcccgca ggcaccgctg ctgacgctgg aaacctaccg tcagattggt 3300

aaaaacgccg cccgctatgc gaaacgagaa tcaccgcagc cggtccctac gctcaatgac 3360

cagatggcac gcccgaagta ccaggcaaag tcggccattt tgcatattaa agagaccaag 3420

tacgtcgtga cgggcaaaaa cccgcaggaa ctgcgcgtgg cgctttgata aaggataact 3480

ccatgaatac cgacgcaatt gaatcgatgg tccgggacgt attgagccgc atgaacagcc 3540

tgcagggcga tgcgccagca gcggctcctg cggcaggcgg cacgtcccgc agcgcaaagg 3600

tcagcgacta cccgctggcg aacaaacacc cggaatgggt gaaaaccgcc accaataaaa 3660

cgctggacga ctttacgctg gaaaacgtgc tgagcaataa agtcaccgct caggatatgc 3720

gtattacccc ggaaaccctg cgcttacagg cctctatcgc caaagatgcg ggtcgcgacc 3780

ggctggcgat gaacttcgaa cgcgccgccg aactgaccgc ggtaccggac gatcgcattc 3840

ttgaaatcta caacgccctt cgtccgtatc gttcaacgaa agaagagctg ctcgctatcg 3900

ccgacgatct cgaaaaccgt tatcaggcaa agatttgcgc agctttcgtt cgtgaagcgg 3960

cagggctgta cgttgagcgt aaaaaactca aaggcgacga ttaaggattt cgtatgcgat 4020

atatagctgg cattgacatc ggtaactcat caacggaagt cgcactggcg cggcaagatg 4080

agactggcgc actgacgatt acacacagcg cgctggcgga aaccaccggg atcaaaggca 4140

cgttgcgtaa cgtgttcggc attcaggaag cgctcgccct cgtcgcaaag cgcgcgggga 4200

tcaatgtcag agatatttcg ctcatccgca ttaacgaagc cacgccggtg attggcgatg 4260

tggcgatgga aaccattacc gaaaccatca tcaccgaatc gacaatgatc ggccataacc 4320

caaaaacgcc gggcggagca ggccttggtg tgggtatcac gattacgccg gaggagctgt 4380

taacccgccc ggcggactcg tcctatattc tggtggtatc gtcagccttt gattttgctg 4440

atatcgccaa tgttatcaac gcctcaatgc gcgccggata ccagattacc ggcgtcattt 4500

tgcagcgcga cgatggcgta ctggtcagca accggctgga aaaatcgcta ccgattgtcg 4560

atgaagttct gtacatcgac cgcattccgc tggggatgct ggcggcgatt gaagtcgccg 4620

tgccgggaaa ggttatcgaa accctctcta acccttacgg catcgccacc gtatttaatc 4680

tcaacgccga tgagacaaaa aacatcgtcc cgatggcgcg cgcgctgatt ggcaaccgtt 4740

ccgccgtggt ggttaaaacg ccatccggcg acgtcaaagc gcgcgcaata cccgccggta 4800

acctggagct gcaggctcag ggtcgtaccg tgcgcgtgga tgttgccgcc ggtgccgaag 4860

ccatcatgaa agcggtggac ggttgcggca agctcgacaa cgtcaccggc gaggccggga 4920

ccaatatcgg cggcatgctg gagcacgtgc gccagaccat ggccgaactg accaacaagc 4980

cgagcagtga gattttcatt caggatctac tggccgttga cacctcggtt ccggtgagcg 5040

tcaccggcgg tctggccggg gagttctcgc tggagcaggc cgtcggcatc gcctcgatgg 5100

tgaaatcaga ccgtctgcag atggcgatga ttgcccgtga aattgagcag aagcttaata 5160

tcgacgtgca gatcggcggc gctgaggctg aagccgccat tctgggcgcg ctgaccacgc 5220

cgggtaccac ccgaccgctg gcgatcctcg acctcggcgc gggctccacc gatgcctcca 5280

tcatcaaccc taaaggcgaa atcatcgcca cccatctcgc cggggcaggc gacatggtca 5340

cgatgattat tgcccgcgaa ctggggctgg aagaccgcta tctggcggaa gagatcaaaa 5400

aatacccgct ggctaaggtc gaaagcctgt tccacttacg ccacgaggac ggcagcgtcc 5460

agttcttccc gacgccgctg cctcccgccg tgttcgcccg cgtctgcgtg gtgaaaccgg 5520

acgaactggt gccgcttccc ggcgacttag cgctggaaaa agtgcgcgcc attcgccgca 5580

gcgctaaaga acgcgtcttt gtcaccaacg ccctgcgcgc gctgcgccag gtcagtccaa 5640

ccggcaacat tcgcgatatt ccgttcgtgg tgctggtcgg cggctcgtcg ctggatttcg 5700

aagttccgca gctggtcacc gatgcgctgg cgcactaccg cctggtcgcc gggcgaggaa 5760

atattcgcgg cagcgaaggc ccaagaaacg cggtggccac cggcctgatt ctctcctggc 5820

ataaggagtt tgcgcatgga cagtaatcac agcgccccgg ctatcgtcat taccgttatc 5880

aacgactgcg ccagcctctg gcacgaagtg ctgctgggca ttgaagagga aggcatccct 5940

ttcctgcttc agcatcaccc ggctggagat atcgttgaca gcgcctggca ggcggcgcgc 6000

agctcgccgc tgctggtcgg cattgcctgc gatcgacact cgctggtcgt gcattacaag 6060

aatttacccg catcggcgcc gctttttacg ctgatgcatc atcaggacag tcaggcccaa 6120

cgcaacaccg gtaataacgc ggcacggctg gtcaaaggga tcccctttcg ggatctccat 6180

gcttaatcac aggagaacgg cagtatgaat aacgcactgg gactggttga aacaaaaggg 6240

ctggtcggcg ccattgaagc cgccgatgca atggttaaat ccgccaacgt acagctggtg 6300

ggctacgaaa aaattggttc tggcctggtg accgtcatgg ttcgcggcga tgtcggcgcg 6360

gttaaagcag ccgtagatgc cggcagcgca gcggcaagcg tcgtgggtga agtgaaatcc 6420

tgccacgtta tcccgcgtcc gcacagcgat gttgaggcca ttttaccgaa atcagcctaa 6480

tcgatggcga ataaggagca ccgcgtgaag caatcactgg gattacttga agtttgtggt 6540

ctggcactgg ctatcagctg cgccgatatc atggcgaaat ccgcttctat cacgctgctc 6600

gccctcgaaa agaccaatgg ttcaggctgg atggtgatta aaattaccgg tgatgtggcc 6660

tccgttcagg cggctatcac caccggtgcg catttcgccg aacagtggaa tggcctggtg 6720

gcccacaagg ttatcgccag gcccggagaa gggatcctgc tcgcagagac accctccccc 6780

tccgtcattg aacctgagcc tgaagcgtca gagatagctg atgtcgtttc tgaagcgcca 6840

gccgaagagg ccccgcagga atcagaactg gtcagctgca atctgtgtct ggatccaaaa 6900

tgtccgcgcc agaagggcga acctcgtacg ctttgcattc attccggcaa gcgaggtgaa 6960

gcgtaatgga taaagagctt ctgcaatcaa cggtccgtaa agttctcgac gagatgcgcc 7020

agcggcctat tccgctgggc gtctcaaacc gtcatatcca cctgtccgca caggattatg 7080

agcggctgtt tcccggccat cccatcagcg agaaaaaagc gctgctgcag ccgggacaat 7140

acgccgcgga gcaaaccgtc actctggtgg gaccgaaagg gcagctcaaa aatgtgcgcc 7200

tgctcggccc gctgcgctcg gtaagccagg tggaaatttc gcgtaccgat gcgagaacgc 7260

tggggattgc cgcgccgctg agaatgtccg gcaacctgaa ggggacgccg ggcatccgcc 7320

tggtcagccc ctttgccgaa ctggagctgc cttccggggt tatcgtcgcg cagcggcata 7380

tccatatgtc gccgcttgat gcgctgatcc tgcgggtttc ccacggcgat atggtctccg 7440

tggcgattga aggcgacgat cgcgggctga ttttcaacaa cgtggcgatt cgcgtttcac 7500

cggacatgcg cctcgaaatg cacattgata ccgatgaagc caacgccgct ggtgccgata 7560

acccgcatgc ctttgcccgg ctggtaggcc cgcgatgaac ggcgaaaccc tgcagcgcat 7620

tgtcgaggag attgtctccc ggctgcatcg ccgtgcccag agcacggcga cgctgagcgt 7680

tacgcaactt cgcgatgccg actgtccagc actgttctgt cagcacgcgt cgctgcgcat 7740

cctgctcatc gacctgccgc tgctgggtca gcttgcggat gcagaaaccg gcgatgccgc 7800

cgcccggaaa attcacgacg cgctggcgtt tggcatccgc gtacagctca gcctgcacag 7860

ccagctgctg ccggttatcc cggtaaaaaa actggctcgt ctgccgctgg tattcaccga 7920

tgagcacgga ctgccgctgg ttctgcatgc gggttcggtg ctgagctatc gcgatgtcgc 7980

gctgctaagc cgggggcgtg tggttgtcca ccgcaaatgc atcgtgaccg ccatggcgcg 8040

tgacgcggcc aatgcgcgga atattcaatt aattaagcag gagtaaatca tgcatctggc 8100

acgagtcacg ggcgcggttg tctccacgca aaaatcacct tctttgattg ggaaaaagct 8160

gctgctggtg cgtcgggtca gcgccgatgg cgaactcccc gcctcgccca cctccggcga 8220

tgaagtggcc gtggactccg tcggcgcggg cgtcggcgaa ctggttttgc tcagcggcgg 8280

ctccagcgcc aggcacgttt tttccgggcc aaatgaggcc attgacctcg ccgttgtcgg 8340

cattgtagat acgctttcgt gttaaggggc ggttatggcg atttataccc gaacaggtga 8400

cgctggcacc acatcactgt ttaccggcca gcgggtgagc aaaacccacc cgcgggttga 8460

agcctacggc acgctggatg agctgaacgc cgcgctgagc ctgtgcgcct gcgccgctgc 8520

ggatgaaaac caccgcacct tactcgaggc catccagcag caactttttt ggtttagcgc 8580

agagctggcc agcgacagcg agcagccatc gcccaaacag cgctacatca gcagcgaaga 8640

gatttcggcc ctggaagccg ctatcgatcg ggcgatggcc cgcgtcgaac cgctgcacag 8700

ctttatttta cccggacgct gcgaagccgc gagccgctta cattttgccc gcacgctggc 8760

gcgccgcgcc gaacgccgtc tggttgaact ggcaactgaa gtcaacgtac gtcaggtgct 8820

gatgcgctat atcaaccgct tatcggactg cctgtacgcc ctggcccgcg cggaagatag 8880

cgatgcgcac caggccaaca tcatccgtga agttagcaag cgctatctgg ctgcctgcca 8940

gccgccccac agcaaggaga cgacgccagt ggccctctca ttccacgatc tgcaccagct 9000

cacccgcgcc gccgttgaac gcgcgcagca actgcaggtt ccggtagtcg tcagcatcgt 9060

tgacgcgcac ggcacggaaa ctgtgacctg gcggatgccg gacgccctgc tggtcagcag 9120

cgagctggcg ccgaaaaagg cctggaccgc agtggcgatg aaaacggcga cccatgagct 9180

gagcgatgtc gttcagccgg gcgccgcgct ttacggtctg gaaagtcatt tacagggaaa 9240

agtggtcacc tttggcggcg gttacgccct gtggcgcgac ggcatattaa ttgggggtct 9300

tggcatcagc ggcggcagcg ttgagcagga catggacata gcacagaccg ccatcgcggc 9360

tattaacgtg ggaactcatc aatgaatact tctgaactcg aaaccctgat tcgcaccatt 9420

cttagcgagc aattaaccac gccggcgcaa acgccggtcc agcctcaggg caaagggatt 9480

ttccagtccg tgagcgaggc catcgacgcc gcgcaccagg cgttcttacg ttatcagcag 9540

tgcccgctaa aaacccgcag cgccattatc agcgcgatgc gtcaggagct gacgccgctg 9600

ctggcgcccc tggcggaaga gagcgccaat gaaacgggga tgggcaacaa agaagataaa 9660

tttctcaaaa acaaggctgc gctggacaac acgccgggcg tagaagatct caccaccacc 9720

gcgctgaccg gcgacggcgg catggtgctg tttgaatact caccgtttgg cgttatcggt 9780

tcggtcgccc caagcaccaa cccgacggaa accatcatca acaacagtat cagcatgctg 9840

gcggcgggca acagtatcta ctttagcccg catccgggag cgaaaaaggt ctctctgaag 9900

ctgattagcc tgattgaaga gattgccttc cgctgctgcg gcatccgcaa tctggtggtg 9960

accgtggcgg aacccacctt cgaagcgacc cagcagatga tggcccaccc gcgaatcgca 10020

gtactggcca ttaccggcgg cccgggcatt gtggcaatgg gcatgaagag cggtaagaag 10080

