A threonine deaminase mutant and its application in the preparation of isoleucine

Abstract

The present invention provides an ilvA gene mutant and its encoded nucleic acid sequence, and the fermentation yield of isoleucine is increased by expressing the ilvA gene mutant in a recombinant strain. The genetically engineered strain containing the ilvA mutant has a significantly improved L-isoleucine production capacity compared with the strain containing the wild-type ilvA gene, and has great industrial application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a threonine deaminase mutant and its application in the preparation of isoleucine. Background Art

[0002] L-isoleucine belongs to branched chain amino acids (BCAAs), and is one of the eight essential amino acids that cannot be synthesized by humans and animals themselves and must be supplied from exogenous sources. It has a variety of physiological functions and is a raw material for synthesizing human hormones and enzymes. It has the effect of promoting protein production and inhibiting its decomposition, and plays an important role in human life activities. Therefore, it has wide applications and commercial value in the food and pharmaceutical industries.

[0003] There are three production methods for L-isoleucine: extraction method, chemical synthesis method and fermentation method. Currently, only the fermentation method is implemented in industrial production. Among the microorganisms used in the fermentation method, the synthesis pathway and regulation mode of L-isoleucine are relatively complex. Threonine deaminase is the key enzyme for the biosynthesis of L-isoleucine, the rate-limiting step for isoleucine synthesis, and also the key limiting factor for the efficient production of L-isoleucine and its derivatives by fermentation method. Threonine dehydratase, also known as threonine deaminase, catalyzes the reaction of threonine dehydration decomposition to generate ammonia and α-ketobutyric acid, and the gene encoding this enzyme is ilvA.

[0004] The threonine deaminase BTD_ilvA encoded by the ilvA gene mainly has three domains, namely the catalytic domain (PALP) at the N-terminus and two regulatory domains (ACT_like) at the C-terminus. Threonine can dock with the PALP domain of ilvA as a substrate, and after isoleucine binds to the C-terminal regulatory domain of ilvA, it will form feedback inhibition on ilvA, inhibiting its binding to the substrate threonine. It is hoped that by studying the structure of ilvA, the affinity with the substrate can be improved, or the feedback inhibition can be solved, thereby improving the activity of the ilvA gene phenotype. Summary of the Invention

[0005] The inventors of the present invention studied the structure of the ilvA gene, performed site-directed mutagenesis on ilvA, transferred the mutated ilvA gene into the host bacterium, increased the yield of isoleucine in the host bacterium, and improved the phenotypic activity of the ilvA gene.

[0006] The present invention provides a mutated ilvA gene, an expression vector containing the mutated gene, an engineered bacterium containing the gene, and their applications in increasing the yield of isoleucine and fermentation production.

[0007] The first aspect of the present invention relates to a polynucleotide molecule, including a threonine deaminase substantially encoded by the nucleic acid molecule that is released from the inhibition of L-isoleucine, and the threonine deaminase is a gene with mutations at the amino acid level relative to the wild type.

[0008] In one embodiment, the gene of the threonine deaminase substantially released from the inhibition of L-isoleucine is a mutated ilvA gene. The ilvA gene of the present invention has mutations (nucleotide sequences shown as Seq No.3, 5 or 7) compared with the wild-type ilvA gene (nucleotide sequence shown as Seq No.1; amino acid sequence shown as Seq No.2), and these mutations release the feedback inhibition encoded by the ilvA gene by L-isoleucine, such as H418Y or H418F or H418W, shown as Seq No.4, 6, 8 in the present application respectively.

[0009] The present invention also relates to a genetically engineered strain, which contains the gene of the mutated threonine deaminase.

[0010] In one embodiment, the gene of the threonine deaminase released from the inhibition of L-isoleucine can be a mutated ilvA gene.

[0011] For the genetically engineered bacterium as described above, after inserting the ilvA gene of the present invention into the vector DNA, it can be introduced into the host. The ilvA gene of the present invention can be retained in the host as plasmid-like extrachromosomal DNA, or the above gene can be incorporated into the chromosome of the host microorganism by using methods such as transduction, transposon, Mu phage or homologous recombination. In order to effectively express the above gene, the ilvA of the present invention can be placed under the control of promoters such as lac, trp, PL and tac that function in microorganisms.

