KR101166026B1 - A microorganism of Enterobacteriacae genus haboring genes associated with L-carintine biosynthesis and method of producing L-carnitine using the microorganism - Google Patents

Abstract

The present invention relates to polynucleotides encoding N-trimethyllysine hydroxylase activity derived from Neurospora crassa , polynucleotides encoding 3-hydroxy-6-N-trimethyllysine aldolase activity. microorganisms belonging to the genus Enteroceracete comprising a polynucleotide encoding γ-trimethylaminoaldehyde dehydrogenase activity and a polynucleotide encoding γ-butylbetaine hydroxylase activity and L-carnitine using the same. It provides a method of manufacturing.

L-Carnitine, Neurospora Krasa

Description

A microorganism of Enterobacteriacae genus haboring genes associated with L-carintine biosynthesis and method of producing L-carnitine using the microorganism}

1 is a diagram showing the biosynthetic pathway of L-carnitine estimated in neurospora Krasa.

2 shows the construction of pT7-7 carB .

3 shows the construction of pT7-7 carC .

4 shows the construction of pT7-7 carD .

5 shows the construction of pT7-7 carE .

Figure 6 is a photograph confirmed by electrophoresis of each gene inserted in pT7-7 carB , pT7-7 carC , pT7-7 carD , and pT7-7 carE . In Figure 6, lane 1: marker, 2: carD , 3: carC , 4: carD, and 5: carE .

7 is a diagram showing the result of the crude extract obtained by culturing Escherichia coli BL21 (DE3) transformed with pT7-7 carB , pT7-7 carC , pT7-7 carD , and pT7-7 carE , respectively, and performing SDS-PAGE. to be. In Figure 7, lane 1: marker, 2: negative control, 3: TMLH (52 KDa), 4: SHMT (53 KDa), 5: TMABADH (55 KDa) and 6: BBH (49 KDa).

Fig. 8 shows the construction of pT7-7BE.

9 is a diagram illustrating a construction of pACYC184CD.

The present invention relates to a microorganism of the genus Enterobacteria containing neurospora Krasa-derived L-carnitine biosynthesis-related genes and a method for producing L-carnitine using the same.

L-carnitine (3-hydroxy-4-trimethylaminobutyrate) is a compound commonly present in organisms that delivers activated long-chain fatty acids across the mitochondrial inner membrane to the mitochondrial matrix, a zwitterionic compound to be. In vivo, L-carnitine is known to be synthesized from lysine or lysine in proteins (hereinafter referred to as protein lysine). In mammals, protein lysine is generally used as a precursor of L-carnitine biosynthesis, but neurospora Krasa is characterized by the use of free lysine. In the biosynthesis process of L-carnitine biosynthesis, ε-N, N, N-trimethyllysine, ε-N, N, N-trimethyl-β-hydroxylysine, N, N, N-trimethylamino butyraldehyde intermediate, and γ-butyrobetaine is formed. γ-butyrobetaine is hydroxylated by γ-butyrobetaine hydroxylase to L-carnitine. 1 is a diagram showing the estimated biosynthetic pathway of L-carnitine in Neurospora Krasa.

L-carnitine can be produced by chemical synthesis, semisynthesis by enzymatic reaction, and production of L-carnitine using microorganisms. However, in the case of the chemical synthesis method, since racemates of DL-carnitine are obtained, there is a problem of separating them. As a semisynthetic method by an enzymatic reaction, for example, US Pat. No. 4,221,869 discloses a process for producing L-carnitine from dehydrocarnitine with carnitine dehydrogenase (EC 1.1.1.108) using NAD as coenzyme. However, dehydrocarnitine is very unstable and spontaneously decomposes into acetonyl trimethylammonium and carbon dioxide. German Patent DE-OS-3123975 also discloses a method for producing L-carnitine from γ-butyrobetaine with γ-butyrobetaine hydroxylase (EC 1.14.11.1) isolated from neurospora krasa. Is disclosed. However, there was a drawback that α-ketoglutarate and a reducing agent (ie, ascorbate) had to be added in the reaction during the hydroxylation reaction.

As a method for producing L-carnitine using microorganisms, U. S. Patent No. 5,028, 538 cultures Escherichia coli 044 K 74 in a medium containing crotonovebetaine (4-N, N, N-triethylamino crotonic acid), followed by culturing. A method for recovering L-carnitine from water is disclosed. In addition, U.S. Patent No. 4,708,936 discloses L-carnitine by culturing Achromobacter xylosoxydans DSM 3225 (HK 1331b) in a medium containing crotonobbetaine and / or γ-butyrobetaine. A method is disclosed. However, these methods require the use of compounds other than precursors or intermediates of L-carnitine biosynthesis, such as crotonovebetaine, and disadvantages of low L-carnitine production efficiency. Therefore, there remains a need to improve the production efficiency in the method for producing L-carnitine using microorganisms.

The inventors of the present invention have been able to use inexpensive precursors, and while trying to manufacture microorganisms with high production efficiency of L-carnitine, the genes involved in the L-carnitine biosynthesis pathway derived from neurospora krasa are well known in microorganisms of the genus Enterocera bacteria. It was found to be expressed and came to complete the present invention.

It is an object of the present invention to provide a microorganism capable of producing L-carnitine with high efficiency.

It is also an object of the present invention to provide a method for producing L-carnitine using the microorganism.

The present invention relates to polynucleotides encoding N-trimethyllysine hydroxylase (TMLH) activity derived from Neurospora crassa ,

Polynucleotides encoding 3-hydroxy-6-N-trimethyllysine aldolase (SHMT) activity,

polynucleotides encoding γ-trimethylaminoaldehyde dehydrogenase (TMABADH) activity and

Provided are microorganisms belonging to the genus Enterobacteriaceae with polynucleotides encoding γ-butyrobetaine hydroxylase (BBH) activity.                     

The microorganism of the present invention includes any of those containing polynucleotides encoding the four kinds of proteins. Preferably, the microorganism is Escherichia coli , more preferably Escherichia coli (Accession No. KCCM-10581).

The four kinds of proteins of the present invention, that is, polynucleotides encoding TMLH, SHMT, TMABADH and BBH, respectively, may be introduced into a microorganism through a vector or as such. When the polynucleotides encoding the four kinds of proteins are introduced into the microorganism through the vector, the polynucleotides encoding the four kinds of proteins are included in a single vector or introduced in one or more vectors. Can be. In the present invention, a vector is used as a meaning well known in the art. Vectors are generally constructs of nucleic acids used to introduce nucleic acids into cells. The construct of such nucleic acid is preferably a nucleic acid construct from a plasmid or viral genome.

The polynucleotide encoding N-trimethyllysine hydroxylase (TMLH) derived from Neurospora crassa used in the present invention is N-trimethyl derived from neurospora crasa. Encodes lysine hydroxylase (TMLH). N-trimethyllysine hydroxylase (TMLH) converts ε-N-trimethyllysine to β-hydroxy-ε-N-trimethyllysine to γ-N-trimethylaminobutyraldehyde in neurospora Krasa cells. Although believed to catalyze the reaction, the scope of the present invention is not limited to this particular mechanism of action. The polynucleotide encoding the N-trimethyllysine hydroxylase (TMLH) is preferably a polynucleotide encoding the amino acid sequence of SEQ ID NO: 13. More preferably, the polynucleotide has a nucleotide sequence of SEQ ID NO.

Poly encoding 3-hydroxy-6-N-trimethyllysine aldolase (SHMT) derived from Neurospora crassa used in the present invention The nucleotides encode 3-hydroxy-6-N-trimethyllysine aldolase (SHMT) from Neurospora Krasa. 3-hydroxy-6-N-trimethyllysine aldolase (SHMT) converts β-hydroxy-ε-N-trimethyllysine to γ-N-trimethylaminobutyraldehyde in neurospora Krasa cells Although believed to catalyze the scope of the invention is not limited to this particular mechanism of action. The polynucleotide encoding the 3-hydroxy-6-N-trimethyllysine aldolase (SHMT) is preferably a polynucleotide encoding the amino acid sequence of SEQ ID NO: 14. More preferably, it is a polynucleotide having the nucleotide sequence of SEQ ID NO: 18.

