# Patent

## Title
Bacterium producing a product of a reaction catalyzed by a protein having 2-oxoglutarate-dependent enzyme activity and a method for manufacturing the product

## Abstract

A method for manufacturing a product of a reaction catalyzed by a protein having 2-oxoglutarate-dependent enzyme activity such as (2S,3R,4S)-4-hydroxy-L-isoleucine or a salt thereof using a bacterium transformed with a DNA fragment containing a gene coding for a protein having 2-oxoglutarate-dependent enzyme activity such as L-isoleucine dioxygenase activity; and wherein said bacterium has the ability to produce a product such as (2S,3R,4S)-4-hydroxy-L-isoleucine.

## Background
This application is a continuation under 35 U.S.C. §120 of PCT Patent Application No. PCT/JP2008/073914, filed Dec. 22, 2008, which claims priority under 35 U.S.C. §119 to Russian Patent Application No. 2007147436, filed on Dec. 21, 2007, which are incorporated in their entireties by reference. The Sequence Listing in electronic format filed herewith is also hereby incorporated by reference in its entirety (File Name: 2010-06-21T_US-351_Seq_List; File Size: 90 KB; Date Created: Jun. 21, 2010).


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for manufacturing a product of a reaction catalyzed by a protein having 2-oxoglutarate-dependent enzyme activity such as 4-hydroxy-L-isoleucine or a salt thereof using a bacterium transformed with a DNA fragment containing a gene coding for a protein having a 2-oxoglutarate-dependent enzyme such as L-isoleucine dioxygenase. This bacterium has also been modified to have enhanced expression of a gene coding for an L-isoleucine transporter, and has the ability to produce (2S,3R,4S)-4-hydroxy-L-isoleucine.
2. Brief Description of the Related Art
4-hydroxy-L-isoleucine is an amino acid which can be extracted and purified from fenugreek seeds (Trigonella foenum-graecum L. leguminosae). 4-hydroxy-L-isoleucine displays an insulinotropic activity, which is of great interest because of its stimulating effect which is clearly dependent on the plasma glucose concentration in the medium. This effect has been demonstrated both in isolated perfused rat pancreas and human pancreatic islets (Sauvaire, Y. et al, Diabetes, 47: 206-210, (1998)). Such a glucose dependency has not been confirmed for sulfonylurea (Drucker, D. J., Diabetes 47: 159-169, (1998)), which is the only insulinotropic drug currently used to treat type II diabetes [or non-insulin-dependent diabetes (NIDD) mellitus (NIDDM)], and as a consequence, hypoglycemia is still a common undesirable side effect of sulfonylurea treatment (Jackson, J., and Bessler, R. Drugs, 22: 211-245; 295-320, (1981); Jennings, A. et al. Diabetes Care, 12: 203-208, (1989)). Methods for improving tolerance to glucose are also known (Am. J. Physiol. Endocrinol., Vol. 287, E463-E471, 2004). This glucometabolism enhancement activity, and its potential application to pharmaceuticals and health foods, have been previously reported (Japanese Patent Application Laid-Open No. Hei 6-157302, US2007-000463A1).
4-hydroxy-L-isoleucine is only found in plants, and due to its particular insulinotropic action, might be considered a novel secretagogue with potential applications for the treatment of type II diabetes, which is characterized by defective insulin secretion associated with various degrees of insulin resistance (Broca, C. et al, Am. J. Physiol. 277 (Endocrinol. Metab. 40): E617-E623, (1999)).
Oxidizing iron, ascorbic acid, 2-oxyglutaric acid, and oxygen-dependent isoleucine by utilizing dioxygenase activity in fenugreek extract has been reported as a method for manufacturing 4-hydroxy-L-isoleucine (Phytochemistry, Vol. 44, No. 4, pp. 563-566, 1997). However, this method is unsatisfactory for manufacturing 4-hydroxy-L-isoleucine because the activity of the enzyme is inhibited by the substrate at isoleucine concentrations of 20 mM and above. Furthermore, the enzyme has not been identified, is derived from plant extracts, is not readily obtained in large quantities, and is unstable.
An efficient eight-step synthesis of optically pure (2S,3R,4S)-4-hydroxyisoleucine with 39% overall yield has been disclosed. The key steps of this synthesis involve the biotransformation of ethyl 2-methylacetoacetate to ethyl(2S,3S)-2-methyl-3-hydroxy-butanoate with Geotrichum candidum and an asymmetric Strecker synthesis (Wang, Q. et al, Eur. J. Org. Chem., 834-839 (2002)).
A short six-step chemoenzymatic synthesis of (2S,3R,4S)-4-hydroxyisoleucine with total control of stereochemistry, the last step being the enzymatic resolution by hydrolysis of a N-phenylacetyl lactone derivative using the commercially available penicillin acylase G immobilized on Eupergit C(E-PAC), has also been disclosed (Rolland-Fulcrand, V. et al, J. Org. Chem., 873-877 (2004)).
But currently, there have been no reports of producing (2S,3R,4S)-4-hydroxy-L-isoleucine by using a bacterium transformed with a DNA fragment containing a gene coding for a protein having L-isoleucine dioxygenase activity; wherein the bacterium is also modified to have enhanced expression of a gene coding for L-isoleucine transporter and has the ability to produce (2S,3R,4S)-4-hydroxy-L-isoleucine.
Besides (2S,3R,4S)-4-hydroxy-L-isoleucine, products which are produced by reactions catalyzed by proteins having 2-oxoglutarate-dependent enzyme activity, and which are industrially important are known. However, there have been no reports of systems for efficiently producing the products by using the proteins having 2-oxoglutarate-dependent enzyme activity.
SUMMARY OF THE INVENTION
An aspect of present invention is to enhance production of a product of a reaction coupled with the formation of succinate from 2-oxoglutarate by a protein having 2-oxoglutarate-dependent enzyme activity. The product includes compounds in both the free form and a salt form thereof. Another aspect of the present invention is to provide a method for manufacturing the product by a reaction coupled with the formation of succinate from 2-oxoglutarate using a bacterium having 2-oxoglutarate-dependent enzyme activity. This bacterium has been modified to attenuate the expression of a gene coding for oxoglutarate dehydrogenase, preferably modified to attenuate the expression of genes coding for oxoglutarate dehydrogenase and isocitrate lyase, more preferably modified to attenuate the expression of genes coding for oxoglutarate dehydrogenase, isocitrate lyase, and isocitrate dehydrogenase phosphatase.
An aspect of present invention is to enhance production of (2S,3R,4S)-4-hydroxy-L-isoleucine, including both the free form and a salt form thereof. This compound may also be referred to as “(2S,3R,4S)-4HIL”. Another aspect of the present invention is to provide a method for manufacturing (2S,3R,4S)-4-hydroxy-L-isoleucine or a salt thereof by direct enzymatic hydroxylation of L-isoleucine using a bacterium having L-isoleucine dioxygenase activity. This bacterium can over-express a gene coding for an L-isoleucine transporter, and is able to produce (2S,3R,4S)-4-hydroxy-L-isoleucine.
A bacterium has previously been isolated from nature having a high level of L-isoleucine dioxygenase activity, and a gene was cloned which encodes L-isoleucine dioxygenase. It was found that L-isoleucine dioxygenase may be used in the synthesis of (2S,3R,4S)-4-hydroxy-L-isoleucine.
Another aspect of the present invention includes providing a method for enhanced production of (2S,3R,4S)-4-hydroxy-L-isoleucine using a bacterium having L-isoleucine dioxygenase activity. The above aspect was achieved by finding that a bacterium having L-isoleucine dioxygenase activity produced more (2S,3R,4S)-4-hydroxy-L-isoleucine if the bacterium is modified to overexpress a gene coding for an L-isoleucine transporter.
It is an aspect of the present invention to provide a bacterium transformed with a DNA fragment comprising a gene coding for a protein having L-isoleucine dioxygenase enzymatic activity, wherein said bacterium has been modified to overexpress a gene coding for an L-isoleucine transporter, and wherein said bacterium is able to produce (2S,3R,4S)-4-hydroxy-L-isoleucine.
It is a further aspect of the present invention to provide the (2S,3R,4S)-4HIL-producing bacterium as described above, wherein the gene coding for a protein having L-isoleucine dioxygenase activity is selected from the group consisting of:

    
    
        (a) a DNA comprising the nucleotide sequence of SEQ ID No: 1;
        (b) a DNA that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID No: 1, and wherein said DNA encodes a protein having L-isoleucine dioxygenase activity;
        (c) a DNA comprising a nucleotide sequence that encodes a protein comprising the amino acid sequence of SEQ ID No: 2;
        (d) a DNA comprising a nucleotide sequence that encodes a protein comprising an amino acid sequence of SEQ ID NO. 2, except that said amino acid sequence contains a substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and wherein said protein has L-isoleucine dioxygenase activity; and
        (e) a DNA comprising a nucleotide sequence that encodes a protein comprising an amino acid sequence that is at least 98% homologous to the amino acid sequence of SEQ ID NO: 2, and wherein said protein has L-isoleucine dioxygenase activity.
    
