Acetohydroxy acid reductoisomerase gene regulatory element mutant and application thereof

Abstract

The invention discloses an acetohydroxy acid reductoisomerase gene regulatory element mutant and application thereof. The invention provides a method for improving the yield of a target product of a microorganism. The method comprises the following steps: mutating the 86th nucleotide of an ilvC regulatory element at the upstream of an acetohydroxy acid reductoisomerase coding gene in genome DNA of a starting microorganism from g to t; the ilvC regulation and control element is as shown in SEQ ID NO: 1. The target product is a product of a synthetic route in which acetohydroxy acid reductoisomerase participates. The method has great application and popularization values in preparation of related products (such as branched chain amino acid, pantoic acid, pantothenic acid and downstream products thereof) of a synthetic route in which acetohydroxy acid reductoisomerase participates.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and relates to a mutant of an acetohydroxyacid reductoisomerase gene regulatory element and its application. Background Art

[0002] Acetohydroxyacid reductoisomerase is a key enzyme shared and essential in the biosynthetic pathways of three essential branched-chain amino acids (isoleucine, valine, and leucine) in the human body, as well as the water-soluble B vitamin (vitamin B5) and the key precursor pantoic acid for vitamin B5 synthesis. The activity of acetohydroxyacid reductoisomerase plays a crucial role in achieving the biosynthesis of these substances.

[0003] Optimizing the catalytic activity or expression state of key enzymes in the biosynthetic pathway to improve the production capacity of target products is one of the key steps in creating engineering strains in synthetic biology. In the production of branched-chain amino acids or pantoic acid by fermentation, modifying the metabolic regulatory gene elements of key enzymes in the metabolic pathway of the production strain to improve the production capacity of the strain is an important research direction at present.

[0004] The synthetic pathways of isoleucine, valine, leucine, and pantothenic acid in organisms are shown in Figure 1 . Summary of the Invention

[0005] The object of the present invention is to provide a mutant of an acetohydroxyacid reductoisomerase gene regulatory element and its application.

[0006] The present invention provides a method for increasing the yield of a target product of a microorganism, comprising the following steps: mutating the 86th nucleotide of the ilvC regulatory element upstream of the acetohydroxyacid reductoisomerase-encoding gene in the genome DNA of the starting microorganism from g to t; the ilvC regulatory element is as shown in SEQ ID NO:1.

[0007] The present invention also provides a method for preparing a recombinant microorganism, comprising the following steps: mutating the 86th nucleotide of the ilvC regulatory element upstream of the acetohydroxyacid reductoisomerase-encoding gene in the genome DNA of the starting microorganism from g to t to obtain a recombinant microorganism; the ilvC regulatory element is as shown in SEQ ID NO:1.

[0008] Compared with the starting microorganism, the yield of the target product of the recombinant microorganism is increased, and thus it can be used as an engineering microorganism for preparing the target product.

[0009] The recombinant microorganism prepared by the above method also belongs to the protection scope of the present invention.

[0010] The present invention also protects the application of the recombinant microorganism in the preparation of the target product.

[0011] The present invention also protects a DNA fragment as shown in SEQ ID NO:2.

[0012] The present invention also protects the use of the said DNA fragment as a regulatory element.

[0013] Specifically, as a regulatory element, the said DNA fragment regulates the transcription level of the gene downstream thereof.

[0014] Specifically, as a regulatory element, the said DNA fragment regulates the expression level of the gene downstream thereof.

[0015] Specifically, the said regulatory element is an element with promoter function.

[0016] Specifically, the said DNA fragment acts as a promoter to initiate the expression of the gene downstream.

[0017] Specifically, the said gene downstream is the gene encoding acetohydroxy acid reductoisomerase.

[0018] The present invention also protects the use of the said DNA fragment in constructing recombinant microorganisms with increased yield of the target product.

[0019] The present invention also protects a recombinant DNA fragment having a regulatory region and a gene region; the said regulatory region is as shown in SEQ ID NO:2.

[0020] Specifically, the said gene is the gene encoding acetohydroxy acid reductoisomerase.

[0021] The present invention also protects the use of the said DNA fragment in constructing recombinant microorganisms with increased yield of the target product.

[0022] Any of the above-mentioned ilvC regulatory elements may be as shown in the reverse complementary sequence of SEQ ID NO:1.

[0023] The coding frame of any of the above-mentioned genes encoding acetohydroxy acid reductoisomerase (ilvC gene) is specifically as shown in SEQ ID NO:6.

