KR100842013B1 - - - -Butyrobetaine hydroxylase derived from Neurospora crassa its gene and processes for preparing L-carnitine using the same - Google Patents

Abstract

The present invention provides a γ-butyrobetaine hydroxylase having an amino acid sequence represented by SEQ ID NO: 1 and generating L-carnitine by hydroxylating γ-butyrobetaine. In addition, the present invention provides a gene encoding the γ-butyrobetaine hydroxylase, an expression vector comprising the same, and a transformant transformed into a Saccharomyces genus microorganism with the expression vector. do. In addition, the present invention provides a method for producing L-carnitine comprising the step of hydroxylating γ-butyrobetaine (γ-butyrobetaine) using the γ-butyrobetaine hydroxylase.

γ-butyrobetaine hydroxylase

Description

Γ-Butyrobetaine hydroxylase derived from Neurospora Krasa, its gene, and method for producing L-carnitine using same (γ-Butyrobetaine hydroxylase derived from Neurospora crassa, its gene, and processes for preparing L-carnitine using the same}

1 shows the amino acid sequence of NCU03802.1 and the results of sequencing of N. crassa TMLH and human γ-butyrobetaine hydroxylase (BBH).

2 shows the construction of expression vectors cloned into genes encoding NCU03802.1.

The present invention is neurospora Krasa ( Neurospora γ-butyrobetaine hydroxylase derived from crassa ), a gene encoding the same, an expression vector comprising the same, and a transformant transformed into a Saccharomyces microorganism with the expression vector will be. In addition, the present invention relates to a method for producing L-carnitine comprising the step of hydroxylating γ-butyrobetaine (γ-butyrobetaine) using the γ-butyrobetaine hydroxylase.

L-carnitine (3-hydroxy-4-trimethylammonium butyrate) is a substance that plays an important role in β-oxidation by transferring activated fatty acids to the matrix through the mitochondrial lining. Since L-carnitine was first commercialized in the 1980s, it has been widely applied in medicine, food, and food additives. While L-carnitine is a physiologically effective form, while D-carnitine acts as a competitive inhibitor of L-carnitine, there is a need in the art to develop a method for selectively producing L-carnitine.

When L-carnitine is prepared by a chemical process, it undergoes the formation of a DL-carnitine mixture, which requires separation of the D- and L- isomers. Therefore, it is more practical to develop an improved manufacturing process based on microbial fermentation. As microorganisms capable of accumulating L-carnitine in cells in small amounts, eukaryotic microorganisms such as Saccharomyces cerevisiae have been reported (Sigma Tau, 1983, Process for enzymatically producing L-carnitine; U.S. Patent No. 4,371,618; Nippon Pet Food, 1990, Method for preparation of L-carnitine; Japanese Patent Application Laid-Open No. 2-069188; Yakult Honsha, 1993, L-carnitine preparation, Japanese Patent Application Laid-open No. Hei 5-199890), several others microorganisms are known to convert the betaine to sikindaneun γ- butyronitrile with L- carnitine. (Jung, H., K. Jung , and HP Klebar. 1993. Synthesis of L-carnitine by microorganisms and isolated enzymes. Adv. Biochem. Eng Biotechnol . 50: 21-44. And Naidu, GSN, IY Lee, EG Lee, GH Kang, and YH Park. 2000. Microbial and enzymatic production of L-carnitine. Bioprocess Eng . 23: 627-635).

Also recently, Escherichia coli), Salmonella typhimurium (Salmonella typhimurium) and Proteus vulgaris (Proteus It has been reported that a species belonging to the genus Enterobacteriaceae , such as vulgaris ), bioconverts crotonobetaine to produce L-carnitine (Caovas, M., JR Maiquez, JM Obon, and JL Iborra. 2002. Modeling of the biotransformation of crotonobetaine into l-(-)-carnitine by Esherichia coli strains. Biotechnol . Bioeng . 77: 764-775; Castellar, MR, M. Canovas, HP Kleber, and JL Iborra. 1998.Biotransformation of d-(+)-carnitine by resting cells of Esherichia coli O44 K74. J. Appl . Microbiol . 85: 883-890; Engemann, CT Elssner, S. Pfeifer, C. Krumbholz, T. Maier, and HP Kebler. 2005. Identification and functional characterization of genes and corresponding enzymes involved in carnitine metabolism of Proteus sp. Arch . Microbiol . 183: 176-189; Kleber, HP 1997. Bacterial carnitine metabolism. FEMS Microbiol . Lett . 147: 1-9; And Obon, JM, JR Maiquez, M. Canovas, HP Kleber, and JL Iborra. 1999.High-density Escherichia coli cultures for continuous l-(-)-carnitine production. Appl . Microbiol . Biotechnol . 51: 760-764)