    


It is a further aspect of the present invention to provide the (2S,3R,4S)-4HIL-producing bacterium as described above, wherein the bacterium has been modified to enhance the activity of L-isoleucine dioxygenase.
It is a further aspect of the present invention to provide the (2S,3R,4S)-4HIL-producing bacterium as described above, wherein the activity of L-isoleucine dioxygenase is enhanced by increasing the expression of the gene encoding L-isoleucine dioxygenase.
It is a further aspect of the present invention to provide the (2S,3R,4S)-4HIL-producing bacterium as described above, wherein the expression of L-isoleucine dioxygenase is increased by modifying an expression control sequence of the gene encoding L-isoleucine dioxygenase or by increasing the copy number of the gene encoding L-isoleucine dioxygenase.
It is a further aspect of the present invention to provide (2S,3R,4S)-4HIL-producing the bacterium as described above, wherein the gene coding for the L-isoleucine transporter is the brnQ gene from Escherichia coli. 
It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium has additionally been modified to attenuate the expression of genes coding for oxoglutarate dehydrogenase, isocitrate lyase, and isocitrate dehydrogenase phosphatase.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said expression is attenuated by inactivating said genes.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium has additionally been modified to attenuate the expression of a gene coding for a branched-chain amino-acid aminotransferase.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said expression is attenuated by inactivating said gene.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium belongs to a genus selected from the group consisting of Escherichia, Pseudomonas, Corynebacterium, Arthrobacter, Aspergillus, and Bacillus. 
It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium is selected from the group consisting of Escherichia coli, Arthrobacter simplex, Corynebacterium glutamicum, Arthrobacter globiformis, Arthrobacter sulfureus, Arthrobacter viscosus, and Bacillus subtilis. 
It is a further aspect of the present invention to provide a method for manufacturing (2S,3R,4S)-4-hydroxy-L-isoleucine or a salt thereof, comprising:
cultivating the (2S,3R,4S)-4HIL-producing bacterium as described above in a culture medium containing L-isoleucine; and
isolating (2S,3R,4S)-4-hydroxy-L-isoleucine.
It is a further aspect of the present invention to provide the method as described above, wherein the culture medium comprises a carbon source selected from the group consisting of a carbohydrate and an alcohol.
It is a further aspect of the present invention to provide the method as described above, wherein said carbohydrate is glucose and said alcohol is glycerol.
An aspect of present invention is to enhance production of 4-hydroxy-L-proline, including both the free form and a salt form thereof. Another aspect of the present invention is to provide a method for manufacturing 4-hydroxy-L-prolinee or a salt thereof by direct enzymatic hydroxylation of L-proline using a bacterium having L-proline hydroxylase activity.
The present invention is described in detail below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows ‘shunting’ of the TCA cycle in the strain MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)_[pELAC-IDO(Lys, 23)] due to simultaneous oxidation of isoleucine and α-ketoglutarate (2-oxoglutarate).
FIG. 2 shows the structure of the recombinant plasmid pET-IlvA.
FIG. 3 shows the structure of the recombinant plasmid pELAC-IDO(Lys, 23).
FIG. 4 shows the construction of the E. coli MG1655(PL-brnQ) strain.
FIG. 5 shows the growth of the strains MG1655 and MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) on the M9-salts medium containing glucose or glycerol with or without addition of lysine, methionine, and diaminopimelate (DAP). Strains were grown in M9-salts medium supplemented with glucose or glycerol and, optionally, lysine, methionine and diaminopimelate (DAP). Abbreviations: Strains WT=MG1655; 2d=MG1655 (ΔsucAB, ΔaceAK, PL-brnQ); Media: A=M9 salts+glucose; B=M9 salts+glucose+(Lys, Met, DAP); C=M9 salts+glycerol; D=M9 salts+glycerol+(Lys, Met, DAP).
FIG. 6 shows the growth of the strains MG1655 [pELAC-IDO(Lys, 23)] and MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) [pELAC-IDO(Lys, 23)] on the M9-salts medium with or without addition of L-isoleucine. Abbreviations: Strains 1=MG1655[pELAC-IDO(Lys, 23)]; 2=MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) [pELAC-IDO(Lys, 23)]; Media: G=M9 salts+glucose (137 mM); GI=M9 salts+glucose (137 mM)+L-isoleucine (137 mM); Y=M9 salts+glycerol (136 mM); YI=M9 salts+glycerol (136 mM)+L-isoleucine (137 mM).



DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Bacterium
The term “bacterium” can include an enzyme-producing bacteria, a mutant, and a genetic recombinant of such bacteria in which the targeted enzymatic activity is present or has been enhanced, and the like.
The term “2-oxoglutarate-dependent enzyme activity” can refer to an enzymatic activity to catalyze a reaction coupled with the formation of succinate from 2-oxoglutarate.
A number of proteins having 2-oxoglutarate-dependent enzyme activity, such as 2-oxoglutarate-dependent dioxygenases have been reported. Other examples include dioxygenases used in the production of useful products, such as a Pro hydroxylase which converts L-Pro to hydroxy-Pro (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, September 1999, p. 4028-4031), a γ-butyrobetaine hydroxylase converting γ-butyrobetaine to L-Carnitine (WO2005/083089). Besides these dioxygenases, a number of dioxygeneases have been reported. For example, refer to: Critical Reviews in Biochemistry and Molecular Biology, 39:21-68, 2004 and NATURE CHEMICAL BIOLOGY, 4 NUMBER 3 March: 152-156, 2008. As for the 2-oxoglutarate-dependent dioxygenases described in the reviews, the bacterium has been modified to attenuate the expression of a gene coding for an oxoglutarate dehydrogenase (such as ΔsucAB, ΔsucA, ΔsucB), and can be modified to attenuate the expression of genes coding for oxoglutarate dehydrogenase and isocitrate lyase (such as ΔsucAB, ΔsucA, or ΔsucB, plus ΔaceA), and can be further modified to attenuate the expression of genes coding for oxoglutarate dehydrogenase, isocitrate lyase, and isocitrate dehydrogenase phosphatase (such as ΔsucAB, ΔsucA, or ΔsucB, plus ΔaceAK), which is represented by E. coli strain MG1655 (ΔsucAB, ΔaceAK) which is described in the Examples, and is considered to be a general host for efficiently using 2-oxoglutarate produced from a carbon source such as D-glucose, in 2-oxoglutarate-dependent enzyme reactions.
As an example, the protein having 2-oxoglutarate-dependent enzyme activity can be a protein having L-isoleucine dioxygenase activity, and the product of the reaction catalyzed by the protein can be (2S,3R,4S)-4-hydroxy-L-isoleucine. However, the present invention is not limited to this example.
The term “(2S,3R,4S)-4-hydroxy-L-isoleucine” or “(2S,3R,4S)-4HIL” or “4HIL” refers to a single chemical compound or a mixture of compounds containing (2S,3R,4S)-4-hydroxyisoleucine.
The term “bacterium” can include an enzyme-producing bacterium, a mutant, and a genetic recombinant of such bacterium in which the targeted enzymatic activity is present or has been enhanced, and the like.
L-isoleucine dioxygenase from microbial cells may be abbreviated “IDO.”
Screening of environmental microorganisms revealed a unique microbe Bacillus thuringiensis strain 2-e-2, which possesses an activity of catalyzing a reaction in which (2S,3R,4S)-4HIL is directly formed from L-isoleucine, both the free form and a salt form thereof. The novel L-isoleucine dioxygenase was purified from the cultivated microbial cells, and may be abbreviated a “IDO(Lys,23).”
Furthermore, the N-terminal amino acid sequence of IDO(Lys,23) was determined by purifying dioxygenase derived from of Bacillus thuringiensis strain 2-e-2. Bacillus thuringiensis strain 2-e-2 was named Bacillus thuringiensis AJ110584 and deposited at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1, Higashi 1-chome, Tsukuba, Ibaraki 305-8566, Japan) on Sep. 27, 2006 and given an accession number of FERM BP-10688 under the provisions of Budapest Treaty.
The DNA encoding IDO(Lys,23) is shown in SEQ ID No: 1. Furthermore, the amino acid sequence of IDO(Lys,23) encoded by the nucleotide sequence of SEQ ID NO: 1 is shown in SEQ ID No: 2. SEQ ID NO: 2 is the amino acid sequence of IDO(Lys,23) encoded by the nucleotide sequence of SEQ ID NO: 1. IDO(Lys,23) of SEQ ID NO: 2 possesses L-isoleucine dioxygenase activity, and catalyzes the reaction in which (2S,3R,4S)-4HIL shown in the following formula (I) is directly synthesized from one molecule of L-isoleucine.