[0024] Specifically, the said target product is the product of the synthesis pathway involving acetohydroxy acid reductoisomerase.

[0025] Specifically, the said target product is a branched-chain amino acid or its downstream product.

[0026] Specifically, the said branched-chain amino acids are isoleucine, valine or leucine.

[0027] The said amino acid is an L-amino acid.

[0028] Specifically, the said target product is pantothenic acid or its downstream product.

[0029] Specifically, the target product is pantothenic acid or its downstream product.

[0030] Specifically, the microorganism is Escherichia coli or recombinant Escherichia coli.

[0031] Exemplarily, the microorganism is valine engineering bacterium Sval030 or pantoic acid engineering bacterium Span050.

[0032] The present invention has great application and popularization value for the preparation of related products (such as branched-chain amino acids, pantoic acid, pantothenic acid and their downstream products) in the synthesis pathway involving acetohydroxy acid reductoisomerase. Description of the Drawings

[0033] Figure 1 It is the biosynthetic pathway of isoleucine, valine, leucine and pantothenic acid. Detailed Embodiments

[0034] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0035] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, 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 embodiments, unless otherwise specified, can all be obtained from commercial channels. Unless otherwise specified, the quantitative tests in the following embodiments are all set with three repeated experiments, and the results are averaged. The ilvC gene: the acetohydroxy acid reductoisomerase gene. Unless otherwise specified, the pH of the culture medium in the embodiments is the natural pH. The pKD46 plasmid: Yale University CGSC Escherichia coli preservation center in the United States, CGSC#7739. The pXZ-CS plasmid is described in the following literature (i.e., plasmid pXZ-CS in the literature): Tan, et al., Appl Environ Microbiol, 2013, 79: 4838 - 4844.

[0036] Example 1. Discovery of Mutant ilvC Regulatory Elements

[0037] The valine engineering bacterium Sval030 is a recombinant Escherichia coli for producing L-valine constructed by the inventor in the early stage, that is, the recombinant strain Sval030 prepared in Example 17 of 202010466347.0 (the application publication number is CN113278641A; the application publication date is August 20, 2021).

[0038] The valine engineering bacterium Sval031 is a recombinant Escherichia coli constructed by the inventor in the early stage for the production of L-valine, that is, the recombinant strain Sval031 prepared in Example 19 of 202010466347.0 (the application publication number is CN113278641A; the application publication date is August 20, 2021).

[0039] Through whole-genome sequencing, it was found that compared with the valine engineering bacterium Sval030, a mutation occurred in the key region -35 of the regulatory element of the Escherichia coli own acetohydroxy acid reductoisomerase gene (EcolC_4228, CP000946.1……4670539-4672014) in the valine synthesis pathway of the valine engineering bacterium Sval031, that is, the nucleotide at position 4672100 on the chromosome (CP000946.1) changed from g to t. The genome DNA of the valine engineering bacterium Sval030 has the ilvC gene, and the DNA fragment adjacent to the upstream of the ilvC gene coding frame is shown as the reverse complementary sequence of SEQ ID NO:1. The genome DNA of the valine engineering bacterium Sval031 has the ilvC gene, and the DNA fragment adjacent to the upstream of the ilvC gene coding frame is shown as the reverse complementary sequence of SEQ ID NO:2. The ilvC gene coding frame is shown as SEQ ID NO:6. The difference between SEQ ID NO:1 and SEQ ID NO:2 is only in the 86th nucleotide, and this difference is the above-mentioned mutation site.

[0040] The DNA fragment shown in SEQ ID NO:1 was named the wild-type ilvC regulatory element. The DNA fragment shown in SEQ ID NO:2 was named the mutant ilvC regulatory element.

[0041] Example 2. Construction of the valine engineering bacterium Sval030P

[0042] 1. Preparation of DNA fragment I

[0043] Using the pXZ-CS plasmid as the template DNA, PCR amplification was carried out with the primer pair composed of primer cat-ilvC*-up and primer sacB-ilvC*-down, and the PCR amplification product was recovered, that is, DNA fragment I (2719bp).

[0044] Reaction system for PCR amplification (50 μl): 10 μl of Phusion 5X buffer (New England Biolabs), 1 μl of dNTP (10 mM each of dNTP), 20 ng of template DNA, 2 μl of primer cat-ilvC*-up (10 μM), 2 μl of primer sacB-ilvC*-down (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), and the balance is distilled water.

[0045] Reaction conditions for PCR amplification: 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).

