Acetohydroxy acid reductoisomerase mutant and application thereof

Abstract

The invention discloses an acetohydroxy acid reductoisomerase mutant and application thereof. The invention provides an acetohydroxy acid reductoisomerase mutant. The amino acid sequence of the acetohydroxy acid reductoisomerase mutant is shown as SEQ ID NO: 1. The invention also provides a method for improving the enzyme activity of the wild-type acetohydroxy acid reductoisomerase. The method comprises the following step: mutating the 105th amino acid residue of the wild-type acetohydroxy acid reductoisomerase from F into L. The present invention is useful in the preparation of products of synthetic pathways in which acetohydroxy acid reductoisomerase is involved, such as branched chain amino acids or downstream products thereof, pantoic acids or downstream products thereof, and pantothenic acids or downstream products thereof.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and relates to an acetohydroxyacid reductoisomerase mutant and application thereof. Background Art

[0002] Isoleucine, valine and leucine together constitute the three essential branched-chain amino acids for the human body and have very wide applications in the fields of medicine, feed, food, etc.

[0003] Pantothenic acid, also known as vitamin B5, is a B vitamin that is produced from pantothenic acid and β-alanine by pantothenate synthase (PanC). As an important water-soluble vitamin, vitamin B5 is widely used in feed, food additives and medicine.

[0004] The synthetic pathways of isoleucine, valine, leucine, pantothenic acid and pantothenic acid in organisms are very similar (synthetic pathways see Figure 1 ). Keto-acid reductoisomerase (KARI) is a key enzyme required in the synthesis pathway of these substances and plays a vital role in the biosynthesis of these substances. Summary of the invention

[0005] The purpose of the present invention is to provide an acetohydroxyacid reductoisomerase mutant and application thereof.

[0006] The present invention provides an acetohydroxy acid reductoisomerase mutant (KARI protein mutant), and its amino acid sequence is shown in SEQ ID NO: 1. The KARI protein mutant provided by the present invention is obtained by mutating the 105th amino acid residue of the wild-type acetohydroxy acid reductoisomerase (wild-type KARI protein) from F to L.

[0007] Nucleic acid molecules encoding the KARI protein mutants also fall within the protection scope of the present invention.

[0008] The nucleic acid molecule can be a DNA fragment or an RNA fragment.

[0009] The DNA fragment encoding the wild-type KARI protein is named the wild-type kari gene. The DNA fragment encoding the mutant KARI protein is named the mutant kari gene. The mutant kari gene is specifically shown in SEQ ID NO: 2. Compared with the wild-type kari gene, the difference of the mutant kari gene is that the nucleotide at position 313 is mutated from T to C.

[0010] The present invention also protects biological materials that are (a1) or (a2) or (a3) ​​or (a4) as follows:

[0011] (a1) a DNA fragment having the kari gene after the mutation modification;

[0012] (a2) an expression cassette having the DNA fragment described in (a1);

[0013] (a3) a recombinant vector having the DNA fragment described in (a1);

[0014] (a4) A recombinant microorganism having the DNA fragment described in (a1).

[0015] Exemplarily, the DNA fragment of the kari gene after the mutation modification is shown in SEQ ID NO:5.

[0016] The present invention also protects the use of the KARI protein mutant as acetohydroxyacid reductoisomerase.

[0017] The present invention also protects the use of the nucleic acid molecule or the biological material, which is as follows (b1) or (b2) or (b3):

[0018] (b1) preparing acetohydroxy acid reductoisomerase;

[0019] (b2) preparing engineered bacteria for producing target products;

[0020] (b3) preparing the target product.

[0021] The present invention also provides a method for improving the enzyme activity of wild-type KARI protein, comprising the following steps: mutating the 105th amino acid residue of the wild-type KARI protein from F to L.

[0022] The present invention also provides a method for improving the production of a target product by a microorganism, comprising the following steps: mutating the codon encoding the 105th amino acid residue of a wild-type KARI protein in the genomic DNA of the starting microorganism from a codon encoding F to a codon encoding L.