gtgattggcg ctggcgcggg taacccgccc tgcatcgttg atgaaacggc ggacctggtg 10140

aaagcggcgg aagatatcat caacggcgcg tcattcgatt acaacctgcc ctgcattgcc 10200

gagaagagcc tgatcgtagt ggagagtgtc gccgaacgtc tggtgcagca aatgcaaacc 10260

ttcggcgcgc tgctgttaag ccctgccgat accgacaaac tccgcgccgt ctgcctgcct 10320

gaaggccagg cgaataaaaa actggtcggc aagagcccat cggccatgct ggaagccgcc 10380

gggatcgctg tccctgcaaa agcgccgcgt ctgctgattg cgctggttaa cgctgacgat 10440

ccgtgggtca ccagcgaaca gttgatgccg atgctgccag tggtaaaagt cagcgatttc 10500

gatagcgcgc tggcgctggc cctgaaggtt gaagaggggc tgcatcatac cgccattatg 10560

cactcgcaga acgtgtcacg cctgaacctc gcggcccgca cgctgcaaac ctcgatattc 10620

gtcaaaaacg gcccctctta tgccgggatc ggcgtcggcg gcgaaggctt taccaccttc 10680

actatcgcca caccaaccgg tgaagggacc acgtcagcgc gtacttttgc ccgttcccgg 10740

cgctgcgtac tgaccaacgg cttttctatt cgctaactga ggtcgcgatg aataccttct 10800

cactacaaac gcggttgtac agcggtcagg gcagcctggc ggtgctcaag cgctttacca 10860

ataagcacat ctggataatc tgcgatggct ttctggcgca ctctccgctg ctggataccc 10920

tgcgtaacgc gctgcccgca gataaccgca tcagcgtctt tagcgagata acgccggacc 10980

ccaccatcca cacagtggtt cagggcattg cgcaaatgca ggctctgcaa ccgcaggtgg 11040

tgatcggttt tggcggcggc tcggcaatgg acgcggcgaa agcgattgtc tggtttagcc 11100

agcagagcgg gatcaacatc gaaacctgcg tggcgatccc gaccaccagc ggcaccggct 11160

cggaagtgac cagcgcctgc gtaattagcg acccggataa aggcatcaag tatccgctgt 11220

tcaacaatgc gctgtatccg gatatggcga tcctcgaccc ggagctggtg gtcagcgtac 11280

cgccgcagat taccgccaac accggcatgg acgtgctgac ccacgccctg gaggcctggg 11340

tgtcaccgcg cgccagcgac tttaccgacg cgctagcgga aaaagccgcc aaactggtgt 11400

tccagtatct gcccacggcg gtggaaaaag gcgactgcgt ggcgacgcgc gggaaaatgc 11460

acaatgcctc aacgctcgcc gggatggcct tcagccaggc ggggctgggg cttaaccacg 11520

cgatagccca ccagcttggc gggcagtttc atctgccgca tggcctggcc aacgccctgc 11580

tgttaaccac ggttatccgt tttaacgccg gcgttccgcg cgccgccaaa cgctacgcgc 11640

ggctggccaa agcctgcggt ttttgcccgg cagaagccaa tgatatcgcg gcgatcaatg 11700

cgctgattca gcaaatcgaa ctgcttaagc aacgctgtgt ccttccctca ctggccgttg 11760

cgcttaaaga aggaagatcc gacttttccg cacgcattcc ggcgatggtg caggctgcgc 11820

tggctgacgt tacgctgcgc actaacccgc gcccggccaa tgccgaagcc attcgggaac 11880

tgctggagga actgctatga gcaccgccat caacagcgtt gaaatgtccc ttagcgccga 11940

tgaaattcgc gagcgcgttc gcgcagcggg cgttgtcggc gcgggcggcg caggttttcc 12000

cgctcacgtc aaactacagg cgcaggtaga gatttttctg gtgaacgccg ccgaatgtga 12060

accgatgctc aaagttgacc agcaactgat gtggcagcaa gctgcgcgcc ttgtgcgcgg 12120

cgtacagtac gccatgacgg caaccggcgc gcgcgaaggg gtgatagccc tgaaagaaaa 12180

ataccgccgg gccatcgacg ccctcacccc actgctgcca gacggtatcc gcctgcatat 12240

cctgccggat gtatatccgg caggcgatga ggtgctgact atctggatgg caaccggtcg 12300

tcgggtcgcc ccggctgcgc taccggccag cgtcggcgtg gtcgtcaata acgtgcaaac 12360

ggtgctaaat attgcccgag cggtcgagca gcggtttccg gtcactcgtc gcacgttgac 12420

cgtgaacggc gcggtcgcca gaccgttgac cgttactgtt cccatcggca tgtcgctgcg 12480

ggaagtactg gcgctggcgg gcggcgcaac ggtcgacgac cctggtttta ttaacggcgg 12540

cccgatgatg ggtggcctga ttacctctct tgataatccg gtgacgaaaa ctaccggcgg 12600

cctgctggtg ctccccaaaa gccatccgct tatccagcgg agaatgcagg acgagcgcac 12660

ggtgctttcc gtcgcgcgca ccgtctgcga acagtgccga ctgtgtaccg atctctgtcc 12720

gcgacacctg atcggccacg aactctctcc gcacctgctg gtgcgggcgg tgaactttca 12780

tcaggctgcc acgccgcagc tgctgctgag cgcccttacc tgttcggaat gcaacgtttg 12840

cgaaagcgtg gcctgtccgg tggggatctc gccgatgcgc atcaaccgca tgttaaaacg 12900

cgagctacgg gcgcaaaacc agcgctacga agggccgctg aatccggccg acgagatggc 12960

gaaatatcgc ctcgtgccgg tgaagcggct gatcgccaaa ctgggactaa gcccctggta 13020

ccaggaagcg ccgctggttg aagaagaacc gtcagtagaa aaagtcactt tgcagctgcg 13080

ccagcatatt ggtgccagcg cggtagcgaa cgttgcggtg ggagaacgcg tgacgcgcgg 13140

gcaatgcgtt gctgatgtcc cgcctggcgc gctcggcgca cccattcacg ccagcatcga 13200

cggcgttgta tcggccatca gcgaacaggc catcactgtt gtaagaggtt aaccatgtct 13260

caggctatag gaattttaga actcaccagc atcgccaaag gcatggagct tggtgatgcc 13320

atgctgaaaa gcgccaacgt cgatctactg gtcagcaaga ccatctgtcc ggggaagttt 13380

ttactgatgc tgggcggcga tatcggcgct atccagcagg ccattgaaac cggtacgtcg 13440

caggccggcg agatgctcgt cgacagcctg gtgctggcga atattcatcc cagcgtactt 13500

ccggccatca gcggcctgaa cagcgtcgat aagcgccagg cggtaggtat cgtcgaaacc 13560

tggagcgtgg cggcctgcat tagcgccgca gaccgggcgg tgaaaggctc aaacgtcacc 13620

ctggtgcggg tgcatatggc cttcggtatc ggcggtaaat gctacatggt ggtggcgggc 13680

gacgtttctg acgtcaataa cgccgtgacg gttgccagcg aaagcgcggg cgagaaaggg 13740

ttgttggttt accgttcggt gatcccacgc ccgcatgaag ccatgtggcg acagatggtg 13800

gaggggtaat ggaaagacaa ccgacaacgg atcgcatgat tcaggaatac gtgccgggga 13860

aacaggtcac tctcgcgcac ctgattgcta atccagggaa agatctcttt aagaagctgg 13920

gcctgcagga tgcagtgtcc gccattggca tccttaccat cacgccgagc gaagcatcga 13980

ttatcgcctg cgatatcgcc accaaatccg gcgcggtgga aattggtttt ctcgatcgct 14040

ttaccggtgc cgtggtgctg accggcgacg tttctgccgt cgagtatgcc ctcaagcagg 14100

taacgcgcac gctgggcgaa atgatgcagt tcaccacttg ctcgatcacc cggacgtaaa 14160

gtcatgaagc gtttgatgtt tatcggcccc agccagtgcg gaaaaacctc gctgacccaa 14220

agcctgcgcg gcgaagcgct ccattataag aaaactcagg ccattgaatg gtcgccgatg 14280

gccatcgata cgccgggcga atatctggag aaccgctgcc tgtacagcgc cctgctgacc 14340

agcgcctgcg aggccgacgt catcgccctg gtgctgaatg ccgacgccca gtggtcgccc 14400

ttttctccgg gttttaccgc cccgatgaac cgcccgacga ttggcctggt caccaaggcc 14460

gatctcgccg agccgcagcg tatttcgctg gttgcggagt ggcttacgca ggctggcgca 14520

cagcagattt ttattaccag cgcgctgaat aactcgggtc tcgacgccgt gctggatttt 14580

ctgaattcga aggaaccttt atgtcttaca aaataatggc cattaacgcc ggtagctcat 14640

cgttaaaatt tcagctgctg gagatgccgc agggcgacat gctctgtcag gggctgattg 14700

agcgtatcgg tatggcagat gcgcaggtga caattaagac gcacagccaa aaatggcagg 14760

aaaccgtccc cgttgccgat catcgcgatg cggtaacgct gcttctggaa aagctgctgg 14820

gttatcaaat cattaacagt ctgcgggaca ttgatggggt gggccatcgt gtggcgcacg 14880

gcggtgagtt ttttaaagac tcgacgctgg tcacggatga aacgttagcg caaatcgaac 14940

gtcttgctga actggccccg cttcacaacc cggtaaacgc gttgggtatt cacgtttttc 15000

gtcaactgtt gcctgatgcc ccttccgtag ccgtgtttga tacggcattt caccagacgc 15060

tggatgaacc cgcctatatt tacccgcttc cctggcatta ttacgctgaa ctggggattc 15120

gccgttatgg ctttcatggc accagccata agtatgtcag cggcgttctg gcggaaaagc 15180

tgggcgtacc actcagcgca ttacgggtga tttgttgtca tcttggaaac ggtagcagca 15240

tctgcgccat caagaacggg cgttcggtca atacgtcgat gggctttacg ccgcagtcag 15300

gcgtgatgat gggcacccgt agcggagata ttgatccctc tattctgccg tggatagccc 15360

agcgcgaaag caaaacgccg caacagttga atcagttatt aaataatgag tcaggcttgc 15420

tgggcgtttc tggcgtgtcg tctgattacc gcgatgtcga acaggcagcc aataccggta 15480

accggcaggc taaactcgct ctcactctgt ttgccgaacg cattcgcgcc accattggca 15540

gctatattat gcaaatggga ggactggatg cgctggtatt cactgggggc attggcgaga 15600

actcagcgcg cgccagatcg gcggtgtgcc ataatttgca atttttgggc ttagccgttg 15660

atgaagagaa aaatcagcgt aatgcgacat ttatccagac ggagaacgcc ctggtgaaag 15720

tcgccgtcat caacaccaac gaagagttaa tgatcgcaca agacgttatg cgcattgcgc 15780

taccggccac ggaagggctt tgtgtaccag cctgagaggg ttatacgacg cattgcggca 15840

agtcatggta ataacgcttt taccatgaca ctatgcgcgc acactattcg tacctgaaag 15900

gtgataatgt ggctgttgcg caatgccccg cctcatgcgg ggaacttatc cagggatgga 15960

ttctgggcag tgagaaactg gtctcctgcc ccgttgactg gtacagcacc gtagcagtca 16020

cggctgcgcc gccgttgata aacgagcgcc cattgtcgcg ggcgatggtg gagcgcgttc 16080

tggcgcactg gcagtatcct gcgcactgga gtaacgagat tcgcgtcgat gtgcgttcgt 16140

caattcccgt tgccaaaggc atggccagca gcaccgcaga tattgccgct acggcggtgg 16200

caacggcgca tcatcttggc cattcgctgg atgaaaccac ccttgcacag ctttgcgtct 16260

caatcgaacc cactgatagc accgtttttc atcagttaac gctgtttgat cataataatg 16320

cggccacgca aatcgcctgc gagccaccgc cgccaatcga tttgctggta ctggaaagcc 16380

cggtcacact gcgcacgcaa gattaccacc gtctccctcg ccagcagaaa ttaatagcaa 16440

gttcagcaac cttgcagcag gcctggaatc tggtgcagga agcctgtata acgcaaaatc 16500

cgctccggct gggtgaggcg gctacgctta gcgctatcgc cagccagacg ctgttaccta 16560

agccgggctt taccgccctg ctgtcgctgg tcgaagagtg tgatttatac ggattgaacg 16620

tggcgcatag cggtagcgtg gtgggtctga tgctggaccg gaaacgtcat gatattgcgc 16680

gtctgaaagg taagctggca gagaaaaaac ttacccgaca ctggccaaaa caacatttac 16740

tcaagatggt cactggcggg gtcaaactgc agtga 16775

<210> 2

<211> 912

<212> DNA

<213> Salmonella (Salmonella enterica subsp. enterica serovar Typhimuriumstr. LT2)