A DNA that encodes IDO which catalyzes the reaction in which (2S,3R,4S)-4HIL is formed from L-isoleucine is not only the DNA shown in SEQ ID No: 1. This is because there may be differences in nucleotide sequences from each species and strains among Bacillus that form IDO which catalyzes the reaction of producing (2S,3R,4S)-4HIL from L-isoleucine.
The DNA not only includes the isolated DNA encoding IDO, but also a DNA in which mutations have been artificially added to the DNA that encodes IDO. This DNA may be isolated from the chromosome of an IDO-producing microorganism. The DNA encodes IDO that is able to catalyze the above-described reaction. Methods for artificially adding mutations include typically used methods for introducing site-specific mutations, such as those described in Method. in Enzymol., 154 (1987).
A DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID No: 1, and encodes a protein having IDO activity is also included in the above-described DNA. The term “stringent conditions” can refer to conditions under which a specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to numerically express these conditions explicitly, by way of example, these conditions include those under which DNA molecules having higher homology e.g. such 70% or more, or in another example 80% or more, or in another example 90% or more, and in another example 95% or more homology, hybridize with each other, while DNA molecules having lower homology do not hybridize with each other, or those under which hybridization occurs under typical washing conditions in Southern hybridization, that is, at a salt concentration corresponding to 0.1×SSC and 0.1% SDS at 37° C., or in another example 0.1×SSC and 0.1% SDS at 60° C., and in another example 0.1×SSC and 0.1% SDS at 65° C. The length of the probe may be suitably selected, depending on the hybridization conditions, and usually varies from 100 bp to 1 kbp. Furthermore, “L-isoleucine dioxygenase activity” may be described as the activity that synthesizes (2S,3R,4S)-4HIL from L-isoleucine. However, when a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID No: 1, it can retain L-isoleucine dioxygenase activity of 10% or more, or in another example 30% or more, or in another example 50% or more, and in another example 70% or more, of the protein having the amino acid sequence of SEQ ID No: 2 at 37° C. and pH 8.
Furthermore, a DNA encoding a protein which is substantially identical to the IDO encoded by the DNA of SEQ ID No: 1 is also included in the above-described DNA. Namely, the following DNAs are also included:
(a) a DNA of the nucleotide sequence of SEQ ID No: 1;
(b) a DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID No: 1 and encodes a protein having L-isoleucine dioxygenase activity;
(c) a DNA that encodes a protein of the amino acid sequence of SEQ ID No: 2;
(d) a DNA that encodes a protein having an amino acid sequence that contains a substitution, deletion, insertion, addition or inversion of one or several amino acid residues in the amino acid sequence of SEQ ID No: 2 and having the L-isoleucine dioxygenase activity; and
(e) a DNA that encodes a protein having an amino acid sequence that is at least 70% homologous, or in another example at least 80% homologous, or in another example at least 90% homologous and still in another example at least 95% homologous to the amino acid sequence of SEQ ID NO:2 and has L-isoleucine dioxygenase activity.
Here, “one or several” refers to the number of changes which do not result in significant changes to the 3D structure of the protein, or a significant reduction of the L-isoleucine dioxygenase activity, and more specifically, can be in the range of 1 to 78, or in another example 1 to 52, or in another example 1 to 26, and still in another example 1 to 13.
The substitution, deletion, insertion, addition, or inversion of one or several amino acid residues should be conservative mutation(s) so that the activity is maintained. The representative conservative mutation is a conservative substitution. Examples of conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His or Lys, substitution of Asn with Glu, Gln, Lys, His or Asp, substitution of Asp with Asn, Glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp or Arg, substitution of Glu with Asn, Gln, Lys or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg or Tyr, substitution of Ile with Leu, Met, Val or Phe, substitution of Leu with Ile, Met, Val or Phe, substitution of Lys with Asn, Glu, Gln, His or Arg, substitution of Met with Ile, Leu, Val or Phe, substitution of Phe with Trp, Tyr, Met, Ile or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe or Trp, and substitution of Val with Met, Ile or Leu.
Furthermore, “L-isoleucine dioxygenase activity” refers to the synthesis of the (2S,3R,4S)-4HIL from L-isoleucine as described above. However, when the amino acid sequence of SEQ ID No: 2 contains a substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, it can retain L-isoleucine dioxygenase activity of 10% or more, or in another example 30% or more, or in another example 50% or more, and still in another example 70% or more, as compared to the protein having the amino acid sequence of SEQ ID No: 2 under conditions of 30° C. and pH 6.0. The L-isoleucine dioxygenase activity of the IDO can be measured by analysis of (2S,3R,4S)-4HIL formation from L-isoleucine by using high-performance liquid chromatography (HPLC).
Furthermore, a homologue DNA of SEQ ID NO: 1 can be used as the gene encoding L-isoleucine dioxygenase. Whether the homologue DNA encodes L-isoleucine dioxygenase or not can be confirmed by measuring L-isoleucine dioxygenase activity of the cell lysate, or cell lysate of the microorganism in which the homologue DNA is overexpressed.
The homologue DNA of SEQ ID NO: 1 can also be prepared from the genome of another Bacillus species, for example, Bacillus cereus, Bacillus weihenstephanensis. 
The phrase “a bacterium belonging to the genus Escherichia” means that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Escherichia can include, but are not limited to, Escherichia coli (E. coli).
The bacterium belonging to the genus Escherichia is not particularly limited; however, e.g., bacteria described by Neidhardt, F. C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) can be used.
The phrase “a bacterium belonging to the genus Pseudomonas” means that the bacterium is classified into the genus Pseudomonas according to the classification known to a person skilled in the art of microbiology.
The phrase “a bacterium belonging to the genus Corynebacterium” means that the bacterium is classified into the genus Corynebacterium according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Corynebacterium can include, but are not limited to, Corynebacterium glutamicum. 
The phrase “a bacterium belonging to the genus Arthrobacter” means that the bacterium is classified into the genus Arthrobacter according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Arthrobacter can include, but are not limited to, Arthrobacter simplex, Arthrobacter globiformis, Arthrobacter sulfureus, and Arthrobacter viscosus. 
The phrase “a bacterium belonging to the genus Aspergillus” means that the bacterium is classified into the genus Aspergillus according to the classification known to a person skilled in the art of microbiology.
The phrase “a bacterium belonging to the genus Bacillus” means that the bacterium is classified into the genus Bacillus according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Bacillus can include, but are not limited to, Bacillus subtilis. 
The brnQ gene from E. coli (synonyms—ECK0395, b0401, hrbA) encodes the branched chain amino acid LIVCS transporter BrnQ (synonyms—B0401, HrbA, LIV-II). The brnQ gene (nucleotides 418815 to 420134; GenBank accession no. NC—000913.2; gi: 16128105) is located between the gene phoR and the gene proY on the chromosome of E. coli K-12. The nucleotide sequence of the brnQ gene and the amino acid sequence of the BrnQ protein encoded by the brnQ gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
The sucA gene from E. coli (synonyms—ECK0714, lys, b0726, lys+met) encodes a subunit of the E1(0) component of the oxoglutarate dehydrogenase complex—SucA (synonyms—B0726, Lys). The sucA gene (nucleotides 757,929 to 760,730; GenBank accession no. NC—000913.2; gi: 16128105) is located between the gene G6388, partially overlapping with it, and the gene sucB on the chromosome of E. coli K-12. The nucleotide sequence of the sucA gene and the amino acid sequence of the SucA protein encoded by the sucA gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
The sucB gene from E. coli (synonyms—ECK0715, b0727) encodes a subunit of the E2(0) component of the oxoglutarate dehydrogenase complex—SucB (synonym B0727). The sucB gene (nucleotides 760,745 to 761,962; GenBank accession no. NC—000913.2; gi: 16128105) is located between the gene sucA and the gene sucC on the chromosome of E. coli K-12. The nucleotide sequence of the sucB gene and the amino acid sequence of the SucB protein encoded by the sucB gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
The aceA gene from E. coli (synonyms—ECK4007, b4015, icI) encodes a subunit of the isocitrate lyase—AceA (synonym B4015, Id). The aceA gene (nucleotides 4,215,132 to 4,216,436; GenBank accession no. NC—000913.2; gi: 16128105) is located between the gene aceB and the gene aceK on the chromosome of E. coli K-12. The nucleotide sequence of the aceA gene and the amino acid sequence of the AceA protein encoded by the aceA gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.
The aceK gene from E. coli (synonyms—ECK4008, b4016) encodes a subunit of the isocitrate dehydrogenase phosphatase—AceK (synonym B4016). The aceK gene (nucleotides 4,216,619 to 4,218,355; GenBank accession no. NC—000913.2; gi: 16128105) is located between the gene aceA and the gene arpA, that is oriented in opposite direction, and partially overlapping with it, on the chromosome of E. coli K-12. The nucleotide sequence of the aceK gene and the amino acid sequence of the AceK protein encoded by the aceK gene are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
The ilvE gene from E. coli (synonyms—ECK3762, b3770) encodes a subunit of the branched-chain amino-acid aminotransferase—IlvE (synonym B3770). The ilvE gene (nucleotides 3,950,507 to 3,951,436; GenBank accession no. NC—000913.2; gi: 16128105) is located between the gene ilvM and the gene ilvD on the chromosome of E. coli K-12. The nucleotide sequence of the ilvE gene and the amino acid sequence of the IlvE protein encoded by the ilvE gene are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
