RU2444568C2 - Bacterium producer of product of reaction catalised by protein showing 2-oxoglutarate-dependent enzyme activity, and method for producing such product - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to a method for producing a product of reaction catalysed by a protein showing 2-oxoglutarate-dependent enzyme activity such as (2S,3R,4S)-4-hydroxy-b-isoleucine or its salt and 4-hydroxy-b-proline or its salt with using a bacterium of a genus selected from a group consisting of Escherichia, Corynebacterium, Arthrobacter, Aspergillus and Bacillus, transformed by a DNA fragment containing a gene coding the protein showing 2-oxoglutarate-dependent enzyme activity with said bacterium modified so that: the gene coding 2-oxoglutarate dehydrogenase is inactivated, or the gene coding 2-oxoglutarate dehydrogenase, and the gene coding aminotransferase with branched-chain amino acid are inactivated.

EFFECT: invention enables high-effective production of said compounds and their salts.

14 cl, 6 dwg, 9 tbl, 9 ex

Description

Technical field

The present invention relates to the microbiological industry, and in particular to a method for producing a reaction product catalyzed by a protein having the activity of a 2-oxoglutarate-dependent enzyme, such as 4-hydroxy-L-isoleucine or its salt, using a bacterium transformed with a DNA fragment containing a gene encoding a protein having activity of a 2-oxoglutarate-dependent enzyme, as well as L-isoleucine dioxygenase activity. This bacterium is also modified to enhance the expression of the gene encoding the L-isoleucine transporter and is capable of producing (2S, 3R, 4S) -4-hydroxy-L-isoleucine.

Description of the Related Art

4-Hydroxy-L-isoleucine is an amino acid that can be extracted and purified from the seeds of Greek fenugreek (Trigonella foenum-graecum L.leguminosae). The drug 4-hydroxyisoleucine exhibits insulinotropic activity, which is of great interest, since this stimulating effect clearly depends on the concentration of glucose in the plasma. This effect is shown both in a model of isolated perfusion pancreatic glands of rats and in pieces of tissue of human pancreas (Sauvaire, Y. et al, Diabetes, 47: 206-210 (1998)). This dependence of the insulinotropic effect on glucose concentration is not confirmed in the case of sulfonylurea (Drucker, DJ, Diabetes 47: 159-169 (1998)), the only insulinotropic drug currently used to treat type II diabetes [non-insulin-dependent diabetes (NIDD) or (NIDDM)], and as a result, hypoglycemia remains the main 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)). The property of 4-hydroxyisoleucine to increase glucose tolerance is also known (Am. J. Physiol. Endocrinol., Vol.287, E463-E471, 2004). The ability of 4-hydroxyisoleucine to accelerate glucose metabolism and the potential for its use as a medicine and a component of functional nutrition were disclosed in Japanese Patent Application Laid-Open No. Hei 6-157302 and in US patent application US 2007-000463 A1.

4-Hydroxy-L-isoleucine is found only in plants and due to its specific insulinotropic activity can be considered as a new secretion stimulator with potential use for the treatment of type II diabetes, because this disease is characterized by insufficient insulin secretion associated with varying degrees of insulin resistance (Broca, C. et al, Am. J. Physiol. 277 (Endocrinol. Metab. 40): E617-E623 (1999)).

The method of oxidizing isoleucine with atmospheric oxygen in the presence of iron, ascorbic acid and 2-oxoglutaric acid using the dioxigenase activity of Greek fenugreek extract has been proposed as a method for producing 4-hydroxy-L-isoleucine (Phytochemistry, Vol.44, No.4, pp.563 -566.1997). However, this method is not suitable for the industrial production of 4-hydroxyisoleucine, since the enzyme activity is inhibited by the substrate at isoleucine concentrations of 20 mM or higher, moreover, the enzyme has not yet been identified, it is obtained from plant extracts in small quantities and its activity rapidly decreases .

To date, an effective method for the synthesis of optically pure (2S, 3R, 4S) -4-hydroxyisoleucine with a total yield of 39%, consisting of eight stages, has been described. A key step in this synthesis process involves the biotransformation of ethyl 2-methylacetoacetate to ethyl (2S, 3S) -2-methyl-3-hydroxybutanoate using Geotrichum candidum and asymmetric Strecker synthesis (Wang, Q. et al, Eur. J. Org. Chem., 834-839 (2002)).

Also described is a short six-step enzyme-chemical synthesis method for (2S, 3R, 4S) -4-hydroxyisoleucine with a general stereochemistry control, the last stage of which is enzymatic decomposition by hydrolysis of the N-phenylacetylactone derivative using commercially available pencillinacylase G immobilized on Eupergit C (E-P-PACIT (E-P-PAC) ) (Rolland-Fulcrand, V. et al, J. Org. Chem., 873-877 (2004)).

However, there are currently no reports of the production of (2S, 3R, 4S) -4-hydroxy-L-isoleucine using a bacterium transformed with a DNA fragment containing a gene encoding a protein with L-isoleucine dioxygenase activity; however, the bacterium is also modified to enhance the expression of a gene encoding the L-isoleucine transporter; and has the ability to produce (2S, 3R, 4S) -4-hydroxy-L-isoleucine.

In addition, (2S, 3R, 4S) -4-hydroxy-L-isoleucine, a reaction product catalyzed by a protein having a 2-oxoglutarate-dependent enzyme activity, as well as an important industrial product, was known. However, there were no reports of effective methods for the preparation of other products using proteins having the activity of a 2-oxoglutarate-dependent enzyme.

Description of the invention

An aspect of the present invention is to increase the production of a reaction product coupled with the formation of succinate from 2-oxoglutarate by the action of a protein having the activity of a 2-oxoglutarate-dependent enzyme. This product includes compounds both in free form and their salts. Another aspect of the present invention is the provision of an industrial method of obtaining a product associated with the formation of succinate from 2-oxoglutarate using a bacterium having the activity of a 2-oxoglutarate-dependent enzyme. This bacterium has been modified so that the expression of the gene encoding oxoglutarate dehydrogenase is attenuated, preferably modified so that the expression of genes encoding the oxoglutarate dehydrogenase and isocitrate lyase is more modified so that the expressions of the genes encoding oxoglutarate dehydrogenase dehydrogenase weakened.

Another aspect of the present invention is the provision of a method for producing 4-hydroxyisoleucine, including both its free form and its salts. This compound may also be referred to as "(2S, 3R, 4S) -4HIL". Another aspect of the present invention is the provision of a method for the production of (2S, 3R, 4S) -4-hydroxy-L-isoleucine or its salt by direct enzymatic hydroxylation of L-isoleucine using bacteria with L-isoleucine dioxygenase activity. Overexpression of the gene encoding the L-isoleucine transporter is also performed in this bacterium, and the bacterium is capable of producing (2S, 3R, 4S) -4-hydroxy-L-isoleucine.

Previously, a bacterium with a high level of L-isoleucine dioxygenase activity was isolated from a natural source, and a gene encoding L-isoleucine dioxygenase was cloned. It was found that L-isoleucine dioxygenase can be used for the synthesis of (2S, 3R, 4S) -4-hydroxy-L-isoleucine.

Another aspect of the present invention includes providing a method for increasing the production of (2S, 3R, 4S) -4-hydroxy-L-isoleucine using bacteria with L-isoleucine dioxygenase activity. The above goal was achieved by the discovery that a bacterium with L-isoleucine dioxygenase activity produces more (2S, 3R, 4S) -4-hydroxy-L-isoleucine if the bacterium is modified so that expression of the gene encoding the L-isoleucine transporter reinforced.

The aim of the present invention is the provision of a bacterium transformed with a DNA fragment containing a gene encoding a protein with L-activity of isoleucine dioxygenase, characterized in that the bacterium is modified so that the expression of the gene encoding the L-isoleucine transporter is enhanced and the bacterium has the ability to produce (2S, 3R, 4S) -4-hydroxy-L-isoleucine.

Another objective of the present invention is the provision of the above-described bacteria producing (2S, 3R, 4S) -4HIL, the gene encoding a protein with L-isoleucine dihydrogenase activity selected from the group consisting of:

(a) DNA comprising the nucleotide sequence shown in the sequence Listing under the number 1 (SEQ ID No: 1);

(b) DNA hybridizing under stringent conditions with DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in Sequence Listing No. 1 (SEQ ID No: 1), said DNA encoding a protein with L-isoleucine dioxygenase activity;

(c) DNA comprising a nucleotide sequence encoding a protein comprising the amino acid sequence shown in the List of sequences under number 2 (SEQ ID No: 2);

(d) a DNA comprising a nucleotide sequence encoding a protein comprising the amino acid sequence shown in the List of sequences under number 2 (SEQ ID No: 2), except that the specified 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 encoding a protein comprising an amino acid sequence homologous to at least 98% of the amino acid sequence shown in Sequence Listing No. 2 (SEQ ID No: 2), wherein said protein has L-isoleucine dioxygenase activity .

It is also an object of the present invention to provide the above-described producing bacteria (2S, 3R, 4S) -4HIL, wherein the bacterium is modified so that the activity of L-isoleucine dioxygenase is increased.

Another objective of the present invention is the provision of the above-described bacteria producing (2S, 3R, 4S) -4HIL, while the activity of L-isoleucine dioxygenase is increased by enhancing the expression of a gene encoding L-isoleucine dioxygenase.

It is also an object of the present invention to provide the (2S, 3R, 4S) -4HIL producing bacterium described above, wherein expression of L-isoleucine dioxygenase is enhanced by modifying the sequence that controls the expression of the gene encoding L-isoleucine dioxygenase or by increasing the number of copies of the gene encoding L -isoleucine dioxygenase.

It is also an object of the present invention to provide the above-described bacteria producing (2S, 3R, 4S) -4HIL, the gene encoding the L-isoleucine transporter is the brnQ gene from Escherichia coli.

It is also an object of the present invention to provide the (2S, 3R, 4S) -4HIL producing bacterium described above, the bacterium being further modified to attenuate the expression of genes encoding oxoglutarate dehydrogenase, isocitrate lyase and isocitrate dehydrogenase phosphatase.

It is also an object of the present invention to provide the bacteria described above, wherein said expression is attenuated by inactivation of said genes.

It is also an object of the present invention to provide the bacteria described above, the bacterium being further modified to attenuate the expression of a gene encoding a branched chain amino acid transferase.

It is also an object of the present invention to provide the bacterium described above, wherein said expression is attenuated by inactivation of said gene.

It is also an object of the present invention to provide a 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 also an object of the present invention to provide a bacterium as described above, the bacterium being selected from the group consisting of Escherichia coli, Arthrobacter simplex, Corynebacterium glutamicum, Arthrobacter globiformis, Arthrobacter sulfureus, Arthrobacter viscosus and Bacillus subtilis.

Another objective of the present invention is the provision of a method of production of (2S, 3R, 4S) -4-hydroxy-L-isoleucine or its salt, including:

the cultivation of the above bacteria producing (2S, 3R, 4S) -4HIL in a nutrient medium containing L-isoleucine; and

isolation of (2S, 3R, 4S) -4-hydroxy-L-isoleucine.