<400> 2

atgatttctg cgagcgctct gaactcagaa ctcattaata aaatcgcaca ggattttgca 60

caagccaccg gtctggcggt tgtggttgtc aatattcatg gtgatgaaat atctgagcta 120

ttcaatttca cgccgttttg tcagttgatg cggcagcatc cccagcatag tacacgttgt 180

cgtatgagcg atcgctgcgg gggactggag gcatcgaagt ccgatcaacc ctgtatttat 240

cgttgtcatg ccggtctaac ggatttttct attccgctgg taatcgcagg gcatttagtt 300

ggctttgttt tatgcgggca ggttcgttta agtaatgacg tggagctggt aaacattctt 360

aatgttgacg atcgctggca ggctgaccct gagctcttga atgaatttcg gaatgtgcca 420

gagatggact attcacgcgt tatcgcttcc gccgatcttc ttaaactgat tgttgagaat 480

tgcctgaaaa aacagctcaa tttcgtggtt atcaaagaca atccgcagca atcggaggca 540

aataaaacca ctcgtggtcc cactccgcat gacagtaaaa tgaaaaaagc gcttcgctat 600

attgatgcgc atttgtccga cgatcttcgt cttgaggatg tcgcctcaca tgtctatctc 660

agcccctatt acttcagtaa gctattcaag aaatatcagg gaatcggatt taatgcgtgg 720

gtaaaccgcc agagaatggt tagcgccaga gaattgcttt gccatagtga ctggagtatt 780

gccagtattg cgcgtaattt aggcttttcg caaaccagct atttttgcaa agtcttccgc 840

cagacctatc aggttacgcc gcaggcttat cgccagcaga taaatgagaa ttctcatcct 900

ccatcgttat ga 912

<210> 3

<211> 16783

<212> DNA

<213> Klebsiella variicola 342

<400> 3

gtctttatgc aacaagaagc attaggaatg gtagaaacca aaggcttaac cgcagccata 60

gaggccgctg acgcaatggt taagtcggcc aatgttttat tagtgggcta cgaaaggatc 120

ggttcaggcc tggtgaccgt catcgtgcgc ggcgacgtgg gggcagttaa agccgctacc 180

gacgccggtg ccgccgcagc ccgccacgtg ggcgaggtga aagccgtaca cgtcattccc 240

cgcccccata ccgatgttga aaaaatctta ccgaagggga atagccaatg agcagcaatg 300

agctggttga acagatcatg gcgcaagtca tagcccgtgt ggcgacgcca gagcagcagg 360

cgcttcccga taccccccat cccacaggag aaacggctat ggcagagaaa agctgcagct 420

taacggaatt tgtcgggacc gccattggcg acaccctcgg gctggtcatc gccaacgtgg 480

atgccgcatt gctggaagcc atgaagcttg aaaaacgcta tcgctctatc ggcatcctcg 540

gtgcgcgcac cggcgccggg ccgcacatta tggccgccga tgaagcggtc aaagccacca 600

acaccgaagt ggtcagcatc gaacttccac gcgacaccaa aggcggcgcc ggccatggct 660

cgctgattgt gctgggcggg aacgatgttt ccgacgtcaa acgcgggatt gaggtcgcgc 720

tcaaagagct ggaccgtact ttcggcgatg tctacggcaa cgaggcgggt catatcgagc 780

tgcagtatac cgcgcgcgcc agctacgccc tggaaaaagc cttcggcgct ccgctgggcc 840

gcgcctgcgg gattatcgtc ggcgcgccgg cgtcagttgg cgtgctgatg gccgataccg 900

cgctgaaatc agccaacgtc gacgtggtgg cttacagctc accggcccac ggcaccagct 960

tcagcaacga agccatcctg gtgatttccg gcgactcagg ggcggtgcgc caggcggtga 1020

tctccgcccg ggagatcggc aaaaccgtac tggggacgct gggcgccgaa ccgaaaaacg 1080

atcgcccctc ctacatttaa tatccgcgag gctgattcat gagatcgaaa agatttgaag 1140

cactggcgaa acgccctgtg aatcaggatg gtttcgttaa ggagtggatt gaagagggct 1200

ttatcgcgat ggaaagccct aacgatccca aaccctctat ccgcatcgtc aacggcgcgg 1260

tgaccgaact cgacggtaaa ccggttgagc agttcgacct gattgaccac tttatcgcgc 1320

gctacggcat taatctcgcc cgggccgaag aagtgatggc catggattcg gttaagctcg 1380

ccaatatgct ctgcgacccg aacgttaaac gcagcgacat cgtgccgctc actaccgcga 1440

tgaccccggc gaaaatcgtc gaagtggtgt cgcatatgaa cgtggtcgag atgatgatgg 1500

cgatgcaaaa aatgcgcgcc cgccgcacgc cgtcccagca ggcgcatgtc accaatatca 1560

aagataaccc ggtacagatt gccgccgacg ccgctgaagg cgcatggcgc ggctttgacg 1620

agcaggagac caccgtcgcc gtggcgcgct acgcgccgtt caacgccatc gccctgctgg 1680

tcggttcaca ggtgggccgc cccggcgtcc tcacccagtg ttccctggaa gaagccaccg 1740

agctgaaact gggcatgctg ggccatacct gctatgccga aaccatttcg gtctacggta 1800

cggaaccggt gtttaccgat ggcgatgaca ccccatggtc aaaaggcttc ctcgcctcct 1860

cctacgcctc gcgcggcctg aaaatgcgct ttacctccgg ctccggctct gaagtgcaga 1920

tgggttatgc cgaaggcaaa tcgatgctgt atctcgaagc gcgctgcatc tacatcacca 1980

aagccgccgg ggtgcaaggc ctgcagaatg gctccgtcag ctgtatcggc gtgccgtccg 2040

ccgtaccgtc gggtatccgc gccgtactgg cggaaaacct gatttgctca gcgctggatc 2100

tggagtgcgc ctcaagtaac gatcaaacct ttacccactc ggatatgcgg cgtaccgcgc 2160

gcctgctgat gcagttcctg ccaggcaccg actttatctc ctccggttac tcggcggtgc 2220

cgaactacga caacatgttc gccggttcca acgaagatgc cgaagacttc gacgactaca 2280

acgtgatcca gcgcgatctg aaggtcgatg gcggcctgcg gccggtgcgt gaagaggacg 2340

tgatcgccat tcgtaataaa gccgcccgcg cgctgcaagc ggtatttgcc ggcatgggtt 2400

tgccgccaat tacggatgaa gaggtggagg ccgccactta cgcccacggt tcaaaagata 2460

tgcctgagcg caacatcgtt gaggacatca agtttgccca ggagatcatc aacaagaacc 2520

gcaacggcct ggaggtggtg aaagccctgg cgaaaggcgg cttccccgat gtcgcccagg 2580

acatgctcaa tattcagaaa gccaagctca ccggcgacta cctgcacacc tccgccatca 2640

tcgtcggcga aggccaggtg ctctcggccg tgaatgacgt gaacgattat gccggtccgg 2700

caacaggcta ccgcctgcaa ggcgagcgct gggaagagat taaaaatatc ccgggcgcgc 2760

tcgatcccaa tgaacttggc taaggggtga aaaatggaaa ttaacgaaac gctgctgcgc 2820

cagattatcg aagaggtgct gtcggagatg aaatcaggcg cagataagcc ggtctccttt 2880

agcgcgcctg ccgcttccgt cgcgtcggcc gctgttgcgc ctgtgtccag cgaaagcttc 2940

ctgacggaaa tcggcgaagc caaacccggc acgcagcagg atgaagtcat cattgccgtc 3000

gggcccgcct ttggcctggc gcaaaccgcc aatatcgtcg gcattccgca taaaaatatc 3060

ctgcgcgaag tgatcgccgg cattgaggaa gaaggcatca aagcccgggt gatccgctgc 3120

tttaaatctt ctgacgtcgc cttcgtggcg gtggaaggca accgcctgag cggctccggc 3180

atctcgatcg gtattcagtc gaaaggcacc accgtcatcc accagcgggg cctgccgccg 3240

ctttccaatc tggaactctt cccgcaggcg ccgctgctga ccctggaaac ctaccgccag 3300

attggcaaaa acgccgcgcg ctacgccaaa cgcgagtcgc cgcagccggt gccgacgctc 3360

aacgatcaga tggctcgtcc caaataccag gcgaagtcgg ccattttgca cattaaagag 3420

accaaatacg tagtgaccgg caaaaacccg caggaactgc gcatggcgct ttaacaaagg 3480

atatcccgat gaataccgac gcaattgaat ccatggtacg cgacgtgctg agccggatga 3540

acagcctaca ggacggggca acgcccgcgc cagccgcacc gacaaacgac accgctcgcc 3600

agcctaaagt cagcgactac ccgttagcca cccgccatcc ggagtgggtc aaaaccgcta 3660

ccaataaaac gctcgatgac ctgacgctgg agaacgtatt aagcgatcgc gttacggcgc 3720

aggacatgcg catcaccccg gaaacgctgc gcatgcaggc ggcgatcgcc caggatgccg 3780

gacgcgatcg gctggcgatg aactttgagc gggccgccga gctcaccgcg gttcccgacg 3840

accggatcct tgagatctac aacgccctgc gcccataccg ttccacccag gcggagctac 3900

tggcgatcgc tgatgacctc gagcatcgct accaggcccg gctctgtgcc gcctttgttc 3960

gggaagcggc cgggctgtac gtcgagcgta aaaaactgaa aggcgacgat taacaggggg 4020

caagcatgcg ctatatcgct ggcattgata ttggcaactc atcgacagag gtcgccctgg 4080

cgacggtcga tgacgcaggt gtgctgaaca tccgccacag cgcgctggct gaaaccaccg 4140

gtattaaagg cacattacgt aatgtgttcg gtattcagga ggcgctaaca caggcggcaa 4200

aagcggcagg cattcaggtc agcgatatct cgcttattcg cattaacgaa gccacgccgg 4260

tcattggcga tgtggcgatg gagaccatca cggaaaccat catcaccgag tccaccatga 4320

tcggccacaa cccgaagaca cccggcggcg tcggactggg ggtcggcatc accatcacac 4380

cggaggcgct gctgtcctgc gctgcggacg ccccctatat tctggtggtc tcctcagcct 4440

ttgactttgc cgatgtcgct gcgatggtca atgcggcgac ggcagcgggc tatcagattt 4500

ccggcattat tttgcagcag gatgacggcg tgctggtcaa taaccggcta cagcaaccgc 4560

tgccggtgat cgatgaagtt cagcatatcg accgtattcc acttggcatg ctggcggcgg 4620

tcgaggtcgc tttacccggc aagattatcg aaacgctctc caacccctat ggtattgcca 4680

ccgttttcaa tctcaacgcc gaagagacca aaaatatcgt gccaatggcg cgggcgctga 4740

ttggcaatcg ttcggccgta gtggtgaaaa ccccctccgg cgacgtcaag gcacggacta 4800

ttccggcggg gaatctgctg ctcatcgctc aggggcgcag cgtacaggtt gatgtggccg 4860

ccggggcgga agccatcatg aaggcggttg acggctgcgg caaactggac aacgtcgcgg 4920

gagaagcggg caccaatatc ggcggcatgc ttgagcatgt gcgccagacc atggctgagc 4980

ttaccaataa gccagctcag gagatcccca ttcaggatct gctggccgtt gatacggcgg 5040

tgccggtcag cgtgaccggt ggcctggcgg gggagttctc gctggagcag gcggtgggta 5100

tcgcctcgat ggtcaagtcg gatcgcctgc agatggccct gatcgcccgt gaaattgagc 5160

acaaactgca gattgcggtt caggtgggtg gcgctgaggc ggaagcggcc attctcgggg 5220

cgctcaccac tcccggcacc acgcgcccgc tggcgatcct cgatctgggc gccgggtcga 5280

ccgacgcctc cattatcaat gcgcaaggag agatcagcgc cactcacctg gccggcgccg 5340

gcgatatggt cacgatgatc atcgcccgcg agctggggct tgaggaccgc tacctggcgg 5400

aagagatcaa aaaatacccg ctggcgaaag tcgaaagcct gtttcatctg cgtcatgaag 5460

acggcagcgt ccagtttttt ccgtcggcct taccaccggc ggtatttgcc cgcgtctgcg 5520

tggtgaaacc ggatgaactg gttcccctgc ccggcgatct gccgctggag aaagtgcgcg 5580

ccattcgccg tagcgccaaa tcacgcgtct ttgtcaccaa cgccctgcgg gcgttacgcc 5640

aggtgagccc taccggcaac attcgcgaca tcccgttcgt agtgctggtg gtcggctcgt 5700

ccctcgattt cgagatcccc cagctggtca ccgacgcgct ggcgcactac cggctggttg 5760