Since there may be some differences in DNA sequences between the genera or strains of the Escherichia genus, the brnQ gene with enhanced expression, or the sucA, sucB, aceA, aceK, ilvE genes with attenuated expression are not limited to the genes shown in SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11 and SEQ ID No: 13, but may include genes which are homologous to SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID No: 9, SEQ ID No: 11 and SEQ ID No: 13, but which encode a variant protein of the BrnQ, SucA, SucB, AceA, AceK and IlvE proteins, respectively. The phrase “variant protein” can mean a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but the activity as the BrnQ/SucA/SucB/AceA/AceK/IlvE protein is maintained. The number of changes in the variant protein depends on the position or the type of amino acid residues in the three dimensional structure of the protein. It may be 1 to 30, or in another example 1 to 15, and in another example 1 to 5 in SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12 and SEQ ID No: 14. These changes in the variants can occur in regions of the protein which are not critical for the function of the protein. This is because some amino acids have high homology to one another so the three dimensional structure or activity is not affected by such a change. Therefore, the protein variant encoded by the brnQ/sucA/sucB/aceA/aceK/ilvE gene may be one which has a homology of not less than 80%, or in another example than 90%, and in another example not less than 95%, with respect to the entire amino acid sequence shown in SEQ ID No: 4, SEQ ID No: 6, SEQ ID No: 8, SEQ ID No: 10, SEQ ID No: 12 and SEQ ID No: 14, as long as the activity of the BrnQ, SucA, SucB, AceA, AceK and IlvE proteins, respectively, is maintained (prior to inactivation of the sucA/sucB/aceA/aceK/ilvE gene).
Homology between two amino acid sequences can be determined using well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
Moreover, the brnQ/sucA/sucB/aceA/aceK/ilvE gene may be a variant which hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID No: 3/SEQ ID No: 5/SEQ ID No: 7/SEQ ID No: 9/SEQ ID No: 11/SEQ ID No: 13, or a probe which can be prepared from the nucleotide sequence under stringent conditions, provided that it encodes a functional BrnQ/SucA/SucB/AceA/AceK/IlvE protein prior to inactivation. “Stringent conditions” can include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, or in another example not less than 70%, or in another example not less than 80%, still in another example not less than 90%, and even in another example not less than 95%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed. For example, stringent conditions are exemplified by washing one time or more, or in another example two or three times at a salt concentration of 1×SSC, 0.1% SDS, or in another example 0.1×SSC, 0.1% SDS at 60° C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the HybondTM N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Washing may be performed 2 to 3 times. The length of the probe may be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
The phrase “enhanced expression of the gene” or “overexpression of a gene” can mean that the expression of the gene is higher than that of a non-modified strain, for example, a wild-type strain. Examples of such modification include increasing the copy number of expressed gene per cell, increasing the expression level of the gene, and so forth. The quantity of the copy number of an expressed gene can be measured, for example, by restricting the chromosomal DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), and the like. The level of gene expression can be measured by various known methods including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein encoded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), and the like. Furthermore, wild-type strains that can act as a control include, for example, Escherichia coli K-12.
The phrase “[t]ransformation of a bacterium with DNA encoding a protein” can mean introduction of the DNA into a bacterium, for example, by conventional methods. Transformation of this DNA will result in an increase in expression of the gene encoding the protein(s), and will enhance the activity of the protein in the bacterial cell. Methods of transformation can include any known methods that have hitherto been reported. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells to DNA has been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) may be used.
Methods of overexpressing a gene, or enhancing the expression of a gene include increasing the gene copy number. Introducing a gene into a vector that is able to function in a bacterium of the Escherichia genus increases the copy number of the gene. The low-copy vectors can be used. Examples of low-copy vectors include but are not limited to pSC101, pMW118, pMW119, and the like. The term “low copy vector” is used for vectors, the copy number of which is up to 5 copies per cell.
Enhancement of gene expression may also be achieved by introduction of multiple copies of the gene into a bacterial chromosome by, for example, homologous recombination, Mu integration, or the like. For example, one act of Mu integration allows for introduction of up to 3 copies of the gene into a bacterial chromosome.
Increasing the copy number of a gene can also be achieved by introducing multiple copies of the gene into the chromosomal DNA of the bacterium. In order to introduce multiple copies of the gene into a bacterial chromosome, homologous recombination can be carried out using a sequence which is present in multiple copies as targets in the chromosomal DNA. Sequences having multiple copies in the chromosomal DNA can include, but are not limited to repetitive DNA, or inverted repeats present at the end of a transposable element.
Enhancing gene expression can also be achieved by placing the objective DNA under the control of a potent promoter. For example, the Ptac promoter, the lac promoter, the trp promoter, the trc promoter, the PR, or the PL promoters of lambda phage are all known examples of potent promoters. The use of a potent promoter can be combined with the multiplication of gene copies.
Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter. Furthermore, it is known that substitution of several nucleotides in the spacer between ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon, can profoundly affect the mRNA translatability.
Moreover, it is also possible to introduce a nucleotide substitution into a promoter region of a gene on the bacterial chromosome, which results in stronger promoter function. The alteration of the expression control sequence can be performed, for example, in the same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed in International Patent Publication WO 00/18935 and Japanese Patent Application Laid-Open No. 1-215280.
It was proposed that attenuating expression of the sucA, sucB, aceA and aceK genes should lead to ‘shunting’ of the TCA cycle in mutant cells due to simultaneous oxidation of isoleucine and α-ketoglutarate (2-oxoglutarate), and may result in enhanced production of 4HIL. At the same time, simultaneous oxidation of isoleucine and α-ketoglutarate by IDO activity will be necessary for both growth of the bacterium and stabilization of the plasmid carrying a gene encoding IDO. In other words, the process of isoleucine hydroxylation will be necessary for cell growth. In this case, the biotransformation of isoleucine into 4-HIL can be achieved during bacterial cell growth without supplementing with antibiotics. This strategy was achieved by constructing a strain which lacks succinyl-CoA due to deletion of sucAB and aceAK genes (FIG. 1, Examples 3-5). This principle can be applied to any reaction which is coupled with the formation of succinate from 2-oxoglutarate. Furthermore, the minimum requirement is attenuating the expression of the gene coding for oxoglutarate dehydrogenase (such as ΔsucAB, ΔsucA, ΔsucB). Preferably the bacterium is further modified to attenuate the expression of the gene coding for isocitrate lyase (such as ΔaceA). Also, the bacterium is further modified to attenuate the expression of the genes coding for isocitrate lyase, and isocitrate dehydrogenase phosphatase (such as ΔaceAK).
By attenuating the expression of the gene coding for oxoglutarate dehydrogenase, the metabolism of 2-oxoglutarate is suppressed and the supply of 2-oxoglutarate to the 2-oxoglutarate-dependent enzyme is enhanced in the cells. The thus-engineered bacterium is then a host suitable for conducting 2-oxoglutarate-dependent enzyme reactions. The attenuation of the gene coding for oxoglutarate dehydrogenase increases 2-oxoglutarate levels in the cells and thereby renders the supply thereof to a 2-oxoglutarate-dependent enzyme to be efficient. The oxoglutarate dehydrogenase converts 2-oxoglutarate to succinyl-CoA in the TCA cycle The attenuation of the gene coding for isocitrate lyase catalyzing the conversion of isocirate to succinate in the glyoxylate cycle further increases the supply of 2-oxoglutarate. By this combination, the pathways from 2-oxoglutarate to succinate in the TCA and glyoxylate cycles are blocked, thereby further increasing the supply of 2-oxoglutarate to the 2-oxoglutarate-dependent enzyme. The attenuation of the gene coding for isocitrate dehydrogenease phosphatase further suppresses the inactivation of isocitrate dehydrogenase producing 2-oxoglutrate from isocirate. As the branched-chain amino acid aminotransferase deaminate 4HIL (Smirnov S. V. et al, FEMS Microbiol Lett.; 273(1):70-7 (2007)), expression of the ilvE gene can be attenuated, and should lead to higher yields of 4HIL due to the prevention of deamination of 4HIL.
The phrase “bacterium has been modified to attenuate expression the gene” can mean that the bacterium has been modified in such a way that the modified bacterium contains a reduced amount of the protein encoded by the gene as compared with an unmodified bacterium, or the modified bacterium is unable to synthesize the protein. The phrase “bacterium has been modified to attenuate expression of the gene” can also mean that the bacterium has been modified in such a way that the modified gene encodes a mutant protein with decreased activity.
The presence or absence of the gene in the chromosome of a bacterium can be detected by well-known methods, including PCR, Southern blotting, and the like.
The phrase “inactivation of the gene” can mean that the modified gene encodes a completely inactive protein. It is also possible that the modified DNA region is unable to naturally express the gene due to deletion of a part of or the entire gene, the shifting of the reading frame of the gene, the introduction of missense/nonsense mutation(s), or the modification of an adjacent region of the gene, including sequences controlling gene expression, such as promoter(s), enhancer(s), attenuator(s), ribosome-binding site(s), etc.
Expression of the gene can be attenuated by introducing a mutation into the gene on the chromosome so that intracellular activity of the protein encoded by the gene is decreased as compared with an unmodified strain. Such a mutation on the gene can be replacement of one base or more to cause an amino acid substitution in the protein encoded by the gene (missense mutation), introduction of a stop codon (nonsense mutation), deletion of one or two bases to cause a frame shift, insertion of a drug-resistance gene, or deletion of a part of the gene or the entire gene (Qiu, Z. and Goodman, M. F., J. Biol. Chem., 272, 8611-8617 (1997); Kwon, D. H. et al, J. Antimicrob. Chemother., 46, 793-796 (2000)). Expression of the gene can also be attenuated by modifying an expression regulating sequence such as the promoter, the Shine-Dalgarno (SD) sequence, etc. (WO95/34672, Carrier, T. A. and Keasling, J. D., Biotechnol Prog 15, 58-64 (1999)).