It is also an object of the present invention to provide the method described above, wherein the culture medium contains a carbon source selected from the group consisting of carbohydrate and alcohol.

It is also an object of the present invention to provide the method described above, wherein said carbohydrate is glucose and said alcohol is glycerin.

Another aspect of the present invention is the provision of a method for producing 4-hydroxy-L-proline, including both its free form and its salts. Another aspect of the present invention is the provision of a method for the production of 4-hydroxy-L-proline or a salt thereof by direct enzymatic hydroxylation of L-proline using bacteria with L-proline hydroxylase activity.

The present invention is described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the 'shunting' of the tricarboxylic acid cycle in strain MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) _ [pELAC-IDO (Lys, 23)] due to the oxidation of isoleucine and α-ketoglutarate (2- oxoglutarate).

Figure 2 shows a schematic of the recombinant plasmid pET-IlvA.

Figure 3 shows a schematic of the recombinant plasmid pELAC-IDO (Lys, 23).

Figure 4 shows the construction of E. coli strain MG1655 (PL-brnQ).

Figure 5 shows the growth of strains MG1655 and MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) on synthetic medium M9 containing glucose or glycerin with or without lysine, methionine and diaminopimelate (DAP-diaminopimelate). The strains were grown on synthetic M9 medium supplemented with glucose or glycerol, as well as lysine, methionine and diaminopimelate (DAP). Abbreviations: Strains WT = MG1655; 2d = MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ); Wednesday: A = M9 + glucose; B = M9 + glucose + (Lys, Met, DAP); C = M9 + glycerin; D = M9 + glycerol + (Lys, Met, DAP).

Figure 6 shows the growth of strains MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) [pELAC-IDO (Lys, 23)] on synthetic medium M9 s with or without L-isoleucine. Abbreviations: Strains 1 = MG1655 [pELAC-IDO (Lys, 23)]; 2 = MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) [pELAC-IDO (Lys, 23)]; Medium: G = M9 + glucose (137 mM); GI = M9 + glucose (137 mM) + L-isoleucine (137 mM); Y = M9 + glycerin (136 mM); YI = M9 + glycerin (136 mM) + L-isoleucine (137 mM).

BEST MODE FOR CARRYING OUT THE INVENTION

1. The bacterium according to the present invention

The term “bacterium” as used herein includes a bacterium producing an enzyme, a mutant and a genetic recombinant of such a bacterium in which the target enzymatic activity is present or increased, and the like.

The term “activity of a 2-oxoglutarate-dependent enzyme,” as used herein, means an enzymatic activity capable of catalyzing the reaction of formation of succinate from 2-oxoglutarate.

Many proteins have the activity of a 2-oxoglutarate-dependent enzyme, as well as 2-oxoglutarate-dependent oxygenases, as previously reported. The following examples include dioxygenases capable of producing the desired products, such as Pro hydroxylase, converting L-Pro to hydroxy-Pro (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1999, p.4028-4031), γ-butyrobetaine hydroxylase, converting γ- butyrobetaine to L-carnitine (WO 2005/083089). In addition, these are dioxigenases, many of which have already been reported. For example, a link 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-oxoglutart-dependent dioxigenases described in the reviews, the bacterium has been modified so that the expression of a gene encoding oxoglutarate dehydrogenase (such as Δsuc A B, Δsuc A , ΔsucB) is impaired; preferably modified in such a way that the expression of genes encoding oxoglutarate dehydrogenase and isocytratliase (such as (Δsuc A B, Δsuc A , or ΔsucB) plus Δ a ce A ) is weakened; more preferably modified so that the expression of genes encoding oxoglutarate dehydrogenase, isocitratlyase and isocitrate dehydrogenase phosphatase (such as (Δsuc A B, Δsuc A or ΔsucB) plus Δ a ce A K) are attenuated, which are represented in E. coli strain MG1655 ( A B, Δ a ce A K), which are described in the examples below, are suitable host cells that efficiently use 2-oxoglutarate, which is produced on media containing carbon as D-glucose, in 2-oxoglutarate-dependent enzymatic reactions.

The present invention will be described with reference to Examples in which a protein with L-isoleucine dioxygenase activity and a reaction product catalyzed by said protein is given as a protein with activity of a 2-oxoglutart-dependent enzyme, is (2S, 3R, 4S) -4-hydroxy- L-isoleucine. However, the present invention is not limited to these examples.

In the present invention, the term “(2S, 3R, 4S) -4-hydroxy-L-isoleucine”, or “(2S, 3R, 4S) -4HIL” or “4HIL” refers to a single chemical compound or to a mixture of compounds containing (2S, 3R, 4S) -4-hydroxyisoleucine.

The term “bacterium” as used herein includes an enzyme-forming bacterium, a mutant and a genetic recombinant of such a bacterium in which the target enzymatic activity is present or increased, and the like.

You can use the abbreviation L-isoleucine dioxygenase - IDO (L-isoleucine dioxygenase).

As a result of screening of natural microorganisms, a unique bacterium was discovered - a strain of Bacillus thuringiensis 2-e-2, which has activity that catalyzes a reaction in which (2S, 3R, 4S) -4HIL is formed directly from L-isoleucine, both in free form and in salt form. After cultivation, L-isoleucine dioxygenase was purified from microbial cells; in short, it can be called IDO (Lys, 23).

In addition, the N-terminal amino acid sequence of IDO (Lys, 23) of the purified dioxigenase of the Bacillus thuringiensis 2-e-2 strain was determined. The strain Bacillus thuringiensis 2-e-2 was named Bacillus thuringiensis AJ110584 and deposited in accordance with the Budapest Treaty at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305- 8566, Japan) September 27, 2006 with inventory number FERM BP-10688.

The DNA encoding IDO (Lys, 23) is presented in the Sequence Listing under SEQ ID No: 1. In addition, the amino acid sequence IDO (Lys, 23) encoded by the nucleotide sequence of SEQ ID NO: 1 is presented in the Sequence Listing under 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) with the sequence SEQ ID NO: 2 has L-isoleucine dioxygenase activity and catalyzes the reaction, which (2S, 3R, 4S) -4HIL, expressed by the following formula (I), is synthesized directly one molecule of L-isoleucine.

DNA encoding an IDO catalyzing a 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 due to the possibility of differences in nucleotide sequences among species and strains Bacillus forming IDO, catalyzing the reaction of formation of (2S, 3R, 4S) -4HIL from L-isoleucine.

The DNA of the present invention includes not only isolated DNA encoding an IDO, but also DNA encoding an IDO into which mutations are artificially introduced. This DNA can be isolated from the chromosome of an IDO-producing microorganism. The DNA of the present invention should encode an IDO capable of catalyzing the above reaction. Methods for artificially introducing mutations include commonly used methods for introducing site-specific mutations described in Method. in Enzymol., 154 (1987).

DNA that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID No: 1 and encoding a protein with IDO activity is also included in the DNA of the present invention. In the framework of the present invention, “stringent conditions” means those conditions under which specific hybrids are formed while non-specific hybrids are not formed. Although a quantitative description of these conditions is difficult, as an example, reference can be made to conditions under which DNA molecules having a higher homology, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% and particularly preferably not less than 95% or more, hybridize with each other, while DNA molecules with lower homology do not hybridize with each other, or such conditions under which hybridization occurs under normal washing conditions during Southern hybridization, which is carried out at a salt concentration of 0.1 × SSC, 0.1% SDS at 37 ° C, preferably 0.1 × SSC, 0.1% SDS at 60 ° C, and more preferably 0.1 × SSC, 0.1% SDS at 65 ° C. The length of the probe can be selected appropriately, depending on hybridization conditions and usually varies from 100 bp. up to 1 thousand bp In addition, "L-isoleucine dioxygenase activity" can be described as an activity that synthesizes (2S, 3R, 4S) -4HIL from L-isoleucine. However, the nucleotide sequence hybridizing under stringent conditions with the nucleotide sequence complementary to the nucleotide sequence of SEQ ID No: 1, preferably retains L-isoleucine dioxygenase activity by 10% or more, preferably 30% or more, more preferably 50% or more, and even more preferably 70 % or more, relative to a protein with the amino acid sequence of SEQ ID No: 2 at 37 ° C and pH 8.

In addition, DNA encoding a protein substantially identical to IDO encoded by SEQ ID No: 1 is also included in the DNA of the present invention. Namely, the following DNAs are also included in the DNAs of the present invention:

(a) DNA with the nucleotide sequence of SEQ ID No: 1;

(b) DNA hybridizing under stringent conditions with DNA with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID No: 1, and encoding a protein with L-isoleucine dioxygenase activity;

(c) DNA encoding a protein with the amino acid sequence of SEQ ID No: 2;

(d) DNA encoding a protein with an amino acid sequence comprising the substitution, deletion, insertion, addition or inversion of one or more amino acid residues in the amino acid sequence of SEQ ID No: 2, having L-isoleucine dioxygenase activity; and

(e) DNA encoding a protein with an amino acid sequence homologous to at least 70%, preferably at least 80%, more preferably at least 90% and even more preferably at least 95% of the amino acid sequence of SEQ ID NO : 2, possessing L-isoleucine dioxygenase activity.

In the framework of the present invention, the phrase "one or more" means a range of changes in which the tertiary structure of the protein or the activity of L-isoleucine dioxygenase does not change significantly and, in particular, corresponds to values from 1 to 78, preferably from 1 to 52, more preferably from 1 to 26 and even more preferably 1 to 13.

Substitution, deletion, insertion, addition or inversion of one or more amino acid residues will be a conservative mutation or conservative mutations, provided that the enzyme activity is maintained. An example of a conservative mutation (s) is (are) a conservative substitution (s). Examples of conservative substitutions include replacing Ala with Ser or Thr, replacing Arg with Gln, His or Lys, replacing Asn with Glu, Gln, Lys, His or Asp, replacing Asp with Asn, Glu or Gln, replacing Cys with Ser or Ala, replacing Gln with Asn, Glu, Lys, His, Asp or Arg, replacing Glu with Asn, Gln, Lys or Asp, replacing Gly with Pro, replacing His with Asn, Lys, Gln, Arg or Tyr, replacing Ile with Leu, Met, Val or Phe, replace Leu with Ile, Met, Val or Phe, replace Lys with Asn, Glu, Gln, His or Arg, replace Met with Ile, Leu, Val or Phe, replace Phe with Trp, Tyr, Met, Ile or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tyr, replacing Tyr with His, Phe or Trp, and replacing Val with Met, Ile or Leu.

In addition, the term “L-isoleucine dioxygenase activity” means the ability to produce (2S, 3R, 4S) -4HIL from L-isoleucine, as described above. However, in the case where the amino acid sequence contains the substitution, deletion, insertion, addition or inversion of one or more amino acid residues in the amino acid sequence of SEQ ID No: 2, the L-isoleucine dioxygenase activity is maintained at least by at least 10%, preferably not less than 30%, more preferably not less than 50% and even more preferably 70% or more of the activity of a protein having the amino acid sequence of SEQ ID No: 2, while maintaining the temperature at 30 ° C and pH 6. L -of leytsindioksigenaznaya IDO activity according to the present invention can be measured by analyzing the formation of (2S, 3R, 4S) -4HIL of L-isoleucine by high performance liquid chromatography (HPLC).