Neurospora , a filamentous fungus crassa ) has revealed the biosynthetic pathway of L-carnitine and its intermediate metabolites (Korean Patent Publication No. 2006-5189). Neurospora Krasa L-carnitine in crassa ) is derived from five enzymes from lysine, namely S-adenosylmethionine-6- N- L-lysine methyltransferase, ε- N -trimethyllysine hydroxylase (EC 1.14.11.8), β- Sequential of hydroxy-ε- N -trimethyllysine aldolase, γ-trimethylaminobutyraldehyde dehydrogenase (EC 1.2.1.47), and γ-butyrobetaine hydroxylase (EC 1.14.11.1) It is synthesized by phosphorus reaction.

The present inventors have while trying to develop transgenic microorganisms genetically engineered to produce an L- carnitine effectively, neuro Spokane LA Crowley four (Neurospora From the genome of crassa ) a new protein with the activity of γ-butyrobetaine hydroxylase and a gene encoding it were found.

Therefore, the present invention is neurospora Krasa ( Neurospora It is an object to provide γ-butyrobetaine hydroxylase derived from crassa ).

The present invention also provides a gene encoding the γ-butyrobetaine hydroxylase, an expression vector comprising the same, and a transformant transformed into a Saccharomyces genus microorganism with the expression vector. For the purpose of

The present invention also provides a method for producing L-carnitine comprising the step of hydroxylating γ-butyrobetaine (γ-butyrobetaine) using the γ-butyrobetaine hydroxylase. It is done.

According to an aspect of the present invention, there is provided a γ-butyrobetaine hydroxylase having an amino acid sequence of SEQ ID NO: 1, which generates L-carnitine by hydroxylating γ-butyrobetaine. do.

According to another aspect of the present invention, a gene encoding said γ-butyrobetaine hydroxylase is provided.

According to another aspect of the present invention, an expression vector comprising the gene is provided.

According to another aspect of the present invention, a transformant transformed into a Saccharomyces microorganism with the expression vector is provided.

According to another aspect of the invention, the method for producing L-carnitine comprising the step of hydroxylating γ-butyrobetaine (γ-butyrobetaine) using the γ-butyrobetaine hydroxylase This is provided.

Hereinafter, the present invention will be described in detail.

We based the known human γ-butyrobetaine hydroxylase on the total genome database of N. crassa (www.broad.mit.edu/annotation/fungi/neurospora_crassa_7/index.html). N. crassa coding for robetine hydroxylase cDNA clones were screened. As a result of BLASTp homology, three open reading frames (ORFs), NCU03802.1, NCU02196.1, and NCU06891.1, were searched for human γ-butyrobetaine hydroxylase, respectively. 23, 24, and 27% homology. The nucleotide sequences of NCU03802.1, NCU02196.1, and NCU06891.1 obtained above were amplified, and a transformant was constructed to measure γ-butyrobetaine hydroxylase activity. ORF sequences of NCU06891.1 were not amplified, but ORF sequences of NCU03802.1 and NCU02196.1 were effectively amplified. Surprisingly, the transformants transformed with the nucleotide sequence encoding NCU02196.1 showed no γ-butyrobetaine hydroxylase activity, but the transformants transformed with the nucleotide sequence encoding NCU03802.1 It exhibited very high γ-butyrobetaine hydroxylase activity. This is very surprising given that the ORF sequences of NCU03802.1 and NCU02196.1 have similar homology (23 and 24%) to human γ-butyrobetaine hydroxylase.

