RU2774071C1 - ESCHERICHIA COLI STRAIN WITH INACTIVATED sdaC GENE - L-THREONINE PRODUCER - Google Patents

Abstract

FIELD: microbiological industry.

SUBSTANCE: invention relates to microbiological industry, in particular to microbiological synthesis of L-threonine. Escherichia coli strain of RNCIM V-14096 with inactivated sdaC gene, producing L-threonine, is proposed.

EFFECT: invention provides for the expansion of the arsenal of Escherichia coli strains producing L-threonine.

1 cl, 1 dwg, 1 tbl, 2 ex

Description

The present invention relates to the microbiological industry, in particular to the microbiological synthesis of the amino acid L-threonine using Escherichia coli.

Traditionally, L-amino acids on an industrial scale can be obtained by fermentation using strains of microorganisms isolated from natural sources, or their mutants, specially modified in order to increase the production of L-amino acids.

Many methods have been described for increasing the production of L-amino acids, for example, by transforming a microorganism with recombinant DNA (US 4278765). These methods are based on increasing the activity of enzymes involved in the biosynthesis of amino acids and/or reducing the sensitivity of the target enzyme to reverse inhibition produced by L-amino acid (WO 9516042, US 5661012, US 6040160).

Various strains are known that are used to produce L-threonine by fermentation. These are strains with increased activities of enzymes involved in the biosynthesis of L-threonine (US 5175107; US 5661012; US 5705371; US 5939307; EP 219027), strains resistant to certain chemicals, such as L-threonine and its analogues (WO 0114525, EP 301572, US 5376538), strains with inactivated enzymes of the threonine degradation system (US 5939307 and US 6297031), strains in which the sensitivity of the target enzyme to inhibition of the produced amino acid or its by-products by the feedback type is eliminated (US 5175107 and US 5661012).

Known strain-producer of L-threonine E. coli VKPM B-3996 (SU 1694643, US 5175107 and US 5705371), obtained by introducing into the strain E. coli VKPM B-7 the following mutations and plasmids:

- mutant thrA gene (mutation thrA ⁴⁴² ) encodes a protein aspartokinase-homoserine dehydrogenase I, which is resistant to feedback inhibition by threonine;

- the mutant ilvA gene (mutation ilvA ⁴⁴² ) encodes a threonine deaminase protein with reduced activity, which is expressed in a reduced level of isoleucine biosynthesis and in a "leaky" isoleucine-deficient phenotype. In bacteria with the ilvA ⁴⁴² mutation, the transcription of the thrABC operon is not repressed by isoleucine, which has a positive effect on the production of L-threonine;

- inactivation of the tdh gene leads to the prevention of the degradation of L-threonine, the genetic determinant of sucrose assimilation (scrKYABR genes) was introduced into this strain;

- an increase in the expression of genes that control the biosynthesis of L-threonine was achieved by introducing into the strain the plasmid pVIC40 containing the mutant threonine operon thrA ⁴⁴² BC.

E. coli strain VKPM B-3996 during fermentation in test tubes produces 13 g/l L-threonine (RU 2182173), when cultivated in a fermenter - 85 g/l L-threonine (US 5175107).

When constructing producers, it is also important to search for new target genes, the modification of which leads to an increase in the production of L-threonine.

The technical problem to be solved by the present invention is the expansion of the arsenal of Escherichia coli strains capable of producing L-threonine.

The problem was solved by obtaining a strain of the bacterium Escherichia coli VKPM B-14096 with an inactivated sdaC gene, which has the ability to produce L-threonine.

The term "sdaC gene is inactivated" means that the target gene is modified such that such a modified gene encodes a mutant protein with reduced activity, or a completely inactive protein. It is also possible that natural expression of the modified DNA region is not possible due to a deletion of the target gene or part of it, a shift in the reading frame of this gene, or the introduction of a missense / nonsense mutation or modification of regions adjacent to the gene that include sequences that control gene expression, such as a promoter ( s), enhancer(s), attenuator(s), ribosome binding site(s), etc.

Gene inactivation can be carried out by any possible method, such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene inactivation by homologous recombination, or/ and insertion-deletion mutagenesis (Yu, D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 5978-83); (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 6640-45), also called Red-Dependent Integration.

In the claimed strain, the inactivation of the sdaC gene is carried out by replacing the open reading frame of the gene with the selective marker aadA, which is a gene for resistance to speccinomycin. The strain thus constructed is unable to synthesize the SdaC protein encoded by the sdaC gene.