In addition, a DNA fragment homologous to the DNA fragment shown in SEQ ID NO: 1 can be used as a gene encoding the L-isoleucine dioxygenase according to the present invention. The fact that a homologous DNA fragment encodes or does not code for L-isoleucine dioxygenase can be confirmed by measuring the L-isoleucine dioxygenase activity in a cell lysate or in a cell lysate of a microorganism in which a homologous DNA fragment is overexpressed.

A DNA fragment homologous to that shown in SEQ ID NO: 1 can also be prepared from the genomic DNA of other bacterial species of the Bacillus genus, for example, Bacillus cereus, Bacillus weihenstephanensis.

The term "bacterium belonging to the genus Escherichia" means that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia used in the present invention, the bacterium Escherichia coli (E. coli) may be mentioned.

The range of bacteria belonging to the genus Escherichia that can be used in the present invention is not limited in any way, however, for example, the bacteria described in Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1), can be included in the number of bacteria according to the present invention.

The term "bacterium belonging to the genus Pseudomonas" means that the bacterium is classified as belonging to the genus Pseudomonas according to the classification known to the specialist in the field of microbiology.

The term "bacterium belonging to the genus Corynebacterium" means that the bacterium is classified as belonging to the genus Corynebacterium according to the classification known to the specialist in the field of microbiology. Examples of bacteria belonging to the genus Corynebacterium as used in the present invention include, but are not limited to, Corynebacterium glutamicum.

The term "bacterium belonging to the genus Arthrobacter" means that the bacterium is classified as belonging to the genus Arthrobacter according to the classification known to the person skilled in the field of microbiology. Examples of bacteria belonging to the genus Arthrobacter as used in the present invention include, but are not limited to, Arthrobacter simplex, Arthrobacter globiformis, Arthrobacter sulfureus, and Arthrobacter viscosus.

The term "bacterium belonging to the genus Aspergillus" means that the bacterium is classified as belonging to the genus Aspergillus according to the classification known to the person skilled in the field of microbiology.

The term "bacterium belonging to the genus Bacillus" means that the bacterium is classified as belonging to the genus Bacillus according to the classification known to the specialist in the field of microbiology. Examples of bacteria belonging to the genus Bacillus as used in the present invention include, but are not limited to, Bacillus subtilis.

The E. coli brnQ gene (synonyms ECK0395, b0401, hrbA) encodes the LIVCS BrnQ branched chain amino acid transporter (synonyms B0401, HrbA, LIV-II). The brnQ gene (nucleotides 418815 to 420134; in sequence with accession number NC_000913.2 in the GenBank database; gi: 16128105) is located between the phoR and proY genes on the chromosome of E. coli K-12 strain. The nucleotide sequence of the brnQ gene and the amino acid sequence of the BrnQ protein encoded by the brnQ gene are shown in the Sequence Listing under the numbers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4), respectively.

The E. coli suc A gene (synonyms - ECK0714, lys, b0726, lys + met) encodes the E1 (0) subunit of the component of the oxoglutarate dehydrogenase complex - Suc A (synonyms - B0726, Lys). The suc A gene (nucleotides 757,929 to 760,730; in sequence with accession number NC_000913.2 in the GenBank database; gi: 16128105) is located between the G6388 gene, partially overlapping with it, and the sucB gene on the chromosome of E. coli K-12 strain. The nucleotide sequence of the suc A gene and the amino acid sequence of the Suc A protein encoded by the suc A gene are shown in the Sequence Listing Nos. 5 (SEQ ID NO: 5) and 6 (SEQ ID NO: 6), respectively.

The E. coli sucB gene (synonyms - ECK0715, b0727) encodes the E2 (0) subunit of the component of the oxoglutarate dehydrogenase complex - SucB (synonym B0727). The sucB gene (nucleotides 760,745 through 761,962; in sequence with accession number NC_000913.2 in the GenBank database; gi: 16128105) is located between the suc A and sucC genes on the chromosome of E. coli K-12 strain. The nucleotide sequence of the sucB gene and the amino acid sequence of the SucB protein encoded by the sucB gene are shown in the Sequence Listing under numbers 7 (SEQ ID NO: 7) and 8 (SEQ ID NO: 8), respectively.

And lo and gene of E. coli (synonyms - ECK4007, b4015, icI) encodes a subunit of the isocitrate - ASEAN (synonyms B4015, IcI). A gene and ce (nucleotides 4,215,132 to 4,216,436 on, in sequence with the accession number NC_000913.2 in the GenBank data base; gi: 16128105) is located between the genes and the sowing and a ceK on the chromosome of E. coli K-12. The nucleotide sequence of the gene and ce A and the amino acid sequence ASEAN protein encoded by the gene and ce A, shown in Sequence Listing Nos 9 (SEQ ID NO: 9) and 10 (SEQ ID NO: 10), respectively.

The E. coli a ceK gene (synonyms ECK4008, b4016) encodes the isocitrate dehydrogenase phosphatase subunit AceK (synonym B4016). Gene a ceK (nucleotides 4,218,355 to 4,216,619 on, in sequence with the accession number NC_000913.2 in the GenBank data base; gi: 16128105) is located between the gene and gene A and lo a rp A, in the opposite orientation, partially overlapping with it, on the chromosome E. coli K-12 strain. The nucleotide sequence of the a ceK gene and the amino acid sequence of the AceK protein encoded by the a ceK gene are listed in the Sequence Listing Nos. 11 (SEQ ID NO: 11) and 12 (SEQ ID NO: 12), respectively.

The E. coli ilvE gene (synonyms - ECK3762, b3770) encodes a branched chain amino acid transferase subunit of IlvE (synonym B3770). The ilvE gene (nucleotides 3,950,507 through 3,951,436; in sequence with accession number NC_000913.2 in the GenBank database; gi: 16128105) is located between the ilvM and ilvD genes on the chromosome of E. coli K-12 strain. The nucleotide sequence of the ilvE gene and the amino acid sequence of the IlvE protein encoded by the ilvE gene are shown in the Sequence Listing Nos. 13 (SEQ ID NO: 13) and 14 (SEQ ID NO: 14), respectively.

As the representatives of different genera or strains of the family Enterobacteriaceae may be some variation in nucleotide sequences, the term brnQ gene with enhanced expression or gene suc A, sucB, and ce A, a ceK, ilvE attenuated expression is not limited to genes, the sequences of which are shown in the Sequence Listing under the numbers 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 also include genes 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, encoding a variant of BrnQ, SucA, SucB, AcAA, AceK and IlvE protein, respectively of course. The term "protein variant", as used in the present invention, means a protein with changes in sequence, be it deletion, insertion, addition or substitution of amino acids, which retains the activity of the protein BrnQ / SucA / SucB / AceA / AceK / IlvE. The number of changes in a protein variant depends on the position or type of amino acid residue in the tertiary structure of the protein. It can be from 1 to 30, preferably from 1 to 15, more preferably from 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 variations in variants may occur in areas not critical to protein function. These changes are possible because some amino acids have a high homology to each other, therefore, such changes do not affect the tertiary structure or activity. Therefore, a variant of the protein encoded by the brnQ / suc A / sucB / a ce A / aceK / ilvE gene can be represented by proteins with a homology of at least 80%, preferably at least 90% and most preferably at least 95%, relative to the total the amino acid sequence shown in the Sequence Listing under the number 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, provided that it remains Activity BrnQ proteins, SucA, sucB, AceA, AceK and ilvE (to inactivate the gene suc A / sucB / a ce A / aceK / ilvE).

Homology between two amino acid sequences can be determined using known methods, for example, the BLAST 2.0 computer program, which counts three parameters: amino acid number, identity, and similarity.

Furthermore, brnQ genes, suc A, sucB, a ce A, aceK, ilvE may be a variant which hybridizes under stringent conditions with a nucleotide sequence in the sequence listing under the number 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 with a probe that can be synthesized based on the indicated nucleotide sequence, provided that it encodes the functional protein BrnQ / Suc before inactivation A / SucB / AceA / AceK / IlvE. "Stringent conditions" include those conditions under which specific hybrids, for example hybrids with a homology of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% and most preferably at least 95%, are formed, and non-specific hybrids, for example hybrids with less homology than indicated above, are not formed. A practical example of harsh conditions is a single wash, preferably two or three times, at a salt concentration of 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at 60 ° C. The duration of washing depends on the type of membrane used for blotting and, as a rule, is as recommended by the manufacturer. For example, the recommended washing time for Hybond ™ N + nylon membrane (Amersham) under stringent conditions is 15 minutes. Two to three times washing is preferred. The length of the probe can be selected depending on hybridization conditions and is usually about 100-1000 bp.

The term “enhanced gene expression” or “overexpression of a gene” means that gene expression is higher than in an unmodified strain, for example, a wild-type strain. Examples of such modifications include an increase in the number of copies of a gene per cell, an increase in the level of gene expression, etc. The number of copies of an expressed gene is determined, for example, by restriction of chromosomal DNA followed by Southern blotting using a probe based on a gene sequence, in situ fluorescence hybridization (FISH fluorescence in situ hybridization), and the like.

Gene expression can be determined using various known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of protein encoded by the gene can be determined using known methods, including SDS-PAGE electrophoresis followed by immunoblotting (Western blotting), etc. In addition, wild-type strains that can be used as controls include, for example, Escherichia coli K-12.

The term "transformation of bacteria using a DNA fragment encoding a protein" means the introduction of a DNA fragment into a bacterium, for example, by traditional methods. Transformation of this DNA will increase expression of the gene encoding the protein of the present invention and increase the activity of the protein in this bacterial cell. Transformation methods include any known methods that have already been described. For example, a method of treating recipient cells with calcium chloride in such a way as to increase cell permeability to DNA can be used. This method has been described for strain Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)).

Methods for overexpressing a gene or enhancing gene expression include increasing the number of copies of a gene. The introduction of a gene into a vector capable of functioning in bacteria of the Enterobacteriaceae family increases the number of copies of the gene. Mostly low copy vectors are 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 number of copies of which in a cell reaches five.

Enhanced gene expression can also be achieved by introducing multiple copies of the gene into the bacterial chromosome, for example, by homologous recombination, Mu integration, etc. For example, one act of Mu integration allows up to 3 copies of a gene to be introduced into the bacterial chromosome.

An increase in the number of copies of the gene can also be achieved by introducing multiple copies of the gene into the chromosomal DNA of the bacterium. To introduce multiple copies of the gene into the bacterial chromosome, homologous recombination is performed using the target sequences present on the chromosome in multiple copies. Multiple copy sequences in chromosomal DNA include, but are not limited to, repetitive DNA or inverted repeats at the ends of transposed elements.

Enhanced gene expression can also be achieved by substituting the DNA of the present invention under the control of a strong promoter. Strong promoters are known to be, for example, the lac promoter, trp promoter, trc promoter, PR or PL phage promoters. Using a strong promoter can be combined with an increase in gene copies.