Furthermore, the amino acid sequence of N ε- NCU03802.1 rather that catalyzes the second step in the L- carnitine biosynthesis-trimethyl lysine hydroxyl sila the (N ε- -trimethyllysine hydoxylase, TMLH) and indicate a homology of 91% (See FIG. 1). Thus, it is surprising that NCU03802.1 exhibits high γ-butyrobetaine hydroxylase activity. The genomic sequences of TMLH and NCU03802.1 are located in different chromosomes on the genome of N. crassa and the amino acid sequences of NCU03802.1 are N. crassa It includes 11 unique residual poly (P) regions that have been found in TMLH. As shown in Figure 1, the amino acid sequence of human γ-butyrobetaine hydroxylase is N. about 25% homology with crassa TMLH. Even considering conservative substitutions, NCU03802.1 and N. The homology of human γ-butyrobetaine hydroxylase to the amino acid sequence of crassa TMLH is only 49 and 50%, respectively. Therefore, the above analysis results are N. It is estimated that duplication of gene fragments or entire genomes occurs in the crassa so that duplicated copies of the genes acquire new functions.

Thus, the present invention provides N. Novel γ-butyrobetaine hydroxylase derived from crassa , ie, amino acid sequence of NCU03802.1 (amino acid sequence of SEQ ID NO: 1), and hydroxylated γ-butyrobetaine (γ-butyrobetaine) To provide γ-butyrobetaine hydroxylase, which produces L-carnitine.

The present invention provides a gene encoding the γ-butyrobetaine hydroxylase. Those skilled in the art can design various base sequences in consideration of degeneracy based on the amino acid sequence of the γ-butyrobetaine hydroxylase, that is, the amino acid sequence of SEQ ID NO: 1. Gene encoding the γ-butyrobetaine hydroxylase is preferably a gene having a nucleotide sequence of SEQ ID NO: 2.

The present invention includes an expression vector comprising a gene encoding the γ-butyrobetaine hydroxylase. As a vector which can be used as a cloning vector of an expression vector in the present invention, various known vectors can be used. For example, pBluescript II KS + (Stratagene, La Jolla, CA, U.S.A) can be used. The expression vector can be prepared by inserting a gene encoding γ-butyrobetaine hydroxylase into a cloning vector digested with an appropriate restriction enzyme by a conventional method. As an expression vector including a gene encoding the γ-butyrobetaine hydroxylase, pRS426 (Christianson, TW, RS Sikorski, M. Dante, JH Shero, and P. Hieter. 1992. Multifunctional yeast high-copy plasmid pSJ420 prepared by cloning the gene of SEQ ID NO: 2 in Gene 110: 119-122).

The present invention includes a transformant transformed Saccharomyces microorganism with the expression vector. The microorganism of the genus Saccharomyces is not particularly limited as long as γ-butyrobetaine hydroxylase can be effectively expressed. For example, Saccharomyces cerevisiae SJ7164 ( S. cerevisiae SJ7164) (ATCC 44774) can be used as the preferred host microorganism.

The present invention includes a method for preparing L-carnitine, comprising the step of hydroxylating γ-butyrobetaine using the γ-butyrobetaine hydroxylase.

The method for preparing L-carnitine of the present invention separates the γ-butyrobetaine hydroxylase of SEQ ID NO: 1 above and reacts in a medium containing γ-butyrobetaine as a substrate (hydroxyl of γ-butyrobetaine Dilation) may be carried out, and the transformant may be cultured in a culture solution containing γ-butyrobetaine to perform hydroxylation of γ-butyrobetaine.

Preferably, the step of hydroxylation is culturing a transformant transformed with S. cerevisiae SJ7164 (ATCC 44774) with plasmid pSJ420 in a medium containing γ-butyrobetaine. And recovering L-carnitine.

The transformant may be cultured in a culture medium for the culture of microorganisms of the genus Saccharomyces , for example, a glucose minimal medium containing yeast nitrogen base (amino acid free), glucose, and uracil. It can culture by containing (gamma) -butyrobetaine in it.

The L-carnitine can be recovered from a conventional cell free extract of crude cells according to a conventional method.