ccgggcgcgg caacatccgc ggcagtgaag gcccgcgcaa tgcggtcgcc agcggattac 5820

tccttgcctg gcaaaaagga ggtacgcatg gagagtagcc tcgtcacccc cgccatcgtc 5880

attgcggtca ctgacgaatg cagcgaacag tggcgcgatg tcctgctggg cattgaagag 5940

gaaggcattc ctttcgttct gcaaccgcgg accggcggcg atcttgtcca tcacgcctgg 6000

caggcggcgc agcgttcgcc gctgctggtg ggcatcgcct gcgaccggga acagctcatc 6060

gtgcactaca aaaatttacc cgcatcaact ccgctgtttt cgctgaggta tcaccaggac 6120

aggctggccc ggcgaaacac tggcaacaat gcggctcgtc tcgtcaaagg gatcccattt 6180

cgggatcgca atgcttaatc cacaggagaa cacaatatga ataacgcact aggactggtt 6240

gaaacgaaag gcctggttgg cgccattgaa gccgctgatg caatggtgaa atccgccaac 6300

gtgcagttga ttggttacga aaaaattggc tccgggctga ttaccgtgat ggtccgcgga 6360

gacgtcggtg cagtgaaagc cgccgttgac gccgggagcg cggcggccag cgtggtcggc 6420

gaagtcaagt ccagccacgt gatcccgcgc ccgcacagcg atgtcgaggc catcctgccg 6480

aaatccgtct gacctggaaa caaggagcac tgcgtgaagc aatcactggg attacttgag 6540

gtcagcggcc tggccctggc catcacctgc gccgacgcca tggcgaaagc ggccgccatc 6600

acgctgctcg ccctggaaaa gaccaacggc tccggctgga tggtggtgaa gatcgtcggt 6660

gatgtggcct ctgtccaggc ggcggtgatg accggggccg aactcgccga ccggcagcag 6720

gggctggtgg cgcagaaggt tatcgccagg cccggcgcag gcctgctgcc agcccgcgtc 6780

gaagcgcctt caccagcacc tgacgctgct ctggaagagg agaacgccac cataatggat 6840

gagcgcagag atcccgcaga caccctcccc cgccctgcgg agcaggtgac ctgcaacctg 6900

tgcctggacc cgcactgtcc acggcaaaaa ggcgaacctc gctcgcagtg cctgcacgcc 6960

ggtaaacgag gtgacgcgta atggataaac aacagctggc ttcaacagtc aacaaagtgc 7020

tggatgagat gcgccagcgt cccatcccaa tcggtatctc cagtcgccat atccatttag 7080

cggcggagga ctatgccagg ctctttcccg ctcagcccat ccagccgaaa aaagcgctgc 7140

tgcagcccgg gcagtatgcc gcagagcaga cggtgactct ggttggcccc aaaggacgat 7200

taaataacgt gcgcctgctc ggccctttgc gccagaccag ccaggtggaa atctcacgca 7260

ccgacgcccg gaccctcggt atcgccgcgc cgctgcgcat gtcaggcaat ctgcaggaca 7320

cgccagggat ccgcctgata agcccctttg ccgaactgga gctgagcagc gggacgatcg 7380

tcgcccaacg acatattcat atgtccccgc tcgatgccct gatcctgcgc gtcgcccatg 7440

gcgatagcgt cgcggtggcg atcgaaggca gcgacaggcg gcttatcttc gacaatgtag 7500

cggtccgcgt agccccggat atgcgtcttg agatgcacat cgatactgac gaagccaatg 7560

cggcaggcgc tgacgctgcg caggcctggg ctacgctggt gacgaagcca tgaaccacgc 7620

gatgctgcag cgtatcgtcg aagaggtcgc cgcccgtctg cgccgtcgcg cccggagtac 7680

ggccacgctc agcgtggccc agctcggcga agcggatatc cacagcctgc tgtgccagca 7740

cgcttcgctg cgcatcgtgc aggtggattt accgctgctg gagcagctta ccggccagcg 7800

cgcgctgacg gccgccggtt gcgcgttgca tgaggcgatg gcctggggcg ttcgggtaca 7860

gctaacgctg cttcccgccc tgctccccgc gctggcggta aaaaaactcg cccgtttgcc 7920

gctgacgttc agcgacccgc ctggccagcc cgtcagactt cacgccgcgt cggtgctcac 7980

ctatgccgat gtagcccgtc tcaggggagg ttatctggtg ctgcgccgcc gccccattat 8040

caccgccctc gcccgcgaag cggccgtcgc gcggcatgtt caattgatta agcaggagtg 8100

aactatgcat ctggcacgcg tgaccggcgc cgtcgtctcg acgcaaaaat cgccctctct 8160

gaacggcaaa aaactgctgc tggtgcgccg ggtaagcaca gacgacgaac atccatttca 8220

gcccgcgagc ggcgatgaag tggcggtaga ttcggttggc gctggcgtcg gcgaactggt 8280

gctgctctgc agcggctcga gcgcacgcca cgtctttagc ggcccgaatg aggccatcga 8340

tcttgccgtt gtgggcattg ttgactcact ttcacgctaa gggggcgcct gtggctattt 8400

atacccgaac cggcgatgcc ggcagtacat cactctttac cggccagcgg gtcagcaaga 8460

cgcatccgcg cgtcgaagcc tacggcaccc tggatgaact gaacgcggtg ctgagcctgt 8520

gcgtctgcgc cgccgctgaa gaggaacatc gcgtcctgct cgaagccctg cagcagcata 8580

ttttctggtt cagcgcggag ctcgccagcg acagcgaaca gccatcaccg ggtaagcgct 8640

atatcagcag cgaagagatt gccctgctgg agcagaccat cgatcgcgag atggcccgcg 8700

tgccggcgct gcaccagttt gtgttgcccg gacggtgcga agccgccagt cgcctgcacc 8760

tggcccgcac cgttgcccgt cgggcggagc gtcggctggt cgagctggcg gcagaggtca 8820

ctatccgtca gatactgctg cgttatctga accgcttatc tgactgtctg tatgccctgg 8880

cgcgcagcga ggatcatgcc gatcaccagc gccggctcgt ggcggagatc gcggcccgct 8940

acctggccgc cagcgggtcg cctgcgtccg acgcgcccaa agcccaggcc ggggcactct 9000

cctttcacga gctgcatcag cttacccgtc aggctatcga acacgcccgc cagctgcagg 9060

tgccggtggt ggtcagcatc gttgacgcac acggtaccga aaccgtgacc tggcgaatgc 9120

ccgatgcgct gctggtcagc agcgagctgg cgccaaaaaa agcctggacc gccgtcgcca 9180

tgaaaaccgc cacccacgag ctcgcgacga ccgtacagcc aggcgctgcg ctgtacggtc 9240

tggagagcca tctgcagggc aaagtcgtca cctttggcgg cggatatccg ctgtggcgcg 9300

atggtcagct aatagccggt ctcgggatta gcggcggcag cgtcgagcag gatatggcta 9360

ttgcacaggc tgccatggcg gccattaacg tgagaacaca tcaatgaata cagcagaact 9420

ggaaaccctt atccgcacca ttctcagcga aaagctcgcg cccgcccccg tttctcagga 9480

acagcagggc atttaccgcg acgtcggcag cgccatcgac gccgcccatc aggcttttct 9540

ccgctatcag cagtgtccgc taaaaacccg cagcgccatt atcagcgccc tgcgggagac 9600

gctggccccc gagctggcga cgctggcgga ggagagcgcc acggagaccg gcatgggcaa 9660

caaagaagat aaatatctga aaaataaagc tgcccttgag aacacaccgg gcattgagga 9720

tctcaccacc agcgccctca ccggcgatgg cgggatggtg ctgtttgagt actcgccgtt 9780

cggggttatt ggcgccgtgg cgcccagcac caacccaacg gaaaccatta tcaacaacag 9840

tatcagcatg ctggcggcgg gtaacagcgt ctatttcagc ccccatcccg gcgcgaaaaa 9900

ggtctcgtta acgcttatcg ccaggatcga agagatcgcc taccgctgca gcgggatccg 9960

taacctggtg gtgaccgttg ccgagccgac ctttgaagcc acccagcaaa tgatggccca 10020

cccgctgatt gccgttctgg ctatcaccgg cggccctggc attgtggcga tgggcatgaa 10080

aagcggtaaa aaagtgatcg gcgctggcgc cggcaatccg ccgtgcatcg ttgatgaaac 10140

ggccgatctc gtcaaagccg ccgaagatat catcagcggc gccgccttcg attacaacct 10200

gccctgtatc gccgaaaaaa gcctgatcgt cgtcgcctcc gtcgctgacc gcctgatcca 10260

gcagatgcag gattttgacg cgctgctgtt gagcccgcag gagaccgata ccctgcgcgc 10320

cgtctgcctg cccgacggcg cggcgaataa aaaactggtt ggtaagagcc cggctgagct 10380

gctggcggcc gccggtctcg ccgtcccttc ccgcccccct cgcctgctga tagccgaggt 10440

gcaggcgaac gacccctggg tgacctgcga gcaactgatg ccggtgctgc cgatcgtccg 10500

ggtcgccgat tttgatagcg ccctggcgct ggccctgcgc gttgaggagg gtctgcacca 10560

caccgccatt atgcactcgc agaatgtctc gcggctcaat ctggcggcac gcacgctgca 10620

gacctccatt tttgtcaaaa atggtccgtc ttacgcgggt atcggcgtcg gcggcgaagg 10680

gtttaccacc ttcaccatcg ccacgccaac cggagaaggc accacctccg cgcggacgtt 10740

cgcccgcctg cggcgctgcg tgttgaccaa cggttttttcc attcgctaac cgggggcgcc 10800

atgcacacct tttctctgca aacgcgcctc tacagcggcc cgggcagcct ggccgcgctg 10860

cagcgcttta gccatcagca catctggatc gtctgcgacg gcttcctggc gcgctcgccg 10920

ctgttcgagc ggctgcgcgc cgcgctgccc gccagcaacc gcgtcagcgt gttcagcgat 10980

attacaccgg atccgaccat tcacaccgtg gcgaaaggga tagcgcagat gcagaccctg 11040

cgtccgcagg tggtgatcgg cttcggcggc ggctcggcga tggacgccgc caaggcgatc 11100

gtctggttca gtcagcaggg cgggctgcct gtcgacacct gcgtagcgat ccccaccacc 11160

agcggtaccg ggtcggaagt gaccagcgcc tgcgtcatca gcgacccgga aaaagggatc 11220

aagtatccgc tgttccatga ggcgctctgt cccgacatgg cgatccttga cccaacgctg 11280

gtggtcagcg taccgcccac catcacagcc cataccgggc tggacgtcct gacccacgcc 11340

cttgaggcat gggtatcgcc gcaggccacc gattttaccg atgcgctggc ggaaaaagcc 11400

gccaggctgg tatttcgcgc cctgcccgtt gcggttcgcc agggcgactg cgttgcgacc 11460

cgcagcaaaa tgcacaatgc ttcaaccctt gccggcatgg cctttagcca ggctggcctt 11520

gggctcaatc atgctatcgc ccatcagctt ggcggccagt ttcacctccc ccatggcctg 11580

gccaacgcgc tgctgctgac cgcggtgatc cgctttaacg ccggcgagcc gcgagcggcg 11640

aagcgctatg cgcgcctggc cagggcctgc cggttctgcc cgccggaagc tggcgaacag 11700

gaggctctcc aggcgctgat tgccgcggtg gaaacactga aacagcagtg cgccattccc 11760

accctcaagg gcgccttgca ggaaaaatat ccccttttct tatcgcgaat tcccgccatg 11820

gtgccggccg cgctggccga cgccaccctg cgcaccaacc cgtgcccggc cgagggcgcg 11880

gccatcgcgc aactgctgga gagcctgcaa tgagtgaagc gatagtgcca ctaccagaca 11940

tggacgcggc ggagatccgc gagcgcgtcc gggctgccgg cgtggtgggc gccggtggcg 12000

caggctttcc gacccacatc aaactgcagg cccgggtgga taccgtgctg gtgaacgccg 12060

ccgaatgcga accgatgctg aaggttgatc aacagctgat ggcgcagcag gctgaccggc 12120

tgattcgcgg cctgggttac gcgatgaccg ccaccggtgc ccgggaagtg atcattgccc 12180

tgaaggcgaa atatgctgcg gcgattgcgg ccttaacccc gcgactcccg gaatgggccc 12240

ggctgcatat tctgcccgat gtctatccgg ccggggatga ggtgcttact atctggctgg 12300

cgacggggcg ccgcgtgccg cctgccgcgc tgccggtcag cgtcggcgtg gtggtcaata 12360

acgtgcagac ggtactcaat atcgccagag ccgtggagca gggctatccg gtgactcgcc 12420

ggacgttgac cgtcaatggc gctgtcgccc gcccgctgac gctgaccgta ccgctgggca 12480