[0046] Sequencing results showed that DNA fragment I was as shown in SEQ ID NO:3. In SEQ ID NO:3, nucleotides 1-50 constituted the upstream homologous arm, nucleotides 247-906 constituted the cat gene, nucleotides 1248-2669 constituted the sacB gene, and nucleotides 2670-2719 constituted the downstream homologous arm.

[0047] In actual operation, DNA fragment I can also be directly prepared by artificial synthesis.

[0048] 2. First homologous recombination

[0049] ① The pKD46 plasmid was introduced into the valine engineering bacterium Sval030 by electrotransformation to obtain Escherichia coli Sval030 with the pKD46 plasmid.

[0050] ② DNA fragment I was introduced into the engineering bacterium obtained in step ①, and after homologous recombination, the recombinant bacterium Sval030-CS was obtained.

[0051] Specific steps: Place 50 μl of electrocompetent cells of Escherichia coli Sval030 with pKD46 plasmid on ice, add 50 ng of DNA fragment I, place it on ice for 2 min, then transfer it to a 0.2 cm Bio-Rad electroporation cuvette, and use a MicroPulser (Bio-Rad) electroporator for electroporation (electroporation parameters: voltage 2.5 kv). Then, quickly add 1 ml of LB liquid medium to the electroporation cuvette, pipette 5 times, and transfer it to a test tube. Incubate at 30 °C and 75 rpm for 2 h. After incubation, take 200 μl of the bacterial solution and spread it on an LB solid medium plate containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol, and culture it overnight at 30 °C. Then, pick single colonies for PCR identification (the primer pair for PCR identification consists of primer ilvC-YZ347-down and primer ilvY-YZ552-down). The amplification product of the correct colonies is 3666 bp, and the correctly obtained recombinant bacterium is named Sval030-CS. The amplification product of the above PCR identification of recombinant bacterium Sval030-CS is as shown in SEQ ID NO:4.

[0052] 3. Preparation of DNA fragment II

[0053] Using the genomic DNA of valine engineering bacterium Sval031 as the template DNA, perform PCR amplification with the primer pair consisting of primer ilvC-YZ347-down and primer ilvY-YZ552-down, and recover the PCR amplification product, namely DNA fragment II (1048 bp).

[0054] The reaction system for PCR amplification is basically the same as in step 1, with the only difference being the replacement of primers and template DNA.

[0055] The reaction conditions for PCR amplification are the same as in step 1.

[0056] The sequencing results show that DNA fragment II is as shown in SEQ ID NO:5.

[0057] In actual operation, DNA fragment II can also be directly prepared by artificial synthesis.

[0058] 4. Second homologous recombination

[0059] Introduce DNA fragment II into recombinant bacterium Sval030-CS, and obtain recombinant bacterium Sval030P after homologous recombination.

[0060] Specific steps: Place 50 μl of electrocompetent cells of recombinant bacterium Sval030-CS on ice, add 50 ng of DNA fragment II, place on ice for 2 min, then transfer to a 0.2 cm Bio-Rad electroporation cuvette, and use a MicroPulser (Bio-Rad) electroporator for electroporation (electroporation parameters: voltage 2.5 kv). Then quickly add 1 ml of LB liquid medium to the electroporation cuvette, pipette 5 times, and transfer to a test tube. Incubate at 30 °C and 75 rpm for 4 h; after incubation, transfer to liquid selection medium and culture for 24 h, then streak and inoculate on solid selection medium and culture overnight; then, pick single colonies for PCR amplification and sequencing. The amplification product of the correct colonies is 1048 bp. The correctly obtained recombinant bacterium is named Sval030P, also known as valine engineering bacterium Sval030P. The amplification product of the above PCR identification of recombinant bacterium Sval030P is as shown in SEQ ID NO:5.

[0061] Liquid selection medium: containing 100 g / L sucrose, 10 g / L tryptone, and 5 g / L yeast extract, with the balance being water.

[0062] Solid selection medium: containing 60 g / L sucrose, 10 g / L tryptone, 5 g / L yeast extract, and 20 g / L agar powder, with the balance being water.

[0063] Verified by sequencing, compared with valine engineering bacterium Sval030, the difference in valine engineering bacterium Sval030P is that the wild-type ilvC regulatory element (as shown in SEQ ID NO:1) in the genomic DNA of valine engineering bacterium Sval030 is replaced by a mutant ilvC regulatory element (as shown in SEQ ID NO:2). The results show that a recombinant bacterium containing a mutant ilvC regulatory element has been successfully constructed.