On the other hand, the action of the promoter can be enhanced, for example, by introducing a mutation into the promoter, leading to an increase in the level of transcription of the gene localized behind the promoter. In addition, it is known that the replacement of several nucleotides in the region between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately before the start codon, significantly affects the mRNA translatability.

In addition, it is possible to introduce a nucleotide substitution in the region of the promoter of the gene on the bacterial chromosome, resulting in an increase in the function of the promoter. Changing the expression control sequence can be accomplished, for example, in the same way as replacing a gene using a temperature-sensitive plasmid, as disclosed in WO 00/18935 and JP 1-215280 A.

The inventors of the present invention have suggested that weakening the expression of suc A , sucB, a ce A, and aceK genes will result in 'shunting' of the tricarboxylic acid cycle in mutant cells due to the simultaneous oxidation of isoleucine and α-ketoglutarate (2-oxoglutarate). This can lead to increased 4HIL production. At the same time, the simultaneous oxidation of isoleucine and α-ketoglutarate due to the action of IDO will be a factor necessary for bacterial growth and for stabilization of a plasmid containing the gene encoding IDO. In other words, the process of hydroxylation of isoleucine will be necessary for cell growth. In this case, the biotransformation of isoleucine into 4-HIL can be achieved during the growth of bacterial cells without the addition of any antibiotics. This approach was implemented by constructing a strain deficient in succinyl-CoA due to deletion of the suc A B and a ce A K genes (Figure 1, Examples 3-5).

Obviously, this principle can be applied to any reactions associated with the formation of succinate from 2-oxoglutarate. It is also obvious that the minimum requirement is a decrease in the expression of a gene encoding oxoglutarate dehydrogenase (such as Δsuc A B, Δsuc A , ΔsucB). Preferably, the bacterium has been further modified so that expression of a gene encoding isocitrate lyase (such as a Δ ce A) has been weakened. Even more preferably, the bacterium is further modified so that the expression of genes encoding isocitratliase and isocitrate dehydrogenase phosphatase (such as Δ a ce A K) is impaired.

When the expression of genes encoding oxoglutarate dehydrogenase is weakened, the metabolism of 2-oxoglutarate in the cell is suppressed and the influx of 2-oxoglutarate to the 2-oxoglutarate-dependent enzyme increases. The bacterium designed in this way is fully adapted to carry out 2-oxoglutarate-dependent enzyme reactions. The weakening of the expression of genes encoding oxoglutarate dehydrogenase increases the level of 2-oxoglutarate, as a result of which its flow to the 2-oxoglutarate-dependent enzyme becomes more effective. Oxoglutarate dehydrogenase is an enzyme that converts 2-oxoglutarate to succinyl-CoA in the tricarboxylic acid cycle. It is preferable to further combine the attenuation of the expression of a gene encoding isocitratlyase catalyzing the conversion of isocitrate to succinate in the glyoxylate cycle, thereby further increasing the influx of 2-oxoglutarate. In the case of such a combination, the pathways from 2-oxoglutarate to succinate in the CTK and glyoxylate cycles are blocked, as a result of which the influx of 2-oxoglutarate to the 2-oxoglutarate-dependent enzyme further increases. Further combination is even more preferred with the attenuation of genes encoding isocitrate dehydrogenase phosphatase, thus inhibiting the inactivation of isocitrate dehydrogenase producing 2-oxoglutarate from isocitrate.

Since the branched chain amino acid aminotransferase deaminates 4HIL (Smirnov SV et al, FEMS Microbiol Lett .; 273 (1): 70-7 (2007)), the inventors of the present invention have suggested that a decrease in ilvE gene expression will result in a higher 4HIL yield by preventing deamination of 4HIL.

The term “bacterium is modified in such a way that the expression of the gene is weakened” means that the bacterium has been modified so that, as a result of the modification, the bacterium contains a reduced amount of protein compared to an unmodified bacterium or the bacterium is not capable of synthesizing the protein. The term “bacterium is modified in such a way that gene expression is weakened” also means that the bacterium has been modified in such a way that the modified gene encodes a mutant protein with reduced activity.

The presence or absence of the yahN gene on the chromosome can be determined by well-known methods, including PCR, Southern blotting, etc.

The term "gene inactivation" means that the modified gene encodes a completely inactive protein. It is also possible that natural expression of the modified DNA region is not possible due to the division of the operon genes, the shift of the reading frame, the introduction of the missense / nonsense mutation (s), or the modification of regions adjacent to the gene, which include sequences that control gene expression, such as promoters, enhancers , attenuators, ribosome binding sites, etc.

Gene expression can be attenuated by introducing mutations into the genes. Such a gene mutation may be the replacement of one or more bases for amino acid substitution in the encoded protein (the Missense mutation), the introduction of a stop codon (the "nonsense" mutation), the deletion of one or more bases for reading frame shift, insertion of the resistance gene an antibiotic or deletion of a gene or part thereof (J. Biol. Chem., 1997, 272 (13); 8611-8617, J. Antimicrobial Chemotherapy, 2000, 46: 793-79). The expression of the yahN gene can also be attenuated by modifying the expression of regulatory sequences, such as a promoter, a Shine-Dalgarno (SD) sequence, etc. (PCT application WO 95/34672; Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods can be used to introduce mutations by gene recombination. A mutant gene is constructed that encodes a mutant protein with reduced activity, and the bacterium for its modification is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced by homologous recombination with the mutant gene, and the resulting strain is selected. Such gene substitution using homologous recombination can be carried out using a linear DNA method known as “Red-dependent integration” or “Red-system integration” (Datsenko, KA, Wanner, BL, Proc.Natl.Acad.Sci. USA , 97, 12, 6640-6645 (2000), PCT application WO 2005/010175), or by a method using a plasmid whose replication is temperature sensitive (US patent 6,303,383 or Japanese patent application JP 05-007491A). Further, the introduction of a site-specific mutation by replacing a gene using the aforementioned homologous recombination can also be carried out using a plasmid with reduced replication ability in the host cell.

Gene expression can also be attenuated by inserting a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107) or by conventional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).

Gene inactivation can also be carried out by traditional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-specific mutagenesis, gene destruction using homologous recombination and / or mutagenesis due to insertion deletions (Yu, D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 5978-83 and Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97:12: 6640-45), also called "Red-Dependent Integration".

Methods for preparing plasmid DNA, DNA restriction and ligation, transformation, selection of nucleotides as a primer, etc. may be conventional methods known to a person skilled in the art. These methods are described, for example, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

2. The method according to the present invention

The method according to the present invention is a method of producing a reaction product catalyzed by a protein having the activity of a 2-oxoglutarate-dependent enzyme by growing the bacteria of the present invention in a culture medium containing a reaction substrate and isolating the resulting product from the culture fluid.

Depending on the product and the specificity of the protein used, having the activity of a 2-oxoglutarate-dependent enzyme, a suitable substrate is 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, the method according to the present invention can be a method for producing (2S, 3R, 4S) -4-hydroxy-L-isoleucine by culturing the bacteria of the present invention in a nutrient medium containing L-isoleucine and isolating the resulting (2S, 3R, 4S) -4-hydroxy-L-isoleucine from the culture fluid.

The nutrient medium used for growing can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, the appropriate amount of nutrient additives necessary for the growth of microorganisms. Carbon sources include various carbohydrates such as glucose and sucrose, as well as various organic acids. Depending on the nature of the assimilation of the microorganism used, alcohols such as ethanol and glycerin may be used. Various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, microorganism fermentolizate, can be used as a nitrogen source. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. As vitamins, thiamine, yeast extract, and the like can be used. The culture medium used in the present invention contained L-isoleucine (20-40 g / l).

The cultivation is preferably carried out under aerobic conditions, such as mixing the culture fluid on a rocking chair, shaking with aeration, at a temperature in the range from 20 to 40 ° C, preferably in the range from 30 to 38 ° C. The pH of the medium is maintained in the range from 5 to 9, preferably from 6.5 to 7.2. The pH of the medium can be adjusted by ammonia, calcium carbonate, various acids, bases and buffer solutions.

Examples of isolation and purification methods may include a method in which (2S, 3R, 4S) -4HIL binds to an ion exchange resin to adsorb basic amino acids, followed by elution and crystallization, and a method in which the product obtained after elution is decolorized and filtered through activated coal followed by crystallization to obtain (2S, 3R, 4S) -4HIL.

For a product other than (2S, 3R, 4S) -4-hydroxy-L-isoleucine, growing conditions, separation and purification methods are selected in accordance with the characteristics of the bacteria and target products used.

Examples

The present invention will be explained in more detail below with reference to Examples, which are not limited to the present invention.

Example 1. Construction of strains MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)]

1.1. Construction of the plasmid pMW119-IDO (Lys, 23)

A DNA fragment of the chromosome of a strain of Bacillus thuringiensis 2-e-2 with a length of 0.8 TPN amplified using oligonucleotides SVS 170 (SEQ ID No: 15) and SVS 169 (SEQ ID No: 16) as primers and purified chromosomal DNA as a template. The PCR scheme was as follows: initial cycle for 30 seconds at 94 ° C; 4 cycles: 40 seconds at 94 ° C; 30 seconds at 49 ° C; 40 seconds at 72 ° C; 35 cycles: 30 seconds at 94 ° C; 30 seconds at 54 ° C; 30 seconds at 72 ° C. The PCR fragment was treated with endonucleases B a mHI and S a cI and then ligated into the vector pMW119 pretreated with the same restriction enzymes.

1.2. Construction of the plasmid pELAC-IDO (Lys, 23)

A DNA fragment of 0.76 kbp excised from the plasmid pMW119-IDO (Lys, 23) using endonucleases Xb a I, S a cI, then cloned in the vector pELAC-ilvA / Xb a I-S a cI (see Reference example 1), the resulting plasmid pELAC-IDO (Lys, 23) (Figure 3).

1.3. Construction of strain MG1655 (P _L -brnQ)

Expression of the Ile BrnQ transporter was enhanced in strain MG1655 to enhance the intake of Ile. A 1.9 kb DNA fragment containing the Cm marker and the P _L promoter was obtained in PCR using oligonucleotides SVS 179 (SEQ ID No: 17) and SVS 180 (SEQ ID No: 18) as primers and DNA strain BW25113 cat-P _L -yddG (EP 1449918 A1, RF patent 2222596) as a matrix. The PCR conditions were as follows: denaturation step for 3 min at 95 ° C; profile for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; profile for the last 25 cycles: 30 s at 95 ° C, 30 s at 54 ° C, 40 s at 72 ° C; final stage: 5 min at 72 ° C.

The resulting PCR product is 1.9 kb. purified on an agarose gel and used for electroporation into E. coli strain MG1655 (ATCC 700926) containing the plasmid pKD46 with a temperature-sensitive origin of replication. Plasmid pKD46 (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 12: 6640-45) contains a phage λ DNA fragment of 2.154 nucleotides (nucleotides 31088 to 33241, in sequence with inventory number J02459 in the GenBank database) and contains the genes of the Red homologous phage λ recombination system (γ, β, exo genes) under the control of the arabinose-induced P _araB promoter. Plasmid pKD46 is required for integration of the PCR product into the chromosome of strain MG1655.