tgtcgctgcg cgaggtgctg gatctggccg gcggcgcgac ggttgccgaa ccggggttta 12540

tcaacggtgg gccgatgatg ggctcgctta tgacctccct cgagacaccg gtcaccaaaa 12600

ccaccggcgg cctgctggtg ctcccgggca accatccgtt gatccagcgg cggcggcagg 12660

atgagcgcac cctgctcgcc atcgcccgca ccgtctgtga acagtgccgt ttatgcacag 12720

atttatgccc ccggcacctg atcggccatg agctttcccc tcatctgctg gtgcgggcgg 12780

tgaactatcg ccaggcggcc accccgtcgc tgctgctcag cgccctgacc tgctcggagt 12840

gcaacgtttg cgagagcgtc gcctgcccgg tgggtatttc ccccatgcgc atcaaccgtc 12900

tgctgaaacg tgagctgagg gcgaaaaacc tgcgctacga gggaccgctg cgtccggcgg 12960

acgagatggc gaagcaccgc ctggtgccgg tcaaacggct tatcagcaaa ctggggcttg 13020

atccctggta tcaggaggcg ccgctgacgg cggtggagcc ggaggtgggc tgcgtcaccc 13080

tgccgctgcg ccagcatatc ggcatcagcg ccgtcccctg cgtcgcgccg ggagagcgag 13140

tcacgcgcgg ccagctgctg gcaggcatcc ctgccgacgc cctcggtgcg ccggtacacg 13200

ccagtattga cggccaggtc tccgccatca cggagcaggc cattacgctt gtaagaggtt 13260

aatcatgacc caggctatcg gaattttaga actgaccagt atcgccaggg gaatggagct 13320

gggcgacgtc atgctgaaaa gcgcgaacgt acagctcctg ctctgcagat ccctttgtcc 13380

cgggaaattc ctgctgatgc tcggcggcga cgtcggcgcc gtgcaacagg caatcgccgc 13440

cggcacggcg cgcgccgggg agatgctggt ggacagcctg gtgctggcca acattcaccc 13500

cagcgtactg ccggcgatca gcggactcaa cgtggtcgag cagcggcagg cggcggggat 13560

cgtcgaaacc tggagcgtgg cagcgtgcat cagcgccgcc gaccgcgcgg tgaaagcggc 13620

caacgtcacc ctggtgcgca tccatatggc gtttggtatc ggcggcaagt gttacatggt 13680

ggtggcgggc gatatcgccg atgtcgacaa cgccgtcacg gtggccagcg acagcgcagg 13740

cgagaaagga ttactggttt accgggcagt gatcccgcga ccgcacgacg cgctgtggca 13800

acagctgatg gagggataat ggaaccgcaa acccctaccg aacgcatgat ccaggagtat 13860

gtcccgggta aacaggtcac tctggcgcat ctcatcgcca atccgggcaa agatctgttc 13920

aaaaagctgg gcctgccgga cgcggtctcc gccatcggca ttctcaccat tacccccagt 13980

gaggcgtcaa tcatcgcctg cgacatcgcc accaagtctg gcgcggtgga gatcggcttt 14040

ctcgatcgct tcaccggcgc ggtggtgttg accggcgatg tctccgcggt ggagtacgcg 14100

ctcaggcagg tcacacgcac ccttggtgag ctgatgcgct tcaccgcctg cccgatcacc 14160

cggacctgag cccatgaagc gcctgatgct tatcggaccc agtcagtgcg gcaaaacctc 14220

gctgacccag gtattgcgtg gcgaaaccct gcgttatcaa aaaacgcagg ccatcgtctg 14280

gacgccggcc gccatcgaca ccccggggga atatctggaa aaccgctgcc tgtacagcgc 14340

actgctcacc agcgcctgcg aggccgatgt catcgcgctg gtactgaatg ccgatgcgcc 14400

ctggtcaccc ttttctccgg ggcttaccgc tccgatgaac cgcccggtca tcggggtgat 14460

caccaaagcc gatttagccg ccccgccccg tctgcaacag gtccgggcat ggctcgaggc 14520

agcaggcgcc gaacatatct ttatcaccag cgcgttaacc ggcgacgggc ttgatgacct 14580

gctcgcctgc ctgaacgcag aggaatatca atgacctata aaatcatggc gatcaacgcc 14640

ggcagctcct ccctgaaatt tcagctgctt aacatgccgc agggagcgtt gctctgccag 14700

gggctgatcg aacgtattgg cctgcccgag gcccgcttca tgctgaaaac gtcggcacag 14760

aaatggcagg agacgctgcc catcgccgat caccatgagg cagtgaccct gctgctggag 14820

gcgctgaccg gccgcgggat cctgagcagc ctgcaggaga tagacggcgt tggccaccgc 14880

gtcgcccacg gcggcgaacg cttcaaagac gccgcgctgg tctgcgacga caccctgcgg 14940

gagatcgaac gcctggcgga gctggcgccg ctgcataacc cggtcaatgc gctgggcatt 15000

cgcctctttc gccagcggct gccggcggta ccggcggtag cggtctttga taccgccttt 15060

caccagaccc tggcgccgga ggcctggctc tatcccctgc cctggcgcta ttacgccgag 15120

ctgggcattc gccgctacgg ttttcacggc accagccatc actatgtcag cagcgcgctg 15180

gcggaaaagc tgggcgtacc gctgagcgcc ctgcgggtag tgagctgcca cctgggcaat 15240

ggctgtagcg tctgcgccat caaaggcggg cagtcggtga atacctcaat gggctttact 15300

ccccagtccg gggtgatgat gggcacccgc agcggcgata tcgatccgtc gattcttccc 15360

tggctggtgg agaaagaggg gaaatccgcc ctgcagctca gccagctgct gaataatgaa 15420

tccgggctgc tgggcgtctc cggcgtgtct tctgattacc gcgacgtcga acaggcagcc 15480

gacgccggca acgaaagggc ggctctggcg ctgtcgctgt ttgccgaacg cattcgcgcg 15540

actatcggca gctatatcat gcagatggga ggcctggatg cgctgatctt taccggcggc 15600

ataggcgagc attccgcccg ggcgcgggcg acgatctgcc gcaatttgca ttttctcggc 15660

ctcgcgctgg acgacgagaa aaaccagcgg agcgcgacct ttattcaggc ggataatgcg 15720

ctggtcaaag tggcggtgat caacaccaat gaagagctaa tgattgcccg cgacgtcatg 15780

cgtcttgcat tgccgcaggc gcgcgagctg acggtgtcgg cataaacgac gcgcctcctc 15840

ttgcggagaa tcatggtaac cggcgggtcg ccatgccact atgcgccctg gcgaaggtaa 15900

gcggaaaggt gaaaacgtgg ctgttgcaca atgtcccgcc tcgtgcggag aacttatcca 15960

gggctggatc ctcggcagcg aaaagctggt ctcctgtccg gtggactggt acagcaccgt 16020

cgaggtcgag accggggtac cgcgaaaaga cgagcggccg ctgtcgcggg cgatggtcga 16080

ccagctgctg gcccactggg gctacccccc agcgctgagc cagcagatcc gcatcaccct 16140

gcactcaacg atcccggtgg ccaaagggat ggccagcagc accgccgata tcgctgccac 16200

cgccgtcgcc accgcccatc atctgggcca tctactggat gagccgacgc tggcgcgcct 16260

gtgcgtcgcg ctggagccta ccgacagcac cctgtttcgc cagctgacgc tgttcgacca 16320

taataccgcc gccacgcaaa tcgcctgtcc ccctccgcca gcgctcgacc tgttagtact 16380

ggagagcccg ctgacgctgc gcaccaccga ctaccaccag ctgccgcgcg aggcgggtct 16440

gttggctaac gcctcccggc tgcagctggc gtgggagaaa gtacagcagg cctgccactg 16500

cggcagccca cagctgctgg gcgaagcggc gaccatcagc gccatcgcca gccagcagct 16560

gttgcccaaa ccggggtttg acgcgctact gcaactggtg gaaagcgcag gattgtatgg 16620

ccttaatgta gcgcacagcg gcagcgtcgt cggcctgctg ctggaccgcc gtcgtcatga 16680

cgtcgaattt ctgcaatggc ggctggcaga gagcgacatc gccgcccact ggccgcagca 16740

gcatctgctg gcgatggtgc ccgggggcgt caccctgcag tag 16783

<210> 4

<211> 912

<212> DNA

<213> Klebsiella variicola 342

<400> 4

atgatttctg cgagtaccct gaactcagaa ctcatcaata aaatcgcaca ggattttgcg 60

caagccacca gtctggcggt tgtggtcgtc aatattcacg gtgatgagat ttctgaactg 120

tttaatttca cgccgttttg ccaactcatg cgtcagcacc cggagcacag cggacgctgt 180

cgcatgagcg accgctgcgg cgggctggaa gcctcgaaaa atgaccagct gtgtatctac 240

cgctgtcacg cggggttaac tgatttttcc atcccgctgg tgatcgccgg ccacctcgtt 300

ggcttcgtgc tgtgcggcca ggtgcgtctg agcaatgata ttgaactggt tgatattctg 360

aacgtggatg accgctggca ggacgatccg gcgctggtcc aggcgtttcg cgatgtgcct 420

gagatggact attcgcgggt tatcgcttcg gcggatctgc tgaagctgat cgtcgagaat 480

tgcctgaaaa aacagctcaa ctttgtggtt atcaaagata acccgtcgca gaacgacccg 540

gcgcaggcct gccgggcggc ctcgccgcac gatggcaaaa tgaagaaggc gctgcgctat 600

atcgacgcgc atctctccga tgagctgcgt ctggaggacg tggcggcgca cgtctatctc 660

agcccgtact acttcagcaa actgtttaaa aagtatcagg ggatcggctt taacgcctgg 720

gtcaatcagc agcggatggc gagcgcaaaa gagctgctat gccacagcga ctggagcatt 780

gccagcatcg cgcggaattt aggtttttcg caaaccagct atttttgcaa agtgtttcgc 840

cagacctatc aggtaacgcc gcaggcattt cgtcagcaaa ttaatgccgg atcacaaact 900

gagtcttttt aa 912

<210> 5

<211> 16761

<212> DNA

<213> Citrobacter freundii str. ballerup 7851

<400> 5

atgcaacaag aagcgttagg aatggtagaa accaaaggct tgactgcagc catagaggcc 60

gcagatgcaa tggtgaagtc agccaatgta atgctggtcg gctacgaaaa aattggttcg 120

gggctggtaa cagtcattgt ccgcggcgat gttggcgcag tcaaagcagc aacagatgca 180

ggtgccgccg cagcacgtaa tgtgggagaa gtgaaagccg tacacgtcat cccacgccct 240

cacaccgatg tagaaaaaat cttaccgaag ggaattagct aatgagcagc aatgagctgg 300

ttgatcagat catggcgcag gtgattgctc gcgtggcaac gccggaacag caggctatcc 360

ctgaaaataa tcctccaaca cgagaaacgg ctatggcaga gaaaagctgc agtttaacgg 420

agtttgtcgg tactgcgatt ggcgacaccg tcggtctggt aatcgccaac gtggacagcg 480

ccctactgga cgcaatgaaa cttgaaaaac ggtatcgctc cattggcatc cttggcgcgc 540

gtactggtgc aggcccgcac atcatggccg cagatgaagc ggtaaaagcc accaatactg 600

aagtcgtcag tattgagttg ccacgtgata ccaaaggcgg cgcgggtcac ggttcgctga 660

ttattctcgg cggcaacgat gtttccgacg tgaaacgcgg aattgaggtt gcgctgaaag 720

aactggatcg cacctttggc gatgtgtatg ccaacgaagc cggtcacatc gagatgcaat 780

acaccgcacg cgccagctac gcgctggaaa aagcctttgg tgcaccgatt ggccgtgcct 840

gtggcgtgat cgtcggcgcc ccggcatccg ttggtgtcct gatggctgat actgcgctga 900

aatccgccaa cgtggaagta gtggcctaca gctcccctgc ccatggcacc agcttcagta 960

acgaagccat tctggtcatt tcaggcgatt ccggcgctgt gcgtcaggcc gttatctccg 1020

cccgcgaaat cggtaaaacc gtactcggga ccctcggctc agaaccgaaa aacgatcgtc 1080

cgtcctacat ctgatatcag tgaggctgat tcatgagatc gaaaagattt gaagcactgg 1140

cgaagcgccc tgtgaatcag gatggcttcg ttaaggagtg gatcgaagaa ggcttcattg 1200

cgatggaaag cccgaacgac cctaaaccgt cgattaaaat cgtcaacggt acagtcacag 1260

aactggacgg caaatccgcc agtgaatttg acttaattga ccatttcatt gcccgctatg 1320

