Construction and application of recombinant kluyveromyces marxianus for producing L-lactic acid
By knocking out specific genes and integrating the L-lactic acid dehydrogenase gene, recombinant Max Kluvier yeast with high yield of L-lactic acid, solving the problem of inhibition of bacterial metabolism caused by lactic acid accumulation in lactic acid production, achieving high yield and high purity L-lactic acid production, which is suitable for industrial applications.
Patent Information
- Application Number
- CN202411892199.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-03-21
AI Technical Summary
Existing lactic acid-producing microorganisms, such as Lactobacillus, will cause inhibition of bacterial metabolism or even death when lactic acid accumulates to a certain concentration, resulting in a decrease in lactic acid production and an increase in production costs.
By knocking out the genes encoding pyruvate decarboxylase, cytochrome oxidase and pyruvate dehydrogenase in Max Kluveryces, and integrating 2 to 3 copies of the L-lactic acid dehydrogenase gene, a recombinant Max Kluveryces with high yield of L-lactic acid was constructed.
Under optimized conditions, L-lactic acid production reaches 130g/L or above, optical purity is greater than 99%, and has good genetic stability, which is suitable for industrial production.
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Figure CN119662435A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the construction and application of a recombinant Kluyveromyces marxianus strain producing L-lactic acid, belonging to the technical field of bioengineering. Background Art
[0002] The scientific name of L-lactic acid is α-hydroxypropionic acid, with a molecular weight of 90.08 and a molecular formula of CH3CHCOOH. Industrially produced lactic acid is usually a mixture, colorless and transparent or light yellow syrupy mucus. Anhydrous lactic acid is usually white crystals when the purity is high, with a melting point of 16.8℃, a boiling point of 122℃ (2Kpa), and a relative density of 1.249. It can be miscible with water in any proportion and is not easy to precipitate.
[0003] At present, the industrial production method of L-lactic acid is divided into microbial fermentation, chemical synthesis and enzymatic conversion. Nowadays, the industrial production of L-lactic acid is mostly produced by microbial fermentation, which can obtain L-lactic acid or D-lactic acid with higher stereospecificity and mixed racemates of two isomers through the selection of excellent strains and the optimization of optimal culture conditions to meet the production needs of polylactic acid. In addition, the fermentation method produces lactic acid, in addition to using monosaccharides such as glucose and lactose as raw materials, it can also use polysaccharides such as starch and cellulose as raw materials for fermentation production, and produces lactic acid with these renewable resources, which will not cause the greenhouse effect. Therefore, the microbial fermentation method of lactic acid has become an important method for industrial production of lactic acid due to its advantages such as a wide range of raw material sources, low production costs, and high safety.
[0004] Kluyveromyces marxianus is a yeast widely used in the field of industrial fermentation. It has significant advantages in food processing, biochemical engineering and biomedicine. As a yeast that has obtained food grade safety certification (GRAS, Generally Recognized as Safe), it has good metabolic safety and can be directly used in food production without complicated detoxification or purification treatment. This gives it a clear advantage in the production of food-grade fermentation products (such as lactic acid, ethanol and enzyme preparations).
[0005] Compared with traditional industrial microorganisms Saccharomyces cerevisiae and Escherichia coli, K. marxianus exhibits many outstanding characteristics. First, K. marxianus has high temperature tolerance, and its optimal fermentation temperature range is 30-45°C, which not only improves production efficiency, but also significantly reduces cooling energy consumption. The optimum temperature of Saccharomyces cerevisiae is usually around 30°C, and its growth and metabolic capacity decrease rapidly under high temperature conditions. At the same time, strict aseptic culture conditions are usually required during the fermentation of Escherichia coli, and harmful metabolites such as endotoxins may be produced during the fermentation process. These problems require additional treatment in food and pharmaceutical production. As a yeast, K. marxianus does not produce endotoxins. In addition, K. marxianus grows faster, with a doubling time of about 1h, while Saccharomyces cerevisiae is usually 1.5-2h, which gives K. marxianus an advantage in fermentation cycle and yield. In addition, although lactic acid bacteria (such as Lactobacillus and Streptococcus) are important microorganisms for traditional production of lactic acid, due to the low tolerance of lactic acid bacteria to lactic acid, when lactic acid accumulates to a certain concentration (usually 50-100g / L), bacterial metabolism is inhibited, and even cell death is caused. This will significantly reduce the final output and yield of lactic acid, and it is necessary to frequently neutralize the acidic environment, which increases the complexity and cost of the production process. Therefore, it is necessary to develop new high-yield L-lactic acid Kluyveromyces marxianus that meets food safety requirements. Summary of the invention
[0006] The invention provides a recombinant Kluyveromyces marxianus for producing L-lactic acid, wherein the gene PDC1 encoding pyruvate decarboxylase, the gene CYB2 encoding cytochrome oxidase, and the gene PDH1 encoding pyruvate dehydrogenase are knocked out, and 2 to 3 copies of the L-lactic dehydrogenase gene are integrated.
