PfkA mutant and application thereof in lactic acid fermentation production

Abstract

The invention discloses a PfkA mutant and application thereof in lactic acid fermentation production. The PfkA mutant is a protein obtained by replacing arginine at the 83rd site in an amino acid sequence of 6-phosphofructokinase pfkA with cysteine. According to the lactic acid engineering strain, pfkA point mutation is introduced into a genome of the lactic acid engineering strain, that is, a basic group C at the 247th site of a nucleotide sequence of a coding region of a wild-type pfkA gene is changed into a basic group T, and the lactic acid engineering strain with significantly improved lactic acid production capacity and biomass is constructed. In practical application, the lactic acid production capacity and biomass of the lactic acid engineering strain can be improved by introducing pfkA gene point mutation into a lactic acid engineering strain genome or introducing a pfkA mutant gene into the lactic acid engineering strain. The method is of great significance to construction of lactic acid production strains and efficient fermentation production of lactic acid.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, in particular to a PfkA mutant and its application in lactic acid fermentation production, and in particular to the application of the PfkA mutant and its related biomaterials in constructing lactic acid engineering bacteria and lactic acid fermentation production. Background Art

[0002] The rapid development of synthetic biology and the demand for green environmental protection have greatly promoted the rapid development of biomanufacturing, making it play an increasingly important role in the manufacture of compounds such as organic acids, amino acids, vitamins and natural products. Lactic acid, as an important organic acid, has a wide range of applications in the food, medicine, tobacco and chemical industries. Especially in recent years, with the rapid growth of demand for biodegradable plastic polylactic acid, the demand for lactic acid, a key monomer for the synthesis of polylactic acid, has increased, and its biomanufacturing has received more and more attention.

[0003] At present, more than 70% of the world's lactic acid is produced by biofermentation with renewable resources, such as corn starch or cassava flour. There are many bacteria that naturally produce lactic acid in nature. These bacterial species are rich and diverse and widely distributed in nature, such as Lactobacillus, Bacillus, Bifidobacterium and Lactococcus. These bacteria are highly acid-resistant and can achieve homotypic fermentation to produce lactic acid after genetic modification. However, these bacterial fermentation processes require rich culture media and cannot utilize pentose, which greatly increases production costs and downstream separation and purification costs. In addition, this type of bacteria usually uses calcium hydroxide or calcium carbonate as a neutralizer to control the fermentation pH, and the final fermentation product is calcium lactate. However, calcium lactate usually needs to be added with sulfuric acid during the separation and extraction process, which will produce a large amount of calcium sulfate waste and is difficult to handle. More importantly, the chiral purity of the lactic acid produced by this type of bacteria usually cannot meet the requirements of polylactic acid production, which limits its application.

[0004] In recent years, with the rapid development of synthetic biology, engineered strains obtained through metabolic engineering have attracted widespread attention and become a research hotspot because they can significantly improve the performance indicators of lactic acid production in terms of lactic acid chirality, engineering bacteria substrate utilization range, strain tolerance, etc. At present, the research on the creation of lactic acid engineered bacteria is more focused on lactate dehydrogenase, the key enzyme in lactic acid production, the supply of reducing power in metabolic balance, and the optimization of fermentation conditions. However, most strains must be domesticated before they can achieve efficient synthesis of lactic acid, and their fermentation capacity still needs to be improved. Summary of the invention

[0005] The purpose of the present invention is to provide a PfkA mutant and its related biological materials for use in constructing lactic acid engineering bacteria and lactic acid fermentation production

[0006] In order to achieve the above object, the present invention first provides a PfkA mutant.

[0007] The PfkA mutant is a protein obtained by replacing the arginine at position 83 in the amino acid sequence of 6-phosphofructokinase PfkA with other amino acids ("other amino acids" refers to any amino acid except arginine);

[0008] The 6-phosphofructokinase PfkA is a protein described in any one of the following (a1)-(a4):

[0009] (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;

[0010] (a2) a protein having the same function as that obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the amino acid residue at position 83 of the amino acid sequence shown in (a1);

[0011] (a3) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the amino acid sequence shown in (a1) or (a2);

[0012] (a4) A protein having 75% or more identity with (a1) and having the same function.