Electrocompetent cells were prepared as follows: MG1655 / pKD46 cells were grown overnight at 30 ° C in LB liquid medium containing ampicillin (100 μg / ml), then diluted 1: 100 with SOB medium (yeast extract, 5 g / L; NaCl, 0.5 g / L; tryptone, 20 g / L; KCl, 2.5 mM; MgCl ₂ , 10 mM) containing ampicillin (100 μg / ml) and L-arabinose (10 mM) (arabinose is used to induce a plasmid containing Red genes system) and grown at 30 ° C until the optical density of the bacterial culture OD ₆₀₀ ≈0.6, cells were then made electrocompetent by concentrating 100-fold and three selfless laundering iced deionized water. Electroporation was performed using 70 μl of cell suspension and ≈100 ng of PCR product.

After electroporation, cells were added to 1 ml of SOC medium (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)), incubated for 2.5 hours at 37 ° C, then plated L-agar containing 30 μg / ml chloramphenicol was grown at 37 ° C to select Cm ^R recombinants. Then, to remove plasmid pKD46, two passages were performed on L-agar with Cm at 42 ° C and the obtained colonies were tested for sensitivity to ampicillin.

Thus, the E. coli strain MG1655 (P _L -brnQ) was constructed (Figure 4).

1.4. Construction of strains MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)]

Cells of strains MG1655 and MG1655 (P _L -brnQ) were transformed with the plasmid pELAC-IDO (Lys, 23). The resulting clones were selected on plates with X-gal / IPTG agar (blue / white test). Thus, strains MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)], respectively, were obtained.

Example 2. Production of 4HIL by E. coli strain MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)]

To assess the effect of enhanced expression of the gene encoding the L-isoleucine transporter on the production of 4HIL cells of strains MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (P _L- brnQ) [pELAC-IDO (Lys, 23)] were grown in medium LB supplemented with ampicillin (200 μg / ml) and IPTG (1 mM) at 37 ° C for 4-5 hours. The specific activity of IDO was determined in a crude cell extract of each recombinant E. coli strain as follows. Cells from 5 ml of culture were harvested by centrifugation at 4 ° C, resuspended in 0.5 ml of A * buffer (50 mM TRIZMA, 5% glycerol, 1 mM EDTA, 1 mM DTT, pH 7 adjusted with HCl) and sonicated at 4 ° C. The reaction mixture (50 μl) contained 50 mM HEPES pH 7.0; 5 mm Il; 0.5 mM α-ketoglutarate; 5 mM ascorbate; 5 mM FeSO ₄ and an aliquot of the protein preparation. The reaction mixture was incubated at 34 ° C for 1 hour with shaking. 4HIL was determined using TLC or HPLC as follows. TLC analysis: a reaction solution was applied to 10 × 15 cm plates coated with a thin layer of silica gel and exposed in the mobile phase of the following composition: propan-2-ol: acetone: ammonia: water = 100: 100: 25: 16, 4HIL s was determined using ninhydrin reagent.

HPLC analysis: a high-pressure chromatograph (Waters, USA) with a 1100 series spectrofluorometer (Agilent, USA) was used. Selected wavelength ranges: excitation wavelength 250 nm, emission wavelength region 320-560 nm. Separation using the accq-tag method was carried out on a Nova-Pak ™ C18 150 × 3.9 mm, 4 μm column (Waters, USA) at + 40 ° C. The sample volume was 5 μl. The formation of amino acid derivatives and their separation was carried out in accordance with the recommendations of the manufacturer Waters (Liu, H. et al, J. Chromatogr. A, 828, 383-395 (1998); Waters accq-tag chemistry package. Instruction manual. Millipore Corporation, pp .1-9 (1993)). Accq-FluorTM kit (Waters, USA) was used to obtain derivatives of amino acids with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Accq-tag analysis was performed using Accq-tag Eluent A concentrated eluent (Waters, USA). All solutions were prepared using Milli-Q water; standard solutions were stored at + 4 ° C. The results of the measurement of IDO activity in the crude extract of the cells of the strains of IDO producing strains are presented in Table 1.

Cells of strains MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (P _L- brnQ) [pELAC-IDO (Lys, 23)] were collected by centrifugation and resuspended in a volume of 2 ml (up to OD ₅₄₀ ≈0.03-0.04) in MI30ch medium (50 mM KH ₂ PO ₄ (pH 7, adjusted with NaOH); 20 mM NH ₄ Cl, 2 mM MgSO ₄ , chalk - 1.25 g / 100 ml, 30 g / l Ile, 2 mM FeSO ₄ , 2 mm ascorbate, ampicillin 200 mg / l) with the addition of L-isoleucine and ketoglutarate, glucose or glycerol in various combinations (see Table 2, Table 3). Cells were cultured for approximately 15 hours at 32 ° C with vigorous shaking. The accumulation of 4HIL was then examined by HPLC as described above. The measurement results of 4HIL formed by strains MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)] depending on the addition of α-ketoglutarate, glucose and glycerol are shown in Table 2 (at least 3 in vitro fermentations). The results of the determination of 4HIL formed by the MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)] strains depending on different glycerol concentrations are shown in Table 3 (at least 3 in vitro fermentations). As follows from Table 2 and Table 3, MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)] accumulated more 4HIL than MG1655 [pELAC-IDO (Lys, 23)].

Example 3. Construction of strain MG1655 (ΔsucAB, Δ a ceAK, PL-brnQ) [pELAC-IDO (Lys, 23)]

3.1. Construction of strain MG1655 (ΔsucAB)

For deletion of sucAB genes, the following manipulations were performed. A 1.8 kb DNA fragment containing the Cm ^R marker and the P _tac promoter was amplified by PCR using oligonucleotides SVS-192 (SEQ ID No: 19) and SVS-193 (SEQ ID No: 20) as primers and chromosomal DNA of strain MG1655 (attR-Cm-attL-P _tac ) (Katashkina JI et al., Molekularnaya biologiya (RU), v. 39. No.5,1-10 (2005)) as a matrix. The PCR conditions were as follows: denaturation step for 3 min at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the last 25 cycles: 30 s at 95 ° C, 30 s at 54 ° C, 40 s at 72 ° C; final stage: 5 min at 72 ° C.

The resulting PCR product is 1.8 kb. purified on an agarose gel and used for electroporation into E. coli strain MG1655 containing plasmid pKD46 with temperature-sensitive replication.

Electroporation was performed as described above. Cells after electroporation were incubated in 1 ml of SOC medium at 37 ° C for 2.5 hours and then plated on L-agar containing chloramphenicol (30 μg / ml) and grown at 37 ° C to select Cm ^R recombinants. Then, to remove the plasmid pKD46, two passages were performed on L-agar with Cm at 42 ° C and the resulting colonies were tested for sensitivity to ampicillin.

Thus, the E. coli strain MG1655 (ΔsucAB) was constructed.

3.2. Construction of strain MG1655 (Δ a ceAK)

The following manipulations were performed for deletion of a ce A K genes. A 1.8 kb DNA fragment containing the Km ^R marker and the P _tac promoter was amplified by PCR using oligonucleotides SVS-199 (SEQ ID No: 21) and SVS-200 (SEQ ID No: 22) as primers and plasmid DNA pMW118- (λattL-Km ^r -λattR) (see Reference Example 2) as a template.

The conditions for PCR were as follows: denaturation step for 3 min at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the last 25 cycles: 30 s at 95 ° C, 30 s at 54 ° C, 40 s at 72 ° C; final stage: 5 min at 72 ° C.

The resulting PCR product is 1.8 kb. purified on an agarose gel and used for electroporation into E. coli strain MG1655 containing the plasmid pKD46 with a temperature-sensitive origin of replication.

Electroporation was performed as described above. Cells after electroporation were incubated in 1 ml of SOC medium at 37 ° C for 2.5 hours and then plated on L-agar containing kanamycin (20 μg / ml) and grown at 37 ° C to select Km ^R recombinants. Then, to remove the plasmid pKD46, two passages were performed on L-agar with Cm at 42 ° C and the resulting colonies were tested for sensitivity to ampicillin.

Thus, the E. coli strain MG1655 (ΔaceAK) was constructed.

3.3. Construction of strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ)

To remove the chloramphenicol resistance marker from strain MG1655 (P _L -brnQ), cells were transformed with plasmid pMW118-int-xis (Ap ^R ) (WO 2005/010175). Ap ^R clones were grown on LB agar plates containing 150 mg / L ampicillin at 30 ° C. Several tens of Ap ^R clones were selected and cloned for sensitivity to chloramphenicol. Plasmid pMW118-int-xis was removed from Cm ^S cells by incubation on LB agar plates at 42 ° C. The resulting strain was used for further construction.

DNA fragments from the chromosome of E. coli strain MG1655 (ΔsucAB) were transferred to the strain obtained after removal of the chloramphenicol resistance marker from strain MG1655 (P _L -brnQ) by transduction P1 (Miller, JH Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY). Thus, the MG1655 strain (ΔsucAB, P _L -brnQ) was constructed. DNA fragments from the chromosome of E. coli strain MG1655 (Δ a ceAK) were transferred to strain MG1655 (ΔsucAB, P _L -brnQ). Thus, the MG1655 strain (ΔsucAB, Δ a ceAK, P _L -brnQ) was constructed.

Cells of strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) were transformed with plasmid pELAC-IDO (Lys, 23). The resulting clones were selected on plates with X-gal / IPTG agar (blue / white test). Thus, strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) [pELAC-IDO (Lys, 23)] was obtained.

3.4. Investigation of the growth of strains MG1655, MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ), MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (ΔsucAB, Δ a ceAK, P _L- brnQ) [pELAC-IDO (Lys , 23)]

Strains MG1655 and MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) were grown on the following nutrient media:

A - M9 salt + glucose (0.4%);

B - M9 salt + glucose (0.4%) + DAP, Met, Lys (40 mg / l each);

С - M9 salts + glycerin (0.4%);

D - M9 salts + glycerin (0.4%) + DAP, Met, Lys (40 mg / L each).

The strains were cultured in test tubes at 37 ° C and the optical density of the cell culture was determined every hour (A ₅₅₅ ). As can be seen in Figure 5, strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) with succinyl CoA deficiency cannot grow on media A or C. Only with the addition of lysine, methionine and diaminopimelate (DAP) did the strain recover.

Next, strains MG1655 [pELAC-IDO (Lys, 23)] and MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) [pELAC-IDO (Lys, 23)] were grown on the following media:

G - M9 salt + glucose (137 mM);

GI - M9 salt + glucose (137 mM) + L-isoleucine (137 mM);

Y - M9 salt + glycerin (136 mmol);

YI - M9 salts + glycerin (136 mM) + L-isoleucine (137 mM).

Each medium contained ampicillin (100 mg / L). The strains were cultured in test tubes at 37 ° C and the optical density of the cell culture was determined every hour (A ₅₅₅ ).

Expression of the IDO gene restores the growth of strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) on M9 synthetic medium supplemented with L-isoleucine (Figure 6). This confirms the idea of the authors of the present invention that the hydroxylation of isoleucine leads to the growth of the strain.