[0007] In one embodiment, the L-lactate dehydrogenase gene is integrated at the PDC1 site encoding pyruvate decarboxylase, the CYB2 site encoding cytochrome oxidase, and the PDH1 site encoding pyruvate dehydrogenase, respectively.
[0008] In one embodiment, the lactate dehydrogenase gene LDH is derived from Lactobacillus plantarum.
[0009] In one embodiment, the nucleotide sequence of the lactate dehydrogenase gene LDH is shown as SEQ ID NO.1.
[0010] The present invention also provides a method for constructing the above-mentioned recombinant bacteria, comprising:
[0011] (1) constructing an integration frame containing the L-lactate dehydrogenase gene; the integration frame includes a DNA fragment with the structure of "integration site upstream homology arm-GPD promoter-LDH gene-CYC1 terminator-integration site downstream homology arm";
[0012] (2) The integration frame of the L-lactate dehydrogenase gene constructed in step (1) is transformed into Kluyveromyces marxianus NBRC1777.
[0013] In one embodiment, the integration sites include a PDC1 site, a CYB2 site, and a PDH1 site.
[0014] In one embodiment, the method comprises the following steps:
[0015] (1) Clone the Lp-LDH gene fragment into the pY26 plasmid to obtain the recombinant plasmid pY26-01;
[0016] (2) Using plasmid pY26-01 as a template, the GPD promoter, CYC1 terminator, and Lp-LDH gene fragments were amplified, respectively, and connected with the upstream and downstream homology arms of PDC1 (PDC1-up / down), the upstream and downstream homology arms of CYB2 (CYB2-up / down), and the upstream and downstream homology arms of PDH1 (PDH1-up / down) to obtain the PDC1 knockout box, the CYB2 knockout box, and the PDH1 knockout box, respectively;
[0017] (3) The PDC1 knockout frame, CYB2 knockout frame and PDH1 knockout frame were transformed into K. marxianus NBRC1777 to construct a recombinant bacterium that produces L-lactic acid.
[0018] In one embodiment, the PDC1 knockout cassette, the CYB2 knockout cassette, and the PDH1 knockout cassette each carry an L-lactate dehydrogenase gene.
[0019] In one embodiment, the knockout box encoding the pyruvate decarboxylase gene PDC1 includes PDC1-up, PDC1-down and the lactate dehydrogenase gene Lp-LDH.
[0020] In one embodiment, the knockout box of the CYB2 gene encoding pyruvate decarboxylase includes CYB2-up, Lp-LDH, and CYB2-down.
[0021] In one embodiment, the knockout box of the gene encoding pyruvate dehydrogenase PDH1 includes PDH1-up, Lp-LDH, and PDH1-down.
[0022] In one embodiment, the knockout box of the gene encoding pyruvate dehydrogenase PDH1 includes PDH1-up, Lp-LDH, and PDH1-down.
[0023] In one embodiment, pY26 is used as an expression vector, and the lactate dehydrogenase gene is cloned into the vector.
[0024] In one embodiment, the recombinant bacteria uses K. marxianus NBRC1777 as a host.
[0025] In one embodiment, the GPD promoter sequence is shown as SEQ ID NO.2.
[0026] In one embodiment, the CYC1 terminator sequence is shown as SEQ ID NO.3.
[0027] In one embodiment, the nucleotide sequence encoding the pyruvate decarboxylase gene PDC1 is shown as SEQ ID NO.4.