[0013] In the above (a2), the substitution and / or deletion and / or addition of one or several amino acid residues is substitution and / or deletion and / or addition of no more than 10 amino acid residues, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or substitution and / or deletion and / or addition of no more than 2 amino acid residues, or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0014] In (a3) ​​above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0015] In (a4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. The 75% or more identity can be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or 100% identity.

[0016] The protein in (a2) or (a3) ​​or (a4) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0017] In a specific embodiment of the present invention, the PfkA mutant is a protein obtained by replacing the 83rd arginine (Arginine, R) in the amino acid sequence of 6-phosphofructokinase PfkA with cysteine ​​(Cysteine, C). The amino acid sequence of the PfkA mutant is shown in Sequence 4 in the sequence table.

[0018] In order to achieve the above object, the present invention further provides a nucleic acid molecule encoding the above PfkA mutant.

[0019] The nucleic acid molecule is a DNA molecule as described in any one of the following (b1)-(b3):

[0020] (b1) the nucleotide sequence is the DNA molecule shown in Sequence 3 in the sequence listing;

[0021] (b2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in (b1) and encoding a PfkA mutant;

[0022] (b3) A DNA molecule that hybridizes to the nucleotide sequence defined in (b1) or (b2) under stringent conditions and encodes a PfkA mutant.

[0023] Wherein, the nucleic acid molecule may be DNA, such as recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA. The nucleic acid molecule may be a nucleic acid molecule formed by a gene encoding the PfkA mutant and its regulatory sequence.

[0024] A person skilled in the art can easily mutate the nucleotide sequence encoding the PfkA mutant of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the PfkA mutant are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the PfkA mutant and have the same function.

[0025] In (b2) above, the identity refers to the sequence similarity with the natural nucleic acid sequence. "Identity" includes nucleotide sequences with 75% or more identity to the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 4 of the present invention. The identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. The 75% or more identity can be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or 100% identity.

[0026] In the above (b3), the stringent conditions may be hybridization in a solution of 2×SSC, 0.1% SDS at 68°C and washing the membrane twice, each time for 5 min; or hybridization in a solution of 0.5×SSC, 0.1% SDS at 68°C and washing the membrane twice, each time for 15 min.

[0027] In order to achieve the above object, the present invention further provides a biomaterial as described in any one of the following (c1)-(c3):

[0028] (c1) an expression cassette containing the above nucleic acid molecule;

[0029] (c2) a recombinant vector containing the above nucleic acid molecule;

[0030] (c3) A recombinant microorganism containing the above nucleic acid molecule.

[0031] In the above (c1), the expression cassette refers to a DNA capable of expressing the PfkA mutant in a host cell, and the DNA may include not only a promoter for initiating transcription of the PfkA mutant encoding gene sequence, but also a terminator for terminating transcription of the PfkA mutant encoding gene sequence. Furthermore, the expression cassette may also include an enhancer sequence.

[0032] In the above (c2), the vector may be a plasmid, cosmid, phage or virus vector.

[0033] In the above (c3), the microorganism may be yeast, bacteria, algae or fungi.

[0034] In order to achieve the above object, the present invention further provides the use of any one of the following (d1)-(d5):

[0035] (d1) Use of the above PfkA mutant as 6-phosphofructokinase;

[0036] (d2) Use of the above nucleic acid molecules or the above biological materials in the preparation of 6-phosphofructokinase;

[0037] (d3) Use of the above PfkA mutant or the above nucleic acid molecule or the above biological material in constructing lactic acid engineering bacteria;

[0038] (d4) Use of the above PfkA mutant or the above nucleic acid molecule or the above biological material in lactic acid production;

[0039] (d5) Use of the above PfkA mutant, the above nucleic acid molecule or the above biological material in increasing lactic acid production.

[0040] In order to achieve the above object, the present invention also provides a recombinant bacterium capable of producing lactic acid.

[0041] The 6-phosphofructokinase involved in the pathway of producing lactic acid through metabolism of the recombinant bacteria capable of producing lactic acid provided by the present invention includes the above-mentioned PfkA mutant.