gcattaacct ggcgcgcgct gaagaagtca tggcgatgga ctcagtaaaa cttgccaata 1380

tgctttgcga ccctaacgtg aagcgcaaag acattgtgcc gctgaccacc gcaatgaccc 1440

cggcaaaaat tgtcgaagtg gtgtcccata tgaacgtcgt tgagatgatg atggccatgc 1500

agaaaatgcg tgctcgtcgt acaccgtctc agcaggcgca cgtcactaac gttaaagata 1560

atccggtgca gattgccgct gatgcggctg aaggcgcctg gcgcggattt gacgaacagg 1620

aaacgacggt tgccgttgcg cgttatgcac cctttaacgc cattgcgctg ttagtcggct 1680

cacaggttgg gcgccctggc gtattaaccc agtgctcgct ggaagaagcg accgaactta 1740

aactcggtat gttaggccac acctgttacg cggaaaccat ctctgtctat ggtactgaac 1800

ccgtctttac cgacggcgat gacacaccat ggtcgaaagg cttcctcgcc tcttcctacg 1860

cctcacgcgg tctgaaaatg cgctttacct ccggctctgg ctcagaagtt cagatgggtt 1920

acgccgaagg taaatcgatg ctttaccttg aagcccgctg tatttacatc accaaagccg 1980

ctggcgtaca gggtttacaa aatggctcag taagctgcat cggcgtgcct tccgctgtgc 2040

cgtcaggcat tcgtgcggtg ttagctgaaa acctgatctg ttcctccctg gatctggagt 2100

gtgcctccag caacgaccaa acctttaccc actcggatat gcgtcgtact gcccgcctgc 2160

tgatgcagtt cctgccgggt actgacttca tttcttccgg ttattccgct gtgccgaact 2220

acgacaacat gtttgcgggt tcgaacgagg atgccgaaga ttttgacgac tacaacgtcc 2280

tccagcgaga cctcaaggta gatgggggtc tgcgtccggt tcgtgaagaa gacgttatcg 2340

ccattcgtaa taaagcggca cgtgcactgc aggccgtctt tgccgggatg ggattaccgc 2400

caatcactga tgaagaagta gaagcggcga cttacgccca cggttcgaaa gatatgccgg 2460

agcgaaacat cgttgaagat atcaagtttg cgcaagagat catcaataaa aaccgtaacg 2520

gtctggaagt ggtgaaagcc cttgctcagg gcgggtttac cgatgtcgct caggacatgc 2580

tcaacatcca gaaagccaag ttaaccgggg attatctgca tacctccgcc attatcgttg 2640

gcgacggaca ggtgctatcc gcagtaaatg acgtcaatga ctatgccggt ccggcaacag 2700

gttatcgcct acaaggcgaa cgctgggaag agatcaaaaa catccctggc gcacttgatc 2760

ccaacgagct tggctaaggg gtgaaaaatg gaaatcaatg aaaagctgct gcgccagata 2820

attgaagacg tactgtctga aatgcaaacc agcgacaaac ccgtctcatt ccgcgcatcg 2880

acagcggcta gcgcaccaca ggctgcagcg gcacagggcg acagctttct gaccgagatt 2940

ggcgaagcca aacaaggcca gcagcaggac gaagtgatta ttgccgtagg tcccgcgttt 3000

ggactgtcgc aaaccgtcaa tatcgtaggg atcccgcata agaacatctt gcgcgaagtg 3060

attgcgggta ttgaagaaga aggcattaaa gcgcgcgtta tccgctgctt taaatcctcc 3120

gatgtggcat ttgtcgccgt ggaaggtaac cgtctgagcg gttccgggat ctctatcggc 3180

atccagtcaa aaggcaccac ggtaatccac cagcaggggt taccgccgct ttctaacctg 3240

gagctgttcc ctcaggcacc gcttctgacg ctggaaacct atcgtcagat cggtaaaaac 3300

gccgcgcgtt atgccaagcg tgaatctccg caaccggtcc ctactctgaa tgaccagatg 3360

gctcgtccta agtaccaggc gaaatcggcc attctgcaca ttaaagagac caaatacgtc 3420

gtgacgggca aaaacccgca ggaactgcgc gtggcgcttt gataaaggat aactccatga 3480

ataccgacgc aattgaatcg atggttcggg atgtgctgag ccggatgaac agcatgcaag 3540

gcgagcccgc gacgccggcc gcggcgagtt catcagcaca taccgcaaaa gtcacggatt 3600

accctctcgc gaataaacat cctgagtggg taaaaaccgc aaccaacaaa actctggatg 3660

acttcacgct cgaaaatgtt ctgagcaaca aagtgacggc gcaggacatg cgcatcaccc 3720

ctgaaacgct gcgccttcag gcggaaatcg ccaaagatgc cggacgtgac agactggcga 3780

tgaacttcga gcgtgccgct gagctaaccg ctgtacctga cgatcgcatt cttgagatct 3840

acaacgcgct gcgcccatac cgctcaacga aagacgagct gatggctatc gctgacgatc 3900

tcgaaaatcg ttatcaggca aagatctgtg cagctttcgt tcgtgaagcg gcagcgctgt 3960

acgttgagcg taaaaaactc aaaggcgacg actaactcag ggacaggatc atgcgatata 4020

tagctggcat tgacataggc aactcatcaa cggaagtcgc tctggctacc ctcgatgagt 4080

ctggcgcact gagcatcacc ggcagtgcgc tggcagaaac caccggaatt aaaggcacat 4140

tacgtaatgt gtttggtatt caggaggcac tttcgctggc agcaaaaaat gccggtatca 4200

atgtcagcga tatttcgctc atccgcatta acgaagccac cccggtcatt ggtgacgtcg 4260

cgatggaaac gataaccgaa accatcatca ctgaatccac catgatcggc cataacccta 4320

aaacgccggg cggcgtcgga ttaggcgttg gcgtcactat tacgccggaa gacctgctca 4380

gccgtccggc ggatacgccc tatattctgg tggtgtcatc ggcctttgat ttcgccgatg 4440

tcgccacgat gattaatgcc tccgtccgcg ccggatacca gttaactggc gttattttgc 4500

agcaggacga tggcgtgttg gtcagtaatc gtctgaccca ccccctgcct atcgtcgatg 4560

aagtccttca tatcgaccgc atcccgttag gtatgctggc tgccattgag gtcgcaattc 4620

caggaaaagt cattgaaacg ctttctaacc cttacgggat tgcgaccgtt tttggcctca 4680

acgccgacga gactaaaaac attgtcccga tggcgcgggc gctgattggc aaccgttctg 4740

ccgtggttgt gaaaacaccg tcaggtgatg tgaaagcacg agccattcct gccggaaatc 4800

tggagctaca gtcgcagggg cgtacggtac gggtcgacgt tgcagctggc gctgaagcga 4860

ttatgaaagc ggttggtgaa tgcccgaaac tggataacgt caccggagag gctggcacca 4920

atattggcgg catgctggag catgtgcgcc agacaatggc tgaattaacc aacaaaccca 4980

gccacgaaat ttttattcag gacctgctgg cagtcgatac ctcggttccc gtcagcgtga 5040

ccggcgggct agccggtgag ttctcgctgg aacaggctgt cggcattgct tccatggtga 5100

aatcagaccg cctgcaaatg gcgatgattg ctcaggaaat cacgcaaaag ctcaatattg 5160

acgtccaggt tggtggcgcc gaagccgaag ctgccattct gggggcatta accacccccg 5220

gcacgacgcg gccactggct attctggacc tcggtgcagg atcgactgac gcctccatca 5280

tcaacccgaa aggcgaaatt atcgccacgc acctggcggg tgcgggcgat atggtcacga 5340

tgattattgc ccgtgaactg gggctggaag accgttatct cgctgaagag atcaaaaagt 5400

acccgctggc taaagtcgag agcttgttcc atcttcgcca cgaagatggc agcgtccagt 5460

tcttccctac acctctccca ccaacggttt ttgcccgcgt atgcgtagta aaacccgatg 5520

aactggtgcc actgccaggc gaactggcgc tggagaaagt ccgggccatt cgccgcagcg 5580

ctaaggaacg cgttttcgtc accaacgcac tcagagcgct acgtcaggtg agtccgaccg 5640

ggaacattcg ggatattccc tttgtggtgc tggttggcgg ctcatccctg gactttgaag 5700

ttccgcaact ggtcaccgat gcgctggcgc actatcgcct ggttgccggg cgcggcaaca 5760

ttcgtggcac cgaaggtccg cgcaacgcgg tcgcgacagg gctgattctc tcctggtaca 5820

aggcattcgc tcatggaaag taatctcacc acgccagcca tcgttatctt cgccactgct 5880

ggatgcactg acgtctggag tgatgtgctg ctgggtattg aagaagaagg gatccccttt 5940

gtgattcagg agagcccgtc aactgacgtt atccacaacg catggctggc agcttgccaa 6000

tcgccactgc tggttggcat tggctgcagt cgcgaaaaac tggtcgtgca ctataaaaac 6060

ttacccacat cagcgccact atttacgctg acgtatcaac aagataacca tgcccgacgc 6120

agcattggta ataacgctgc gcggctggtt aaaggcattc catttcggga atgccactca 6180

taacccacag gagaaaagca gtatgaataa cgcactggga ctggttgaaa caaaagggct 6240

cgtcggcgct attgaagccg ctgatgccat ggtgaaatcc gcaaacgtgc agttggttgg 6300

ttacgaaaaa atcggctcag gccttatcac cgttatggtt cgcggcgatg tcggcgcagt 6360

gaaagctgcc gtagatgcag gaagcgccgc agcaagcgcc gttggcgagg tgaaatcctg 6420

ccacgttatc ccgcgtccgc acagcgacgt tgaagccatt ttacctaaat ccgcataaat 6480

cgttgcaacc aaggagcaca gcgtgaagca atcactggga ttacttgaag ttagtggtct 6540

ggcattagcc atcagttgcg cggacgtcat ggcgaaagcc gcctccatca cgctggtggg 6600

cctcgaaaaa accaacggtt caggctggat ggtgatcaaa ataatcgggg atgtggcctc 6660

cgtccaggcg gccatttcca ccggtgtcag tttcgctgac cagcgagatg gactggtggc 6720

tcacaaagtc atatccagac caggggatgg cattctgtca catagcgtca ccccggagtc 6780

tgagccaacg cccgccccga caccggtcgt accacatgaa gagatccctg aggaccatgc 6840

agcgcccgaa gcgccgcaag atgcagagtt gattagctgc aatctgtgtc ttgaccctgc 6900

ctgcccccgt caaaaaggcg agccgcgctc tctttgtctg cactcaggca aacgaggtga 6960

agcgtgatgg ataaacagca actggagaca acggtcacca aagttctgga tgaaatgcgt 7020

gagcgcccta ttccgctagg gatctctaat cgtcatattc atctatgcgc tgaagattac 7080

gaccgcttgt ttcccaatca ccctattagt gagaaaaaag agctgctgca gcccggacaa 7140

tatgcagcag accagaccgt cacactggtg ggaccaaaag gtcagttaaa aaatgtgcgc 7200

ttactcggcc cactgcgtag caccagtcag gtcgaaattt ctcgtaccga cgctcgtacg 7260

ttgggaattg ctgctccact acggatgtca ggcagtatcg agggcacgcc aggcgttcgc 7320

cttatcagtc catttgctga actggactta gcatccgggg taatcgtcgc ccaacgccat 7380

atccatatgt cgccgctcga tgccctaatc ctgcgcgttt cccatggcga taaagtctct 7440

gttgccatta atggcgatga acgtcgactg attttcgata acgtcgccgt acgcgtttcg 7500

ccggatatgc gccttgaaat gcatatcgat accgacgaag ccaatgctgc gggcgcagat 7560

aatccgcagg catttgccac actggtgagc ccacgatgaa caacgaactg ctgcaacgca 7620

ttatcgagga ggttgtttcc cgattaaaaa aacgcgcgga aagcacgctc tcgcttagcg 7680

tcgcgcagtt acgggaaatc gaaccacgga cattgtgctg ccagtactcg tcgctccacc 7740

tcctgcaggc ggacctgcca ttgctggagc aaattgccga gggctgcgcg gacaacatat 7800

cggtggtaac gatccacgaa gcgctggcat gcggagtccg cgtgaaaatt tcactacaac 7860

accgattgtt acccgcaatc cccgttagaa aacttgcgcg tctgcctctg gagttcagtg 7920

acgagctggg ccgaatcatc gttttgcatc ctgacaaact tctcagctat gccgacgtcg 7980

cccaactgaa gggcggcgtt ctggtgctac gtcgtcgatg tgtcgtgacg gctctggctc 8040