The polynucleotide encoding γ-trimethylaminoaldehyde dehydrogenase activity (TMABADH) derived from Neurospora crassa used in the present invention is γ derived from Neurospora Krasa. -Encodes trimethylaminoaldehyde dehydrogenase activity (TMABADH). Although γ-trimethylaminoaldehyde dehydrogenase activity (TMABADH) is believed to catalyze the reaction of converting γ-N-trimethylaminobutyraldehyde to γ-butyrobetaine in neurospora Krasa cells, The scope is not limited to this particular mechanism of action. The polynucleotide encoding the γ-trimethylaminoaldehyde dehydrogenase activity (TMABADH) is preferably a polynucleotide encoding the amino acid sequence of SEQ ID NO: 15. More preferably, they are polynucleotides having the nucleotide sequence of SEQ ID NO: 19.

Polynucleotides encoding γ-butyrobetaine hydroxylase (BBH) activity derived from Neurospora crassa used in the present invention may be derived from Neurospora Krasa . encodes γ-butylobetaine hydroxylase (BBH). Although γ-butylobetaine hydroxylase (BBH) is believed to catalyze the reaction of converting γ-butyrobetaine to L-carnitine in neurospora Krasa cells, the scope of the present invention is directed to this specific mechanism of action. It is not limited to. The polynucleotide encoding the γ-butylobetaine hydroxylase (BBH) is preferably a polynucleotide encoding the amino acid sequence of SEQ ID NO. More preferably, the polynucleotide has a nucleotide sequence of SEQ ID NO: 20.

The invention also relates to microorganisms according to the invention comprising ε-N-trimethyllysine, β-hydroxy-N-trimethyllysine, γ-N-trimethylaminobutyraldehyde, γ-butyrobetaine and mixtures thereof. It provides a method for producing L-carnitine, comprising the step of producing L-carnitine in the culture by culturing in the presence of a substrate selected from the group consisting of.

In the L-carnitine production method of the present invention, the microorganism according to the present invention is as described above.

In the method for producing L-carnitine of the present invention, the group consisting of the above-mentioned N-trimethyllysine, β-hydroxy-N-trimethyllysine, γ-N-trimethylaminobutyraldehyde, γ-butyrobetaine and mixtures thereof The concentration of the substrate selected from is not particularly limited, but is preferably 0.1 to 10% by weight relative to the weight of the culture medium.

In the method of the present invention, L-carnitine in culture can be separated and purified by a recovery process. Such separation and purification procedures are well known in the art. In this method, for example, cells are separated from cultures such as ultrafiltration, centrifugation and decantation to obtain a supernatant, and the obtained supernatant is recovered by L-carnitine by cation exchange chromatography or recrystallization after electrodialysis. Methods include, but are not limited to:

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

In this example, polynucleotides encoding four kinds of proteins related to the biosynthesis of L-carnitine were selected from neurospora krasa, and a nucleic acid construct comprising the same was prepared. Next, E. coli was transformed into these constructs, and the transformed E. coli obtained therefrom was cultured in a medium containing an intermediate product of the L-carnitine production route to produce L-carnitine and then recovered.                     

Example 1 Isolation of Polynucleotides Encoding TMLH, SHMT, TMABADH and BBH from Neurospora Krasa

In this example, polynucleotides encoding TMLH, SHMT, TMABADH and BBH were isolated from neurospora krasa, cloned, and their nucleotide sequences were analyzed.

(1) Construction of cDNA Library of Neurospora Krasa

Total mRNA was isolated from cultures containing neurospora Krasa cells (including spores), reverse transcription was carried out using poly T primers, and then PCR reactions were performed to amplify cDNA. The amplified cDNA was treated with EcoRI and XhoI, and then inserted into the EcoRI and XhoI sites of the λAD5 cloning vector to prepare a neurospora Krasa-derived cDNA library.

Next, the cDNA library was infected with E. coli BNN322, and then cultured and amplified E. coli BNN322. First, E. coli BNN322 was incubated overnight in LB medium containing 50 μg / ml of kanamycin and 0.2% glucose. The resulting culture was centrifuged, then the supernatant was removed and the cell pellet was resuspended in 1 ml of 10 mM MgSO ₄ . The resulting suspension and the λ cDNA library of 5 × 10 ⁷ PFU were incubated at 30 ° C. for 30 minutes without shaking. 2 ml of LB medium was further added to the culture and shaken in a shake incubator at 30 ° C. for 1 hour. Cultured cells were plated in LB medium plates containing ampicillin (75 μg / ml) and incubated at 37 ° C. for 8 hours. CDNA library pools were isolated from the colonies of the plates using the Wizard kit. [Lambda] comprising such isolated cDNA library pool was used as a template for amplifying polynucleotides encoding TMLH, SHMT, TMABADH and BBH.

(2) Confirmation of amplification, cloning and TMLH production of polynucleotides encoding TMLH ( carB gene)

(a) Amplification and cloning of polynucleotides encoding the TMLH ( carB gene)

PCR was performed using λ containing the cDNA library pool of (1) as a template and SEQ ID NOs: 1 and 2 as primers. The resulting PCR product was subjected to agarose gel electrophoresis, confirming the desired product of about 1.4 kb. The primers of SEQ ID NOs: 1 and 2 include sequences estimated to be sequences encoding start codons and stop codons of neurospora Krasa-derived TMLH, which are known as aminospores of human and rat-derived TMLH. By searching for homology with the amino acid sequence of the entire protein expressed from the genome, neurospora Krasa derived TMLH was designed from this.

The PCR product was digested with EcoRI and SalI, linked to pBS KS ⁺ (Stratagene Inc.) treated with the same enzyme, and transformed into E. coli DH5α with pBS KS ⁺ carB inserted with the obtained PCR product. The transformed E. coli DH5α was incubated at 37 ° C. for 8 hours, and then pBS KS ⁺ carB was isolated and treated with EcoRI and SalI to confirm that the PCR product was correctly inserted. Next, the isolated pBS KS ⁺ carB was treated with NdeI and SalI, followed by electrophoresis on an agarose gel, and then NdeI and SalI fragments were separated. The fragment was linked to the expression vector pT7-7 treated with the same enzyme to obtain pT7-7 carB (see FIG. 2). E. coli BL21 (DE3) was transformed with the pT7-7 carB .

(b) Confirmation of TMLH Generation

E. coli BL21 (DE3) transformed with pT7-7 carB thus obtained was incubated at 37 ° C. in a 250 ml baffle flask filled with 50 ml of LB medium supplemented with ampicillin (100 μg / ml) until the OD ₆₀₀ value was 0.6. Then, IPTG (1 mM) was added, followed by further 4 hours of incubation. PT7-7 carB was isolated from the culture and treated with NdeI and SalI and electrophoresed on agarose gel. The results are shown in FIG. As shown in FIG. 6, the bands corresponding to the NdeI and SalI fragments were identified (lane 2). Next, pT7-7 carB was isolated and analyzed for the nucleotide sequence of carB , and found to be identical to the sequence stored in the neurospora Krasa genomic database of NCBI (SEQ ID NO: 17).

In addition, the activity of TMLH expressed in the culture of Escherichia coli BL21 (DE3) transformed with pT7-7 carB was investigated. First, the culture was centrifuged at 4,000 xg for 15 minutes to recover cell pellets. The resulting cell pellet was added to 1 ml of lysis buffer (140 mM NaCl in 200 mM sodium phosphate buffer pH 7.4, 200 g / l glycerol, and 1 mM DTT) and resuspended. The cell suspension was immersed in an ice bath and sonicated five times for 10 seconds using an ultrasonic mill to disrupt cells. The cell debris was centrifuged at 10,000 g at 4 ° C. for 20 to 30 minutes, then cell debris was removed and the supernatant was recovered to obtain a crude crude extract. Samples were taken from the obtained crude cell extracts and subjected to 8% SDS-PAGE (see FIG. 7). SDS-PAGE showed a band corresponding to a TMLH of about 52 KDa.