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

Example  One. N. crassa of  Genome analysis

Human γ-butyrobetaine hydroxylase (Vaz, FM, S. van Gool, R. Ofman, L. Ijlst, and RJA Wanders. 1998. Carnitine biosynthesis: Identification of the cDNA encoding human γ-butyrobetaine hydroxylase, .... Biochem Biophys Res Commun 250: 506-510.), A basis in which the hydroxyl N. sila beta -butyrolactone as γ- encoding a claim on the total genome database (www.broad.mit.edu/annotation/fungi/neurospora_crassa_7/index.html) of N. crassa crassa cDNA clones were screened. As a result of BLASTp homology, three open reading frames (ORFs), NCU03802.1, NCU02196.1, and NCU06891.1, were searched for human γ-butyrobetaine hydroxylase, respectively. 23, 24, and 27% homology. The proteins consist of sequences of 1,407, 1,278, and 3,786 bp, respectively. When the ORFs and N. crassa genomic sequences are aligned, the nucleotide sequences of NCU03802.1, NCU02196.1, and NCU06891.1 are It confirmed that it consists of 8, 2, and 5 exons, respectively.

1 shows the amino acid sequence of NCU03802.1 and the results of sequencing of N. crassa TMLH and human γ-butyrobetaine hydroxylase (BBH). Amino acid sequences are aligned by the T-COFFEE program (Notredame, C., D. Higgins, and J. Heringa. 2000. A novel method for multiple sequence alignments. J. Mol . Biol . 302: 205-217.) Dashes in the sequence represent gaps introduced to maximize alignment. (*: Identical residues,;: conserved residues).

Also the amino acid sequence of NCU03802.1 As can be seen in 1 ε- N - trimethyl lysine has a hydroxyl sila claim homology (ε- N -trimethyllysine hydoxylase, TMLH) and 91%. Furthermore, the amino acid sequence of human γ-butyrobetaine hydroxylase is N. 25% homology with crassa TMLH, and NCU03802.1 and N. were considered when conservative substitutions were considered. The homology of human γ-butyrobetaine hydroxylase to the amino acid sequence of crassa TMLH is 49 and 50%, respectively. The genomic sequences of TMLH and NCU03802.1 are located in different chromosomes on the genome of N. crassa and the amino acid sequences of NCU03802.1 are N. crassa Found TMLH (Swiegers, JH, FM Vaz , IS Pretorius, RJA Wanders, and FF Bauer 2002. Carnitine biosynthesis in Neurospora crassa:.. Identification of a cDNA coding for ε- N -trimethyllysine hydoxylase and its functional expression in Saccharomyces cerevisiae FEMS Microbiol . Lett . 210: 19-23.), Including 11 unique residual poly (P) regions.

Example  2. Polymerase Chain Reaction

Based on the expected coding sequences of NCU03802.1, NCU02196.1, and NCU06891.1, a primer set as shown in Table 1, ie 3802.1F and 3802.1R, 2196.1F and 2196.1R, 6891.1F, and 6891.1R, was prepared. , They overlap with the start and stop codons.

primer Sequence number Sequence ^a Restriction enzyme 3802.1F 3 5'-attcc gatatc catATG AGACCGCAAGTGGTAGG-3 ' Eco RV and Nde I 3802.1R 4 5'-ccgat gaattc TCAGTCCTTAACCAGTAACCC-3 ' Eco RI 2196.1F 5 5'-actgat aagcttc ATGGCCACGGCAGCGGTTCAGG-3 ' Hin dIII 2196.1R 6 5'-ccgat gaattc TCAATACCCTCCCCCACCCTGCG-3 ' Eco RI 6891.1F 7 5'-attcc gatatc ATGGGGTTCCTCGCTACTCTCATCG-3 ' Eco RV 6891.1R 8 5'-attcc gatatc TTATGCGTTCCAGTTCACCGTGCC-3 ' Eco RV

^The underlined sequence indicates the position cleaved by the restriction enzyme.

Using the primer set, ORFs derived from N. crassa cDNA yeast expression library (Lambda g15-NC cDNA; Fungal Genetic Stock Center, Kansas City, KS, USA) were amplified by polymerase chain reaction (PCR). .