For example, the following methods may be employed to introduce a mutation by gene recombination. A mutant gene encoding a mutant protein having a decreased activity can be prepared, and a bacterium can be transformed with a DNA fragment containing the mutant gene. Then, the native gene on the chromosome can be replaced with the mutant gene by homologous recombination, and the resulting strain can be selected. Such gene replacement using homologous recombination can be conducted by the method employing a linear DNA, which is known as “Red-driven integration” (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by methods employing a plasmid containing a temperature-sensitive replication (U.S. Pat. No. 6,303,383 or JP 05-007491A). Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted with a plasmid which is unable to replicate in the host.
Expression of the gene can also be attenuated by insertion of a transposon or an IS factor into the coding region of the gene (U.S. Pat. No. 5,175,107), or by conventional methods, such as mutagenesis treatment by UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine).
Inactivation of the gene can also be performed by conventional methods, such as a mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, or/and insertion-deletion mutagenesis (Yu, D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 5978-83 and Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 6640-45), also called “Red-driven integration”.
Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989).
2. Method
The method can be a method for producing a product of a reaction catalyzed by a protein having 2-oxoglutarate-dependent enzyme activity by cultivating the bacterium as described above in a culture medium containing a substrate of the reaction, and isolating the produced product from the medium.
According to the product and the specificity of the protein having 2-oxoglutarate-dependent enzyme activity, the substrate is suitably selected. For example, when the product is (2S,3R,4S)-4-hydroxy-L-isoleucine and the protein has L-isoleucine dioxygenase activity, the substrate may be L-leucine.
Thus, a method for producing (2S,3R,4S)-4-hydroxy-L-isoleucine by cultivating the bacterium as described herein in a culture medium containing L-isoleucine, and isolating produced (2S,3R,4S)-4-hydroxy-L-isoleucine from the medium can be used.
The medium chosen for the culture may be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the chosen microorganism, alcohol, including ethanol and glycerol, can be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. As vitamins, thiamine, yeast extract, and the like, can be used. The medium can contain L-isoleucine (20-40 g/l).
The cultivation is preferably performed under aerobic conditions, such as a shaking culture, or a stirring culture with aeration, at a temperature of 20 to 40° C., or in another example 30 to 38° C. The pH of the culture is usually between 5 and 9, or in another example between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers.
Examples of separation and purification methods can include a method in which the (2S,3R,4S)-4HIL is contacted with an ion exchange resin to adsorb basic amino acids followed by elution and crystallization, and a method in which the product obtained by elution is discolored and filtrated with activated charcoal followed by crystallization to obtain (2S,3R,4S)-4HIL.
With respect to the product other than (2S,3R,4S)-4-hydroxy-L-isoleucine, the culture condition, the separation and purification methods and the line are similarly selected depending on the nature of the chosen bacterium and the target product.
EXAMPLES
The present invention will be explained in further detail with reference to the following examples; however, the invention is not limited thereto.
Example 1
Construction of the MG1655 [pELAC-IDO(Lys, 23)] and MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)] Strains
1.1. Construction of the pMW119-IDO(Lys, 23) Plasmid.
An 0.8 kb DNA fragment of the chromosome of the Bacillus thuringiensis strain 2-e-2 was amplified using oligonucleotides SVS 170 (SEQ ID No:15) and SVS 169 (SEQ ID No:16) as a primers and purified chromosomal DNA as a template. The PCR protocol was as follows: initial cycle for 30 seconds at 94° C.; 4 cycles for 40 seconds at 94° C.; 30 seconds at 49° C.; 40 seconds at 72° C.; 35 cycles for 30 seconds at 94° C.; 30 seconds at 54° C.; 30 seconds at 72° C. The PCR fragment was digested with BamHI and SacI endonucleases and then ligated into the pMW119 vector which had been previously treated with the same restrictases.
1.2. Construction of the pELAC-IDO (Lys, 23) Plasmid.
A 0.76 kb DNA fragment was excised from the pMW119-IDO(Lys, 23) plasmid with the XbaI, SacI endonucleases, and then cloned into the pELAC-ilvA/XbaI-SacI vector (see Reference example 1), resulting in the recombinant plasmid pELAC-IDO (Lys, 23) (FIG. 3).
1.3. Construction of the MG1655 (PL-brnQ) Strain.
The expression of the Ile-transporter BrnQ was increased in the MG1655 strain to improve the Ile influx. A 1.9 kbp DNA fragment harboring a Cm marker and the PL-promoter was PCR-amplified using oligonucleotides SVS 179 (SEQ ID No:17) and SVS 180 (SEQ ID No:18) as primers and chromosomal DNA of the BW25113 cat-PL-yddG (EP1449918A1, Russian patent RU2222596) strain as a template. Conditions for PCR were as follows: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30 sec at 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.
A 1.9 kbp PCR product was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MG1655 (ATCC 700926), which contains the plasmid pKD46 which has a temperature-sensitive replication origin. The plasmid pKD46 (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nucleotide DNA fragment of phage λ (nucleotide positions 31088 to 33241, GenBank accession no. J02459), and contains genes of the λ Red homologous recombination system (γ, β, exo genes) under the control of the arabinose-inducible ParaB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655.
Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30° C. in LB medium containing ampicillin (100 mg/l), and the culture was diluted 100 times with 5 ml of SOB medium (Sambrook et al, “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30° C. to an OD600 of ≈0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H2O. Electroporation was performed using 70 μl of cells and ≈100 ng of the PCR product. Cells after electroporation were incubated with 1 ml of SOC medium (Sambrook et al, “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, 1989) at 37° C. for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37° C. to select CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
Thus, the E. coli MG1655 (PL-brnQ) strain was constructed (FIG. 4).
1.4. Construction of the MG1655 [pELAC-IDO(Lys, 23)] and MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)] Strains.
The cells of the strains MG1655 and MG1655 (PL-brnQ) were each transformed with plasmid pELAC-IDO (Lys, 23). Resulting clones were selected on a X-gal/IPTG agar-plate (blue/white test). Thus, the strains MG1655 [pELAC-IDO(Lys, 23)] and MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)], respectively, were obtained.
Example 2
Production of 4HIL by E. coli Strain MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)]
To test the effect of enhanced expression of a gene coding for L-isoleucine transporter on 4HIL production, cells of the MG1655 [pELAC-IDO(Lys, 23)] and MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)] strains were grown in LB medium supplemented with ampicillin (200 μg/ml) and IPTG (1 mM) at 37° C. for about 4-5 hours. Specific IDO activity was measured in crude protein extracts for each grown recombinant E. coli strain as follows. Cells from 5 ml of culture were harvested by centrifugation at 4° C., re-suspended in 0.5 ml of buffer A*(50 mM TRIZMA, 5% glycerol, 1 mM EDTA, 1 mM DTT, pH 7 adjusted by HCl) and disrupted by sonication at 4° C. The reaction mixture (50 μl) contained 50 mM HEPES pH 7.0; 5 mM Ile; 0.5 mM α-ketoglutarate; 5 mM ascorbate; 5 mM FeSO4 and an aliquot of the protein preparation. The reaction was incubated at 34° C. for 1 hour with shaking. 4HIL was detected using TLC or HPLC analysis as follows. TLC analysis: a thin-layer silica gel plate (10×15 cm) spotted with an aliquot (1-2 μl) of the reaction solution was developed with a developing solvent (2-propanol:acetone:ammonia:water=100:100:25:16) and 4HIL was detected with the ninhydrin reagent. HPLC analysis: High pressure chromatograph (Waters, USA) using a spectrofluorimeter 1100 series (Agilent, USA) was conducted. The chosen detection wave range: excitation wavelength at 250 nm, range of emission wavelengths were 320-560 nm. The separation by the accq-tag method was performed in a column Nova-PakTM C18 150×3.9 mm, 4 μm (Waters, USA) at +40° C. The injection volume of the sample was 5 μl. The formation of amino acid derivatives and their separation was performed according to Waters manufacturer's recommendation (Liu, H. et al, J. Chromatogr. A, 828, 383-395 (1998); Waters accq-tag chemistry package. Instruction manual. Millipore Corporation, pp. 1-9 (1993)). To obtain amino acid derivatives with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, the kit Accq-FluorTM (Waters, USA) was used. The analysis by accq-tag method was performed using concentrated Accq-tag Eluent A (Waters, USA). All solutions were prepared using Milli-Q water, standard solutions were stored at +4° C. Results of measuring of IDO activity in crude extract of IDO-producing strains are shown in Table 1.
Cells of the MG1655 [pELAC-IDO(Lys, 23)] and MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)] strains were harvested by centrifugation and re-suspended up to a final volume of 2 ml (to an OD540≈0.03-0.04) in the medium MI30ch (50 mM KH2PO4 (pH7, adjusted by NaOH); 20 mM NH4Cl, 2 mM MgSO4, chalk—1.25 g/100 ml, 30 g/l Ile, 2 mM FeSO4, 2 mM ascorbate, ampicillin 200 mg/l) supplemented with L-isoleucine and ketoglutarate, glucose or glycerol in various combinations (see Table 2, Table 3). Cells were cultivated for about 15 hours at 32° C. with vigorous agitation. Then, accumulation of 4HIL was investigated by HPLC-analysis as described above. The results of measuring the 4HIL produced by the MG1655 [pELAC-IDO(Lys, 23)] and MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)] strains depending on the α-ketoglutarate, glucose, and glycerol are shown in Table 2 (at least 3 test tubes). The results of measuring the 4HIL produced by the MG1655 [pELAC-IDO(Lys, 23)] and MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)] strains depending on the different concentrations of glycerol are shown in Table 3 (at least 3 test tubes). As follows from Table 2 and Table 3, MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)] produced a higher amount of 4HIL, as compared with MG1655 [pELAC-IDO(Lys, 23)].