[0064] The primer-related information used in Example 2 is shown in Table 1.

[0065] Table 1

[0066]

[0067] Example 3. Production of valine using valine engineering bacterium Sval030P and valine engineering bacterium Sval030

[0068] Seed culture 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 μg / L of FeCl3·6H2O, 0.1 μg / L of CoCl2·6H2O, 0.1 μg / L of CuCl2·2H2O, 0.1 μg / L of ZnCl2, 0.1 μg / L of Na2MoO4·2H2O, 0.2 μg / L of MnCl2·4H2O and 0.05 μg / L of H3BO3, with the balance being water.

[0069] Compared with the seed culture medium, the fermentation culture medium is only different in that: the glucose content is 50 g / L.

[0070] The test bacteria are respectively: valine engineering bacteria Sval030P and valine engineering bacteria Sval030.

[0071] 1. Seed culture

[0072] Inoculate the monoclonal of the test bacteria into 4 ml of the seed culture medium, and shake culture overnight at 37 °C and 250 rpm. Then transfer it to 30 ml of the seed culture medium at an inoculation amount of 2%, and shake culture at 37 °C and 250 rpm for 12 hours to obtain a seed solution.

[0073] 2. Fermentation culture

[0074] Add 250 ml of the fermentation culture medium to an anaerobic fermenter with a capacity of 500 ml, and then inoculate the seed solution obtained in step 1 (at the initial moment of completing inoculation, the OD 550nm value is 0.1), 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.

[0075] 3. Detection of L-valine yield

[0076] 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.

[0077] The L-valine content in the fermentation broth obtained from the valine engineering bacterium Sval030 was 2.8 g / L. The L-valine content in the fermentation broth obtained from the valine engineering bacterium Sval030P was 8.6 g / L. Compared with the valine engineering bacterium Sval030, the L-valine production of the valine engineering bacterium Sval030P increased by 207%. Compared with the valine engineering bacterium Sval030, the difference in the valine engineering bacterium Sval030P was only that the wild-type ilvC regulatory element shown in SEQ ID NO:1 was replaced by the mutant ilvC regulatory element shown in SEQ ID NO:2. Therefore, the increase in the above L-valine production was caused by the increased promoter activity of the mutant ilvC regulatory element relative to the wild-type ilvC regulatory element on the ilvC gene.

[0078] Example 4. Construction of the pantothenic acid engineering bacterium Span050P

[0079] The pantothenic acid engineering bacterium Span050 is the Escherichia coli Span050 for producing pantothenic acid constructed by the inventor in the early stage, that is, the recombinant strain Span050 described in 202110391896.0 (the application publication number is CN115109736A; the application publication date is September 27, 2022). Escherichia coli Span050 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, address: No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on January 22, 2021, and the deposit registration number is CGMCC No. 21699. The genomic DNA of the pantothenic acid engineering bacterium Span050 has an ilvC gene, and the DNA fragment adjacent to the upstream of the coding frame of the ilvC gene is shown as the reverse complementary sequence of SEQ ID NO:1. The coding frame of the ilvC gene is shown as SEQ ID NO:6.

[0080] Replace the valine engineering bacterium Sval030 with the pantothenic acid engineering bacterium Span050, and the others are the same as in Example 2.

[0081] The pantothenic acid engineering bacterium Span050P was obtained.

[0082] Verified by sequencing, compared with the pantothenic acid engineering bacterium Span050, the difference in the pantothenic acid engineering bacterium Span050P was that the wild-type ilvC regulatory element (shown in SEQ ID NO:1) in the genomic DNA of the pantothenic acid engineering bacterium Span050 was replaced by the mutant ilvC regulatory element (shown in SEQ ID NO:2). The results showed that a recombinant bacterium containing the mutant ilvC regulatory element was successfully constructed.

[0083] Example 5: Production of DL-pantothenic acid using engineered bacteria Span050P and Span050 for DL-pantothenic acid

[0084] Seed culture medium: containing 20 g / L glucose, 3.5 g / L (NH4)2HPO4, 3.91 g / L KH2PO4, 4.48 g / L K2HPO4, 0.18 g / L MgSO4·7H2O, 0.15 g / L betaine hydrochloride, 1.5 μg / L FeCl3·6H2O, 0.1 μg / L CoCl2·6H2O, 0.1 μg / L CuCl2·2H2O, 0.1 μg / L ZnCl2, 0.1 μg / L Na2MoO4·2H2O, 0.2 μg / L MnCl2·4H2O, 0.05 μg / L H3BO3, with the balance being water.