50 μl of PCR reaction was performed with 10 μl of 10 × reaction buffer [100 mM KCl, 100 mM (NH ₄ ) ₂ SO ₄ , 200 mM Tris-Cl (pH 8.75), 20 mM MgSO ₄ , 1% Triton ^™ X-100, 1000 ug / ml BSA], 200 uM of each dNTP, 100 pmol of each primer, 2.5 units of Top - Pfu DNA polymerase (Solgent, Daejeon, Korea), and 250 ng of Lambda as template g15-NC cDNA.

Each sample was placed in a PTC-100 ^™ Programmable Thermal Controller (MJ Research, Waltham, Mass., USA), initially 5 minutes at 95 ° C., then 1 minute at 94 ° C., 1 minute at 56 ° C., and 5 minutes at 72 ° C. 30 cycles were carried out under the conditions. Using the N. crassa cDNA library as a template, 1,407 and 1,278 bp PCR products containing ORFs of NCU03802.1 and NCU02196.1 cDNA were successfully amplified, respectively, but PCR products using primers 6891.1F and 6891.1R were amplified. It wasn't.

Example  3. Preparation of Expression Vectors

The ORF sequence obtained in Example 2 S. cerevisiae -. E. coli shuttle vector pRS426 (Christianson, TW, Sikorski RS, Dante M., Shero JH, and Hieter P. 1992. Multifunctional yeast high-copy-number shuttle vectors. Gene 110: 119-122 .) Was cloned between the glucose inducible adh1 promoter and adh1 transcription terminator.

The 990 bp adh1 promoter region and 706 bp terminator region were first amplified using genomic DNA of primers P1 and P2, T1 and T2, and S. cerevisiae SJ7164 as a template, respectively. The primer sequence is shown in Table 2 below.

primer Sequence number Sequence ^a Restriction enzyme P1 9 5'-ta ggtacc ACGCGCATAACCGCTAGAGTA-3 ' Kpn I P2 10 5'-ta ctcgag TGAGATAGTTGATTGTATGCTTGG-3 ' Xho I T1 11 5'-tc actagt CGTTGTTGACACTTCTAAATAAGCG-3 ' Spe i T2 12 5'-ta gagctc TACCAGGATAGGACACGGTGTT-3 ' Sac i

The resulting 990 bp product containing adh1 promoter sequence was digested with Kpn I and Xho I and subcloned into the corresponding site of pBluescript II KS + (Stratagene, La Jolla, CA, USA). Cutting the resulting plasmid with Spe I and Sac I, and, Spe I and Sac I and the PCR product was joined (ligation) of 706 bp containing the terminator sequence adh1 cut into. The resulting plasmid (named pSJ400) was cut into Eco RV and Eco RI, followed by conjugation with a 1,407 bp Eco RV- Eco RI PCR fragment containing an ORF of NCU03802.1 cDNA to prepare plasmid pSJ401. adh1 promoter and inserted into the Kpn I- Sac I fragment of pSJ401 contains the ORF of the cDNA NCU03802.1 between adh1 terminator to the corresponding region of pRS426 was prepared plasmid pSJ420. To construct an expression plasmid that overexpresses the protein of NCU02196.1, the internal Hin dIII - Eco RI fragment of pSJ420 was replaced with a 1,278 bp PCR fragment containing the ORF of NCU02196.1 cDNA digested with Hin dIII - Eco RI. Was prepared. Sequencing confirmed that it was an ORF of 1,407 bp encoding a protein with 468 amino acids of NCU03802.1 and an 1,278 bp ORF encoding a protein with 425 amino acids of NCU02196.1.

Example  4. Preparation and Cultivation of Transformant

Using the expression vector prepared in Example 3, a lithium acetate method (Gietz, RD, RH Schiestl, AR Williams, and RA Woods. 1995. Studies on the transformation of intact yeast cells by the LiAc / SS-DNA / PEG procedure . Yeast 11:... Lett 355-360 and Kleber, HP 1997. Bacterial carnitine metabolism FEMS Microbiol 147: 1-9), the non-zero My process celebrity SJ7164 (S. cerevisiae SJ7164) (ATCC 44774) (his3 a saccharide-by transforming a delta1, leu2 -3, leu2 -112, -52 ura3, trp1 -289), Transformants transformed with pSJ420 and pSJ404, respectively, were prepared.