(3) Confirmation of amplification, cloning and SHMT production of polynucleotide ( carC ) encoding 3-hydroxy-6-N-trimethyllysine aldolase (SHMT)

(a) Amplification and cloning of polynucleotides ( carC ) encoding 3-hydroxy-6-N-trimethyllysine aldolase (SHMT)

PCR was performed using λ including the cDNA library pool of (1) as a template and SEQ ID NOs: 3 and 4 as primers. The resulting PCR product was subjected to agarose gel electrophoresis, confirming the desired product of about 1.4 kb. The primers of SEQ ID NOs: 3 and 4 include sequences estimated to be sequences encoding start codons and stop codons of neurospora Krasa-derived SHMTs, which are known as neurospora amino acid sequences of human and rat-derived SHMTs. By searching for homology with the amino acid sequence of the whole protein expressed from the genome, SHSP derived neurospora Krasa was designed from this.                     

The PCR product was digested with EcoRI and SalI, linked to pBS KS ⁺ (Stratagene Inc.) treated with the same enzyme, and transformed into E. coli DH5α with pBS KS ⁺ carC inserted with the resulting PCR product. The transformed E. coli DH5α was incubated at 37 ° C. for 8 hours, and then pBS KS ⁺ carC was isolated and treated with EcoRI and SalI to confirm that the PCR product was properly inserted. Next, the isolated pBS KS ⁺ carC was treated with NdeI and SalI, followed by electrophoresis on an agarose gel, and then NdeI and SalI fragments were separated. The fragment was linked to expression pT7-7 treated with the same enzyme to obtain pT7-7 carC (see FIG. 3). E. coli BL21 (DE3) was transformed with the pT7-7 carC .

(b) Confirmation of SHMT Generation

E. coli BL21 (DE3) transformed with pT7-7 carC thus obtained was incubated at 37 ° C. in a 250 ml baffle flask filled with 50 ml of LB medium supplemented with ampicillin (100 μg / ml) until the OD ₆₀₀ value was 0.6. Then, IPTG (1 mM) was added, followed by further 4 hours of incubation. PT7-7 carC was isolated from the culture and treated with NdeI and SalI and electrophoresed on agarose gel. The results are shown in FIG. As shown in FIG. 6, the bands corresponding to the NdeI and SalI fragments were identified (lane 3). Next, pT7-7 carC was isolated and analyzed for the nucleotide sequence of carC . As a result, it was confirmed that it was identical to the sequence stored in the neurospora Krasa genomic database of NCBI (SEQ ID NO: 18).

In addition, the activity of SHMT expressed in the culture of Escherichia coli BL21 (DE3) transformed with pT7-7 carC was investigated. First, the culture was centrifuged at 4,000 xg for 15 minutes to recover cell pellets. The resulting cell pellet was added to 1 ml of lysis buffer (140 mM NaCl in 200 mM sodium phosphate buffer pH 7.4, 200 g / l glycerol, and 1 mM DTT) and resuspended. The cell suspension was immersed in an ice bath and sonicated five times for 10 seconds using an ultrasonic mill to disrupt cells. The cell debris was centrifuged at 10,000 g at 4 ° C. for 20 to 30 minutes, then cell debris was removed and the supernatant was recovered to obtain a crude crude extract. Samples were taken from the obtained crude cell extracts and subjected to 8% SDS-PAGE (see FIG. 7). SDS-PAGE showed a band corresponding to SHMT of about 53 KDa.

(4) Amplification, cloning of polynucleotide ( carD ) encoding γ-trimethylaminoaldehyde dehydrogenase (TMABADH), confirmation of TMABADH production

(a) Amplification and cloning of polynucleotides ( carD ) encoding γ-trimethylaminoaldehyde dehydrogenase (TMABADH)

PCR was performed using λ comprising the cDNA library pool of (1) as a template and SEQ ID NOs: 5 and 6 as primers. The resulting PCR product was subjected to agarose gel electrophoresis, confirming the desired product of about 1.5 kb. The primers of SEQ ID NOs: 5 and 6 include sequences estimated to be sequences encoding initiation and termination codons of neurospora Krasa-derived TMABDH. Through homology with amino acid sequences of whole proteins expressed from the genome, neurospora Krasa-derived TMABDH was designed and designed therefrom.

The PCR product was digested with EcoRI and SalI, linked to pBS KS ⁺ (Stratagene Inc.) treated with the same enzyme, and transformed into E. coli DH5α with pBS KS ⁺ carD inserted with the obtained PCR product. The transformed E. coli DH5α was incubated at 37 ° C. for 8 hours, and then pBS KS ⁺ carD was isolated and treated with EcoRI and SalI to confirm that the PCR product was properly inserted. Next, the isolated pBS KS ⁺ carD was treated with NdeI and SalI, followed by electrophoresis on an agarose gel, and then NdeI and SalI fragments were separated. The fragment was linked to expression pT7-7 treated with the same enzyme to obtain pT7-7 carD (see FIG. 4). E. coli BL21 (DE3) was transformed with the pT7-7 carD .

(b) Confirmation of TMABADH production

E. coli BL21 (DE3) transformed with pT7-7 carD thus obtained was cultured in a 250 ml baffle flask filled with 50 ml of LB medium supplemented with ampicillin at 37 ° C. until the OD ₆₀₀ value was 0.6, followed by IPTG (1 mM) was added and then incubated for another 4 hours. PT7-7 carD was isolated from the culture and treated with NdeI and SalI and electrophoresed on agarose gel. The results are shown in FIG. As shown in FIG. 6, the bands corresponding to the NdeI and SalI fragments were identified (lane 4). Next, pT7-7 carD was isolated and analyzed for the nucleotide sequence of carD , and it was confirmed that it was identical to the sequence stored in the neurospora Krasage genome database of NCBI (SEQ ID NO: 19).

In addition, the activity of TMABADH expressed in the culture of Escherichia coli BL21 (DE3) transformed with pT7-7 carD was investigated. First, the culture was centrifuged at 4,000 xg for 15 minutes to recover cell pellets. The resulting cell pellet was added to 1 ml of lysis buffer (140 mM NaCl in 200 mM sodium phosphate buffer pH 7.4, 200 g / l glycerol, and 1 mM DTT) and resuspended. The cell suspension was immersed in an ice bath and sonicated five times for 10 seconds using an ultrasonic mill to disrupt cells. The cell debris was centrifuged at 10,000 g at 4 ° C. for 20 to 30 minutes, then cell debris was removed and the supernatant was recovered to obtain a crude crude extract. Samples were taken from the obtained crude cell extracts and subjected to 8% SDS-PAGE (see FIG. 7). SDS-PAGE showed a band corresponding to TMABADH of about 55 KDa.

(5) Confirmation of amplification, cloning and titer of polynucleotide ( carE ) encoding γ-butyrobetaine hydroxylase (BBH)

(a) Amplification and cloning of polynucleotides ( carE ) encoding γ-butyrobetaine hydroxylase (BBH)

PCR was performed using λ comprising the cDNA library pool of (1) as a template and SEQ ID NOs. 7 and 8 as primers. The resulting PCR product was subjected to agarose gel electrophoresis, confirming the desired product of about 1.3 kb. The primers of SEQ ID NOs: 7 and 8 include sequences estimated to be sequences encoding start codons and stop codons of neurospora Krasa-derived BBH, which are known as neurospora amino acid sequences of known human and rat-derived BBHs. By searching for homology with the amino acid sequence of the whole protein expressed from the genome, neurospora Krasa-derived BBH was estimated and designed therefrom.