Example 4. Production of 4HIL by E. coli strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) [pELAC-IDO (Lys, 23)]

To assess the effect of attenuation of the expression of genes encoding oxoglutarate dehydrogenase, isocitratlyase and isocitrate dehydrogenase phosphatase, cells of strains MG1655 (P _L- brnQ) [pELAC-IDO (Lys, 23)] and MG1655 (ΔsucAB, Δ a ceAK, P _L- brn -IDO (Lys, 23)] was grown in medium A [(NH ₄ ) ₂ SO ₄ - 1.5 g / 100 ml; KH ₂ PO ₄ - 0.15 g / 100 ml; MgSO ₄ - 0.1 g / 100 ml (MgSO ₄ · 7H ₂ O - 0.205 g / 100 ml); Ile - 220 mm; FeCl ₂ - 2 mm, 1 mm IPTG, chalk - 2 g / 100 ml] with the addition of glucose (300 mm) or glycerol (500 mm) at 32 ° C for 72 hours with vigorous shaking. The accumulation of 4HIL was then investigated using HPLC as described above. The measurement results of 4HIL formed by strains MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)] and MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) [pELAC-IDO (Lys, 23)] are shown in Table 4 (at least 3 fermentation assay tubes). As follows from Table 4, MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) [pELAC-IDO (Lys, 23)] formed more 4HIL than MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23) ].

Example 5. Production of 4HIL by E. coli strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) * [pEL-IDO (Lys, 23)]

5.1. Construction of the plasmid pEL-IDO (Lys, 23)

Due to the presence of the lacI gene on plasmid pELAC-IDO (Lys, 23), the addition of IPTG is necessary to induce IDO expression during the biotransformation process of 4-HIL production. To avoid this IPTG dependence, most of the lacI gene was removed from the pELAC-IDO plasmid (Lys, 23) (Figure 1) by excision of the SphI-EcoRV DNA fragment using appropriate restriction enzymes followed by ligation of the remaining plasmid. Thus, the plasmid pEL-IDO was constructed (Lys, 23).

5.2. 4HIL Products

MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) * strain was obtained by sequentially excising Cm and Kn resistance markers from strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) using plasmid pMW118-int-xis (Ap ^R ) (WO 2005/010175) as described above. Plasmids pELAC-IDO (Lys, 23) and pEL-IDO (Lys, 23) were introduced into the resulting strain. Thus, strains MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) * [pELAC-IDO (Lys, 23)] and MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) * [pEL-IDO (Lys , 23)].

Part of the biomass of strain MG1655 freshly grown on LB agar (ΔsucAB, Δ a ceAK, P _L -brnQ) * [pELAC-IDO (Lys, 23)] and MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) * [pEL- IDO (Lys, 23)] was inoculated in 50 ml of LB broth supplemented with Ap (100 mg / L) and cultured at 37 ° C. for approximately 4 hours in 750 ml flasks. The obtained cell cultures were used as seed for biotransformation carried out on Marubischi fermenters. The following cultivation parameters were used: initial culture volume - 500 ml; shaking - 1200 rpm; air - 1: 1; cultivation temperature - 34 ° C; pH 7.0 was maintained using 2.5 M NH ₄ OH.

Inoculum of strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) * [pELAC-IDO] was added to 450 ml of medium containing (NH ₄ ) ₂ SO ₄ - 5 g / l; KH ₂ PO ₄ - 1.5 g / l; MgSO ₄ 7H ₂ O - 1 g / l; FeSO ₄ 7H ₂ O - 0.01 g / l; isoleucine - 22-23 g / l (≈170 mm); glucose - 50 g / l; 1 mM IPTG (pH 7 adjusted with KOH, final volume = 500 ml), and cultured for 20 hours.

Inoculum of strain MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) * [pEL-IDO] was added to 450 ml of the above medium without IPTG and cultured for approximately 20 hours. The concentrations of 4HIL and L-isoleucine were determined using the HPLC method as described above. As can be seen from Table 5, the use of the plasmid pEL-IDO avoids IPTG dependence.

Reference example 1. Construction of the plasmid pELAC-ilvA

Plasmid pET-ilvA was constructed as follows. A fragment of the chromosomal DNA strain E. coli MG1655 size 1.5 TPN amplified in PCR using IlvA-5 oligonucleotides (SEQ ID NO: 23) and ilvA-3 (SEQ ID NO: 24) as primers. Part of the cell culture of E. coli strain MG1655 with LB agar was used as the source of the template DNA for the PCR procedure. The resulting DNA fragment contained the ilvA gene and was flanked by the NdeI and B a mHI restriction sites. The fragment was cloned in pET22b (+) vector (Novagen, Germany) between NdeI-B and mHI sites. Thus, the recombinant plasmid pET-ilvA was constructed (Figure 2).

Plasmid pELAC-ilvA was constructed on the basis of pET-ilvA by replacing the BglII-Xb a I fragment containing the T7 promoter, a fragment BglII-Xb a I, containing promoter P _lac (SEQ ID NO: 25).

Reference example 2. Construction of the plasmid pMW118- (λattL-Km ^r -λattR)

Plasmid pMW118- (λattL-Km ^r- λattR) was constructed on the basis of plasmid pMW118-attL-Tc-attR (WO 2005/010175) by replacing the tetracycline resistance marker gene with the kanamycin resistance marker gene from pUC4K plasmid (Vieira, J . and Messing, J., Gene, 19 (3); 259-68 (1982)).

For this purpose, a large EcoRI - HindIII fragment from the plasmid pMW118-attL-Tc-attR was leader with two fragments of the pUC4K plasmid: the HindIII fragment - PstI (676 bp) and the EcoRI - HindIII fragment (585 bp).

The base plasmid pMW118-attL-Tc-attR was obtained by ligation of the following four fragments:

1) a BglII-EcoRI fragment (114 bp) containing attL (SEQ ID NO: 26) obtained by PCR amplification of the corresponding region of the E. coli W3350 chromosome (containing λ prophage) using oligonucleotides P3 and P4 (SEQ ID NOS : 27 and 28) as primers (these primers contained auxiliary recognition sites for BglII and EcoRI endonucleases);

2) a PstI-HindIII fragment (182 bp) containing attR (SEQ ID NO: 29) obtained by PCR amplification of the corresponding region of the E. coli W3350 chromosome (containing λ prophage) using oligonucleotides P5 and P6 (SEQ ID NOS : 30 and 31) as primers (these primers contained auxiliary recognition sites for the endonucleases PstI and HindIII);

3) a large BglII-HindIII fragment (3916 bp) of the plasmid pMW118-ter_rrnB. Plasmid pMW118-ter_rrnB was obtained by ligation of the following three DNA fragments:

a) a large DNA fragment (2359 bp) containing the AatII-EcoRI fragment of plasmid pMW118, prepared as follows: pMW118 was digested with EcoRI restriction enzyme, digested with a Klenov fragment of DNA polymerase I, and then digested with restriction enzyme Aa tII;

b) a small AatII-BglII fragment (1194 bp) of the pUC19 vector containing the bla gene for ampicillin resistance (Ap ^R ) was obtained by PCR amplification of the corresponding region of the plasmid pUC19 using oligonucleotides P7 and P8 (SEQ ID NOS: 32 and 33) as primers (these primers contained auxiliary recognition sites for endonucleases Aa tII and BglII);

c) a small BglII-PstIpol fragment (363 bp) of the ter_rrnB transcription terminator was obtained by PCR amplification of the corresponding region of the E. coli chromosome MG1655 using oligonucleotides P9 and P10 (SEQ ID NOS: 34 and 35) as primers (these primers contained auxiliary recognition sites for the endonucleases BglII and PstI);

4) a small EcoRI-PstI fragment (1388 bp) (SEQ ID NO: 36) of the plasmid pML-Tc-ter_thrL containing the tetracycline resistance gene and ter_thrL transcription terminator; plasmid pML-Tc-ter_thrL was obtained in two stages:

- the plasmid pML-ter_thrL was obtained by processing the plasmid pML-MCS (Mashko, SV et al., Biotekhnologiya (in Russian), 2001, no.5, 3-20) with restriction enzymes Xb a I and B a mHI followed by ligation of a large fragment ( 3342 bp) with an Xb a I-B a mHI fragment (68 bp) containing the term ter_thrL obtained by PCR amplification of the corresponding region of the E. coli chromosome MG1655 using oligonucleotides P11 and P12 (SEQ ID NOS: 37 and 38 ) as primers (these primers contained auxiliary recognition sites for endonucleases Xb a I and BamHI);

- the plasmid pML-Tc-ter_thrL was obtained by treating the plasmid pML-ter_thrL with restriction enzymes KpnI and Xb a I, followed by treatment with the Maple fragment of DNA polymerase I and ligation with a small EcoRI-Van91I fragment (1317 bp) of the plasmid pBR322 containing the gene tetracycline resistance (pBR322 was treated with restriction enzymes EcoRI and Van91I and then treated with a Maple fragment of DNA polymerase I).

Example 6. Obtaining hydroxyproline using E. coli strain MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) *

The synthesis of the DNA fragment shown in SEQ ID NO: 39 in the Sequence Listing was assigned to Invitrogen and this DNA fragment encoding L-prolyl-4-hydroxygenase (PDO) from Dactylosporangium sp. with the amino acid sequence of SEQ ID NO: 40, was obtained as the BlueHeron plasmid pUCminusMCS into which the DNA fragment was ligated. As for the plasmid, recognition sequences for the NdeI and HindIII restriction enzymes located at the 5'- and 3'-ends of the DNA, respectively, were introduced into the DNA sequence, and this plasmid was treated with the restriction enzymes NdeI and HindIII. In parallel, the ptrp4 vector (Journal of Molecular Catalysis B: Enzymatic 32 (2005) 205-211) was treated with the same restriction enzymes NdeI and HindIII. Two obtained fragments treated with restriction enzymes NdeI and HindIII were leading and the resulting plasmid was introduced into the E. coli strain JM109 to obtain the JM109 / ptrp4_PDO transformant.

The plasmid was isolated from the obtained transformants and introduced into E. coli strains MG1655 and MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) * to obtain transformants MG1655 / ptrp4_PDO and MG1655 (Δsuc A B, Δ a ce A K , P _L -brnQ) * / ptrp4_PDO, respectively.

Strains MG1655 / ptrp4_PDO and MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) * / ptrp4_PDO were grown in LB growth medium containing 100 mg / L ampicillin at 30 ° C for 24 hours and 300 μl of culture liquids were added to 3 ml of LB medium containing 100 mg / L ampicillin and grown at 37 ° C for 6 hours. The tubes containing L-proline and calcium carbonate with the amount necessary for their final concentration to be 10 g / l and 20 g / l, respectively, were added the obtained culture fluid (0.3 ml) and culture medium (2.7 ml) of the following composition:

culture medium A (300 ml): D-glucose (20 g / l), MgSO ₄ · 7H ₂ O (1 g / l);

culture medium B (700 ml): ammonium sulfate (5 g / l), KH ₂ PO ₄ (1.5 g / l), sodium ascorbate (0.01 g / l), FeSO ₄ 7H ₂ O (0.1 g / l), pH brought to 7.0 using KOH.

Media A and B were autoclaved separately at 120 ° C for 20 minutes and mixed after cooling.