[0042] In order to achieve the above object, the present invention also provides a method for constructing the above recombinant bacteria.

[0043] The method for constructing the above-mentioned recombinant bacteria provided by the present invention comprises the following steps: increasing the content and / or activity of the above-mentioned PfkA mutant in the lactic acid engineering bacteria.

[0044] Furthermore, the method for increasing the content and / or activity of the above-mentioned PfkA mutant in the lactic acid engineering bacteria is to replace the wild-type pfkA gene (sequence 1) in the genomic DNA of the lactic acid engineering bacteria with the mutant pfkA* gene (sequence 3).

[0045] Furthermore, the method for replacing the wild-type pfkA gene (sequence 1) in the genomic DNA of the lactic acid engineering bacteria with the mutant pfkA* gene (sequence 3) is to change the base C at the 247th position of the coding region of the wild-type pfkA gene in the lactic acid engineering bacteria to the base T.

[0046] The method of changing the base C at position 247 of the coding region of the wild-type pfkA gene in the lactic acid engineered bacteria to the base T may be homologous recombination.

[0047] Furthermore, the engineered lactic acid bacteria may be engineered L-lactic acid producing bacteria or engineered D-lactic acid producing bacteria.

[0048] In a specific embodiment of the present invention, the L-lactic acid-producing engineered bacteria is an Escherichia coli Slac006 strain. The Escherichia coli Slac006 strain is a strain obtained by introducing an L-lactate dehydrogenase gene ldhL expression cassette into the fumarate reductase encoding gene (frd for short) site of Escherichia coli, knocking out the D-lactate dehydrogenase gene ldhA in the Escherichia coli, knocking out the pyruvate formate lyase gene pflB in the Escherichia coli, and knocking out the methylglyoxal synthase gene mgsA in the Escherichia coli, and the genotype of the strain is frd::M1-93::ldhL, △ldhA, △pflB, △mgsA. The Escherichia coli can specifically be an Escherichia coli ATCC8739 strain.

[0049] In another specific embodiment of the present invention, the D-lactic acid-producing engineered bacteria is an Escherichia coli Dlac006 strain. The Escherichia coli Dlac006 strain is a strain obtained by knocking out the fumarate reductase gene frdABCD in the Escherichia coli, knocking out the pyruvate formate lyase gene pflB in the Escherichia coli, and knocking out the methylglyoxal synthase gene mgsA in the Escherichia coli, and the genotype of the strain is △pflB, △frdABCD, △mgsA. The Escherichia coli can specifically be an Escherichia coli ATCC8739 strain.

[0050] The use of the above-mentioned recombinant bacteria or the recombinant bacteria constructed according to the above-mentioned method in lactic acid production or increasing lactic acid production also falls within the protection scope of the present invention.

[0051] In order to achieve the above object, the present invention finally provides a method for producing lactic acid.

[0052] The method for producing lactic acid provided by the present invention comprises the steps of fermenting and culturing the above-mentioned recombinant bacteria or the recombinant bacteria constructed according to the above-mentioned method.

[0053] Furthermore, the method for producing lactic acid may include the following steps:

[0054] 1) inoculating the above-mentioned recombinant bacteria or the recombinant bacteria constructed according to the above-mentioned method into a seed culture medium for culturing to obtain a seed solution;

[0055] 2) inoculating the seed liquid into a fermentation medium for fermentation to obtain a fermentation liquid;

[0056] 3) Separating and obtaining lactic acid from the fermentation broth.

[0057] Furthermore, in 1), the culture condition may be culturing at 37°C and 100 rpm for 12 hours. The seed culture medium may be composed of the following components (the solvent is water): glucose 20 g / L, NH 4 H2 PO 4 0.87g / L, (NH 4 ) 2 HPO 4 2.63g / L, MgSO 4 7H 2 O 0.18g / L, betaine-HCl 0.15g / L, FeCl 3 6H 2 O 1.5μg / L, CoCl 2 6H 2 O0.1μg / L, CuCl 2 ·2H 2 O 0.1μg / L, ZnCl 2 0.1μg / L, Na 2 MoO 4 ·2H 2 O 0.1 μg / L, MnCl 2 ·4H 2 O 0.2 μg / L, H 3 BO 3 0.05μg / L.