[0023] The present invention also provides a method for preparing a recombinant microorganism, comprising the following steps: mutating the codon encoding the 105th amino acid residue of the wild-type KARI protein in the genomic DNA of the starting microorganism from a codon encoding F to a codon encoding L to obtain a recombinant microorganism.

[0024] The recombinant microorganism prepared by the method also falls within the protection scope of the present invention.

[0025] The present invention also protects the use of the recombinant microorganism in preparing a target product.

[0026] Specifically, any of the above wild-type KARI proteins is shown in SEQ ID NO:3.

[0027] Specifically, any of the above wild-type kari genes is shown in SEQ ID NO:4.

[0028] Illustratively, in any of the above methods, the codon encoding the 105th amino acid residue of the wild-type KARI protein in the genomic DNA of the starting microorganism is mutated from TTT to CTT.

[0029] Exemplarily, in any of the above methods, the wild-type kari gene (as shown in SEQ ID NO:4) in the genomic DNA of the starting microorganism is replaced with the mutated kari gene (as shown in SEQ ID NO:2).

[0030] As an example, the microorganism may be Escherichia coli or a recombinant Escherichia coli.

[0031] As an example, the starting microorganism may be the valine-engineered bacteria Sval064 or the pantothenic acid-engineered bacteria Span096.

[0032] Specifically, any of the above target products is a product of a synthetic pathway involving acetohydroxy acid reductoisomerase.

[0033] Specifically, any of the target products described above is a branched-chain amino acid or a downstream product thereof.

[0034] Specifically, the branched-chain amino acid is isoleucine, valine or leucine.

[0035] Specifically, any of the above target products is pantoic acid or its downstream products.

[0036] Specifically, any of the above target products is pantothenic acid or its downstream products.

[0037] The invention has great application and promotion value for the preparation of related products (such as branched-chain amino acids, pantothenic acid, pantothenic acid and their downstream products) of the synthetic pathway involving acetohydroxy acid reductoisomerase. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the biosynthetic pathway for isoleucine, valine, leucine and pantothenic acid. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0040] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are all repeated three times, and the results are averaged. Unless otherwise specified, the pH of the culture medium in the examples is natural pH. pKD46 plasmid: CGSC Escherichia coli Collection Center, Yale University, USA, CGSC#7739. pUC57-RBS5-kari plasmid is recorded in ZL202010401422.5 (authorization announcement number is CN113278655B; authorization announcement date is 2022.05.17). pXZ-CS plasmid is recorded in the following document (i.e., plasmid pXZ-CS in the document): Tan, et al., Appl Environ Microbiol, 2013, 79: 4838-4844.

[0041] Example 1: Construction of an L-valine engineered strain expressing an acetohydroxyacid reductoisomerase mutant

[0042] 1. Preliminary preparations

[0043] In the previous study, the inventor obtained a batch of engineered bacteria that can efficiently produce L-valine through domestication screening. After simultaneous genome sequencing and comparison of the starting strain and the domesticated strain, it was found that a point mutation occurred in the wild-type kari gene in the genomic DNA of a domesticated strain. The mutation was located at the 313th nucleotide in the coding region of the gene, from T to C. This mutation caused the 105th amino acid residue of the wild-type acetohydroxyacid reductoisomerase to mutate from F to L.

[0044] The wild-type KARI protein is shown in SEQ ID NO: 3. The wild-type kari gene is shown in SEQ ID NO: 4. The KARI protein mutant is shown in SEQ ID NO: 1. The mutant kari gene is shown in SEQ ID NO: 2.

[0045] The valine engineering bacteria Sval064 is a recombinant Escherichia coli for producing L-valine constructed by the inventors in the early stage, that is, the recombinant strain Sval064 prepared in Example 17 of ZL 202010401422.5 (authorization announcement number is CN113278655B; authorization announcement date is 2022.05.17). The genomic DNA of the valine engineering bacteria Sval064 has a DNA segment shown in SEQ ID NO:4.