[0012] In one embodiment, the isoleucine fermentation engineering strain containing the mutated ilvA gene has an isoleucine production rate 43% higher than that of the engineering strain containing the wild-type ilvA gene.

[0013] The genetically engineered strain of the present invention can be used for the fermentation production of L-isoleucine.

[0014] Compared with the prior art, the present invention has the following beneficial technical effects:

[0015] 1. The present invention provides a mutated ilvA gene and its encoded nucleic acid sequence

[0016] 2. The present invention provides a method for increasing the production of isoleucine, which improves the fermentation yield of isoleucine by expressing a mutated ilvA gene in a recombinant strain and is used for the construction of an L-isoleucine engineering strain and the high-efficiency fermentation of L-isoleucine. The genetically engineered strain containing ilvA H418W has a 43% increase in L-isoleucine production capacity compared to the strain containing the wild-type ilvA gene, and has great industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a molecular docking diagram of the catalytic domain PALP of the wild-type WT of BTD_ilvA and the substrate threonine.

[0018] Figure 2 It is a molecular docking diagram of the regulatory domain of the wild-type WT of BTD_ilvA and the substrate isoleucine.

[0019] Figure 3 It is a molecular docking diagram of the catalytic domain PALP of the mutant H418Y of BTD_ilvA and the substrate threonine.

[0020] Figure 4 It is a molecular docking diagram of the regulatory domain of the mutant H418Y of BTD_ilvA and the substrate isoleucine.

[0021] Figure 5 It is a molecular docking diagram of the catalytic domain PALP of the mutant H418F of BTD_ilvA and the substrate threonine.

[0022] Figure 6 It is a molecular docking diagram of the regulatory domain of the mutant H418F of BTD_ilvA and the substrate isoleucine.

[0023] Figure 7 It is a molecular docking diagram of the catalytic domain PALP of the mutant H418W of BTD_ilvA and the substrate threonine.

[0024] Figure 8 It is a molecular docking diagram of the regulatory domain of the mutant H418W of BTD_ilvA and the substrate isoleucine. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation manner of the present invention. The scope of the present invention is defined by the appended claims. Combining this specification and general common knowledge in the art, those of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modification or change to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0027] Example 1: Bioinformatics Structure Simulation of ilvA Gene - AutoDock Vina Molecular Docking

[0028] The threonine deaminase BTD_ilvA encoded by the ilvA gene mainly has three domains, namely the catalytic domain (PALP) at the N-terminus and two regulatory domains (ACT_like) at the C-terminus. Among them, using threonine as the substrate to dock with the PALP domain of BTD_ilvA to observe their interaction; after isoleucine binds to the C-terminal regulatory domain of BTD_ilvA, it will form a feedback inhibition on BTD_ilvA and inhibit its binding to the substrate threonine. According to its structural analysis, the binding region of isoleucine to BTD_ilvA is between the interfaces of the two C-terminal regulatory domains, which are named Ile1 region and Ile2 region. Among them, the Ile 1 region is closer to the PALP catalytic domain, and it is speculated that the binding of isoleucine to this region has a greater impact on the feedback inhibition of BTD.

[0029] The wild type WT of BTD_ilvA and its three mutants H418Y, H418F, and H418W were respectively subjected to molecular docking in AutoDockVina, and the substrates were selected as threonine and isoleucine. After docking, they were visualized in Pymol (see Appendix Figures 1 - 8 ). The results are shown in Table 1. The affinity between the catalytic domain PALP of the wild type WT of BTD_ilvA and the substrate threonine is -4.6 kcal / mol, and the affinities between the regulatory domain of WT and isoleucine are -4.8 kcal / mol and -6.5 kcal / mol respectively. The affinities between the catalytic domains of the two mutants H418F and H418W and the substrate threonine have been improved to -4.8 kcal / mol, and the binding site of threonine to H418W is at a shallower position in the binding pocket compared to WT (as shown in Figure 1 and Figure 7 ), making it easier to bind. At the same time, the affinities between the regulatory domain Ile1 of these two mutants and isoleucine have decreased significantly. The affinity of H418F with isoleucine has decreased to -4.5 kcal / mol, and the affinity of H418W with isoleucine has decreased to -4.4 kcal / mol. The decrease in the affinity of H418F and H418W with isoleucine means that the binding ability of isoleucine to the mutants has decreased, thereby reducing the feedback inhibition of isoleucine on BTD_ilvA.