The concentrations of D-glucose (g / l) and 4-hydroxyproline (mM) are shown in Table 6. The concentration of D-glucose was determined using a glucose analyzer (SAKURA SI, Japan). The concentration of 4-hydroxyproline was determined using Sumichiral OA-5000 HPLC (Sumika Analysis Center, Japan) (mobile phase composition: 2 mM copper sulfate aqueous solution, column temperature: 30 ° C, flow rate: 1 ml / min, detector: UV 254 nm).

As a result, it was shown that strain MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) * / ptrp4_PDO is able to utilize D-glucose in a hydroxyproline production reaction more efficiently than strain MG1655 / ptrp4_PDO.

Example 7-1 Construction of E. coli strain MG1655 (Δsuc A , Δ a ce A K) and MG1655 (ΔsucB, Δ a ce A K)

In order to verify whether the modification of P _L -brnQ is necessary for the efficient utilization of D-glucose, the following experiment was carried out.

In order to obtain a strain deficient in 2-oxoglutarate dehydrogenase (suc A and sucB), isocitrate lyase ( a ce A ) and kinase / phosphatase isocitrate dehydrogenase (aceK) without modification of P _L -brnQ, deletion Δ a ce A K was introduced into E. strains. coli MG1655 Δsuc A (JW0715) and MG1655 ΔsucB (JW0716) from the Keio Knockout Collection (http://ecoli.naist.jp) to obtain strains MG1655 (Δsuc A , Δ a ce A K) and MG1655 (ΔsucB, Δ a ce A K). A deficiency of one of the suc A and sucB genes led to a deficiency of 2-oxoglutarate dehydrogenase activity.

To obtain a deletion of the a ce A K gene, the following manipulations were performed. A 1.6 kb DNA fragment containing the Cm ^R marker was amplified by PCR using primers SVS-199 (SEQ ID NO: 21) and SVS-200 (SEQ ID NO: 22) and plasmid pMW118- (λattL -Cmr-λattR) (WO 2005/010175) as a matrix. PCR conditions are as follows: denaturation for 3 min at 94 ° C; 30 cycles: 30 s at 94 ° C, 30 s at 50 ° C, 2 min at 72 ° C.

The resulting PCR product of 1.6 kbp purified on an agarose gel and used for electroporation of E. coli strains MG1655 (Δsuc A ) and MG1655 (ΔsucB) containing plasmid pKD46 (WO 2005/010175) with a heat-sensitive replicon.

Electroporation was performed as described above. After electroporation, the cells were incubated in 1 ml of SOC growth medium at 37 ° C for 1 hour, and then placed on a L-agar plate containing chloramphenicol (25 μg / ml) and grown at 37 ° C to select the Cm ^R recombinant. Then, two passages were performed on plates with chloramphenicol-containing agar at 42 ° C to remove the plasmid pKD46, and the obtained colonies were tested for sensitivity to ampicillin.

The Cm ^R marker was then removed using the plasmid pMW118-int-xis-ts (Ap ^R ) (WO 2005/010175) as described above.

Thus, E. coli strains MG1655 (Δsuc A , Δ a ce A K) and MG1655 (ΔsucB, Δ a ce A K) were constructed.

Example 7-2 Preparation of 4HIL by E. coli strains MG1655 [pEL-IDO (Lys, 23)], MG1655 (Δsuc A , Δ a ce A K) [pEL-IDO (Lys, 23)] and MG1655 (ΔsucB, Δ a ce A K) [pEL-IDO (Lys, 23)]

Plasmid pEL-IDO (Lys, 23) was introduced into each of the strains MG1655, MG1655 (Δsuc A , ΔaceAK) and MG1655 (Δsuc A B, ΔaceAK) to obtain transformants MG1655 [pEL-IDO (Lys, 23)], MG1655 (Δsuc A , Δ a ce A K) [pEL-IDO (Lys, 23)] and MG1655 (ΔsucB, Δ a ce A K) [pEL-IDO (Lys, 23)].

The strains were grown in LB medium containing 100 mg / L ampicillin at 30 ° C for 24 hours and 300 μl of culture fluid was added to 3 ml of LB medium containing 100 mg / L ampicillin and grown at 37 ° C for 6 hours.

The tubes containing L-isoleucine and calcium carbonate with the amount necessary for their final concentration to be 10 g / l and 20 g / l, respectively, were added the obtained culture fluid (0.3 ml) and culture medium (2.7 ml) of the following composition:

culture medium A (300 ml): D-glucose (20 g / l), MgSO ₄ · 7H ₂ O (1 g / l);

culture medium B (700 ml): ammonium sulfate (5 g / l), KH ₂ PO ₄ (1.5 g / l), sodium ascorbate (0.01 g / l), FeSO ₄ 7H ₂ O (0.1 g / l), pH brought to 7.0 using KOH.

Media A and B were autoclaved separately at 120 ° C for 20 minutes and mixed after cooling.

The concentrations of D-glucose (g / l) and 4-hydroxy-L-isoleucine (mm) are shown in Table 7. The concentration of D-glucose was determined on a glucose analyzer (SAKURA SI, Japan). The concentration of 4-hydroxy-L-isoleucine was determined using HPLC MCI GEL CRS10W (Mitsubishi Kagaku, Japan) (mobile phase composition: 2 mm solution of copper sulfate in 5% MeOH, column temperature: 30 ° C, flow rate: 1 ml / min detector: UV 254 nm).

As a result, it was shown that using E. coli strains MG1655 (Δsuc A , Δ a ce A K) [pEL-IDO (Lys, 23)] and MG1655 (ΔsucB, Δ a ce A K) [pEL-IDO (Lys , 23)] without modification of P _L -brnQ, D-glucose is more efficiently utilized in the reaction with isoleucine dioxygenase compared to MG1655 [pEL-IDO (Lys, 23)].

The above results confirm that strains deficient in the Δsuc A B, Δsuc A , ΔsucB and ΔaceAK genes, such as E. coli MG1655 (Δsuc A B, Δ a ce A K), MG1655 (Δsuc A , Δ a ce A K) and MG1655 (ΔsucB, Δ a ce A K), can be widely used for the use of 2-oxoglutarate-dependent enzymes.

Sequencing

1: L-isoleucine dioxygenase gene from Bacillus thuringiensis 2-e-2 strain

2: L-Isoleucine dioxygenase from Bacillus thuringiensis 2-e-2 strain

3: Genn brnQ from E. coli

4: BrnQ Protein from E. coli

5: suc A gene from E. coli

6: SucA protein from E. coli

7: sucB gene from E. coli

8: SucB Protein from E. coli

9: And lo a gene from E.coli

10: ACEA protein from E. coli

11: a ceK gene from E. coli

12: AceK protein from E. coli

13: E. coli ilvE gene

14: Protein 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: Fragment of BglII-Xb a I containing the promoter P _lac

26: Fragment a ttL

27: Primer P3

28: Primer P4

29: Fragment a ttR

30: Primer P5

31: Primer P6

32: Primer P7

33: Primer P8

34: Primer P9

35: Primer P10

36: Fragment pML-Tc-ter_thrL

37: Primer P11

38: Primer P12

39: L-proline-4-hydroxygenase gene from Dactylosporangium sp.

40: L-Proline-4-hydroxygenase from Dactylosporangium sp.

Although the invention has been described in detail with reference to the Best Mode for Carrying Out the Invention, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not beyond the scope of the present invention.

Each of the above documents has a link, and all cited documents are part of the description of the present invention.

Industrial applicability

According to the present invention, the production of (2S, 3R, 4S) -4-hydroxy-L-isoleucine can be increased. This compound is useful as a component of pharmaceutical compositions with insulinotropic activity. The production of (2S, 3R, 4S) -4-hydroxy-L-isoleucine is increased using a bacterium transformed with a DNA fragment containing a gene encoding a protein with L-isoleucine dioxygenase activity.

Table 1 Strain IDO activity (nmol / min · mg) MG1655 [pELAC-IDO (Lys, 23)] 300 ± 12 MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)] 280 ± 10

table 2 Strain Initial culture fluid ^a) The final culture fluid Ile (mm) KG ^b) (mm) Gluc. (mm) Glyc. (mm) 4HIL (mm) MG1655 / pELAC-IDO (Lys, 23) one hundred 55 0 115 ± 6 0 55 0 17 ± 1 one hundred 0 136 129 ± 6 144 0 0 136 77 ± 4 MG1655 (P _L -brnQ) / pELAC-IDO (Lys, 23) one hundred 55 0 128 ± 6 0 55 0 34 ± 2 one hundred 0 136 131 ± 6 0 0 136 106 ± 5 a) - described in the text b) abbreviations: KG - α-ketoglutarate; Gluc. - glucose; Glyc. - glycerin

Table 3 Strain Initial culture fluid ^a) The final culture fluid Ile (mm) Glycerol ^b) (mm) 4HIL (mm) MG1655 / pELAC-IDO (Lys, 23) 0 6 ± 0.3 136 86 ± 4 272 146 ± 7 220 408 164 ± 8 MG1655 (P _L -brnQ) / pELAC-IDO (Lys, 23) 0 7 ± 0.4 136 93 ± 5 272 158 ± 8 408 203 ± 10 a) - described in the text

Table 4 Strain Initial culture fluid The final culture fluid Glucose (mm) Glycerin (mm) Ile (mm) 4HIL (mm) MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)] 300 - 220 134 MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) [pELAC-IDO (Lys, 23)] 300 - 220 189 MG1655 (P _L -brnQ) [pELAC-IDO (Lys, 23)] - 500 220 one hundred MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) [pELAC-IDO (Lys, 23)] - 500 220 195

Table 5 Strain Initial culture fluid The final culture fluid Glucose (g / l) Ile (mm) Glucose (g / l) Ile (mm) 4HIL (mm) MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) [pELAC-IDO (Lys, 23)] fifty 179 Nd Nd 132 MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) [pEL-IDO (Lys, 23)] fifty 174 Nd 2.3 143 ND - not determined (≤0.1 g / l)

Table 6 Strain 0 h 22 h 66 h Glucose (g / l) Pro ** (mm) Glucose (g / l) Hydroxy-Pro (mm) Glucose (g / l) Hydroxy-Pro (mm) MG1655 / ptrp4_PDO eighteen 87 7 fifteen 0 31 MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) / ptrp4_PDO eighteen 87 eleven fifteen 3 58 ** estimated amount

Table 7 Strain 0 h 48 h 66 h Glucose (g / l) Ile (mm) Glucose (g / l) Ile (mm) 4HIL (mm) Glucose (g / l) Ile (mm) 4HIL (mm) MG1655 [pEL-IDO (Lys, 23)] eighteen 38 0 35 10 0 34 9 MG1655 (ΔsucA, Δ a ceAK) [pEL-IDO (Lys, 23)] eighteen 38 fourteen 45 one 0 17 26 MG1655 (ΔsucB, Δ a ceAK) [pEL-IDO (Lys, 23)] eighteen 38 fourteen 44 one 0 26 17

Additional example 1

In order to verify the sufficiency of deletion of genes encoding oxoglutarate dehydrogenase for 4HIL production, E. coli strain MG1655 (Δsuc A B, Δ a ce A K) was constructed. For this, DNA fragments from the chromosome of E. coli strain MG1655 (Δ a ce A K) (see Example 3.2) were transferred to strain MG1655 (Δsuc A B) (see Example 3.1) by transduction P1 (Miller, JH Experiments in Molecular Genetics , Cold Spring Harbor Lab. Press, 1972, Plainview, NY). Thus, the MG1655 strain (ΔsucAB, Δ a ceAK) was constructed.