The PCR product was digested with EcoRI and SalI, linked to pUC19 treated with the same enzyme, and transformed into E. coli DH5α with pUC19 carE inserted with the obtained PCR product. The transformed Escherichia coli DH5α was incubated for 8 hours at 37 ° C. in LB medium to which ampicillin (100 μg / ml) was added, and then the pUC19 carE was isolated and treated with EcoRI and SalI to confirm that the PCR product was correctly inserted. . Next, the isolated pUC19 carE was treated with NdeI and SalI, followed by electrophoresis on an agarose gel, and then NdeI and SalI fragments were separated. The fragment was linked to expression pT7-7 treated with the same enzyme to obtain pT7-7 carE (see FIG. 5). E. coli BL21 (DE3) was transformed with the pT7-7 carE .

E. coli BL21 (DE3) transformed with pT7-7 carE thus obtained was incubated at 37 ° C. in a 250 ml baffle flask filled with 50 ml of LB medium supplemented with ampicillin (100 μg / ml) until the OD ₆₀₀ value was 0.6. Then, IPTG (1 mM) was added, followed by further 4 hours of incubation. PT7-7 carE was isolated from the culture and treated with NdeI and SalI and electrophoresed on 0.8% agarose gel. The results are shown in FIG. As shown in FIG. 6, bands corresponding to the NdeI and SalI fragments were identified. Next, pT7-7 carE was isolated and the nucleotide sequence (1278 bp) of carE was analyzed, and it was confirmed that it was identical to the sequence stored in NCBI's Neurospora Krasa genomic database (SEQ ID NO: 20).

(b) Confirmation of BBH protein production

The activity of BBH expressed in culture of Escherichia coli BL21 (DE3) transformed with pT7-7 carE was investigated. First, the culture was centrifuged at 4,000 xg for 15 minutes to recover cell pellets. The resulting cell pellet was added to 1 ml of lysis buffer (140 mM NaCl in 200 mM sodium phosphate buffer pH 7.4, 200 g / l glycerol, and 1 mM DTT) and resuspended. The cell suspension was immersed in an ice bath and sonicated five times for 10 seconds using an ultrasonic mill to disrupt cells. Cell debris was centrifuged at 10,000 g at 4 ° C. for 20 to 30 minutes, then cell debris was removed and the supernatant was recovered to obtain cell crude extract. Samples were taken from the obtained crude cell extracts and subjected to 8% SDS-PAGE (see FIG. 7). SDS-PAGE showed a BBH of about 49 KDa.

Example 2 Preparation of Host Cells Containing carB , carC , carD and carE

In this example, carB and carE genes were amplified from the neurospora Krasa cDNA library prepared in Example 1, and pT7-7BE having these two genes was prepared. In addition, carC and carD genes were amplified from the neurospora Krasa cDNA library prepared in Example 1, and pACYC184CD having these two genes was prepared. PT7-7BE and pACYC184CD thus prepared were introduced into E. coli strains to prepare transformed strain cells having all of carB , carC , carD and carE genes. The strain was named Escherichia coli DH5α CJ2004, and was deposited on January 27, 2004 at the Korea Microorganism Conservation Center (Accession No. KCCM-10581).

(1) Preparation of pT7-7BE Having CarB and CarE Genes Simultaneously

First, the neurospora Krasa cDNA library was used as a template, and carB was amplified using oligonucleotides of SEQ ID NOs: 1 and 2 as primers. Next, carE containing a stop codon was amplified from the T7 promoter using a neurospora Krasa cDNA library as a template and oligonucleotides of SEQ ID NOs: 7 and 8 as primers. The amplified products of carB and E thus amplified were introduced into pT7-7. First, the carE amplification product was treated with BamHI and SalI to obtain BamHI and SalI fragments, which were then linked with pT7-7 treated with the same enzyme to obtain pT7-7 carE . Next, carB amplification products were treated with NdeI and EcoRI to obtain NdeI and EcoRI fragments, which were then linked with pT7-7 carE treated with the same enzyme to obtain pT7-7BE (see FIG. 8).

(2) Preparation of pACYC184CD Having CarC and CarD Genes Simultaneously

First, carC containing a stop codon was amplified from the T7 promoter using a neurospora Krasa cDNA library as a template and oligonucleotides of SEQ ID NOs: 3 and 4 as primers. Next, the neurospora Krasa cDNA library was used as a template, and the carD containing the stop codon was amplified from the T7 promoter using oligonucleotides of SEQ ID NOs: 5 and 6 as primers. The amplified products of carC and D thus amplified were introduced into pACYC184. First, the carC amplification product was treated with BamHI and HIndIII to obtain BamHI and HindIII fragments, which were linked with pACYC184 treated with the same enzyme to obtain pACYC184 carC . Next, the carD amplification product was treated with BamHI and SalI to obtain BamHI and SalI fragments, which were then linked with pACYC184 carC treated with the same enzyme to obtain pACYC184CD (see FIG. 9).

Example 3 Production of L-Carnitine Using Strains Comprising Polynucleotides Encoding TMLH, SHMT, TMABADH and BBH

In this example, E. coli BL21 (DE3) transformed with pT7-7 carB , pT7-7 carC , pT7-7 carD , and pT7-7 carE produced in Example 1, respectively, was mixed and cultured in a medium containing trimethyllysine. L-carnitine production was confirmed. In addition, L-carnitine production was confirmed by culturing Escherichia coli BL21 (DE3) transformed simultaneously with pT7-7BE and pACYC184CD prepared in Example 2.

(1) Mixed culture of Escherichia coli BL21 (DE3) transformed with pT7-7 carB , pT7-7 carC , pT7-7 carD , and pT7-7 carE , respectively

First, each strain was plated and cultured in LB solid plate medium to which ampicillin (100 μg / ml) was added. Colonies of each strain were incubated for 12 hours at 37 ° C. in a flask containing 20 ml of LB medium to which ampicillin (100 μg / ml) was added until OD ₆₀₀ became 1.0. Cultures of each of these cultured strains were then added in equal amounts (0.1 ml each) to a 250 ml baffle flask containing 20 ml of LB medium supplemented with ampicillin (100 μg / ml) containing 2 mM trimethyllysine and 37 The cells were incubated at an OD ₆₀₀ value of 0.6. In case of adding IPTG, after the OD ₆₀₀ value was 0.6, IPTG (1 mM) was added and incubated for 4 hours. LB medium without trimethyl lysine was used as a control, and cultured by adding IPTG in the same manner.

After the incubation was completed, the content of L-carnitine in the culture was measured. 500 μl of culture supernatant was taken and mixed with 500 μl of 1.2 M perchloric acid. The mixed solution was incubated at room temperature for 10 minutes and then centrifuged for 5 minutes. 600 μl of supernatant and 320 μl of 0.7 M K ₃ PO ₄ were mixed and left in an ice bath for 20 minutes. The mixture is centrifuged for 5 minutes, only 750 μl of supernatant is taken and diluted with 250 μl of sterile distilled water. 100 μl of DNTB / H ₂ O ₂ was added to the solution and left for 10 minutes. 50 μl of the catalase solution was added and allowed to stand at room temperature for 30 minutes, followed by centrifugation to recover 1 ml of the supernatant. 50 [mu] l of acetyl CoA was added thereto and allowed to stand at room temperature for 5 minutes. 2.26 μl of carnitine acetyl transferase was added thereto and allowed to stand at room temperature for 10 minutes. After measuring absorbance at 405 nm, the amount of L-carnitine was calculated. The results are shown in Table 1 below.