[0028] In one embodiment, the nucleotide sequence encoding the cytochrome oxidase gene CYB2 is shown as SEQ ID NO.5.
[0029] In one embodiment, the nucleotide sequence encoding the cytochrome oxidase gene PDH1 is shown as SEQ ID NO.6.
[0030] The present invention also provides application of the recombinant bacteria in fermentation production of L-lactic acid.
[0031] In one embodiment, the fermentation uses glucose as a substrate.
[0032] In one embodiment, the fermentation is carried out at 30-37° C. for 60-72 hours.
[0033] In one embodiment, the fermentation conditions are 37°C, 200 rpm, and the initial OD of the strain is 600 is 1.0, fermentation 72h.
[0034] In one embodiment, the fermentation conditions are 37°C, 300 rpm, an inoculation amount of 10%, calcium carbonate is added during the fermentation process, the pH is maintained at 5.0-6.0, and the initial OD of the strain is 600 is 0.8-1.0, the ventilation volume is 1vvm, and the fermentation is 72h.
[0035] The present invention also provides the use of the recombinant bacteria in the fields of biology, pharmacy, food or chemical industry in producing products containing L-lactic acid or its derivatives.
[0036] Beneficial effects:
[0037] The present invention uses K.marxianus NBRC1777 as the starting strain, and obtains a Kluyveromyces marxianus strain with high L-lactic acid production through metabolic engineering transformation, which is named FMME-LA01. Under the optimized conditions of the shake flask, the L-lactic acid production can reach 130g / L or more, and the L-lactic acid production in a 5L fermentation tank can reach more than 140g / L, the optical purity is greater than 99%, and it has good genetic stability, which is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 The L-lactic acid production pathway in the engineered strain of Kluyveromyces marxianus.
[0039] Figure 2 This is the pY26 plasmid map.
[0040] Figure 3 This is the plasmid map of PUCC01.
[0041] Figure 4 The change of L-lactic acid content in the shake flask.
[0042] Figure 5 The change of L-lactic acid content in the 5-L fermenter. DETAILED DESCRIPTION
[0043] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
[0044] The materials and methods involved in the following embodiments are as follows:
[0045] Plasmid construction was carried out using classical molecular biological methods.
[0046] Table 1. Relevant characteristics of expression vectors
[0047]
[0048] Table 2 Primers used for PCR amplification
[0049]
[0050]
[0051] Seed culture medium: YPD medium, containing 20 g / L peptone, 10 g / L yeast powder, and 20 g / L glucose.
[0052] Fermentation medium: contains 5g / L ammonium sulfate, 3g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, 100g / L glucose; 15mg / L ethylenediaminetetraacetic acid, 4.5mg / L zinc sulfate heptahydrate, 0.3mg / L cobalt chloride hexahydrate, 1.0mg / L manganese chloride tetrahydrate, 0.3mg / L copper sulfate pentahydrate, 4.5mg / L calcium chloride dihydrate, 3.0mg / L ferrous sulfate heptahydrate, 0.4mg / L sodium molybdate dihydrate, 1.0mg / L boric acid, 0.1mg / L potassium iodide, filtered and sterilized; 0.05mg / L biotin, 1.0mg / L calcium pantothenate, 1.0mg / L niacin, 25mg / L inositol, 1.0mg / L vitamin B1, 1.0mg / L pyridoxine, 0.2mg / L p-aminobenzoic acid, 20mg / L uracil.
[0053] L-lactic acid detection method:
[0054] Sample pretreatment method: take the fermentation liquid and centrifuge it at 12000r / min for 10min to take the supernatant. After diluting it to an appropriate multiple, the sample is processed by membrane and tested by HPLC. The peak area is substituted into the linear regression equation, and the result multiplied by the dilution multiple is the L-lactic acid concentration in the fermentation liquid.
[0055] Determination of L-lactic acid: Prepare L-lactic acid solution with a concentration of 10g / L, dilute to 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0g / L respectively, and use high performance liquid chromatography (HPLC) for detection to obtain the peak time and the peak area corresponding to different concentrations of L-lactic acid. Use the concentration of L-lactic acid as the horizontal coordinate and the peak area as the vertical coordinate to draw a standard curve to obtain the linear regression equation. The regression coefficient of the linear regression equation should be above 0.99 before it can be used. The instrument is Agilent high performance liquid chromatograph, and the chromatographic column uses Aminex HPX-87H column; the mobile phase is 5mM dilute sulfuric acid; the flow rate is set to 0.6mL / min; the detector is a UV detector, the detection wavelength is 210nm, and the column temperature is 52℃.