[0030] Table 1: Analysis of the Affinity of ilvA Gene Mutants with Substrates

[0031]

[0032]

[0033] Example 2: Construction of ilvA Gene Mutant Strains

[0034] The genetically engineered strain with ilvA mutation constructed in the present invention can convert threonine in the culture medium into isoleucine. Using Escherichia coli CGMCC No. 19458 as the initial strain (see the strain Sval065 in Chinese Patent CN202010401422.5, with the preservation number CGMCC NO. 19458, which contains the wild-type ilvA gene), by knocking out the gene encoding threonine dehydratase (tdh gene) and introducing the gene encoding threonine deaminase (ilvA), a recombinant bacterium for fermentative production of isoleucine is constructed. Knocking out the gene encoding threonine dehydratase (Tdh protein) can prevent the consumption of threonine in the culture medium to generate the by-product glycine, and introducing the gene encoding threonine deaminase, which can convert threonine into α-ketoglutaric acid, a key precursor for isoleucine synthesis, thus increasing the yield of isoleucine.

[0035] Table 2: Strains and Plasmids Used in the Present Invention

[0036]

[0037]

[0038] Table 3: Primers Used in the Present Invention

[0039]

[0040]

[0041] (I). Knockout of Threonine Dehydrogenase tdh in CGMCC No. 19458 Strain

[0042] Starting from Escherichia coli CGMCC No. 19458, the threonine dehydrogenase tdh was knocked out by a two-step homologous recombination method. The specific steps are as follows:

[0043] In the first step, using the pRE112 plasmid DNA as a template, a 3569-bp DNA fragment I was amplified using the primers tdh-cs-up / tdh-cs-down for the first-step homologous recombination.

[0044] The amplification system is as follows: 10 μl of Phusion 5X buffer (New England Biolabs), 1 μl of dNTP (10 mM each of the four dNTPs), 20 ng of DNA template, 2 μl each of the primers (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), and 33.5 μl of distilled water, with a total volume of 50 μl.

[0045] The amplification conditions are as follows: pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 4 minutes (30 cycles); extension at 72°C for 10 minutes (1 cycle).

[0046] The above DNA fragment I was used for the first homologous recombination: First, the pKD46 plasmid (purchased from the E. coli Stock Center of Yale University, USA, CGSC #7739) was transformed into E. coli CGMCC No. 19458 by electroporation, and then DNA fragment I was electroporated into E. coli CGMCC No. 19458 carrying pKD46.

[0047] The electroporation conditions are as follows: First, prepare the electrocompetent cells of E. coli CGMCC No. 19458 carrying the pKD46 plasmid; place 50 μl of the competent cells on ice, add 50 ng of DNA fragment I, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB medium to the electroporation cuvette, pipette 5 times and then transfer to a test tube, incubate at 75 rpm and 30°C for 2 hours. Take 200 μl of the bacterial solution and spread it on an LB plate containing ampicillin (final concentration 100 μg / ml) and chloramphenicol (final concentration 34 μg / ml), culture overnight at 30°C, pick single colonies for PCR verification. The primers used are XZ-tdh-up / XZ-tdh-down. The correct colony amplification product is a 4449 bp fragment. Pick one correct single colony and name it mIVL001.

[0048] In the second step, using the genomic DNA of E. coli CGMCC No. 19458 as a template, a 532 bp DNA fragment II was amplified with the primers XZ-tdh-up / tdh-del-down. DNA fragment II was used for the second homologous recombination. The amplification conditions and system are the same as those described in the first step. DNA fragment II was electroporated into strain Sval001.

[0049] The electrotransformation conditions are as follows: First, prepare electrocompetent cells of mIVL001 with the pKD46 plasmid; place 50 μl of electrocompetent cells on ice, add 50 ng of DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV voltage. Immediately after electroporation, transfer 1 ml of LB medium to the electroporation cuvette, pipette 5 times and then transfer to a test tube, rotate at 75 rpm, and incubate at 30 °C for 4 hours. Transfer the bacterial solution to an LB liquid medium without sodium chloride containing 10% sucrose (50 ml of medium in a 250 ml flask), and after culturing for 24 hours, streak plate on an LB solid medium without sodium chloride containing 6% sucrose. After PCR verification, the primers used are XZ-tdh-up / tdh-del-down, and the correct colony amplification product is a 532 bp fragment. Select a correct single colony and name it mIVL002.