Table 1. Production of L-Carnitine by Mixed Culture

Culture condition Concentration (㎍ / ml) LB medium (IPTG induction) 0 LB medium containing 2 mM trimethyllysine (no IPTG induction) 0.16 LB medium containing 2 mM trimethyllysine (IPTG induction) 0.97

As shown in Table 1, L-carnitine was produced at high efficiency by mixing and culturing strains containing polynucleotides encoding TMLH, SHMT, TMABADH and BBH in a medium containing trimethyllysine.

(2) Production of L-carnitine by culturing Escherichia coli BL21 (DE3) simultaneously transformed with pT7-7BE and pACYC184CD prepared in Example 2

First, each strain was incubated in LB solid plate medium to which ampicillin (100 μg / ml) and chloramphenicol (50 μg / ml) were added. Colonies of each strain were incubated at 37 ° C. for 12 hours in a flask containing 20 ml of LB medium supplemented with ampicillin (100 μg / ml) and chloramphenicol (50 μg / ml), respectively, to give an OD ₆₀₀ of 1.0. Incubated until. 0.1 ml of each strain cultured in this way was added to a 250 ml baffle flask containing 20 ml of LB medium containing 2 mM trimethyllysine and incubated at 37 ° C. until the OD ₆₀₀ value was 0.6. In case of adding IPTG, after the OD ₆₀₀ value was 0.6, IPTG (1 mM) was added and incubated for 4 hours. LB medium without trimethyl lysine was used as a control, and cultured by adding IPTG in the same manner.

After the incubation was completed, the content of L-carnitine in the culture was measured in the same manner as in (1) above. The results are shown in Table 2 below.

Table 2. Production of L-Carnitine by Single Culture

Culture condition Concentration (㎍ / ml) LB medium (IPTG induction) 0 LB medium containing 2 mM trimethyllysine (no IPTG induction) 0.65 LB medium containing 2 mM trimethyllysine (IPTG induction) 1.12

As shown in Table 2, L-carnitine could be produced with high efficiency by culturing a strain containing all polynucleotides encoding TMLH, SHMT, TMABADH and BBH in a medium containing trimethyllysine. Moreover, when compared with the production amount of L-carnitine shown in Table 1, it turned out that the production efficiency is high by the single culture.

According to the microorganism according to the present invention, it is excellent in L-carnitine production ability can be usefully used in the method for producing L-carnitine through fermentation.

According to the method for producing L-carnitine according to the present invention, L-carnitine can be produced with high efficiency by using microorganisms belonging to the genus Enterobacteriaceae.