TABLE 1








IDO activity



Strain
(nmoles/min * mg)









MG1655 [pELAC-IDO(Lys, 23)]
300 ± 12



MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)]
280 ± 10




















TABLE 2









Initial culture mediuma)














Ile
KGb)
Gluc.
Glyc.
Final culture broth


Strain
(mM)
(mM)
(mM)
(mM)
4HIL (mM)















MG1655/
144
100
55
0
115 ± 6


pELAC-IDO(Lys, 23)

0
55
0
 17 ± 1




100
0
136
129 ± 6




0
0
136
 77 ± 4


MG1655(PL-brnQ)/

100
55
0
128 ± 6


pELAC-IDO(Lys, 23)

0
55
0
 34 ± 2




100
0
136
131 ± 6




0
0
136
106 ± 5





a)described in the text


b)Abbreviations: KG, α-ketoglutarate; Gluc., glucose; Glyc., glycerol
















TABLE 3









Initial culture mediuma)
Final culture broth











Ile
Glycerolb)
4HIL


Strain
(mM)
(mM)
(mM)













MG1655/
220
0
   6 ± 0.3


pELAC-IDO(Lys, 23)

136
 86 ± 4




272
146 ± 7




408
164 ± 8


MG1655(PL-brnQ)/

0
   7 ± 0.4


pELAC-IDO(Lys, 23)

136
 93 ± 5




272
158 ± 8




408
 203 ± 10





a)described in the text






Example 3
Construction of the MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) [pELAC-IDO(Lys, 23)] Strain
3.1. Construction of the MG1655 (ΔsucAB) Strain.
To delete the sucAB genes, the following manipulations were carried out. A 1.8 kb DNA fragment containing a CmR-marker and the Ptac promoter was amplified by PCR with oligonucleotides SVS-192 (SEQ ID No:19) and SVS-193 (SEQ ID No:20) as primers and chromosomal DNA of the MG1655(attR-Cm-attL-Ptac) strain (Katashkina J. I. et al., Molekularnaya biologiya(RU), v. 39, No. 5, 1-10 (2005)) as the template. Conditions for PCR were as follows: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30 sec at 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.
A 1.8 kbp PCR product was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MG1655, which contains the plasmid pKD46 having a temperature-sensitive replication origin.
Electroporation was performed as described above. Cells after electroporation were incubated with 1 ml of SOC medium at 37° C. for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37° C. to select CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
Thus, the E. coli MG1655 (ΔsucAB) strain was constructed.
3.2. Construction of the MG1655 (ΔaceAK) Strain.
To delete the aceAK genes, the following manipulations were carried out. A 1.8 kb DNA fragment containing the KmR-marker and Ptac promoter was amplified by PCR with oligonucleotides SVS-199 (SEQ ID No:21) and SVS-200 (SEQ ID No:22) as primers and pMW118-(λattL-Kmr-λattR) (see Reference example 2) plasmid DNA as the template.
Conditions for PCR were as follows: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30 sec at 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.
A 1.8 kbp PCR product was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MG1655, which contains the plasmid pKD46 which has a temperature-sensitive replication origin.
Electroporation was performed as described above. Cells after electroporation were incubated with 1 ml of SOC medium at 37° C. for 2.5 hours, and then were plated onto L-agar containing kanamycine (20 μg/ml) and grown at 37° C. to select KmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Km at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
Thus, the E. coli MG1655 (Δ aceAK) strain was constructed.
3.3. Construction of MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) Strain.
To eliminate the chloramphenicol resistance marker from the strain MG1655 (PL-brnQ), cells were transformed with the plasmid pMW118-int-xis (ApR) (WO2005/010175). ApR clones were grown on LB agar plates containing 150 mg/l ampicillin at 30° C. Several tens of ApR clones were picked up and tested for chloramphenicol sensitivity. The plasmid pMW118-int-xis was eliminated from the CmS cells by incubation on LB agar plates at 42° C. The obtained strain was used for further construction.
DNA fragments from the chromosome of the E. coli MG1655(ΔsucAB) strain were transferred to the strain obtained after elimination of the chloramphenicol resistance marker from the strain MG1655 (PL-brnQ) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.). Thus, MG1655 (ΔsucAB, PL-brnQ) strain was constructed. DNA fragments from the chromosome of the E. coli MG1655(ΔaceAK) strain were transferred to the strain MG1655 (ΔsucAB, PL-brnQ). Thus, the MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) strain was constructed.
The cells of the strain MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) were transformed with plasmid pELAC-IDO (Lys, 23). The resulting clones were selected on a X-gal/IPTG agar-plate (blue/white test). Thus, the strain MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) [pELAC-IDO(Lys, 23)] was obtained.
3.4. Investigation of the Growth of Strains MG1655, MG1655 (ΔsucAB, ΔaceAK, PL-brnQ), MG1655 [pELAC-IDO(Lys, 23)], and MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) [pELAC-IDO(Lys, 23)].
Strains MG1655 and MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) were grown in the following cultivation media:
A—M9 salts+glucose (0.4%);
B—M9 salts+glucose (0.4%)+DAP, Met, Lys (40 mg/l of each);
C—M9 salts+glycerol (0.4%);
D—M9 salts+glycerol (0.4%)+DAP, Met, Lys (40 mg/l of each).
The strains were cultivated in test tubes at 37° C. and optical density of the culture (A555) was measured every hour. As it can be seen from FIG. 5, strain MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) lacks succinyl-CoA and cannot grow in the media A or C. Only the addition of lysine, methionine, and diaminopimelate (DAP) restored the growth of the strain.
Furthermore, the MG1655[pELAC-IDO(Lys, 23)] and MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) [pELAC-IDO(Lys, 23)] strains were grown in following media:
G—M9 salts+glucose (137 mM)
GI—M9 salts+glucose (137 mM)+L-isoleucine (137 mM)
Y—M9 salts+glycerol (136 mM)
YI—M9 salts+glycerol (136 mM)+L-isoleucine (137 mM)
Each medium contained ampicillin (100 mg/l). The strains were cultivated in test tubes at 37° C. and the optical density of the culture (A555) was measured every hour.
Expression of the IDO gene restored the growth of the MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) strain on the M9-salts medium supplemented with L-isoleucine (FIG. 6). This data demonstrates that isoleucine hydroxylation leads to the growth of the strain.
Example 4
Production of 4HIL by the E. coli Strain MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) [pELAC-IDO(Lys, 23)]
To test the effect of attenuated expression of genes coding for oxoglutarate dehydrogenase, isocitrate lyase, and isocitrate dehydrogenase phosphatase, cells of the MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)] and MG1655(ΔsucAB, ΔaceAK, PL-brnQ)[pELAC-IDO(Lys, 23)] strains were each grown in medium A [(NH4)2SO4—1.5 g/100 ml; KH2PO4—0.15 g/100 ml; MgSO4—0.1 g/100 ml (MgSO4.7H2O—0.205 g/100 ml); Ile—220 mM; FeCl2—2 mM, 1 mM IPTG, chalk—2 g/100 ml] supplemented with glucose (300 mM) or glycerol (500 mM) at 32° C. for 72 hours with vigorous agitation. Then, the accumulation of 4HIL was determined by HPLC-analysis as described above. The results of measuring the 4HIL produced by the MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)] and MG1655(A sucAB, ΔaceAK, PL-brnQ)[pELAC-IDO(Lys, 23)] strains are shown in Table 4 (at least 3 test tubes). As follows from Table 4, MG1655(ΔsucAB, ΔaceAK, PL-brnQ)[pELAC-IDO(Lys, 23)] produced a higher amount of 4HIL, as compared with MG1655(PL-brnQ)[pELAC-IDO(Lys, 23)].










TABLE 4










Final




culture



Initial culture broth
broth












Glucose
Glycerol
Ile
4HIL


Strain
(mM)
(mM)
(mM)
(mM)





MG1655(PL-brnQ)
300
—
220
134


[pELAC-IDO(Lys, 23)]


MG1655(Δ sucAB, Δ aceAK,
300
—
220
189


PL-brnQ)[pELAC-IDO(Lys, 23)]


MG1655(PL-brnQ)
—
500
220
100


[pELAC-IDO(Lys, 23)]


MG1655(Δ sucAB, Δ aceAK,
—
500
220
195


PL-brnQ)[pELAC-IDO(Lys, 23)]









Example 5
Production of 4HIL by E. coli Strain MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)* [pEL-IDO(Lys, 23)]
5.1. Construction of the pEL-IDO(Lys, 23) Plasmid.
Due to the presence of the lacI gene in the pELAC-IDO(Lys, 23) plasmid, addition of IPTG is necessary to induce IDO expression during the biotransformation process of 4-HIL production. To avoid such IPTG-dependence, a large part of the lacI gene was deleted from pELAC-IDO(Lys, 23) plasmid (FIG. 1) by excision of the SphI-EcoRV DNA fragment using corresponding restrictases followed by ligation of the remaining part of the plasmid. Thus, the pEL-IDO(Lys, 23) plasmid was constructed.
5.2. Production of 4HIL
The MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)* strain was obtained by sequential excisions of Cm and Kn markers from the MG1655 (ΔsucAB, ΔaceAK, PL-brnQ) strain using plasmid pMW118-int-xis (ApR) (WO2005/010175) as described above. Plasmids pELAC-IDO(Lys, 23) and pEL-IDO(Lys, 23) were introduced into resulted strain. Thus, the MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)* [pELAC-IDO(Lys, 23)] and MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)*[pEL-IDO(Lys, 23)] strains were obtained.
A piece of biomass of the MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)* [pELAC-IDO(Lys, 23)] or MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)*[pEL-IDO(Lys, 23)] strain from a freshly made LB-agar plate was separately inoculated in 50 ml of LB broth supplemented with Ap (100 mg/l) and cultivated at 37° C. for about 4 hours in 750 ml-flask. The obtained cell cultures were used as inoculums in the biotransformation carried out in “Marubischi” fermenters. The following cultivation parameters were used: starting culture volume—500 ml; agitation—1200 rev/min; air—1:1; cultivation temperature—34° C.; pH 7.0 was stabilized using 2.5 M NH4OH.
An inoculum of the MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)* [pELAC-IDO] strain was added to 450 ml of medium containing (NH4)2SO4—5 g/l; KH2PO4—1.5 g/l; MgSO47H2O—1 g/l; FeSO47H2O—0.01 g/l; isoleucine—22-23 g/l (≈170 mM); glucose—50 g/l; 1 mM IPTG (pH 7 adjusted by KOH, final volume=500 ml) and cultivated for about 20 hours.
An inoculum of the MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)* [pEL-IDO] strain was added to 450 ml of the above medium without IPTG and cultivated for about 20 hours. Concentrations of 4HIL and L-isoleucine were determined using HPLC analysis as above. As seen from the Table 5, the use of the pEL-IDO plasmid avoids dependence on IPTG.