[0050] (2). Mutation of the threonine deaminase gene ilvA

[0051] Introduce mutations into the ilvA gene by two-step homologous recombination to relieve the feedback inhibition of L-isoleucine. The specific steps are as follows:

[0052] First step, using pRE112 plasmid DNA as a template, amplify a 3539 bp DNA fragment I with primers ilvA-mut-cat-up / ilvA-mut-cat-down for the first step of homologous recombination.

[0053] The amplification system is: 10 μl of Phusion 5X buffer (New England Biolabs), 1 μl of dNTP (10 mM each dNTP), 20 ng of DNA template, 2 μl of each primer (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), 33.5 μl of distilled water, with a total volume of 50 μl.

[0054] The amplification conditions are: pre-denaturation at 98 °C for 2 minutes (1 cycle); denaturation at 98 °C for 10 seconds, annealing at 56 °C for 10 seconds, extension at 72 °C for 4 minutes (30 cycles); extension at 72 °C for 10 minutes (1 cycle).

[0055] Use the above DNA fragment I for the first homologous recombination: First, transform the pKD46 plasmid - (purchased from the E. coli Stock Center of Yale University, CGSC#7739) into E. coli mIVL002 by electroporation, and then electrotransfer DNA fragment I into E. coli mIVL002 with pKD46.

[0056] The electrotransformation conditions are as follows: First, prepare electrocompetent cells of Escherichia coli mIVL002 carrying the pKD46 plasmid; place 50 μl of the electrocompetent cells on ice, add 50 ng of DNA fragment I, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV voltage. Immediately after electroporation, transfer 1 ml of LB medium to the electroporation cuvette, pipette 5 times and then transfer to a test tube, incubate at 75 rpm and 30 °C for 2 hours. Take 200 μl of the bacterial solution and spread it on an LB plate containing ampicillin (final concentration 100 μg / ml) and chloramphenicol (final concentration 34 μg / ml), culture overnight at 30 °C, pick single colonies for PCR verification, using primers XZ-ilvA-mut-up / XZ-ilvA-mut-down, the correct colony amplification product is a 4746 bp fragment, pick one correct single colony and name it mIVL003.

[0057] In the second step, using the DNA of wild-type Escherichia coli ATCC 8739 as a template, amplify a 2528 bp DNA fragment II with primers ilvA-ilvA-H418Y-F / ilvA-mut-cs-R. DNA fragment II is used for the second homologous recombination. Electroporate DNA fragment II into strain mIVL003.

[0058] Using the DNA of wild-type Escherichia coli ATCC 8739 as a template, amplify a 2528 bp DNA fragment II with primers ilvA-ilvA-H418F-F / ilvA-mut-cs-R. DNA fragment II is used for the second homologous recombination. Electroporate DNA fragment II into strain mIVL003.

[0059] Using the DNA of wild-type Escherichia coli ATCC 8739 as a template, amplify a 2528 bp DNA fragment II with primers ilvA-ilvA-H418W-F / ilvA-mut-cs-R. DNA fragment II is used for the second homologous recombination. Electroporate DNA fragment II into strain mIVL003.

[0060] The electrotransformation conditions are as follows: First, prepare electrocompetent cells of Sval001 with the pKD46 plasmid; place 50 μl of electrocompetent cells on ice, add 50 ng of DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV voltage. Immediately after electroporation, transfer 1 ml of LB medium to the electroporation cuvette and incubate at 30 °C for 4 hours. Transfer the bacterial solution to an LB liquid medium without sodium chloride containing 10% sucrose (50 ml of medium in a 250 ml flask), culture for 24 hours, and then streak on an LB solid medium without sodium chloride containing 6% sucrose. After PCR verification, the primers used are XZ-ilvA-mut-up / ilvA-mut-ilvA-down, and the correct colony amplification product is a 2528 bp fragment. Select a single colony in which the 418th amino acid of the wild-type ilvA gene has been successfully mutated to tyrosine, named mIVL004, select a single colony in which the 418th amino acid of the wild-type ilvA gene has been successfully mutated to phenylalanine, named mIVL005, and select a single colony in which the 418th amino acid of the wild-type ilvA gene has been successfully mutated to tryptophan, named mIVL006.