[0058] In the above 2), the culture conditions may be culturing at 37° C., 150 rpm, and pH 7.0 for 2 days.

[0059] The fermentation medium may be composed of the following components (the solvent is water): glucose 100 g / L, NH 4 H 2 PO 4 0.87g / L, (NH 4 ) 2 HPO 4 2.63g / L, MgSO 4 7H 2 O 0.18g / L, betaine-HCl 0.15g / L, FeCl 3 6H 2 O 1.5μg / L, CoCl 2 6H 2 O 0.1μg / L, CuCl 2 ·2H 2 O 0.1μg / L, ZnCl 2 0.1μg / L, Na 2 MoO 4 ·2H 2 O 0.1 μg / L, MnCl 2 ·4H 2 O 0.2 μg / L, H 3BO 3 0.05μg / L.

[0060] Any of the above-mentioned lactic acids include L-lactic acid and D-lactic acid.

[0061] In order to improve the lactic acid synthesis ability of lactic acid engineering strains, the present invention provides a PfkA mutant, and experimentally confirms that the PfkA mutant can significantly improve the lactic acid synthesis ability in different lactic acid-producing Escherichia coli engineering bacteria. Therefore, in practical applications, the lactic acid production capacity and biomass of lactic acid engineering strains can be improved by introducing pfkA gene point mutations into the lactic acid engineering strain genome or by introducing pfkA mutant genes into the lactic acid engineering strains. The present invention is of great significance to the construction of lactic acid production strains and efficient fermentation production of lactic acid. DETAILED DESCRIPTION

[0062] 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.

[0063] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the 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 channels.

[0064] The strains involved in the following examples and their related characteristics and sources are detailed in Table 1.

[0065] Table 1. Strains and their related characteristics and sources

[0066]

[0067]

[0068] The primers and their sequences involved in the following examples are shown in Table 2.

[0069] Table 2. Primers and their sequences

[0070]

[0071] The pXZ-CS plasmid involved in the following examples is described in the document “Tan, et al., Appl Environ Microbiol, 2013, 79: 4838-4844”.

[0072] The pKD46 plasmid used in the following examples is described in the document "Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97: 6640-6645", and is a product of the Yale University CGSC Escherichia coli Collection (CGSC#7739).

[0073] Example 1: Obtaining PfkA mutant and its encoding gene

[0074] Escherichia coli is one of the most important model strains in metabolic engineering, with advantages such as clear genetic background, convenient genetic manipulation tools, wide substrate utilization, fast growth and easy cultivation. At present, the industrial production of compounds such as amino acids, organic acids and vitamins has been achieved using modified Escherichia coli engineering bacteria.

[0075] The inventors of the present application screened out a batch of recombinant strains that can efficiently produce lactic acid through strain domestication, and by sequencing the genomes of the starting strains and these recombinant strains respectively, they found that the pfkA gene encoding 6-phosphofructokinase located at position EcolC_4102 on the chromosome DNA in the genomes of some recombinant strains had a point mutation, resulting in a pfkA mutant gene, which was named pfkA* gene.

[0076] The pfkA* gene is a gene obtained by mutating the base C at position 247 in the nucleotide sequence of the wild-type pfkA gene (sequence 1) to base T, and its nucleotide sequence is shown in sequence 3 in the sequence listing.

[0077] The amino acid sequence of the protein encoded by the pfkA* gene (denoted as PfkA mutant) is shown in Sequence 4 in the sequence table. The PfkA mutant is a protein obtained by replacing the arginine (Arginine, R) at position 83 in the amino acid sequence of the wild-type 6-phosphofructokinase PfkA (Sequence 3) with cysteine ​​(Cysteine, C).