TABLE 5









Initial culture broth
Final culture broth













Glucose,

Glucose,
Ile,
4HIL,


Strain
(g/l)
Ile, (mM)
(g/l)
(mM)
(mM)





MG1655(ΔsucAB, ΔaceAK,
50
179
ND
ND
132


PL-brnQ) [pELAC-IDO(Lys, 23)]


MG1655(ΔsucAB, ΔaceAK,
50
174
ND
2.3
143


PL-brnQ) [pEL-IDO(Lys, 23)]





ND—non detected (≦0.1 g/l)






Reference Example 1
Construction of the pELAC-ilvA Plasmid
The plasmid pET-ilvA was constructed as follows. A 1.5 kb fragment of the E. coli MG1655 strain chromosomal DNA was amplified using PCR with oligonucleotides ilvA-5 (SEQ ID NO: 23) and ilvA-3 (SEQ ID NO: 24) as primers. A piece of cell culture of the E. coli MG1655 strain from an LB-agar plate was used as the source of the template DNA for the PCR procedure. As a result, the DNA fragment contained the ilvA gene and was flanked by the NdeI and BamHI restriction sites. It was cloned into the pET22 (b+) vector (Novagen, Germany) between the NdeI-BamHI sites. Thus, recombinant plasmid pET-ilvA was constructed (FIG. 2).
The pELAC-ilvA plasmid was constructed from pET-ilvA by replacing the BglII-XbaI fragment containing the T7 promoter with the BglII-XbaI fragment containing the Plac promoter (SEQ ID NO: 25).
Reference Example 2
Construction of the pMW118-(λattL-Kmr-λattR) Plasmid
The pMW118-(λattL-Kmr-λattR) plasmid was constructed based on the pMW118-attL-Tc-attR (WO2005/010175) plasmid by substitution of the tetracycline resistance marker gene with the kanamycin resistance marker gene from the pUC4K plasmid (Vieira, J. and Messing, J., Gene, 19(3): 259-68 (1982)).
For that purpose, the large EcoRI-HindIII fragment from pMW118-attL-Tc-attR plasmid was ligated to two fragments from the pUC4K plasmid: HindIII-PstI fragment (676 bp) and EcoRI-HindIII fragment (585 bp).
Basic pMW118-attL-Tc-attR was obtained by ligation of the following four DNA fragments:

    
    
        1) the BglII-EcoRI fragment (114 bp) carrying attL (SEQ ID NO: 26) which was obtained by PCR amplification of the corresponding region of the E. coli W3350 (containing λ prophage) chromosome using oligonucleotides P3 and P6 (SEQ ID NOS: 27 and 28) as primers (these primers contained the subsidiary recognition sites for BglII and EcoRI endonucleases);
        2) the PstI-HindIII fragment (182 bp) carrying attR (SEQ ID NO: 29) which was obtained by PCR amplification of the corresponding region of the E. coli W3350 (containing λ prophage) chromosome using the oligonucleotides P5 and P6 (SEQ ID NOS: 30 and 31) as primers (these primers contained the subsidiary recognition sites for PstI and HindIII endonucleases);
        3) the large BglII-HindIII fragment (3916 bp) of pMW118-ter_rrnB. The plasmid pMW118-ter_rrnB was obtained by ligation of the following three DNA fragments:
        a) the large DNA fragment (2359 bp) carrying the AatII-EcoRI fragment of pMW118 that was obtained in the following way: pMW118 was digested with EcoRI restriction endonuclease, treated with Klenow fragment of DNA polymerase I, and then digested with AatII restriction endonuclease;
        b) the small AatII-BglII fragment (1194 bp) of pUC19 carrying the bla gene for ampicillin resistance (ApR) was obtained by PCR amplification of the corresponding region of the pUC19 plasmid using oligonucleotides P7 and P8 (SEQ ID NOS: 32 and 33) as primers (these primers contained the subsidiary recognition sites for AatII and BglII endonucleases);
        c) the small BglII-PstIpol fragment (363 bp) of the transcription terminator ter_rrnB was obtained by PCR amplification of the corresponding region of the E. coli MG1655 chromosome using oligonucleotides P9 and P10 (SEQ ID NOS: 34 and 35) as primers (these primers contained the subsidiary recognition sites for BglII and PstI endonucleases);
        4) the small EcoRI-PstI fragment (1388 bp) (SEQ ID NO: 36) of pML-Tc-ter_thrL bearing the tetracycline resistance gene and the ter_thrL transcription terminator; the pML-Tc-ter_thrL plasmid was obtained in two steps:
        the pML-ter_thrL plasmid was obtained by digesting the pML-MCS plasmid (Mashko, S. V. et al., Biotekhnologiya (in Russian), 2001, no. 5, 3-20) with the XbaI and BamHI restriction endonucleases, followed by ligation of the large fragment (3342 bp) with the XbaI-BamHI fragment (68 bp) carrying terminator ter_thrL obtained by PCR amplification of the corresponding region of the E. coli MG1655 chromosome using oligonucleotides P11 and P12 (SEQ ID NOS: 37 and 38) as primers (these primers contained the subsidiary recognition sites for the XbaI and BamHI endonucleases);
        the pML-Tc-ter_thrL plasmid was obtained by digesting the pML-ter_thrL plasmid with the KpnI and XbaI restriction endonucleases followed by treatment with Klenow fragment of DNA polymerase I and ligation with the small EcoRI-Van91I fragment (1317 bp) of pBR322 bearing the tetracycline resistance gene (pBR322 was digested with EcoRI and Van91I restriction endonucleases and then treated with Klenow fragment of DNA polymerase I).
    
    


Example 6
Production of Hydroxyl-Pro by the E. coli Strain MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)*
The synthesis of DNA of SEQ ID NO: 39 was consigned (Invitrogen), and a DNA encoding an amino acid sequence (SEQ ID NO: 40) of L-prolyl 4-hydroxylase from Dactylosporangium sp. was obtained in the form of a plasmid in which the DNA was ligated to BlueHeron pUCminusMCS. To the plasmid, sequences were inserted so that a sequence recognized by NdeI and a sequence recognized by HindIII were positioned at the 5′ terminal and 3′ terminal sides of the DNA, respectively, and the plasmid was digested with NdeI/HindIII. Separately, the ptrp4 vector (Journal of Molecular Catalysis B: Enzymatic 32 (2005)205-211) was digested with NdeI/HindIII. The two fragments digested with NdeI/HindIII were ligated, and introduced into the E. coli strain JM109 to obtain a transformant JM109/ptrp4_PDO.
Plasmid DNA was extracted from the obtained transformant, and introduced into the E. coli strain MG1655 and E. coli strain MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)* to obtain transformants MG1655/ptrp4_PDO and MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)*/ptrp4_PDO, respectively.
Strains MG1655/ptrp4_PDO and MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)*/ptrp4_PDO were cultivated in LB medium containing 100 mg/l ampicillin at 30° C. for 24 hours, and 300 μL of the culture was inoculated into 3 ml of LB medium containing 100 mg/l ampicillin and cultivated at 37° C. for 6 hours. To a test tube containing L-Pro and calcium carbonate so that their final concentrations were 10 g/l and 20 g/l, respectively, the obtained culture (0.3 ml) and the following cultivation media (2.7 ml) were added:
Medium Composition:
Medium A (300 ml): D-glucose (20 g/L), MgSO4.7H2O (1 g/L);
Medium B (700 ml): ammonium sulfate (5 g/L), KH2PO4 (1.5 g/L), sodium ascorbate (0.01 g/L), FeSO47H2O (0.1 g/L), adjusted pH to 7.0 with KOH.
Media A and B were separately autoclaved at 120° C. for 20 min, and mixed after cooling.
The D-glucose concentration (g/L) and the 4-hydroxy-proline concentration (mM) are shown in Table 6. D-glucose was analyzed with a glucose analyzer (SAKURA SI, Japan). 4-Hydroxy-proline was analyzed by HPLC with Sumichiral OA-5000 (Sumika Analysis Center, Japan) (Mobile phase: 2 mM copper sulfate aqueous solution, column temperature: 30° C., flow rate: 1 ml/min, detection: UV 254 nm).
It was confirmed that MG1655 (ΔsucAB, ΔaceAK, PL-brnQ)*/ptrp4_PDO could utilize D-glucose more efficiently for the production reaction of hydroxyproline compared with MG1655/ptrp4_PDO.