Plasmid pEL-IDO was introduced into E. coli MG1655 strains (ΔsucAB), E. coli MG1655 (Δsuc A B, Δ a ce A K) and into E. coli MG1655 strain as described above. Thus, strains of E. coli MG1655 (ΔsucAB) [pEL-IDO], E. coli MG1655 [pEL-IDO] and E. coli MG1655 [pEL-IDO] were obtained.

The obtained strains were individually cultured in medium A of the following composition: [(NH ₄ ) ₂ SO ₄ - 1.5 g / 100 ml; KH ₂ PO ₄ - 0.15 g / 100 ml; MgSO ₄ - 0.1 g / 100 ml (MgSO ₄ · 7H ₂ O - 0.2 g / 100 ml); Ile - 100 mM; FeCl ₂ - 2 mm, chalk - 2 g / 100 ml, Ap - 100 μg / ml], with the addition of glucose (150 mm). Part of the cell cultures grown on agar plates was transferred to 2 ml of medium A. Cells were cultured in 50 ml tubes at 32 ° C for 72 hours. The concentrations of 4HIL and L-isoleucine were determined using the HPLC method as described above. The results are presented in Table 8. As can be seen from Table 8, the E. coli strain MG1655 (ΔsucAB) [pEL-IDO] with inactivated genes encoding oxoglutarate dehydrogenase accumulates 2.5 times more 4-HIL than the control strain MG1655 [pEL -IDO], approximately the same amount of 4-HIL as strain MG1655 Δsuc A B, Δ a ce A K [pEL-IDO].

Table 8 Strain Final concentration 4-HIL (mm) L-Ile (mm) MG1655 [pEL-IDO] 36 ± 0.6 59 ± 1 MG1655 Δsuc A B [pEL-IDO] 90 ± 3 <3 MG1655 Δsuc A B, Δ a ce A K [pEL-IDO] 90 ± 5

Additional example 2

2.1. Construction of E. coli strain MG1655 (Δsuc A B, Δ a ce A K, ΔilvE, P _L -brnQ) *

The following manipulations were performed for deletion of the ilvE gene. A 1.8 kb DNA fragment containing the Cm ^R marker and the P _tac promoter was PCR amplified using oligonucleotides SVS_9 (SEQ ID NO: 41) and SVS_11 (SEQ ID NO: 42) as primers and chromosomal DNA strain MG1655 (attR-Cm-attL-P _tac ) (Katashkina JI et al., Molekularnaya biologiya (RU), v. 39, No.5, 1-10 (2005)) as a matrix. The PCR conditions were as follows: denaturation step for 3 min at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the last 25 cycles: 30 s at 95 ° C, 30 s at 54 ° C, 40 s at 72 ° C; final stage: 5 min at 72 ° C.

The resulting PCR product is 1.8 kb. purified on an agarose gel and used for electroporation into E. coli strain MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) * containing plasmid pKD46 with temperature-sensitive replication.

Electroporation was performed as described above. Cells after electroporation were incubated in 1 ml of SOC medium at 37 ° C for 2.5 hours and then plated on L-agar containing chloramphenicol (30 μg / ml) and grown at 37 ° C to select Cm ^R recombinants. Then, to remove the plasmid pKD46, two passages were performed on L-agar with Cm at 42 ° C and the resulting colonies were tested for sensitivity to ampicillin.

Thus, the E. coli strain MG1655 (Δsuc A B, Δ a ce A K, ΔilvE, P _L -brnQ) * was constructed.

2.2. 4HIL Products

Plasmid pELAC-IDO (Lys, 23) was introduced into the obtained E. coli strain MG1655 (Δsuc A B, Δ a ce A K, ΔilvE, P _L -brnQ) * as described above. Thus, E. coli strain MG1655 (Δsuc A B, Δ a ce A K, ΔilvE, P _L -brnQ) * [pELAC-IDO (Lys, 23)] was obtained.

Part of the biomass of strain MG1655 freshly grown on LB agar (ΔsucAB, Δ a ceAK, ΔilvE, P _L -brnQ) * [pELAC-IDO (Lys, 23)] and MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) * [ pEL-IDO (Lys, 23)] was inoculated in 50 ml of LB broth supplemented with Ap (100 mg / L) and cultured at 37 ° C. for approximately 4 hours in 750 ml flasks. The obtained cell cultures were used as seed for biotransformation carried out on Marubischi fermenters. The following cultivation parameters were used: initial culture volume - 500 ml; shaking - 1200 rpm; air - 1: 1; cultivation temperature - 34 ° C; pH 7.0 was maintained using 2.5 M NH ₄ OH.

Inoculums of strains MG1655 (ΔsucAB, Δ a ceAK, ΔilvE, P _L -brnQ) * [pELAC-IDO (Lys, 23)] and MG1655 (ΔsucAB, Δ a ceAK, P _L -brnQ) * [pELAC-IDO] were added to 450 ml of medium containing (NH ₄ ) ₂ SO ₄ - 5 g / l; KH ₂ PO ₄ - 1.5 g / l; MgSO ₄ · 7H ₂ O - 1 g / l; FeSO ₄ · 7H ₂ O — 0.01 g / l; isoleucine - 22-23 g / l (= 170 mm); glucose - 50 g / l; 1 mM IPTG (pH 7 adjusted with KOH, final volume = 500 ml), and separately cultured for 20 hours. The concentrations of 4HIL and L-isoleucine were determined using the HPLC method as described above. The results are shown in Table 9. As can be seen from Table 9, the introduction of a deletion of the ilvE gene in the E. coli strain MG1655 (Δsuc A B, Δ a ce A K) [pELAC-IDO (Lys, 23)] leads to an additional increase in 4HIL production.

Table 9 Strain Initial culture fluid ^a) The final culture fluid ^b) Gluc. (g / l) Ile (mm) 4HIL (mm) Gluc. (g / l) Ile (mm) 4HIL (mm) Yield ^e) (%) MG1655 (Δsuc A B, Δ a ce A K, P _L -brnQ) * [pELAC-IDO (Lys, 23)] fifty 179 0 Nd ^c) Nd ^d) 132 74 MG1655 (Δsuc A B, Δ a ce A K, ΔilvE, P _L -brnQ) * [pELAC-IDO (Lys, 23)] fifty 169 0 Nd Nd 149 88 ^a) - The measurement was carried out in the culture fluid before the start of cultivation (in the fermenter) ^b) - The measurement was carried out in the culture fluid after the completion of the cultivation process (20 hours) ^c) - ND - not detected (≤0.1 g / l) ^d) - ND - not detected (≤0.1 g / l) ^f) - Calculated as 100 · [4HIL (received) / Ile (source)] Gluc. - glucose

Claims (14)

1. A bacterium belonging to a genus selected from the group consisting of Escherichia, Corynebacterium, Arthrobacter, Aspergillus and Bacillus, transformed with a DNA fragment containing a gene encoding a protein having a 2-oxoglutarate-dependent enzyme activity, characterized in that the bacterium is modified so that:
a gene encoding 2-oxoglutarate dehydrogenase is inactivated; or
a gene encoding 2-oxoglutarate dehydrogenase and a gene encoding a branched chain amino acid aminotransferase are inactivated,
and said bacterium is capable of producing a reaction product catalyzed by said protein. 2. The bacterium according to claim 1, characterized in that the bacterium is selected from the group consisting of Escherichia coli, Arthrobacter simplex, Corynebacterium glutamicum, Arthrobacter globiformis, Arthrobacter sulfureus, Arthrobacter viscosus and Bacillus subtilis. 3. The bacterium according to claim 1, characterized in that the specified gene encoding 2-oxoglutarate dehydrogenase is inactivated by deletion of the specified gene in the bacterial chromosome. 4. The bacterium according to claim 1, characterized in that said gene encoding a branched chain amino acid aminotransferase is inactivated due to deletion of said gene on the bacterial chromosome. 5. A method of obtaining a reaction product catalyzed by a protein having a 2-oxoglutarate-dependent enzyme activity, comprising:
growing bacteria according to any one of claims 1 to 4 in a nutrient medium containing a substrate of the specified reaction; and
the selection of the specified product from the culture fluid. 6. The bacterium according to claim 1, characterized in that said protein has L-isoleucine dioxygenase activity, and said product is (2S, 3R, 4S) -4-hydroxy-L-isoleucine. 7. The bacterium according to claim 1, characterized in that said bacterium is modified in such a way that expression of the gene encoding the L-isoleucine transporter is enhanced in it. 8. The bacterium according to claim 6, characterized in that the gene encoding a protein with L-isoleucine dioxygenase activity is selected from the group consisting of:
(a) DNA comprising the nucleotide sequence shown in the List of sequences under number 1 (SEQ ID NO: 1);
(b) DNA hybridizing under stringent conditions with DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in Sequence Listing No. 1 (SEQ ID NO: 1), said DNA encoding a protein with L-isoleucine dioxygenase activity;
(c) a DNA comprising a nucleotide sequence encoding a protein comprising the amino acid sequence shown in the List of sequences under number 2 (SEQ ID NO: 2);
(d) a DNA comprising a nucleotide sequence encoding a protein comprising the amino acid sequence shown in the List of sequences under number 2 (SEQ ID NO: 2), except that the specified amino acid sequence contains a substitution, deletion, insertion, addition or inversion of one or several amino acid residues, and wherein said protein exhibits L-isoleucine dioxygenase activity; and
(e) a DNA comprising a nucleotide sequence encoding a protein comprising an amino acid sequence homologous to at least 98% of the amino acid sequence shown in Sequence Listing No. 2 (SEQ ID NO: 2), wherein said protein exhibits L-isoleucine dioxygenase activity . 9. The bacterium according to claim 7, characterized in that the gene encoding the L-isoleucine transporter is the brnQ gene from Escherichia coli. 10. A method of obtaining (2S, 3R, 4S) -4-hydroxy-L-isoleucine or its salt, including:
growing bacteria according to any one of claims 6 to 9 in a nutrient medium containing L-isoleucine; and
isolation of (2S, 3R, 4S) -4-hydroxy-L-isoleucine from the culture fluid. 11. The method according to claim 10, characterized in that the nutrient medium contains a carbon source selected from the group consisting of carbohydrate and alcohol. 12. The method according to claim 11, characterized in that said carbohydrate is glucose, and said alcohol is glycerin. 13. The bacterium according to claim 1, characterized in that said protein has the activity of L-proline hydroxylase, and said product is 4-hydroxy-L-proline. 14. A method for producing 4-hydroxy-L-proline or a salt thereof, comprising:
growing bacteria according to item 13 in a nutrient medium containing L-proline; and
isolation of 4-hydroxy-L-proline from the culture fluid.

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