<110> CJ Corporation <120> A microorganism of Enterobacteriacae genus haboring genes          associated with L-carintine biosynthesis and method of producing          L-carnitine using the microorganism <130> PN054899 <160> 20 <170> Kopatentin 1.71 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 atgaattcca tatgagaccg caagtggtag gg 32 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 atgaattctc attttccgct ggtttctttc cg 32 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 atgaattcca tatgtctacc tactccctct cc 32 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 attgtcgact tagagaccgg catcgtatct 30 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 atgaattcca tatggaagtc gagcttacgg cc 32 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 attgtcgact catgccgcca ggtttacatg gat 33 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 atgaattcca tatgatggcc acggcagcgg ttcag 35 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 attagtcgac tcaataccct cccccaccct g 31 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 9 atggatccta atacgactca ctataggga 29 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 10 attaagcttt tagagaccgg catcgtatct 30 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 atggatccta atacgactca ctataggga 29 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 12 atggatccta atacgactca ctataggga 29 <210> 13 <211> 471 <212> PRT <213> Neuropora crassa <400> 13 Met Arg Pro Gln Val Val Gly Ala Ile Leu Arg Ser Arg Ala Val Val   1 5 10 15 Ser Arg Gln Pro Leu Ser Arg Thr His Ile Phe Ala Ala Val Thr Val              20 25 30 Ala Lys Ser Ser Ser Pro Ala Gln Asn Ser Arg Arg Thr Phe Ser Ser          35 40 45 Ser Phe Arg Arg Leu Tyr Glu Pro Lys Ala Glu Ile Thr Ala Glu Gly      50 55 60 Leu Glu Leu Ser Pro Pro Gln Ala Val Thr Gly Gly Lys Arg Thr Val  65 70 75 80 Leu Pro Asn Phe Trp Leu Arg Asp Asn Cys Arg Cys Thr Lys Cys Val                  85 90 95 Asn Gln Asp Thr Leu Gln Arg Asn Phe Asn Thr Phe Ala Ile Pro Ser             100 105 110 Asp Ile His Pro Thr Lys Val Glu Ala Thr Lys Glu Asn Val Thr Val         115 120 125 Gln Trp Ser Asp Asn His Thr Ser Thr Tyr Pro Trp Pro Phe Leu Ser     130 135 140 Phe Tyr Leu Thr Ser Asn Ala Arg Gly His Glu Asn Asp Gln Ile Ser 145 150 155 160 Leu Trp Gly Ser Glu Ala Gly Ser Arg Pro Pro Thr Val Pro Phe Pro                 165 170 175 Arg Val Met Ala Ser Asp Gln Gly Val Ala Asp Leu Thr Ala Met Ile             180 185 190 Lys Glu Phe Gly Phe Cys Phe Val Lys Asp Thr Pro His Asp Asp Pro         195 200 205 Asp Val Thr Arg Gln Leu Leu Glu Arg Ile Ala Phe Ile Arg Val Thr     210 215 220 His Tyr Gly Gly Phe Tyr Asp Phe Thr Pro Asp Leu Ala Met Ala Asp 225 230 235 240 Thr Ala Tyr Thr Asn Leu Ala Leu Pro Ala His Thr Asp Thr Thr Tyr                 245 250 255 Phe Thr Asp Pro Ala Gly Leu Gln Ala Phe His Leu Leu Glu His Lys             260 265 270 Ala Ala Pro Ser Arg Pro Pro Pro Pro Pro Pro Pro Pro Pro         275 280 285 Ser Glu Glu Lys Glu Ala Ala Gly Ser Ala Ala Gly Glu Ala Ala Ala     290 295 300 Ala Ala Glu Gly Gly Lys Ser Leu Leu Val Asp Gly Phe Asn Ala Ala 305 310 315 320 Arg Ile Leu Lys Glu Glu Asp Pro Arg Ala Tyr Glu Ile Leu Ser Ser                 325 330 335 Val Arg Leu Pro Trp His Ala Ser Gly Asn Glu Gly Ile Thr Ile Ala             340 345 350 Pro Asp Lys Leu Tyr Pro Val Leu Glu Leu Asn Glu Asp Thr Gly Glu         355 360 365 Leu His Arg Val Arg Trp Asn Asn Asp Asp Arg Gly Val Val Pro Phe     370 375 380 Gly Glu Lys Tyr Ser Pro Ser Glu Trp Tyr Glu Ala Ala Arg Lys Trp 385 390 395 400 Asp Gly Ile Leu Arg Arg Lys Ser Ser Glu Leu Trp Val Gln Leu Glu                 405 410 415 Pro Gly Lys Pro Leu Ile Phe Asp Asn Trp Arg Val Leu His Gly Arg             420 425 430 Ser Ala Phe Ser Gly Ile Arg Arg Ile Cys Gly Gly Tyr Ile Asn Arg         435 440 445 Asp Asp Phe Ile Ser Arg Trp Arg Asn Thr Asn Tyr Pro Arg Ser Glu     450 455 460 Val Leu Pro Arg Val Thr Gly 465 470 <210> 14 <211> 480 <212> PRT <213> Neurospora crassa <400> 14 Met Ser Thr Tyr Ser Leu Ser Glu Thr His Lys Ala Met Leu Glu His   1 5 10 15 Ser Leu Val Glu Ser Asp Pro Gln Val Ala Glu Ile Met Lys Lys Glu              20 25 30 Val Gln Arg Gln Arg Glu Ser Ile Ile Leu Ile Ala Ser Glu Asn Val          35 40 45 Thr Ser Arg Ala Val Phe Asp Ala Leu Gly Ser Pro Met Ser Asn Lys      50 55 60 Tyr Ser Glu Gly Leu Pro Gly Ala Arg Tyr Tyr Gly Gly Asn Gln His  65 70 75 80 Ile Asp Glu Ile Glu Val Leu Cys Gln Asn Arg Ala Leu Glu Ala Phe                  85 90 95 His Leu Asp Pro Lys Gln Trp Gly Val Asn Val Gln Cys Leu Ser Gly             100 105 110 Ser Pro Ala Asn Leu Gln Val Tyr Gln Ala Ile Met Pro Val His Gly         115 120 125 Arg Leu Met Gly Leu Asp Leu Pro His Gly Gly His Leu Ser His Gly     130 135 140 Tyr Gln Thr Pro Gln Arg Lys Ile Ser Ala Val Ser Thr Tyr Phe Glu 145 150 155 160 Thr Met Pro Tyr Arg Val Asn Ile Asp Thr Gly Leu Ile Asp Tyr Asp                 165 170 175 Thr Leu Glu Lys Asn Ala Gln Leu Phe Arg Pro Lys Val Leu Val Ala             180 185 190 Gly Thr Ser Ala Tyr Cys Arg Leu Ile Asp Tyr Glu Arg Met Arg Lys         195 200 205 Ile Ala Asp Ser Val Gly Ala Tyr Leu Val Val Asp Met Ala His Ile     210 215 220 Ser Gly Leu Ile Ala Ser Glu Val Ile Pro Ser Pro Phe Leu Tyr Ala 225 230 235 240 Asp Val Val Thr Thr Thr Thr His Lys Ser Leu Arg Gly Pro Arg Gly                 245 250 255 Ala Met Ile Phe Phe Arg Arg Gly Val Arg Ser Val Asp Ala Lys Thr             260 265 270 Gly Lys Glu Thr Leu Tyr Asp Leu Glu Asp Lys Ile Asn Phe Ser Val         275 280 285 Phe Pro Gly His Gln Gly Gly Pro His Asn His Thr Ile Thr Ala Leu     290 295 300 Ala Val Ala Leu Lys Gln Ala Ala Ser Pro Glu Phe Lys Glu Tyr Gln 305 310 315 320 Gln Lys Val Val Ala Asn Ala Lys Ala Leu Glu Lys Lys Leu Lys Glu                 325 330 335 Leu Gly Tyr Lys Leu Val Ser Asp Gly Thr Asp Ser His Met Val Leu             340 345 350 Val Asp Leu Arg Pro Ile Gly Val Asp Gly Ala Arg Val Glu Phe Leu         355 360 365 Leu Glu Gln Ile Asn Ile Thr Cys Asn Lys Asn Ala Val Pro Gly Asp     370 375 380 Lys Ser Ala Leu Thr Pro Gly Gly Leu Arg Ile Gly Thr Pro Ala Met 385 390 395 400 Thr Ser Arg Gly Phe Gly Glu Ala Asp Phe Glu Lys Val Ala Val Phe                 405 410 415 Val Asp Glu Ala Val Lys Leu Cys Lys Glu Ile Gln Ala Ser Leu Pro             420 425 430 Lys Glu Ala Asn Lys Gln Lys Asp Phe Lys Ala Lys Ile Ala Thr Ser         435 440 445 Asp Ile Pro Arg Ile Asn Glu Leu Lys Gln Glu Ile Ala Ala Trp Ser     450 455 460 Asn Thr Phe Pro Leu Pro Val Glu Gly Trp Arg Tyr Asp Ala Gly Leu 465 470 475 480 <210> 15 <211> 495 <212> PRT <213> Neurospora crassa <400> 15 Met Glu Val Glu Leu Thr Ala Pro Asn Gly Lys Lys Trp Met Gln Pro   1 5 10 15 Leu Gly Leu Phe Ile Asn Asn Glu Phe Val Lys Ser Ala Asn Glu Gln              20 25 30 Lys Leu Ile Ser Ile Asn Pro Thr Thr Glu Glu Glu Ile Cys Ser Val          35 40 45 Tyr Ala Ala Thr Ala Glu Asp Val Asp Ala Ala Val Ser Ala Ala Arg      50 55 60 Lys Ala Phe Arg His Glu Ser Trp Lys Ser Leu Ser Gly Thr Glu Arg  65 70 75 80 Gly Ala Leu Met Arg Lys Leu Ala Asp Leu Val Ala Glu Asn Ala Glu                  85 90 95 Ile Leu Ala Thr Ile Glu Cys Leu Asp Asn Gly Lys Pro Tyr Gln Thr             100 105 110 Ala Leu Asn Glu Asn Val Pro Glu Val Ile Asn Val Leu Arg Tyr Tyr         115 120 125 Ala Gly Tyr Ala Asp Lys Asn Phe Gly Gln Val Ile Asp Val Gly Pro     130 135 140 Ala Lys Phe Ala Tyr Thr Val Lys Glu Pro Leu Gly Val Cys Gly Gln 145 150 155 160 Ile Ile Pro Trp Asn Tyr Pro Leu Asp Met Ala Ala Trp Lys Leu Gly                 165 170 175 Pro Ala Leu Cys Cys Gly Asn Thr Val Val Leu Lys Leu Ala Glu Gln             180 185 190 Thr Pro Leu Ser Val Leu Tyr Leu Ala Lys Leu Ile Lys Glu Ala Gly         195 200 205 Phe Pro Pro Gly Val Ile Asn Ile Ile Asn Gly His Gly Arg Glu Ala     210 215 220 Gly Ala Ala Leu Val Gln His Pro Gln Val Asp Lys Ile Ala Phe Thr 225 230 235 240 Gly Ser Thr Thr Thr Gly Lys Glu Ile Met Lys Met Ala Ser Tyr Thr                 245 250 255 Met Lys Asn Ile Thr Leu Glu Thr Gly Gly Lys Ser Pro Leu Ile Val             260 265 270 Phe Glu Asp Ala Asp Leu Glu Leu Ala Ala Thr Trp Ser His Ile Gly         275 280 285 Ile Met Ser Asn Gln Gly Gln Ile Cys Thr Ala Thr Ser Arg Ile Leu     290 295 300 Val His Glu Lys Ile Tyr Asp Glu Phe Val Glu Lys Phe Lys Ala Lys 305 310 315 320 Val Gln Glu Val Ser Val Leu Gly Asp Pro Phe Glu Glu Ser Thr Phe                 325 330 335 His Gly Pro Gln Val Thr Lys Ala Gln Tyr Glu Arg Val Leu Gly Tyr             340 345 350 Ile Asn Val Gly Lys Glu Glu Gly Ala Thr Val Met Met Gly Gly Glu         355 360 365 Pro Ala Pro Gln Asn Gly Lys Gly Phe Phe Val Ala Pro Thr Val Phe     370 375 380 Thr Asn Val Lys Pro Thr Met Lys Ile Phe Arg Glu Glu Ile Phe Gly 385 390 395 400 Pro Cys Val Ala Ile Thr Thr Phe Lys Thr Glu Glu Glu Ala Leu Thr                 405 410 415 Leu Ala Asn Asp Ser Met Tyr Gly Leu Gly Ala Ala Leu Phe Thr Lys             420 425 430 Asp Leu Thr Arg Ala His Arg Val Ala Arg Glu Ile Glu Ala Gly Met         435 440 445 Val Trp Val Asn Ser Ser Asn Asp Ser Asp Phe Arg Ile Pro Phe Gly     450 455 460 Gly Val Lys Gln Ser Gly Ile Gly Arg Glu Leu Gly Glu Ala Gly Leu 465 470 475 480 Ala Pro Tyr Cys Asn Val Lys Ser Ile His Val Asn Leu Ala Ala                 485 490 495 <210> 16 <211> 425 <212> PRT <213> Neurospora crassa <400> 16 Met Ala Thr Ala Ala Val Gln Val Ser Val Pro Ala Pro Val Gly Gln   1 5 10 15 Pro Asp Ile Gly Tyr Ala Pro Asp His Asp Lys Tyr Leu Ala Arg Val              20 25 30 Lys Arg Arg Arg Glu Asn Glu Lys Leu Glu Ser Ser Leu Pro Pro Gly          35 40 45 Phe Pro Arg Arg Leu Asp Ser Asp Leu Val Trp Asp Gly Asn Thr Leu      50 55 60 Ala Glu Thr Tyr Asp Trp Thr Tyr Arg Leu Thr Glu Glu Ala Ile Asp  65 70 75 80 Glu Ile Glu Ala Ala Leu Arg His Phe Lys Ser Leu Asn Lys Pro Leu                  85 90 95 Gly Tyr Ile Asn Gln Glu Thr Phe Pro Leu Pro Arg Leu His His Thr             100 105 110 Leu Arg Ser Leu Ser His Glu Leu His His Gly His Gly Phe Lys Val         115 120 125 Leu Arg Gly Leu Pro Val Thr Ser His Thr Arg Glu Glu Asn Ile Ile     130 135 140 Ile Tyr Ala Gly Val Ser Ser His Val Ala Pro Ile Arg Gly Arg Gln 145 150 155 160 Asp Asn Gln His Asn Gly His Pro Ala Asp Val Val Leu Ala His Ile                 165 170 175 Lys Asp Leu Ser Thr Thr Val Ser Asp Val Ser Lys Ile Gly Ala Pro             180 185 190 Ala Tyr Thr Thr Glu Lys Gln Val Phe His Thr Asp Ala Gly Asp Ile         195 200 205 Val Ala Leu Phe Cys Leu Gly Glu Ala Ala Glu Gly Gly Gln Ser Tyr     210 215 220 Leu Ser Ser Ser Trp Lys Val Tyr Asn Glu Leu Ala Ala Thr Arg Pro 225 230 235 240 Asp Leu Val Arg Thr Leu Ala Glu Pro Trp Val Ala Asp Glu Phe Gly                 245 250 255 Lys Glu Gly Arg Lys Phe Ser Val Arg Pro Leu Leu His Phe Gln Ser             260 265 270 Thr Ala Ala Ala Ala Ser Arg Glu Ala Lys Pro Glu Ser Glu Arg Leu         275 280 285 Ile Ile Gln Tyr Ala Arg Arg Thr Phe Thr Gly Tyr Trp Gly Leu Pro     290 295 300 Arg Ser Ala Asp Ile Pro Pro Ile Thr Glu Ala Gln Ala Glu Ala Leu 305 310 315 320 Asp Ala Leu His Phe Thr Ala Glu Lys Tyr Ala Val Ala Leu Asp Phe                 325 330 335 Arg Gln Gly Asp Val Gln Phe Val Asn Asn Leu Ser Val Phe His Ser             340 345 350 Arg Ala Gly Phe Arg Asp Glu Gly Glu Lys Gln Arg His Leu Val Arg         355 360 365 Leu Trp Leu Arg Asp Pro Glu Asn Ala Trp Glu Thr Pro Glu Ala Leu     370 375 380 Lys Glu Arg Trp Glu Arg Val Tyr Gly Gly Val Ser Pro Glu Arg Glu 385 390 395 400 Val Phe Pro Leu Glu Pro Gln Ile Arg Ser Ala Ser Lys Gly Glu Ser                 405 410 415 Val Gly Thr Gln Gly Gly Gly Gly Tyr             420 425 <210> 17 <211> 1407 <212> DNA <213> Neurospora crassa <400> 17 atgagaccgc aagtggtagg ggcaatcctc cgctctagag ctgttgtcag cagacaacct 60 ctttcgagga cccatatctt tgctgccgtc actgttgcaa agtcctcatc acctgcccag 120 aactcgagaa gaaccttttc atcctctttc cgacggttgt atgagccaaa ggcggagata 180 acagctgagg gacttgagtt gagccctcca caggctgtta cgggtggaaa gcggactgtt 240 ttacccaact tctggctacg tgacaactgc cggtgtacga aatgcgtgaa ccaagatact 300 ctccagagaa acttcaacac ttttgccatc ccctccgaca tccacccaac aaaggttgaa 360 gccaccaagg agaacgtcac cgtccaatgg tccgacaacc acacatccac ctacccctgg 420 cccttcctct ctttctacct cacctccaac gcgcgcgggc acgaaaacga ccagatctcc 480 ctctggggct ccgaagccgg ctcccgcccg ccaaccgtct ccttccctcg cgtgatggca 540 tcagaccagg gcgtcgccga cctaaccgcc atgatcaaag agttcggctt ctgtttcgtc 600 aaagacacac cccatgacga cccggacgtg acccgccagc ttctggagag aatcgccttt 660 atccgagtga cccattacgg cggcttttac gatttcacgc ccgacctcgc gatggccgac 720 acggcgtaca cgaacctggc gctgccggcg catacggata cgacgtactt cacggacccg 780 gcggggttgc aggcttttca cttgttggag cataaggccg ctccttctcg tcctcctcct 840 cctcctcctc ctcctcctcc tccttctgaa gaaaaagaag ctgcaggctc agcagcaggg 900 gaggcggcgg cggcagcaga agggggaaag tcgttgttgg tcgatgggtt caacgccgcg 960 aggattctga aggaggagga tccccgggct tatgagatct tgagcagcgt gagactgccg 1020 tggcatgcga gtggaaacga agggatcacg attgcgcccg ataagcttta tccggtgctg 1080 gaactgaatg aggataccgg ggaactgcat agggttaggt ggaataatga tgataggggt 1140 gtggtgccgt ttggggagaa gtacagcccg tcagagtggt atgaggcggc gaggaagtgg 1200 gatgggattt tgaggaggaa gagcagcgag ttgtgggtgc agttggagcc ggggaagccg 1260 ttgaggttct tcatggacgg agcgcgttct cgggtattag gaggatttgt ggagggtata 1320 tcaaccgcga tgacttcatc tctcggtgga ggaacacgaa ttacccaagg agcgaggttc 1380 ttccgagggt tactggttaa ggactga 1407 <210> 18 <211> 1443 <212> DNA <213> Neurospora crassa <400> 18 atgtctacct actccctctc cgagactcac aaggccatgc tcgagcatag cttggtcgag 60 tccgaccccc aggtcgccga gatcatgaag aaggaggttc agcgccagcg cgagtccatc 120 atcctcatcg cctccgagaa cgtcacctcg cgtgccgtct tcgatgccct cggctccccc 180 atgtccaaca agtactcgga gggtcttccc ggcgcccgct actatggtgg caaccagcac 240 atcgacgaga tcgaggttct ctgccagaac cgtgcccttg aggccttcca cctcgacccc 300 aagcagtggg gtgtcaatgt tcagtgcttg tccggcagcc ctgccaacct ccaggtctac 360 caggccatca tgcccgtcca cggcagactc atgggtcttg acctccccca cggtggccat 420 ctttcccacg gttaccagac cccccagcgc aagatctctg ctgtctctac ctacttcgag 480 accatgccct accgcgtcaa cattgacact ggtctcatcg actacgatac cctcgagaag 540 aacgcccagc tcttccgccc caaggtcctc gtcgccggta cctctgccta ctgccgtctg 600 attgactacg agcgcatgcg caagattgcc gactccgttg gcgcttacct tgtcgtcgat 660 atggctcaca tttccggcct cattgcctcc gaggttatcc cctcgccctt cctctacgcc 720 gatgtcgtca ccaccaccac tcacaagtct ctccgtggcc ctcgtggcgc catgatcttc 780 ttccgccgcg gtgtccgctc cgttgacgcc aagaccggca aggagaccct ctacgacctt 840 gaggacaaga tcaacttctc cgtcttccct ggtcaccagg gtggccccca caaccacacc 900 atcaccgccc ttgccgttgc cctcaagcag gctgcctccc ccgagttcaa ggagtaccag 960 cagaaggtcg ttgccaacgc caaggctctc gagaagaagc tcaaggagct cggctacaag 1020 ctcgtctctg acggcactga ctctcacatg gtcctcgttg accttcgccc catcggcgtc 1080 gatggtgccc gtgttgagtt cctccttgag cagatcaaca ttacctgcaa caagaacgcc 1140 gttcccggcg acaagagcgc cctcaccccc ggcggtctcc gtattggtac ccccgctatg 1200 acctcccgtg gcttcggcga ggccgacttc gagaaggtcg ccgtcttcgt cgatgaggct 1260 gtcaagctct gcaaggagat ccaggcttcc ctccccaagg aggctaacaa gcagaaggac 1320 ttcaaggcca agatcgccac cagcgatatt ccccgcatca acgagctcaa gcaggagatt 1380 gccgcctgga gcaacacctt ccccctcccc gttgagggct ggagatacga tgccggtctc 1440 taa 1443 <210> 19 <211> 1488 <212> DNA <213> Neurospora crassa <400> 19 atggaagtcg agcttacggc ccccaacggc aagaagtgga tgcagccact gggcttgttc 60 attaataacg agtttgtcaa aagtgccaat gagcagaagt tgatttccat caacccaact 120 accgaagagg agatctgctc ggtatacgcc gcaaccgccg aggatgttga cgccgcagta 180 tcagcagccc gcaaggcctt taggcacgaa tcatggaagt cgctatccgg cactgagcgc 240 ggcgccctga tgcgcaagct ggccgaccta gtggccgaga atgccgaaat cctagccacc 300 atcgagtgcc tggacaacgg caagccgtat cagacagccc ttaacgagaa cgtgcccgaa 360 gtgatcaacg tcctcaggta ctatgccggc tatgcggaca agaactttgg ccaagtgatt 420 gacgttggcc ccgccaagtt tgcctacacg gtcaaggagc ctctcggcgt atgtggccag 480 atcatcccct ggaactaccc gctagatatg gccgcctgga agctggggcc agctctctgc 540 tgcggcaaca ccgtggtcct caagctggcc gagcagactc ccctgtccgt gttgtacttg 600 gctaagctca ttaaggaggc cggcttccct cccggtgtga tcaatatcat caacggacac 660 ggcagggaag cgggtgccgc acttgtgcaa catcctcagg tggacaagat tgcctttacc 720 ggcagcacca ctacgggcaa ggagatcatg aagatggctt cctataccat gaagaacatc 780 accctggaga ctggcggcaa gtcaccgttg atcgtgtttg aggatgccga ccttgagctg 840 gcggcgacat ggtcacacat cggcatcatg agcaaccagg gccaaatctg cacagccact 900 tcacgcattc tcgtgcacga gaagatctac gacgagtttg tcgaaaaatt caaggccaaa 960 gtccaggagg tttcggtact cggcgacccc ttcgaggaga gcacgttcca cggacctcag 1020 gtcaccaaag cgcagtatga gcgtgttctg ggctatatca atgtcggaaa ggaagagggt 1080 gccacggtga tgatgggtgg tgagccggct ccgcagaacg gtaaaggttt ctttgtggcc 1140 ccgactgtct tcacgaacgt caagccgacc atgaagatct tcagggagga gatctttggg 1200 ccctgcgtgg ccattaccac gttcaaaacg gaggaggagg cgttgacgct ggccaacgac 1260 agcatgtatg gcctgggagc ggctctgttc accaaggacc taaccagggc acacagagtg 1320 gcgcgggaga tcgaggccgg catggtctgg gtcaacagca gcaacgattc agactttagg 1380 attccatttg gaggcgtgaa gcagtctggt attgggaggg agttgggaga ggcaggtctg 1440 gcaccttatt gcaacgtcaa gagtatccat gtaaacctgg cggcatga 1488 <210> 20 <211> 1278 <212> DNA <213> Neurospora crassa <400> 20 atggccacgg cagcggttca ggtttcagtc ccagctccgg ttggacaacc agatatcggg 60 tacgctcctg accacgacaa gtacctcgca agagtcaaaa gacgacgaga aaacgagaag 120 ctggagtcgt ctcttccgcc aggtttccct cgaagactag actcggacct tgtgtgggac 180 ggcaacaccc tcgccgagac gtacgactgg acctacagac tgacagaaga ggccattgat 240 gaaatcgagg ccgcgcttcg tcattttaag agcctcaaca agcccctagg ctacatcaac 300 caagaaacct tcccccttcc ccgcctacac cacactctcc gctccctctc ccacgagctc 360 caccacggcc acggcttcaa agtcctccgc gggctccccg tcacctccca tacacgcgag 420 gaaaacatca tcatctacgc cggcgtctcc tcgcatgtcg ctcctatccg cggccgccag 480 gacaaccagc acaacggcca cccagccgac gtagtcctag cacacatcaa agacctgtcc 540 acgactgttt ctgacgtgag caaaatcggt gcacccgcct acaccaccga gaaacaagtc 600 ttccacaccg acgcaggcga catcgtcgcc ctcttttgct tgggagaggc cgccgagggc 660 ggacagagtt acctgtccag cagctggaag gtgtacaacg agctggcagc cactcggccc 720 gatctggttc gcacgctggc ggagccgtgg gtggcggacg agtttggcaa ggaagggagg 780 aagttttctg tgcgaccgct tttgcatttt cagtctactg ctgctgctgc ttctagggaa 840 gcaaagcccg agtctgaacg gctcatcatc cagtacgccc gccgcacgtt tacggggtat 900 tggggattac cgaggtcggc ggatatcccg cccattacgg aggcgcaggc ggaggcgttg 960 gatgcgctgc actttacggc ggagaagtac gcggtggcgc tggatttcag gcagggggat 1020 gtccagtttg tgaataactt gagtgtgttc cattcgaggg cggggtttag agatgagggg 1080 gagaagcaga ggcatttggt taggttgtgg ttgagagatc cggagaatgc gtgggagacg 1140 cccgaggcgt tgaaggaacg gtgggaacgc gtgtatggcg gggtgagtcc ggagagggag 1200 gtgtttccgc ttgagccgca gattaggagc gcgagtaagg gggagagcgt ggggacgcag 1260 ggtgggggag ggtattga 1278  