[0078] Example 2: Introduction of pfkA point mutation in L-lactic acid engineered bacteria

[0079] In order to verify that the production capacity and biomass of the lactic acid engineering strain can be significantly improved by introducing the point mutation found in Example 1 into the genome of the lactic acid engineering strain, the inventors of the present application started with the Escherichia coli Slac006 strain (the strain is an engineering bacterium that can produce L-lactic acid under anaerobic conditions by fermentation obtained by the inventors of the present application through metabolic engineering and genetic modification in the early stage), and replaced the wild-type pfkA gene in the genomic DNA with the mutant pfkA* gene by a two-step homologous recombination method, that is, a point mutation was introduced at position 247 in the coding region of the pfkA gene in the Escherichia coli Slac006 strain, so that the base C was changed to the base T, and the arginine (Arginine, R) at position 83 in the amino acid sequence of the 6-phosphofructokinase encoded by it was replaced by cysteine ​​(Cysteine, C), and the engineering strain Slac019 was obtained. The specific steps are as follows:

[0080] 1. Obtaining DNA fragment I

[0081] Using pXZ-CS plasmid DNA as a template, PCR amplification was performed using primers pfkA-CS-up / pfkA-CS-down to amplify a DNA fragment I with a size of 2719 bp. Positions 1-50 of the fragment are the upper homology arm of pfkA, positions 51-2619 are the expression frame of cat-sacB, and positions 2670-2719 are the lower homology arm of pfkA, which are used for the first step of homologous recombination.

[0082] The PCR amplification system was: 10 μL of NewEngland Biolabs Phusion 5X buffer, 1 μL of dNTP (10 mM of 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), and 33.5 μL of distilled water, with a total volume of 50 μL.

[0083] PCR amplification conditions were 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, and extension at 72°C for 2 minutes (30 cycles); and extension at 72°C for 10 minutes (1 cycle).

[0084] 2. First step: homologous recombination

[0085] The above DNA fragment I was electroporated into Escherichia coli Slac006 for the first step of homologous recombination. The specific steps are as follows: the pKD46 plasmid was transformed into Escherichia coli Slac006 by electroporation to obtain Escherichia coli Slac006 carrying the pKD46 plasmid; then the DNA fragment I was transformed into Escherichia coli Slac006 carrying the pKD46 plasmid by electroporation. The electroporation method includes the following steps: first, prepare electroporation competent cells of Escherichia coli Slac006 carrying the pKD46 plasmid; place 50 μL of 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 cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameter is a voltage of 2.5 kV. After electroporation, quickly transfer 1 mL of LB culture medium to the electroporation cup, blow 5 times, and then transfer to a test tube, incubate at 75 rpm and 30°C for 2 hours.

[0086] Take 200 μL of bacterial solution and spread it on LB plate containing ampicillin (final concentration of 100 μg / mL) and chloramphenicol (final concentration of 34 μg / mL). After culturing overnight at 30°C, select a single colony for PCR verification. The primers used for PCR verification are pfkA-YZ85-up / pfkA-YZ894-down. The correct colony amplification product is a 4560 bp fragment. Select a correct single colony and name it Slac018.

[0087] 3. Obtaining DNA fragment II

[0088] Using E. coli ATCC8739 genomic DNA as a template, PCR amplification was performed using primers pfkAmut-up / pfkA-down to amplify a 1561 bp DNA fragment II. Positions 1-767 of the fragment were part of the pfkA gene containing the mutation site, and positions 768-1561 were the downstream homology arms of pfkA, which were used for the second step of homologous recombination. The PCR amplification conditions and system were the same as in step 1.

[0089] 4. Second step: homologous recombination

[0090] The above DNA fragment II was electroporated to the Slac018 strain for the second step of homologous recombination. The specific steps are as follows: the pKD46 plasmid was transformed into the Slac018 strain by electroporation to obtain the Slac018 strain carrying the pKD46 plasmid; then the DNA fragment II was transformed into the Slac018 strain carrying the pKD46 plasmid by electroporation. The electroporation method includes the following steps: first, prepare electroporation competent cells of Slac018 carrying the pKD46 plasmid; place 50 μL of competent 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 cup. Use the MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV. After electroporation, quickly transfer 1 mL of LB culture medium to the electroporation cup, blow 5 times, and then transfer to a test tube, incubate at 75 rpm and 30 ° C for 4 hours.