[0046] 2. Preparation of recombinant bacteria Sval064K

[0047] 1. The DNA fragment I was electroporated into the valine engineering bacteria Sval064 to obtain the recombinant bacteria Sval064-CS.

[0048] (1) Preparation of DNA fragment I

[0049] Using pXZ-CS plasmid as template DNA, PCR amplification was performed using a primer pair consisting of primer adhE-cat-up and primer adhE-sacB-down, and the PCR amplification product, namely DNA fragment I (2719 bp), was recovered.

[0050] The reaction system for PCR amplification (50 μl): Phusion 5X buffer (New England Biolabs) 10 μl, dNTP (10 mM of each dNTP) 1 μl, template DNA 20 ng, primer adhE-cat-up (10 μM) 2 μl, primer adhE-sacB-down (10 μM) 2 μl, Phusion High-Fidelity DNA polymerase (2.5 U / μl) 0.5 μl, and the balance is distilled water.

[0051] The reaction conditions of PCR amplification were as follows: pre-denaturation at 98°C for 2 min (1 cycle); denaturation at 98°C for 10 s, annealing at 56°C for 10 s, extension at 72°C for 2 min (30 cycles); extension at 72°C for 10 min (1 cycle).

[0052] (2) First homologous recombination

[0053] ① The pKD46 plasmid was introduced into the valine engineering bacteria Sval064 by electroporation to obtain Sval064 containing the pKD46 plasmid.

[0054] ②Introduce DNA fragment I into the recombinant bacteria obtained in step ①, and obtain the recombinant bacteria Sval064-CS after homologous recombination.

[0055] Specific steps: 50 μl of Sval064 competent cells with pKD46 plasmid were placed on ice, 50 ng of DNA fragment I was added, and after being placed on ice for 2 min, the cells were transferred to a 0.2 cm Bio-Rad electroporation cup and electroporated using a MicroPulser (Bio-Rad) electroporator (electroporation parameters: voltage 2.5 kV), and then 1 ml of LB liquid culture medium was pipetted 5 times and then transferred to a test tube, and incubated at 30°C and 75rpm for 2h. After the incubation, 200μl of the bacterial solution was spread on an LB solid culture medium plate containing 100μg / ml ampicillin and 34μg / ml chloramphenicol, and cultured at 30°C overnight. Then, a single colony was selected for PCR identification (the primer pair for PCR identification consisted of primer adhE-YZ-up-W and primer XZ-adhE-down, and the colony with an amplification product of 3762bp was a PCR-positive colony) to obtain the recombinant bacterium Sval064-CS.

[0056] (3) Preparation of DNA fragment II

[0057] ① Using pUC57-RBS5-kari plasmid as template DNA, PCR amplification was performed using a primer pair consisting of primer RBS5-adhE-up and primer karimut-down, and the PCR amplification product, namely mutant fragment 1 (475 bp), was recovered.

[0058] The reaction system for PCR amplification is basically the same as step (1), the only difference being the replacement of primers and template.

[0059] The reaction conditions for PCR amplification are the same as in step (1).

[0060] ② Using pUC57-RBS5-kari plasmid as template, PCR amplification was performed using a primer pair consisting of primer karimut-up and primer kari-adhE-down, and the PCR amplification product, namely mutant fragment 2 (757 bp), was recovered.

[0061] The reaction system for PCR amplification is basically the same as step (1), the only difference being the replacement of primers and template.

[0062] The reaction conditions for PCR amplification are the same as in step (1).

[0063] ③ Using mutant fragment 1 and mutant fragment 2 as template DNA, PCR amplification was performed using a primer pair consisting of primer RBS5-adhE-up and primer kari-adhE-down, and the PCR amplification product, namely DNA fragment II, was recovered.

[0064] The reaction system of PCR amplification is basically the same as step (1), the only difference is the replacement of primers and template DNA (the template DNA consists of 20 ng of mutant fragment 1 and 20 ng of mutant fragment 2).