Claims (9)

Polynucleotides encoding N-trimethyllysine hydroxylase activity having the amino acid sequence of SEQ ID NO: 13 derived from Neurospora crassa , A polynucleotide encoding 3-hydroxy-6-N-trimethyllysine aldolase activity having the amino acid sequence of SEQ ID NO: 14, A polynucleotide encoding γ-trimethylaminoaldehyde dehydrogenase activity having the amino acid sequence of SEQ ID NO: 15 and A microorganism belonging to an Escherichia coli species comprising a polynucleotide encoding a γ-butyllobetaine hydroxylase activity having the amino acid sequence of SEQ ID NO: 16. delete The microorganism of claim 1, wherein the microorganism is Escherichia coli accession No. KCCM-10581. delete delete delete delete The microorganism of claim 1 or 3 is selected from the group consisting of N-trimethyllysine, β-hydroxy-N-trimethyllysine, γ-N-trimethylaminobutyraldehyde, γ-butyrobetaine and mixtures thereof. A method for producing L-carnitine comprising culturing in the presence of a substrate to produce L-carnitine in a culture. The substrate according to claim 8, wherein the substrate is selected from the group consisting of N-trimethyllysine, β-hydroxy-N-trimethyllysine, γ-N-trimethylaminobutyraldehyde, γ-butyrobetaine and mixtures thereof. The concentration is from 0.1 to 10% by weight relative to the weight of the culture medium.

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