aggacgccgt cggtacgcga aatgtccaat taattaagca ggagtgagct atgcatctgg 8100

cacgggttac aggcgttgtg gtttccacgc aaaaatcttc atcactggtg gggaagaaac 8160

tgttgctggt acgtcgggtg agtgcggacg gagaacttcc cgcgtcccct gtgagtggag 8220

atgaagtcgc ggttgattct gtcggcgctg gaactggaga actggtatta ctcagcagtg 8280

gctccagcgc cagacacgtt ttttccggcc ctaatgaggc catcgatctg gctatcgtcg 8340

gcattgtcga cacgctttct cgttaggagg aactatggcg atttataccc gcactggcga 8400

tgctggcaca acggccctct ttaccgggca acgcgtgagc aaaacgcacc cgcgcgtcga 8460

ggcttacggt acgctggacg aacttaatgc ggcgcttagc ctgtgtgttt gcgcagcgaa 8520

aaacccgcag caccgccagt tactcgaaaa cattcaactg cagctttttt ggttcagcgc 8580

cgagctggca agcgaaagcg aacagccagc gccagagcag cgctacatca gctcagaaga 8640

aattgccgcg ctggaagccg ccattgatac cgcgatgggg agagtgcctc ccctacgcag 8700

ctttatttta cctggccgca gcgaagccgc cagccgtctg cactttgccc gcacgctggc 8760

ccgccgcgca gagaggcgac tggtcgaact ctccaccgag atttccgtca gacacgtgct 8820

gatgcgttat atcaatcgcc tctccgactg cttatatgcg cttgcacgag cagaagatca 8880

tgacgcccat caaaacaaca ttattcagaa agtggcggag cgttatctcg cagccatccg 8940

tacatcagcc accagggagc cggctatgtc tctgtctttt caggaactcc accaactcac 9000

tcgcgctgcg gtaacgagag cagaagaact tcaggttccg gtcgtcatca gcattgttga 9060

tgcgaatggg acccaaacgg tgacctggcg catgcctgac gcattactcg tcagtagcga 9120

actggcaccg aaaaaagcct ggaccgccgt ggcaatgaaa accgccacgc acgagctgac 9180

atcagcagtg cagccaggcg ccgcgcttta cggtctggaa agccatatgc agggaaaagt 9240

cgtcaccttc ggtggcgggt acgcattatg gcgtgagggg ttactccttg ggggtcttgg 9300

catcagcggg ggaagcgttg agcaggatat ggacattgca gaaacggcca ttgcggcaat 9360

taacgtgaga acacatcaat gaacacttca gaacttgaaa cccttattcg taacattttg 9420

agtgagcaac ttgccccggc aaaagcagaa gttaagggta atggaatttt tccgtccgtt 9480

agcgaagcga tcgatgccgc tcatcaggct tttctgcgtt atcagcaatg tccattgaaa 9540

acccgcagcg ccatcattaa cgccctgcga gaagagctga cgccttacct tgcttcattg 9600

gctgctgaaa gcgctgccga aacgggaatg ggcaacaaag aggataagtt ccttaaaaat 9660

aaagctgcgt tggacaatac cccgggtatc gaagatctca ccaccacggc gcttaccggc 9720

gacggcggca tggtgttatt cgagtattca ccctttggcg tgattggttc cgtcgccccc 9780

agcaccaacc cgacggaaac cattattaac aacagcatca gtatgctggc tgcgggaaat 9840

agcgtttatt ttagcccaca tccaggtgca aaagcggtct cactgaagct gattaccatg 9900

attgaagata ttgcttttcg ctgctgcggg attcggaatc ttgtcgtcac ggtcacagaa 9960

cccacttttg aggctaccca gcagatgatg gcgcacccta aaatcgcagt tctggcgatt 10020

accggtggtc caggcattgt tgctatgggc atgaaaagcg ggaaaaaagt cattggcgca 10080

ggcgcaggca atcctccctg cattgttgat gaaacagccg acctggttaa ggcggcggaa 10140

gatatcatca atggcgcatc ctttgattac aacctcccct gcattgctga gaagagtctg 10200

attgttgttg atgccgttgc cgaacgactg gttcaacaaa tgcagtcttt tggcgcgatg 10260

cgactcaata gcgaagagat agacaaactg cgcgcagttt gtctgccaga aggaatagcc 10320

aataaacaac tggtagggaa aagcccggca accttactgg aagccgcggg aataccggtt 10380

ccggcaaaag cacctcgatt actgataggt atcgtcaagg ctgacgaccc atgggttacc 10440

agcgagcaac tgatgccaat gctccccatc gtaacggtga gcgattttga cagcgcgtta 10500

acgttggccc tgaaggttga agagggattg catcacaccg ccattatgca ttcgcaaaat 10560

gtgtcacgtc tgaaccttgc cgcgcgcacg cttcagacct ccatttttgt gaaaaatggc 10620

ccctcttatg ctgggattgg cgttggtggt gaaggcttta ccaccttcac cattgcaacg 10680

ccgaccggcg aaggaacgac atctgcgcgc actttcgcac gctctcgtcg ttgcgtgctg 10740

acaaacggtt tttctattcg ctaatggggc cgttatgaaa agcttttcac tgcaaactcg 10800

attgtacagc ggccagggaa acctggatgt gcttaagcgc ttcacgaata agcatatctg 10860

gattatctgc gatggttttc tcgcgcgttc tcccctgatt gaaaagctgc gtgacgcgct 10920

gccagcggat aaccgcatca gcattttcag cgatatcacc cccgatccca ccattagcac 10980

ggtagtacaa gggattgcgc aaatgcagtc cctacaaccg gacgtcgtaa ttggttttgg 11040

cggtggctct gcactggacg ccgcaaaagc catcgtctgg ttcagccgac agttcggtat 11100

tgagattgaa acctgcgttg caattcccac caccagcggc acgggatcgg aggtgaccag 11160

cgcctgcgtg attagcgatc cggataaagg cattaagtat ccattattta ataatgcgct 11220

ttatccggac atggccatcc ttgatccgac gctggtcgtc agcgttccac cagccataac 11280

agcgaatacc ggcatggacg tattaacgca tgcgctggag gcttacgtgt ccacgaaagc 11340

cagcgacttc accgatgcgc tggctgaaaa agcggcacaa attgtcttcc agtacctgcc 11400

tgtcgccgtc agcaaaggcg actgcctggc aaccagagga aaaatgcaca acgcgtcaac 11460

gctcgcggga atggcattca gtcaggctgg tctgggtatc aatcatgcga ttgcccatca 11520

gttgggcggc cagtttcatc tgccccacgg acttgccaat gcgctgctac tgacccatgt 11580

cattcatttt aatgcccgcg atccgcgggc tgcgaagcgc tatgcccgtt tcgctaaagc 11640

ctgccaccta tgcccggata acgcgaacga tacggcggca ctgaatgcgc taattcgtca 11700

tatagagcta ctcaaaaaac agtgtgcgtt accttctttt gctgacgcgc ttaaagacgg 11760

aaagcaagct tggtcacaac gtataccgtc gatggttcag gccgcgctgg ctgatgtcac 11820

gctgcaaacc aatcctcgtg ttgccgacgc cagcgccatg caggaactgc ttgaggaatt 11880

attatgaaga cagctatgac ggcagagtcc acgctttatg atgcgcagac aattcgcgaa 11940

cgcgttcgcg cagcgggtgt tgtcggggcc ggaggcgcag gctttcctgc ccacgtaaaa 12000

ttacaggcgc aggtcgatac gtttctggtc aatgcggcag aatgtgagcc gatgctgaaa 12060

gttgaccagc aattgatggc cgttcaggct gagcgactta tccgcggcgt tcaatatgcc 12120

atgacggcta ccggggccag ggccggtatt attgcgctga aagagaaata tcaaagggct 12180

atcaatgcat taacgcccct tctgcctgcc gggatccgct tacatatttt gcctgatgtc 12240

tacccggctg gtgacgaggt tttgaccatc tggatggcaa cgggccggag agttccgcct 12300

gccgcgctgc cggtcagcgt cggcgtggtg gtcaataacg tccagactgt actcaacatc 12360

gccagagccg ttgagcagca gtatccggtt acgcgccgta ccctgaccgt taatggcgcg 12420

gtcgccagac caatcactct taccgtccct atcggaatgt ctttacgtga ggttctggcg 12480

ctggccggtg gtgctaccgt tgacgatccc ggttttatta acggtggtcc gatgatgggc 12540

ggcctgatta cctcactgga taccccagtc agcaagacca ccgggggact gctggtttta 12600

ccaaagtctc atgcgttgat tcagcggcgg atgcaggatg aacgcaccgt gctttccgtc 12660

gcaaaaaccg tttgcgagca gtgtcggttg tgtacggatt tgtgcccaag acatttgatc 12720

ggccatgaat tatcgccgca cctgctggtt cgcgcggtta attatcagca ggcggccaca 12780

ccgcagttac tgctcacggc cctgacctgt tcagaatgca atgtatgtga aagcgtagcc 12840

tgtccggttg ggatttcacc gatgcgaatc aaccgcatgc ttaaacgcga gctacgggcg 12900

ctcaaccatc gttatgaagg gccgttaaat cctgaggatg aaatggcgaa gtatcggctt 12960

attccggtga agcgccttat taccaaactg ggccttaacg actggtatca cgatgcgcca 13020

ctgactgaga ctgattatcc cacggataaa acgacgctgt tgctacgcca gcatattggt 13080

gccagcgcca tcccatgtgt actgcagggc gaacacgttg ttcgcggtca atgcgttgcc 13140

gatgtgccct ccggtgcatt aggcgcacct gtccacgcca gtattgacgg tattgtcagc 13200

gaaattactg agcaatccat cacggtaata agaggttaat catgtctcag gctataggga 13260

ttttagaact caccagcatt gccaaaggaa tggaagcggg cgatgccatg ttaaaaagcg 13320

cgaatgtgaa tttacttgtc agcaagacca tctgcccggg taaatttctg ctcatgctcg 13380

gtggtgacgt aggcgcagta caacaggcga tcgccaccgg aacttctctt gctggcgata 13440

tgctcgttga tagtctggta ctacccaaca ttcacgccag cgtacttcct gcgatcagtg 13500

gacttaacag cgtggataag cgtcaggccg ttggcattgt tgaaacatgg agcgttgcgg 13560

cctgcatctg cgccgccgac cgtgcggtta aagcctccaa tgtcacactg gtacgcgtcc 13620

atatggcatt tggtatcggt ggcaagtgct acatggttgt cgctggcgat gtgtctgatg 13680

tgaataacgc agtgacagtt gccagtgaaa gtgcgggtga aaaaggcctg ttggtttacc 13740

gttcggtcat ccctcgtccg catgaaagca tgtggcgaca gatggtggag ggataatgga 13800

aagacaaccc accacggatc gtatgattca ggaatatgtt cctggcaagc aggttacgct 13860

ggcgcatctt atcgctaatc cgggtaaaga tttgttcaag aaactgggat taccagagtc 13920

ggtttccgca atcggtattc tgacgattac ccccagcgaa gcctcaatca tcgcctgtga 13980

tatcgccacg aaatccgggg cggtagagat tggttttctc gaccgtttta ccggcgcagt 14040

ggtactgaca ggcgatgttt ccgctgttga gtacgcgctg aaacaggtaa cccggacgct 14100

gggcgaaatg atgcgtttta ccgcttgtcc catcacccgg acataatctc atgaaacgca 14160

taatgctaat tggccccagc cagtgcggta aaacgtcact cacgcagtgc atgcggggag 14220

aggcgcttca ctatcagaag acccaggcca ttgtctggtc tcccacaacg atagacacac 14280

cgggtgaata tctcgaaaac cgctgcctgt acagcgcgct gctggccagc gcctgtgaag 14340

cagacatcat cgcgctggtt ctcaatgctg acgcgccgtg gtcacctttt tctccaggtt 14400

ttactggccc gatgaaccga cccgttatcg gcctcgtcac caaagccgat ttagccatcc 14460

cgcagcgtat ttctctcgtg gagagttggt tagttcaggc tggcgcgcaa aaagtatttg 14520

tcaccagcgc gctggagaat accggggtcg atgagatgtt catttttctg aatgcaaagg 14580

aaccttcatg tctcacaaaa taatggccat taatgcaggc agttcctcgc ttaagtttca 14640

gcttctggcg atgccgcagg gtgaaatgat ttgccaggga ctgatcgagc gtatcggtat 14700