[0065] The reaction conditions for PCR amplification are the same as in step (1).

[0066] The sequencing result showed that the DNA fragment II was as shown in SEQ ID NO:5.

[0067] In actual operation, DNA fragment II can also be directly prepared by artificial synthesis.

[0068] (4) Second homologous recombination

[0069] The DNA fragment II was introduced into the recombinant bacterium Sval064-CS, and the recombinant bacterium Sval064K was obtained after homologous recombination.

[0070] Specific steps: 50 μl of Sval064-CS competent cells containing pKD46 were placed on ice, 50 ng of DNA fragment II was added, and after being placed on ice for 2 min, the cells were transferred to a 0.2 cm Bio-Rad electroporation cup and electroporated using a MicroPulser (Bio-Rad) electroporator (electroporation parameters: voltage 2.5 kV), and then 1 ml of LB liquid culture medium was pipetted 5 times and then transferred to a test tube and incubated at 30°C and 75rpm for 4h. After the incubation, the culture was transferred to a liquid screening culture medium and cultured for 24h. Then, the culture was streaked into a solid screening culture medium and cultured overnight. Then, a single colony was selected for PCR identification (the primer pair for PCR identification consisted of primer adhE-YZ-up-W and primer XZ-adhE-down, and the colony with an amplification product of 2231bp was a PCR-positive colony) and sequencing to obtain the recombinant bacterium Sval064K, also known as the valine engineered bacterium Sval064K.

[0071] Liquid screening medium: contains 100 g / L sucrose, 10 g / L tryptone and 5 g / L yeast extract, and the balance is water.

[0072] Solid screening medium: contains 60g / L sucrose, 10g / L tryptone, 5g / L yeast extract and 15g / L agar powder, and the balance is water.

[0073] Sequencing verification showed that compared with the valine engineering bacterium Sval064, the recombinant bacterium Sval064K differed in that the wild-type kari gene (as shown in SEQ ID NO: 4) in the genome DNA of the valine engineering bacterium Sval064 was replaced with the mutated and modified kari gene (as shown in SEQ ID NO: 2). The results indicated that a recombinant bacterium expressing the acetohydroxy acid reductoisomerase mutant (F105L) was successfully constructed.

[0074] The relevant information of the primers used in Example 1 is shown in Table 1.

[0075] Table 1

[0076]

[0077] Example 2. Production of valine using the valine engineering bacterium Sval064K and the valine engineering bacterium Sval064

[0078] Seed medium: containing 20 g / L of glucose, 0.87 g / L of NH4H2PO4, 2.63 g / L of (NH4)2HPO4, 0.18 g / L of MgSO4·7H2O, 0.15 g / L of betaine hydrochloride, 1.5 g / L of FeCl3·6H2O, 0.1 g / L of CoCl2·6H2O, 0.1 g / L of CuCl2·2H2O, 0.1 g / L of ZnCl2, 0.1 g / L of Na2MoO4·2H2O, 0.2 g / L of MnCl2·4H2O, and 0.05 g / L of H3BO3, with the balance being water.

[0079] Compared with the seed medium, the fermentation medium differed only in that the glucose content was 50 g / L.

[0080] The test bacteria were respectively the valine engineering bacterium Sval064K and the valine engineering bacterium Sval064.

[0081] 1. Seed culture

[0082] Inoculate a monoclonal of the test bacteria into 4 ml of the seed medium and shake culture overnight at 37°C and 250 rpm. Then transfer it to 30 ml of the seed medium at an inoculation amount of 2% and shake culture at 37°C and 250 rpm for 12 hours to obtain a seed solution.

[0083] 2. Fermentation culture

[0084] Add 250 ml of fermentation medium to an anaerobic fermenter with a capacity of 500 ml, and then inoculate the seed liquid obtained in step 1 (at the initial moment of inoculation completion, the OD550nm value of the system is 0.1), and ferment at 37 °C and 150 rpm for 6 days to obtain a fermentation broth (the fermentation broth refers to all the contents in the fermenter). During the fermentation process, control the system pH to 7.0 by adding 5 M ammonia water. During the fermentation process, no air is introduced throughout the process.