The invention is illustrated by the following figures of the graphic image.

Fig. 1. Scheme of the pISA plasmid.

Example 1. Construction of a strain with an inactivated sdaC gene

Gene inactivation is carried out in Escherichia coli strain VKPM B-13207, which was obtained on the basis of the wild strain Escherichia coli K-12 MG1655 (ATCC 47076) as a result of several rounds of mutagesis with various mutating agents in order to select the mutants most resistant to L-threonine, and then introduction of targeted genetic modifications: replacement of the promoter of the threonine operon genes, introduction of a desensitizing mutation in the thrA gene, overexpression of rhtA, inactivation of the sstT gene, inactivation of the roxB gene encoding pyruvate oxidase.

The deletion of the sdaC gene is carried out by replacing its ORF (sequence number according to the GenBank database NC_000913.3 U00096.3: 4,383,839 -> 4,385,341), with a selective marker of resistance to spectinomycin, by PCR using the following oligonucleotides:

The pISA plasmid is used as a template for synthesis (Biotechnology 2019 T. 35 No. 4 pp. 42-54). In its composition, this plasmid (figure 1) carries the following genetic elements: the aadA gene, which determines the resistance of cells to spectinomycin for direct selection of transformants; phage sequences λ attL and attR - recognition sites of the Int/Xis recombination system; repA - replicon; the bla gene encoding beta-lactamase, which confers resistance to ampicillin.

Fragment amplification was performed using KAPA polymerase (KAPAbiosystems) in order to avoid sequence errors. Use the following temperature profile for PCR: denaturation at 95°C for 3 min; 25 cycles: 20 sec at 98°C, 20 sec at 60°C, 2 min at 72°C; and final polymerization: 2 min at 72°C. PCR product size 2036 p. O. purified by extraction from agarose gel and then used to transform the strain E. coli VKPM B-13207, which is preliminarily transformed with the plasmid pKD46, which is necessary for the integration of the PCR product into the chromosome of the strain. Plasmid pKD46 (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) contains a DNA fragment of phage λ (sequence accession number J02459 in the GenBank database) of length 2.154 nucleotides (31088-33241), and also contains the Red genes of the homologous recombination system (β, γ, exo genes) under the control of the ParaB promoter; induced by arabinose.

Electrocompetent cells are obtained as follows: an overnight culture of E. coli strain VKPM B-13207 is grown at 30°C in a liquid LB medium (wt.%): tryptone -1, NaCl -1, yeast extract - 0.5, water - the rest, pH 7.0 with the addition of ampicillin (125 mg / l), diluted 100 times with 5 ml of SOB medium (wt.%): tryptone 2, yeast extract - 0.5, MgCl ₂ - 0.0956, MgSO ₄ ⋅ 7H ₂ O - 0.252, NaCl - 0.0058, KCl - 0.0185, the rest is water with the addition of ampicillin (100 mg/l) and L-arabinose (1 mM). The resulting culture is grown with stirring at 30°C until an optical density of OD _{660 nm} of 0.6 units is reached, after which the cells are made electrocompetent by concentrating 100 times and washing three times with ice-cold deionized H ₂ O. Electroporation is carried out using 40 μl of cells and 1 µg of the PCR product. After electroporation, the cells are incubated in 1 ml of SOC medium (wt.%): tryptone 2, yeast extract - 0.5, glucose - 0.36, MgCl ₂ - 0.0956, MgSO ₄ -7H ₂ O - 0.252, NaCl - 0 0058, KCl - 0.0185, water - the rest, at 37°C for 2.5 hours, after which they are sown on plates with LA medium (wt.%): agar-agar - 2, tryptone - 1, NaCl - 1, yeast extract - 0.5, water - the rest, pH 7.0 with the addition of spectinomycin (75 μg / ml).

The selection of transformants carrying a deletion in the sdaC gene is carried out on plates with LA medium supplemented with spectinomycin. The presence of an insertion at the sdaC locus in selected clones was confirmed by PCR. To amplify the fragment, Taq polymerase (Thermo Fisher Scientific) and the following oligonucleotides were used:

For PCR use the following temperature profile: denaturation at 94°C for 4 min; 25 cycles: 20 sec at 94°C, 20 sec at 55°C, 2 min 30 sec at 72°C; and final polymerization: 5 min at 72°C.