The resulting transformants were incubated in a glucose minimal medium [6.7 g yeast nitrogen base (without amino acid), 20 g glucose, and 20 mg uracil per liter].

Example  5. γ- Butyrobetaine Hydroxylase  Activity analysis

γ-butyrobetaine hydroxylase activity is a two-step process from a crude cell free extract of a transformant comprising N. crassa ORF against γ-butyrobetaine hydroxylase. In this process, L-carnitine, which was enzymatically generated from γ-butyrobetaine hydroxylase, was measured colorimetrically (Vaz, FM, S. van Gool, R. Ofman, L). . Ijlst, and RJA Wanders 1998. Carnitine biosynthesis:.... Identification of the cDNA encoding human γ-butyrobetaine hydoxylase Biochem Biophys Res Commun 250:... 506-510 and Wieland, OH, T. Deufel, and I. Paetzke . -Brunner 1985. Free and esterified carnitine: .. colometric method, pp 481-488 In HU Bergmeyer (. ed), Methods of Enzymatic Analysis , Vol. 8, 3 ^rd Ed. VCH, Weinheim. 23, 25).

Cell-free extracts for assaying γ-butyrobetaine hydroxylase activity were obtained from algebraically grown cultures. The reaction mixture for assaying γ-butyrobetaine hydroxylase activity in a final volume of 500 μl was 20 mM KCl, 3 mM α-ketoglutarate, 10 mM sodium ascorbate, 2 g / liter Triton X 20 mM potassium phosphate buffer at pH 7.0 containing -100, 0.25 mM of (NH ₄ ) ₂ Fe (SO ₄ ) ₂ , and 0.2 mM γ-butyrobetaine. The L-carnitine content enzymatically produced by γ-butyrobetaine hydroxylase was calculated from a standard curve created using known L-carnitine. The results are shown in Table 3 below. In Table 3 below, protein concentrations in cell extracts were measured using bovine serum albumin Bradford, MM 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal . Biochem . 72: measured by the method disclosed in 31-37.

host Plasmid Specific activity (umol / min / mg protein) Saccharomyces cerevisiae sj7164 ( S. cerevisiae SJ7164) pRS426 ND pSJ404 ND pSJ420 0.036 ± 0.003

ND: not detected

As can be seen in Table 3, crude cell free extracts of transformants prepared to overexpress the protein product of NCU03802.1 (pSJ420) in the presence of exogenus γ-butyrobetaine Γ-butyrobetaine hydroxylase activity measured from) shows high γ-butyrobetaine hydroxylase activity, whereas cells of the transformant to overexpress the protein product of NCU02196.1 (pSJ404) No γ-butyrobetaine hydroxylase activity was determined from crude cell free extracts. In addition, the activity from crude cell free extracts of transformants comprising pRS426 (negative control) was not detected in the presence of external γ-butyrobetaine. This indicates that Saccharomyces cerevisiae SJ7164 ( S. cerevisiae SJ7164) itself lacks γ-butyrobetaine hydroxylase activity.

Polypeptides having the amino acid sequence of SEQ ID NO: 1 have γ-butyrobetaine hydroxylase activity, and when cultured Saccharomyces microorganisms transformed with the gene encoding it, L-carnitine can be prepared from lovetaine.

Attach sequence list electronic file

Claims (10)

delete delete delete delete delete delete delete delete A method for producing L-carnitine comprising the step of hydroxylating γ-butyrobetaine using γ-butyrobetaine hydroxylase composed of the amino acid sequence of SEQ ID NO: 1. 10. The transformation according to claim 9, wherein the hydroxylating step is a transformation of S. cerevisiae SJ7164 (ATCC 44774) with plasmid pSJ420 in a medium containing γ-butyrobetaine. A method for producing L-carnitine, which is carried out by culturing a sieve and recovering L-carnitine.

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