[0061] Example 3: Fermentation production of L-isoleucine using recombinant strains

[0062] The seed medium consists of the following components (the solvent is water):

[0063] Glucose 20 g / L, corn steep liquor dry powder 10 g / L, KH2PO4 8.8 g / L, (NH4)2SO4 2.5 g / L, MgSO4·7H2O 2 g / L.

[0064] The fermentation medium is mostly the same as the seed medium, except that the glucose concentration is 50 g / L and 15 g / L of threonine is additionally added.

[0065] The anaerobic fermentation of mIVL002, mIVL004, mIVL005, and mIVL006 includes the following steps:

[0066] (1) Seed culture: Inoculate fresh clones on an LB plate into a test tube containing 4 ml of seed medium and culture overnight at 37 °C with shaking at 250 rpm. Then, transfer the culture to a 250 ml Erlenmeyer flask containing 30 ml of seed medium at an inoculation amount of 2% (V / V) and culture with shaking at 37 °C and 250 rpm for 12 hours to obtain a seed culture solution for inoculating the fermentation medium.

[0067] (2) Fermentation culture: The volume of the fermentation medium in a 500 ml anaerobic tank is 250 ml. The seed culture solution is inoculated into the fermentation medium at an inoculum size with a final OD550 of 0.1, and fermented at 37 °C and 150 rpm for 4 days to obtain a fermentation broth. The neutralizing agent is 5 M ammonia water to control the pH of the fermentation tank at 7.0. No gas is introduced during the culture process.

[0068] Analysis method: An Agilent-1260 high performance liquid chromatograph was used to determine the components in the fermentation broth after 3 days of fermentation. The concentration of glucose and organic acids in the fermentation broth was determined using an Aminex HPX–87H organic acid analysis column from Biorad. A Sielc amino acid analysis column primesep 100 250×4.6 mm was used for amino acid determination.

[0069] The experiment was repeated 3 times, and the average value of the analysis results is presented in Table 4 below.

[0070] Table 4: Comparison of the production capacity of L-isoleucine strains

[0071] strain characteristic L-isoleucine concentration (g / L) mIVL002 CGMCC No.19458△tdh (wild-type ilvA gene) 10.2 mIVL004 CGMCC No.19458△tdh△ilvA::ilvA H418Y 11.3 mIVL005 CGMCC No.19458△tdh△ilvA::ilvA H418F 13.5 mIVL006 CGMCC No.19458△tdh△ilvA::ilvA H418W 14.6

[0072] As presented in Table 4, in the medium containing threonine, compared with the control group mIVL002, mIVL004, mIVL005, and mIVL006 have increased isoleucine yields. In particular, the mIVL006 (ilvA H418W) strain has a 43% increase in the L-isoleucine production capacity compared to the strain containing mIVL002 (wild-type ilvA). The experimental data shows that the Ile yield of H418W is 14.6 g / L, which is consistent with the results of molecular docking analysis.

[0073] The above is only the preferred embodiment of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and refinements made without departing from the principle of the present invention should be regarded as within the protection scope of the present invention.

Claims

1. An ilvA gene mutant, characterized in that: the amino acid sequence of the mutant is shown in SEQ ID NO: 4, 6, or 8 in the sequence listing.

2. A polynucleotide encoding the ilvA gene mutant according to claim 1.

3. The polynucleotide according to claim 2, characterized in that: the polynucleotide sequence is shown in SEQ ID NO: 3, 5, or 7 in the sequence listing.

4. An expression vector containing the polynucleotide encoding the ilvA gene mutant according to claim 1.

5. The expression vector according to claim 4, characterized in that: the polynucleotide is shown in SEQ ID NO: 3, 5, or 7 in the sequence listing.

6. A recombinant strain comprising the ilvA gene mutant according to claim 1 or the polynucleotide encoding the ilvA gene mutant, or an expression vector containing the polynucleotide encoding the ilvA gene mutant.

7. Use of the recombinant strain according to claim 6 in the preparation of isoleucine.

8. Application of the ilvA gene mutant according to claim 1, or the polynucleotide according to any one of claims 2 - 3, or the expression vector according to any one of claims 4 - 5 in the preparation of an isoleucine recombinant strain.

9. A method for increasing the yield of isoleucine, characterized in that: fermenting to produce isoleucine using a recombinant strain, and the recombinant strain used contains the ilvA gene mutant according to claim 1.

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