[0085] 3. Detect the L-valine yield

[0086] Use an Agilent (Agilent-1260) high-performance liquid chromatograph to determine the components in the fermentation broth. The amino acid concentration in the fermentation broth is determined using a Sielc amino acid analysis column primesep 100 25×4.6 mm.

[0087] The L-valine content in the fermentation broth obtained by the valine engineering bacterium Sval064 is 2.5 g / L. The L-valine content in the fermentation broth obtained by the valine engineering bacterium Sval064K is 3.6 g / L. Compared with the valine engineering bacterium Sval064, the L-valine yield of the valine engineering bacterium Sval064K has increased by 44%. Compared with the valine engineering bacterium Sval064, the difference in the valine engineering bacterium Sval064K is only that the gene encoding the KARI protein mutant replaces the gene encoding the wild-type KARI protein. Therefore, the above increase in L-valine yield is caused by the increased activity of the KARI protein mutant relative to the wild-type KARI protein.

[0088] Example 3. Construct a pantothenic acid engineering strain expressing a mutant of acetohydroxy acid reductoisomerase

[0089] The pantothenic acid engineering bacterium Span096 is Escherichia coli Span096 constructed by the inventor for the production of pantothenic acid in the early stage. Escherichia coli Span096 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC, address: No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on December 26, 2022, and the deposit registration number is CGMCC No. 26276. The genomic DNA of the pantothenic acid engineering bacterium Span096 has the DNA segment shown in SEQ ID NO: 4.

[0090] Replace the valine engineering bacterium Sval064 with the pantothenic acid engineering bacterium Span096, and the others are the same as in Example 1.

[0091] Obtain the pantothenic acid engineering bacterium Span096K.

[0092] Sequencing verification showed that compared with the pantothenic acid engineering strain Span096, the recombinant strain Span096K differed in that the wild-type kari gene (as shown in SEQ ID NO:4) in the genomic DNA of the pantothenic acid engineering strain Span096 was replaced with the mutated and modified kari gene (as shown in SEQ ID NO:2). The results indicated that a recombinant strain expressing the acetohydroxy acid reductoisomerase mutant (F105L) was successfully constructed.

[0093] Example 4: Production of pantothenic acid using the pantothenic acid engineering strain Span096K and the pantothenic acid engineering strain Span096

[0094] Seed medium: containing 20 g / L of glucose, 3.5 g / L of (NH4)2HPO4, 3.91 g / L of KH2PO4, 4.48 g / L of K2HPO4, 0.18 g / L of MgSO4·7H2O, 0.15 g / L of betaine hydrochloride, 1.5 g / L of FeCl3·6H2O, 0.1 g / L of CoCl2·6H2O, 0.1 g / L of CuCl2·2H2O, 0.1 g / L of ZnCl2, 0.1 g / L of Na2MoO4·2H2O, 0.2 g / L of MnCl2·4H2O, 0.05 g / L of H3BO3, and the balance being water.

[0095] Fermentation medium: containing 5 g / L of serine, 50 g / L of glucose, 3.5 g / L of (NH4)2HPO4, 3.91 g / L of KH2PO4, 4.48 g / L of K2HPO4, 0.18 g / L of MgSO4·7H2O, 0.15 g / L of betaine hydrochloride, 1.5 g / L of FeCl3·6H2O, 0.1 g / L of CoCl2·6H2O, 0.1 g / L of CuCl2·2H2O, 0.1 g / L of ZnCl2, 0.1 g / L of Na2MoO4·2H2O, 0.2 g / L of MnCl2·4H2O, 0.05 g / L of H3BO3, and the balance being water.

[0096] The test strains were respectively the pantothenic acid engineering strain Span096K and the pantothenic acid engineering strain Span096.