TABLE 6









0 min
22 hr
66 hr














Glucose,
Pro**
Glucose,
Hydroxy-
Glucose,
Hydroxy-


Strain
(g/l)
(mM)
(g/l)
Pro
(g/l)
Pro
















MG1655/ptrp4_PDO
18
87
7
15
0
31


MG1655 (Δ sucAB, Δ
18
87
11
15
3
58


aceAK, PL-brnQ)*/





**calculated value






Example 7-1
Construction of E. coli Strain MG1655 (ΔsucA, ΔaceAK) and E. coli Strain MG1655 (ΔsucB, ΔaceAK)
To confirm whether the PL-brnQ mutation is required for the efficient utilization of D-glucose or not, the following experiments were carried out.
To construct a strain deficient in 2-oxoglutarate dehydrogenase (sucA and sucB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK) without any mutation in PL-brnQ, ΔaceAK was introduced into E. coli strain MG1655 ΔsucA strain (JW0715) and E. coli strain MG1655 ΔsucB strain (JW0716) of E. coli Keio Knockout Collection (http://ecoli.naist.jp) to obtain the E. coli strain MG1655 (ΔsucA, ΔaceAK) and E. coli strain MG1655 (ΔsucB, ΔaceAK). A deficiency of one of sucA and sucB results in the deficiency of 2-oxoglutarate dehydrogenase activity.
To delete the aceAK genes, the following manipulations were carried out. A 1.6 kb DNA fragment containing the CmR-marker was amplified by PCR with oligonucleotides SVS-199 (SEQ ID NO:21) and SVS-200 (SEQ ID NO:22) as primers and pMW118-(λattL-Cmr-λattR) (WO2005/010175) plasmid DNA as the template.
Conditions for PCR were as follows: denaturation step for 3 min at 94° C.; profile for 30 cycles: 30 sec at 94° C., 30 sec at 50° C., 2 min at 72° C.
A 1.6 kbp PCR product was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MG1655 (ΔsucA), E. coli strain MG1655 (ΔsucB), which contains the plasmid pKD46 (WO2005/010175) which has a temperature-sensitive replication origin.
Electroporation was performed as described above. Cells after electroporation were incubated with 1 ml of SOC medium at 37° C. for 1 hour and then were plated onto L-agar containing Cm (25 μg/ml) and grown at 37° C. to select for CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
After that, the CmR-marker was removed using plasmid pMW118-int-xis-ts (ApR) (WO2005/010175) as described above.
Thus E. coli strain MG1655 (ΔsucA, ΔaceAK) and E. coli strain MG1655 (ΔsucB, ΔaceAK) strain were constructed.
Example 7-2
Production of 4HIL by E. coli Strain MG1655[pEL-IDO(Lys, 23)], E. coli Strain MG1655 (ΔsucA, ΔaceAK)[pEL-IDO(Lys, 23)], and E. coli Strain MG1655 (ΔsucB, ΔaceAK) [pEL-IDO(Lys, 23)]
The pEL-IDO(Lys, 23) plasmid was introduced into E. coli strain MG1655 as well as E. coli strain MG1655 (ΔsucA, ΔaceAK) and E. coli strain MG1655 (ΔsucB, ΔaceAK) obtained by the above method to obtain the transformants E. coli strain MG1655[pEL-IDO(Lys, 23)], E. coli strain MG1655 (ΔsucA, ΔaceAK)[pEL-IDO(Lys, 23)], and E. coli strain MG1655 (ΔsucB, ΔaceAK) [pEL-IDO(Lys, 23)].
These strains were cultivated in LB medium containing 100 mg/l ampicillin at 30° C. for 24 hours, and 300 μL of the culture was inoculated into 3 ml of LB medium containing 100 mg/l ampicillin and cultivated at 37° C. for 6 hours. To a test tube containing L-Ile and calcium carbonate so that their final concentrations are 10 g/l and 20 g/l, respectively, the obtained culture (0.3 ml) and the following cultivation media (2.7 ml) were added:
Medium Composition:
Medium A (300 ml): D-glucose (20 g/L), MgSO4.7H2O (1 g/L);
Medium B (700 ml): ammonium sulfate (5 g/L), KH2PO4 (1.5 g/L), sodium ascorbate (0.01 g/L), FeSO47H2O (0.1 g/L), adjusted pH to 7.0 with KOH.
The media A and B were separately autoclaved at 120° C. for 20 min, and mixed after cooling.
The remaining D-glucose and the produced 4-hydroxy-isoleucine in the medium are shown in Table 7. D-glucose was analyzed with a glucose analyzer (SAKURA SI, Japan). 4-Hydroxy-isoleucine was analyzed by HPLC with MCI GEL CRS10W (Mitsubishi Kagaku, Japan) (Mobile phase: 1 mM CuSO4, 5% MeOH, column temperature: 30° C., flow rate: 1 ml/min, detection: UV 254 nm).
As a result, it was confirmed that in the reaction using E. coli strain MG1655 (ΔsucA, ΔaceAK)[pEL-IDO(Lys, 23)], and E. coli strain MG1655 (ΔsucB, ΔaceAK) [pEL-IDO(Lys, 23)] without the PL-brnQ mutation, D-glucose could be more efficiently utilized for the conversion reaction by Ile dioxygenase compared with MG1655[pEL-IDO(Lys, 23)].
From these results, it is confirmed that strains deficient in the ΔsucAB, ΔsucA, ΔsucB, and ΔaceAK genes, such as E. coli MG1655 (ΔsucAB, ΔaceAK), E. coli MG1655 (ΔsucA, ΔaceAK), and E. coli MG1655 (ΔsucB, ΔaceAK), can be widely used for the 2-oxoglutrate-dependent enzymes.











TABLE 7









0 min
48 hr
96 hr
















Glucose,

Glucose,

4HIL,
Glucose,

4HIL,


Strain
(g/l)
Ile, (mM)
(g/l)
Ile, (mM)
(mM)
(g/l)
Ile, (mM)
(mM)


















MG1655 [pEL-IDO(Lys, 23)]
18
38
0
35
10
0
34
9


MG1655(Δsuc A, Δace AK)
18
38
14
45
1
0
17
26


[pEL-IDO(Lys, 23)]


MG1655(ΔsucB, Δace AK)
18
38
14
44
1
0
26
17


[pEL-IDO(Lys, 23)]









EXPLANATION OF SEQUENCES
1: L-isoleucine dioxygenase gene from Bacillus thuringiensis strain 2-e-2
2: L-isoleucine dioxygenase from Bacillus thuringiensis strain 2-e-2
3: brnQ gene from E. coli 
4: BrnQ from E. coli 
5: sucA gene from E. coli 
6: SucA from E. coli 
7: sucB gene from E. coli 
8: SucB from E. coli 
9: aceA gene from E. coli 
10: AceA from E. coli 
11: aceK gene from E. coli 
12: AceK from E. coli 
13: ilvE gene from E. coli 
14: IlvE from E. Coli 
15: primer SVS 170
16: primer SVS 169
17: primer SVS 179
18: primer SVS 180
19: primer SVS 192
20: primer SVS 193
21: primer SVS 199
22: primer SVS 200
23: primer IlvA-5
24: primer ilvA-3
25: BglII-XbaI fragment containing the Plac promoter
26: fragment attL
27: primer P3
28: primer P4
29: fragment attR
30: primer P5
31: primer P6
32: primer P7
33: primer P8
34: primer P9
35: primer P10
36: fragment of pML-Tc-ter_thrL
37: primer P11
38: primer P12
39: L-proline 4-hydroxylase gene from Dactylosporangium sp.
40: L-proline 4-hydroxylase from Dactylosporangium sp.
While the invention has been described in detail with reference to the preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated as a part of this application by reference.
INDUSTRIAL APPLICABILITY
According to the present invention, production of (2S,3R,4S)-4-hydroxy-L-isoleucine can be enhanced. This compound is useful as a component of pharmaceutical compositions with insulinotropic activity. Production of (2S,3R,4S)-4-hydroxy-L-isoleucine is enhanced by using a bacterium transformed with a DNA fragment containing a gene coding for a protein having L-isoleucine dioxygenase activity.

## Claims

1. A method for manufacturing a product of a reaction catalyzed by a protein having 2-oxoglutarate-dependent enzyme activity, comprising:
A) cultivating a bacterium transformed with a DNA fragment comprising a gene coding for a protein having 2-oxo-glutarate-dependent enzymatic activity, wherein said bacterium has been modified to attenuate the expression of a gene coding for oxoglutarate dehydrogenase, in a culture medium containing a substrate of the reaction; and wherein said protein has L-isoleucine dioxygenase activity and catalyzes a reaction coupled with the formation of succinate from 2-oxoglutarate; and
B) isolating the product, wherein said product is (2S,3R,4S)-4-hydroxy-L-isoleucine or a salt thereof.

2. The method according to claim 1, wherein said expression is attenuated by inactivating said genes coding for oxoglutarate dehydrogenase.

3. The method according to claim 1, wherein said bacterium has been modified to attenuate the expression of genes coding for oxoglutarate dehydrogenase and isocitrate lyase.

4. The method according to claim 3, wherein said expression is attenuated by inactivating said genes coding for oxoglutarate dehydrogenase and isocitrate lyase.

5. The method according to claim 1, wherein said bacterium has been modified to attenuate the expression of genes coding for oxoglutarate dehydrogenase, isocitrate lyase, and isocitrate dehydrogenase phosphatase.

6. The method according to claim 5, wherein said expression is attenuated by inactivating said genes coding for oxoglutarate dehydrogenase, isocitrate lyase, and isocitrate dehydrogenase phosphatase.

7. The method according to claim 1, wherein the bacterium belongs to a genus selected from the group consisting of Escherichia, Pseudomonas, Corynebacterium, Arthrobacter, Aspergillus, and Bacillus.

8. The method according to claim 1, wherein the bacterium is selected from the group consisting of Escherichia coli, Arthrobacter simplex, Corynebacterium glutamicum, Arthrobacter globiformis, Arthrobacter sulfureus, Arthrobacter viscosus, and Bacillus subtilis.

9. The method according to claim 6, wherein said bacterium has been modified to overexpress a gene coding for an Escherichia coli L-isoleucine transporter.

10. The method according to claim 1, wherein the culture medium contains a carbon source selected from the group consisting of a carbohydrate, and an alcohol.

11. The method according to claim 10, wherein said carbohydrate is glucose, and said alcohol is glycerol.

12. The method according to claim 1, wherein said protein is L-isoleucine dioxygenase.