The presence of a product with a size of 2344 p. interpreted as integration of the cassette into the target locus. To remove the helper plasmid pKD46, spend 2 passages on LA medium with spectinomycin at 42°C and the resulting colonies are tested for sensitivity to ampicillin.

As a result, 5 clones carrying a deletion in the sdaC gene were selected.

Example 2. Selection of the most productive clone

To test the effect of the deletion of the sdaC gene on the production of L-threonine, test-tube fermentation of five selected clones is carried out, the parent strain of E. coli VKPM B-13207 is used as a control strain.

The strains are grown on LA plates for 24 hours at 37°C. To prepare the inoculum, an inoculation medium of the following composition is used (wt %):

yeast extract 3.5, glucose 0.25, NaCl 0.25, KH ₂ PO ₄ 0.25, water the rest.

In test tubes with a volume of 50 ml with a working volume of 5 ml of seed medium, cell biomass is added to the starting value of optical density equal to OD _600nm 0.1 units. The tubes are incubated on a rotary shaker for 5 hours at 37°C and agitation speed of 220 rpm. For the main fermentation process, a medium of the following composition is used (wt.%):

Glucose 4.0 (NH ₄ ) ₂ SO ₄ 3.0 Corn extract 1.0 _{KH2PO4 _} 0.25 MgSO ₄ ⋅7H ₂ O 0.2 Lemon acid 0.0192 FeSO ₄ ⋅7H ₂ O 0.003 MnSO ₄ ⋅H ₂ O 0.0021 CaCO ₃ 2.0 Water Rest

Solutions of glucose, manganese sulfate and magnesium sulfate are sterilized separately by autoclaving at 121° C. for 40 minutes. 40 mg portions of CaCO ₃ are sterilized in glass test tubes by autoclaving at 121°C for 20 minutes. The pH is adjusted to a value of 7.0. The ferrous sulfate solution is sterilized by filtration through a membrane with a pore diameter of 22 μm.

The resulting inoculum is introduced into test tubes with a volume of 50 ml with a working volume of 2 ml of the fermentation medium to a starting optical density of OD ₆₆₀ nm of 0.1 units. Cells are cultured for 24 hours at 37°C on a rotary shaker - 220 rpm.

The amount of L-threonine accumulated in the medium is determined by HPLC (Waters 2695, Alliance). The results of the fermentation of the five clones are shown in table. one.

As can be seen from Table. 1, clone 3 containing the deletion of the sdaC gene accumulates more L-threonine than the control E. coli strain VKPM B-13207. The selected clone 3 was deposited at the Bioresource Center All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center "Kurchatov Institute" - GosNIIgenetika as Escherichia coli VKPM V-14096.

The inventive strain of Escherichia coli VKPM B-14096 is characterized by the following features.

Cultural and morphological characteristics

Gram-negative bacterium. The daily culture in liquid LB is represented by weakly motile cells of a rounded shape 1 µm in diameter. When cultivated on L-agar for 18-24 hours at 37°C, it forms round, whitish, translucent to light colonies 1-2 mm, the surface of the colony is smooth, the edges are even or slightly wavy, the center of the colonies is raised, the structure is homogeneous, the consistency is pasty, easily emulsified. When cultivating on Endo agar medium (wt.%: peptone - 1, lactose - 1, Na ₂ SO ₃ - 0.33, K ₂ HPO ₄ - 0.25, basic fuchsin - 0.03, agar-agar - 2% , water - the rest) at 37 ° C, colonies are formed, round in shape with a smooth, clearly defined edge of a raspberry-red color with a metallic sheen. The diameter of the colonies is 0.5-1.5 mm.

Physiological and biochemical characteristics.

Facultative anaerobe. Sugar does not ferment. Assimilates: D-glucose, L-arabinose, D-mannitol, D-xylose, to a lesser extent: D-galactose, D-lactose. There is no ability to hydrolyze starch. There is no need for growth factors. The strain is resistant to chloramphenicol. The optimal pH value for growth is 7.2-7.4. Does not grow at temperatures above 45°C. The optimum growth temperature is 37°C. The strain is not pathogenic.

Thus, a strain of Escherichia coli VKPM B-14096 with an inactivated sdaC gene was obtained, capable of synthesizing up to 17.1 g/l of L-threonine when cultivated in test tubes.

Claims (1)

Escherichia coli strain VKPM B-14096 with an inactivated sdaC gene is a producer of L-threonine.

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