[0097] 1. Seed culture

[0098] Inoculate a monoclonal of the test strain into 4 ml of the seed medium and culture it overnight at 37°C with shaking at 250 rpm. Then transfer it to 30 ml of the seed medium at an inoculation amount of 2% and culture it at 37°C with shaking at 250 rpm for 12 hours to obtain a seed solution.

[0099] 2. Fermentation culture

[0100] Add 25 ml of fermentation medium into an Erlenmeyer flask with a capacity of 250 ml, and then inoculate the seed liquid obtained in step 1 (at the initial moment of completing inoculation, the OD550nm value of the system is 0.1), and ferment at 37 °C and 250 rpm for 3 days to obtain a fermentation broth (the fermentation broth refers to all the contents in the Erlenmeyer flask).

[0101] 3. Detect the pantothenic acid yield

[0102] Use an Agilent-1260 high performance liquid chromatograph to determine the components in the fermentation broth. The determination of the pantothenic acid concentration in the fermentation broth uses an Aminex HPX–87H organic acid analysis column from Biorad company.

[0103] The pantothenic acid content in the fermentation broth obtained by the pantothenic acid engineering bacterium Span096 is 1.8 g / L. The pantothenic acid content in the fermentation broth obtained by the pantothenic acid engineering bacterium Span096K is 2.5 g / L. Compared with the pantothenic acid engineering bacterium Span096, the pantothenic acid yield of the pantothenic acid engineering bacterium Span096K has increased by 39%. Compared with the pantothenic acid engineering bacterium Span096, the difference in the pantothenic acid engineering bacterium Span096K is only that the gene encoding the KARI protein mutant replaces the gene encoding the wild-type KARI protein. Therefore, the above increase in pantothenic acid yield is caused by the increased activity of the KARI protein mutant relative to the wild-type KARI protein.

[0104] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art. According to the scope of the following appended claims, some basic features can be applied.

Claims

1. An acetohydroxyacid reductoisomerase mutant, as shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the acetohydroxyacid reductoisomerase mutant according to claim 1.

3. A biological material, which is any one of the following (a1), (a2), (a3), or (a4): (a1) A DNA fragment having the encoding gene of the acetohydroxyacid reductoisomerase mutant according to claim 1; (a2) An expression cassette having the DNA fragment described in (a1); (a3) A recombinant vector having the DNA fragment described in (a1); (a4) A recombinant microorganism having the DNA fragment described in (a1).

4. Use of the acetohydroxyacid reductoisomerase mutant according to claim 1 as an acetohydroxyacid reductoisomerase.

5. Use of the nucleic acid molecule according to claim 2 or the biological material according to claim 3, which is any one of the following (b1), (b2), or (b3): (b1) Preparing an acetohydroxyacid reductoisomerase; (b2) Preparing an engineered bacterium for producing a target product; (b3) Preparing a target product; The target product is a product of a synthesis pathway involving acetohydroxyacid reductoisomerase.

6. A method for increasing the enzyme activity of wild-type acetohydroxyacid reductoisomerase, comprising the following step: mutating the 105th amino acid residue of wild-type acetohydroxyacid reductoisomerase from F to L.

7. A method for increasing the production of a target product by a microorganism, comprising the following step: mutating the codon encoding the 105th amino acid residue of wild-type acetohydroxyacid reductoisomerase in the genomic DNA of the starting microorganism from the codon encoding F to the codon encoding L; the target product is a product of a synthesis pathway involving acetohydroxyacid reductoisomerase.

8. A method for preparing a recombinant microorganism, comprising the following step: mutating the codon encoding the 105th amino acid residue of wild-type acetohydroxyacid reductoisomerase in the genomic DNA of the starting microorganism from the codon encoding F to the codon encoding L to obtain a recombinant microorganism.

9. A recombinant microorganism prepared by the method according to claim 8.

10. Use of the recombinant microorganism according to claim 9 in the preparation of a target product; the target product is a product of a synthesis pathway involving acetohydroxyacid reductoisomerase.

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