[0091] The bacterial liquid was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 mL medium in a 250 mL flask), and after culturing for 24 hours, it was streaked on LB solid medium without sodium chloride containing 6% sucrose. A single clone was picked for PCR amplification and sequencing using primers pfkA-YZ85-up / pfkA-YZ894-down. The correct colony amplification product was a 1941 bp fragment. A single colony with correct sequencing was selected and named as the Slac019 strain.

[0092] Example 3: Fermentation of L-lactic acid engineered bacteria

[0093] Test strains: Escherichia coli Slac006 and Slac019.

[0094] Fermentation involves the following steps:

[0095] (1) Seed culture: 100 mL of seed culture medium was placed in a 250 mL Erlenmeyer flask and sterilized at 115°C for 15 min. After cooling, the test strains were inoculated into the seed culture medium at an inoculum rate of 1% (V / V) and cultured at 37°C and 100 rpm for 12 hours to obtain seed solution for inoculation of the fermentation medium.

[0096] The seed culture medium consists of the following components (the solvent is water):

[0097] Macroelements: glucose 20g / L, NH 4 H 2 PO 4 0.87g / L, (NH 4 ) 2 HPO 4 2.63g / L, MgSO 47H 2 O0.18g / L, betaine-HCl 0.15g / L.

[0098] Trace elements: FeCl 3 6H 2 O 1.5μg / L, CoCl 2 6H 2 O 0.1μg / L, CuCl 2 ·2H 2 O 0.1μg / L, ZnCl 2 0.1μg / L, Na 2 MoO 4 ·2H 2 O 0.1 μg / L, MnCl 2 ·4H 2 O 0.2 μg / L, H 3 BO 3 0.05μg / L.

[0099] (2) Fermentation culture: The volume of the fermentation medium in a 500 mL anaerobic tank was 250 mL, and the seed solution was added according to the final concentration OD 550 =0.1 inoculation amount was inoculated into the fermentation medium, and fermented at 37°C, 150rpm for 2 days to obtain a fermentation liquid. The neutralizer was 5M ammonia water, and the pH of the fermentation tank was controlled at 7.0. The fermentation liquid was all the substances in the fermentation tank. No gas was passed during the culture process.

[0100] The only difference between the fermentation medium and the seed medium is that the glucose concentration is 100 g / L, and the other components and concentrations remain unchanged.

[0101] (3) Analysis method: Agilent-1200 high performance liquid chromatograph was used to measure the components in the fermentation broth after 48 hours. The concentrations of glucose and organic acids in the fermentation broth were determined using an Aminex HPX-87H organic acid analysis column from Biorad. The optical purity of lactic acid was analyzed using a SUMICHIRAL OA-6000 chiral column from Sumika Chemical Analysis Service, Japan.

[0102] The results are shown in Table 3. For the engineered bacteria producing L-lactic acid, the engineered strain Slac019 obtained by introducing the mutant pfkA* gene into the genomic DNA had an L-lactic acid yield that was 18% higher than that of the starting strain Slac006 after 48 hours of fermentation, and the optical purity was close to 100% (greater than 99.9%). This indicates that the PfkA mutant can significantly improve the production capacity and biomass of the L-lactic acid engineered strain.

[0103] Table 3. L-lactic acid engineering bacteria fermentation

[0104] strain L-lactic acid production (g / L) Change in L-lactic acid production (%) Slac006 60.6 0 Slac019 71.5 17.99

[0105] Example 4: Introduction of pfkA point mutation in D-lactic acid engineered bacteria

[0106] The inventors of the present application started with the Escherichia coli Dlac006 strain (this strain is an engineered bacterium that can produce D-lactic acid by fermentation under anaerobic conditions obtained by the inventors of the present application through metabolic engineering and genetic modification in the early stage), and used the same two-step homologous recombination method as in Example 2 to replace the wild-type pfkA gene in the genomic DNA with the mutant pfkA* gene, that is, a point mutation was introduced at position 247 in the coding region of the pfkA gene in the Escherichia coli Dlac006 strain, so that the base C was changed to the base T, and the arginine (Arginine, R) at position 83 in the amino acid sequence of the 6-phosphofructokinase encoded by it was replaced by cysteine ​​(C), and the engineered strain Slac021 was obtained.