gtctgatgcg caggtcaccc taaaagcacc agcgcaaaaa tggcaggaga ctttgcccgt 14760

agccgatcac cgtgaagcag taacgctgct actggaaaaa ctgctcagcc acaatattat 14820

cagcagtctg gaagagatcg acggcgttgg ccatcgcgtc gcacacggtg gagaatcctt 14880

caaagattcc gcgcgggtaa ctgatgaaac actggcggaa atcgaacgcc ttgcggaact 14940

ggcaccgctt cataacccgg ttaatgcgtt ggggattgcg atatttcgcc agctaatgcc 15000

gaaaacgcca gccgttgcgg tttttgacac ggcttttcat cagacccttg atgagccttc 15060

tttcatctat cccctgccct ggcgctacta ctcagaactc ggtattcgtc gttatggttt 15120

tcatggtacc agccacaagt atgtcagcgc tcagcttgct gaaaagctgg gggtaccgct 15180

cagcgccctg cgggttgttt gttgtcatct gggcaacggc agcagcatct gcgctatcaa 15240

aggcggacac tcggtgaata cctccatggg atttacgccg cagtctggcg taatgatggg 15300

cacgcgaagt ggcgatattg atccatccat tctgccgtgg attgccctgc gtgaaggcaa 15360

aaccccgcag cagctcaacc aattgctcaa caatgagtcc ggattactcg gagtttctgg 15420

tgtttcaccc gactaccggg acgttgaaca tgccgcagat acgggcaatc accaggcagc 15480

actggcgttg acgctttttg ccgagcggat tcgcgccact attggcagct atattatgca 15540

gatgggcggt ctggatgcgc tggtgtttac cgggggaatt ggtgaaaatt cagcgcgtgc 15600

tcgcgccgcg atttgccaca acctgaattt cttaggtctg gccgttgatg aagaaaaaaa 15660

tcagcgtaac gccaccttta ttcagacgga aaatgcggtg gttaaagtgg cggtgattaa 15720

taccaatgaa gagctgatga tagcccagga cgttatgcgt ttagcgatat cagaaactgt 15780

gacattgggc attcccgcct gatattgccg caccgcaaat tgcggcaaat catggtaata 15840

gcgcgatcgc catgacacta tgcgccgagg attgctgaac acaaaggtga agacgtggct 15900

gttgcgcaat gtcccgcatc gtgcggagaa cttatccagg gctggattct gggcagtgag 15960

aaactggtct cctgcccggt cgaatggtat agcaccgttg aggtcacttc aggctcaccg 16020

ctgacagatg aacgtccact ttctcgggca atggttgaca ggttattaca gcactggcag 16080

tacccggcgc atctcagtca ggatatccgg atagatgttc agtcaaccat tcccatcgcg 16140

aaaggaatgg ccagcagcac tgcggatatt gccgccacgg ccatcgccgc agcgcattat 16200

ctgggtcatc aactggacga gccaacactg gcacagcttt gcgtttctct ggagccaacc 16260

gacagcaccg tttttcgtaa gctaaccctt ttcgatcata acaacgcttc cacgcaaatt 16320

ggttgtgaag ctcagcctca actcgatctg ctggttctcg aaagccctga aacattgcgt 16380

accgcggatt atcaccgtat tccacgtcat tccggcttgc aggcgggagc tgcggccctc 16440

aaacgggcat gggaaaaagt acaggaagcc tgtattagcc aaaatcctta ccggttggga 16500

gaagcggcta cgcttagcgc aatcgccagc caactgctgt taccaaagcc ggattttgat 16560

tcactattag cactggtgga agaatgtgat ttatacggcg tcaacgtcgc gcacagcggc 16620

agcgtagtcg gactgatgct ggacagaaat cgtcatgatg tggattacat taaatggatg 16680

ctgacgcaga aaaaacttac gattcactgg ccggaacagc atttgttacg catggtaaca 16740

ggcggggtcg aactgcaata a 16761

<210> 6

<211> 912

<212> DNA

<213> Citrobacter freundii str. ballerup 7851

<400> 6

atgatttctg cgagtgccct gaactcagaa ctcattaata aaatcgcaca ggattttgcg 60

caggccacca gtctggccgt tgtggttgtc aatattcacg gtgatgaaat ttctgagctt 120

tttaatttca ccccttttttg ccagttgatg cgtcagcatc ctcagcacag tacccgttgt 180

cgtatgagcg atcgctgtgg agggctggaa gcctctaaat ccgatcaacc ctgcatctat 240

cgctgtcacg ccggacttac cgatttctca attccgctgg tcattgcggg gcacctggta 300

gggtttgtgc tttgtggcca ggtacgctta agtaacgatg ttggtttggt tgatattctg 360

aatgtagatg accgctggca agccgatccc gaactggtca acgagtttcg taatgtaccg 420

gaaatggact attcgcgagt catcgcctca gcggatctgc tgaagctcat cgtagaaaat 480

tgccttaaga aacaacttaa ttttgtggtt atcaaagata acccgcagca gtcagagccg 540

gtacgcgcta accgcagtac caatcctcat gacagcaaaa tgaaaaaagc actgcgctat 600

attgatgcgc atttatcaga cgaattgcgt ctggaggatg tcgcctctca tgtttacctt 660

agcccttact atttcagcaa attgttcaaa aaatatcatg gtatcggttt taacgcatgg 720

gtaaaccagc agcgaatggc cagtgcaaaa gagctgcttt gccacagtga ctggagcata 780

gccagcattg cgcgtaattt aggattttcg caaaccagtt atttttgcaa agtttttcgt 840

caaacgtatc aggtcacgcc gcaggcattt cgccaacaaa taaatgtcat ttcacgtggc 900

gaatccgctt aa 912

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 7

atcagtactc ctagggtacc agctg 25

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 8

caccttcatg gtggtcagtg c 21

<210> 9

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 9

ggatcgccat cagatctgat atgcaacaag aagcgttagg aat 43

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 10

ggtaccctag gagtactgat ttattgcagt tcgaccccgc 40

<210> 11

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 11

cccggggatc ctctagagat ccgacgtcag atagacggca 40

<210> 12

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 12

ctccagccta caggataaga taaaattgtc gtttatttat ttaa 44

<210> 13

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 13

gaggaagtgg tatatgcaac aagaagcact aggaatg 37

<210> 14

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 14

catgcctgca ggtcgacgat cagtgttggt ggctttcacc g 41

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 15

tcttatcctg taggctggag ctgcttcg 28

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 16

aaggaattcc ggggatccgt cga 23

<210> 17

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 17

acggatcccc ggaattcctt ggcggcctgg gtg 33

<210> 18

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 18

gttgcatata ccacttcctc gttcaacaaa tat 33

<210> 19

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 19

atcgtcgacc tgcaggca 18

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 20

atctctagag gatccccggg t 21

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 21

aagcttggct gttttggcg 19

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 22

gaattctgtt tcctgtgtga aattg 25

<210> 23

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 23

tcacacagga aacagaattc atgatttctg cgagcacact ga 42

<210> 24

<211> 46

<212> DNA

<213> Artificial Sequence

<400> 24

ccgccaaaac agccaagctt ttaaactgac tcagtttgtg atctgg 46

Claims (8)

1. The modified 1,3-propanediol production strain is characterized in that: the modified bacterial strain refers to an engineered strain with improved 1,3-propanediol production performance by increasing the pdu microchamber content in the cells of the strain by manual operation. 2. the modified 1,3-propanediol production strain as claimed in claim 1, is characterized in that: the starting bacteria of the modified strain is a wild strain with 1,3-propanediol production capacity or is used for 1 after genetic modification , 3-propanediol production of engineering bacteria. 3. the modified 1,3-propanediol production strain of claim 1, wherein the starting bacteria of the modified strain is selected from Klebsiella, Citrobacter freundii, Clostridium butyricum, Roche Lactobacillus, artificially constructed Escherichia coli or Corynebacterium glutamicum with the ability to produce 1,3-propanediol. 4. The modified 1,3-propanediol production strain of claim 1, wherein the method for improving the pdu microchamber content in the cells of the strain adopts at least one of the following methods: (1) The pdu microchamber encoding gene is connected to the expression plasmid through molecular biological operations, and the expression plasmid is transferred into the 1,3-propanediol production strain; (2) Recombining the pdu microchamber-encoding gene into the chromosome of the 1,3-propanediol-producing strain by molecular manipulation; (3) by replacing the promoter of the pdu microchamber-encoding gene on the chromosome of the 1,3-propanediol-producing strain with a high-level promoter; (4) PocR, a regulatory gene that expresses pdu microchamber-encoding genes at a high level. 5. The modified 1,3-propanediol production strain according to any one of claims 1 to 4, wherein the pdu microchamber is a microchamber that metabolizes 1,2-propanediol in bacteria. 6. Application of the modified 1,3-propanediol producing strain of any one of claims 1-4 in the production of 1,3-propanediol. 7. The method for producing 1,3-propanediol by the modified 1,3-propanediol production strain of any one of claims 1-4, wherein the modified strain is inoculated into a medium containing a carbon source to carry out Fermentation culture, the bacteria convert the carbon source in the medium into 1,3-propanediol. 8 . The method for producing 1,3-propanediol by the modified 1,3-propanediol production strain of claim 7 , wherein the culture medium uses glycerol or glucose as the main carbon source. 9 .