[0085] Fermentation medium: containing 5 g / L serine, 50 g / L glucose, 3.5 g / L (NH4)2HPO4, 3.91 g / L KH2PO4, 4.48 g / L K2HPO4, 0.18 g / L MgSO4·7H2O, 0.15 g / L betaine hydrochloride, 1.5 μg / L FeCl3·6H2O, 0.1 μg / L CoCl2·6H2O, 0.1 μg / L CuCl2·2H2O, 0.1 μg / L ZnCl2, 0.1 μg / L Na2MoO4·2H2O, 0.2 μg / L MnCl2·4H2O, 0.05 μg / L H3BO3, with the balance being water.

[0086] The test bacteria were respectively: engineered bacteria Span050P and Span050 for DL-pantothenic acid.

[0087] 1. Seed culture

[0088] Inoculate a monoclonal of the test bacteria into 4 ml of the seed culture medium and shake-culture overnight at 37°C and 250 rpm. Then transfer it to 30 ml of the seed culture medium at an inoculation amount of 2% and shake-culture at 37°C and 250 rpm for 12 hours to obtain a seed solution.

[0089] 2. Fermentation culture

[0090] Add 25 ml of the fermentation medium to a 250-ml Erlenmeyer flask, and then inoculate the seed solution obtained in step 1 (at the initial moment of completing inoculation, the OD 550nm value 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).

[0091] 3. Detection of the yield of DL-pantothenic acid

[0092] The components in the fermentation broth were determined using an Agilent-1260 high performance liquid chromatograph. The concentration of pantothenic acid in the fermentation broth was determined using an Aminex HPX–87H organic acid analysis column from Biorad.

[0093] The content of pantothenic acid in the fermentation broth obtained from the pantothenic acid engineering bacterium Span050 was 1.2 g / L. The content of pantothenic acid in the fermentation broth obtained from the pantothenic acid engineering bacterium Span050P was 2.1 g / L. Compared with the pantothenic acid engineering bacterium Span050, the pantothenic acid production of the pantothenic acid engineering bacterium Span050P increased by 75%. Compared with the pantothenic acid engineering bacterium Span050, the only difference in the pantothenic acid engineering bacterium Span050P was that the wild-type ilvC regulatory element shown in SEQ ID NO:1 was replaced by the mutant ilvC regulatory element shown in SEQ ID NO:2. Therefore, the increase in the above-mentioned pantothenic acid production was caused by the increased promoter activity of the mutant ilvC regulatory element relative to the wild-type ilvC regulatory element on the ilvC gene.

[0094] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, 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 cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A method for increasing the yield of a target product of a microorganism, comprising the following steps: mutating the 86th nucleotide of the ilvC regulatory element upstream of the acetohydroxy acid reductoisomerase-encoding gene in the genome DNA of the starting microorganism from g to t; the ilvC regulatory element is as shown in SEQ ID NO:1; the target product is a product of the synthesis pathway involving acetohydroxy acid reductoisomerase.

2. A method for preparing a recombinant microorganism, comprising the following steps: mutating the 86th nucleotide of the ilvC regulatory element upstream of the acetohydroxy acid reductoisomerase-encoding gene in the genome DNA of the starting microorganism from g to t to obtain a recombinant microorganism; the ilvC regulatory element is as shown in SEQ ID NO:

1.

3. The method according to claim 2, characterized in that: Compared with the starting microorganism, the yield of the target product of the recombinant microorganism is increased; the target product is a product of the synthesis pathway involving acetohydroxy acid reductoisomerase.

4. The recombinant microorganism prepared by the method according to claim 2 or 3.

5. The use of the recombinant microorganism according to claim 4 in the preparation of a target product; the target product is a product of the synthesis pathway involving acetohydroxy acid reductoisomerase.

6. A DNA fragment as shown in SEQ ID NO:

2.

7. The use of the DNA fragment according to claim 6 as a regulatory element.

8. The use of the DNA fragment according to claim 6 in constructing a recombinant microorganism with increased yield of a target product; the target product is a product of the synthesis pathway involving acetohydroxy acid reductoisomerase.

9. A recombinant DNA fragment having a regulatory region and a gene region; the regulatory region is as shown in SEQ ID NO:

2.

10. The use of the DNA fragment according to claim 9 in constructing a recombinant microorganism with increased yield of a target product; the target product is a product of the synthesis pathway involving acetohydroxy acid reductoisomerase.

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