[0107] Example 5: Fermentation of D-lactic acid engineered bacteria

[0108] Test strains: Escherichia coli Dlac006 and Slac021 strains.

[0109] The same fermentation method as in Example 3 was used to perform anaerobic fermentation on the test strain. The seed culture medium and the fermentation culture medium were prepared in the same manner as in Example 3. The same detection method as in Example 3 was used to analyze D-lactic acid in the fermentation broth.

[0110] The results are shown in Table 4. For the engineered bacteria producing D-lactic acid, the engineered strain Slac021 obtained by introducing the mutant pfkA* gene into the genomic DNA had a D-lactic acid yield increased by 29% after 2 days of fermentation compared with the Dlac006 strain, and the optical purity was close to 100% (greater than 99.9%). This shows that the PfkA mutant can significantly improve the production capacity and biomass of the D-lactic acid engineered strain.

[0111] Table 4. D-lactic acid engineering bacteria fermentation

[0112] strain D-lactic acid production (g / L) Change in D-lactic acid production (%) Dlac006 28.5 0 Slac021 34.6 21.1

[0113] From the above analysis, it can be seen that the PfkA mutant has a significant effect on improving the fermentation performance of both L-lactic acid and D-lactic acid Escherichia coli engineered bacteria.

[0114] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A PfkA mutant, wherein the PfkA mutant is a protein obtained by replacing the arginine at position 83 in the amino acid sequence of 6-phosphofructokinase PfkA with other amino acids; The 6-phosphofructokinase PfkA is a protein described in any one of the following (a1)-(a4): (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; (a2) a protein having the same function as that obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the amino acid residue at position 83 of the amino acid sequence shown in (a1); (a3) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the amino acid sequence shown in (a1) or (a2); (a4) A protein having 75% or more identity with (a1) or (a2) or (a3) ​​and having the same function.

2. The PfkA mutant according to claim 1, characterized in that: The PfkA mutant is a protein obtained by replacing the 83rd arginine in the amino acid sequence of 6-phosphofructokinase PfkA with cysteine.

3. A nucleic acid molecule encoding the PfkA mutant according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleic acid molecule is a DNA molecule as described in any one of the following (b1)-(b3): (b1) the nucleotide sequence is the DNA molecule shown in Sequence 3 in the sequence listing; (b2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in (b1) and encoding the PfkA mutant of claim 1 or 2; (b3) A DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under stringent conditions and encodes the PfkA mutant according to claim 1 or 2.

5. The biological material described in any one of the following (c1) to (c3): (c1) an expression cassette containing the nucleic acid molecule according to claim 3 or 4; (c2) a recombinant vector containing the nucleic acid molecule according to claim 3 or 4; (c3) A recombinant microorganism containing the nucleic acid molecule according to claim 3 or 4.

6. The use described in any one of the following (d1) to (d5): (d1) Use of the PfkA mutant according to claim 1 or 2 as 6-phosphofructokinase; (d2) Use of the nucleic acid molecule according to claim 3 or 4 or the biological material according to claim 5 in the preparation of 6-phosphofructokinase; (d3) Use of the PfkA mutant according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, or the biomaterial according to claim 5 in constructing engineered lactic acid bacteria; (d4) Use of the PfkA mutant according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, or the biomaterial according to claim 5 in lactic acid production; (d5) Use of the PfkA mutant according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, or the biomaterial according to claim 5 for increasing lactic acid production.

7. A recombinant bacterium capable of producing lactic acid, characterized in that: The 6-phosphofructokinase involved in the metabolic pathway for producing lactate includes the PfkA mutant according to claim 1 or 2.

8. The method for constructing the recombinant bacteria according to claim 7, comprising the following steps: increasing the content and / or activity of the PfkA mutant according to claim 1 or 2 in the lactic acid engineering bacteria.

9. Use of the recombinant bacterium according to claim 7 or the recombinant bacterium constructed according to the method according to claim 8 in producing lactic acid or increasing lactic acid yield.

10. A method for producing lactic acid, comprising the step of fermenting and culturing the recombinant bacteria according to claim 7 or the recombinant bacteria constructed according to the method according to claim 8.

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