[0056] Example 1: Construction of recombinant plasmid pY26-01
[0057] The plasmid pY26 was double-digested with BamHI and EcoRI endonucleases, and the fragment LDH shown in SEQ ID NO.1 was amplified by pY26-LDH-F / R primers, and connected to the double-digested plasmid pY26 by homologous recombination to obtain the connected recombinant plasmid pY26-01. The ligation product was transformed into E. coli JM109 competent cells by chemical transformation, screened on LB plates containing ampicillin antibiotics, and positive transformants were picked and verified by colony PCR. The verified correct transformants were cultured, and the plasmids were extracted and sequenced for verification. The correct plasmid was named pY26-01.
[0058] Example 2: Construction of recombinant plasmid PUCC01-PDC1
[0059] Using plasmid PUCC01 (nucleotide sequence as shown in SEQ ID NO.7) as a template, sgRNA with the targeted recognition gene PDC1 was reversely amplified by sgRNA-F and sgRNA-PDC1-R primers, and then the amplified product was digested with Dpn I digestive enzyme and connected to the PUCC01 plasmid by homologous recombination. The ligation product was transformed into E.coli JM109 competent cells by chemical transformation, screened on LB plates containing ampicillin antibiotics, and positive transformants were picked and verified by colony PCR. After plasmid extraction, sequencing verification was performed. After sequencing verification, the recombinant plasmid PUCC01-PDC1 targeting the recognition gene PDC1 was successfully constructed.
[0060] Example 3: Construction of recombinant plasmid PUCC01-CYB2
[0061] Using plasmid PUCC01 as a template, sgRNA with the targeted recognition gene CYB2 was reversely amplified by sgRNA-F and sgRNA-CYB2-R primers. After transformation, colony PCR was used for verification on a plate containing ampicillin antibiotics. After the plasmid was extracted, sequencing was performed for verification, and the correct plasmid was named PUCC01-CYB2.
[0062] Example 4: Construction of recombinant Kluyveromyces marxianus FMME LA02 producing lactic acid
[0063] The sequence SEQ ID NO.4 encoding the pyruvate decarboxylase gene PDC1 was retrieved from the database, and then the genome of K. marxianus NBRC1777 was used as a template to amplify 500 bp of PDC1-up and PDC1-down using primers PDC1-U-F1 / R1 and PDC1-D-F1 / R1, respectively. PDC1-up and PDC1-down were connected by fusion PCR to obtain the PDC1 knockout cassette.
[0064] The recombinant plasmid PUCC01-PDC1 with Cas9 protein constructed in Example 2 and the PDC1 knockout box were introduced into K.marxianus NBRC1777 by the method of lithium acetate chemical transformation, screened on YNB plates, picked positive transformants, boiled and verified using 20mM NaOH, and sequenced and verified after genome extraction. The fragment of PDC1 knocked out was 1000bp, and the fragment of PDC1 not knocked out was 2695bp. The positive transformant was transferred to YPD medium for subculture, and the thalline grew normally on the YNB plates added with 1.0mg / mL 5-fluoroorotic acid and 2g / L uracil, and the thalline did not grow on the YNB plates, indicating that the plasmid had been successfully eliminated. Knockout strain FMME LA01 was obtained.
[0065] Using pY26-01 plasmid as a template, the fragments shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 were amplified by PDC1-LDH-F / R primers to obtain the LDH integration frame. PDC1-up, LDH integration frame, and PDC1-down were connected by fusion PCR to obtain the PDC1 knockout frame. The recombinant plasmid PUCC01-PDC1 constructed in Example 2 and the PDC1 knockout frame were introduced into the strain FMME LA01 by chemical transformation to construct a ΔPDC1::Lp-LDH strain, named FMME LA02. Among them, the strain carries one copy of the L-lactate dehydrogenase gene.
[0066] Example 5: Construction of recombinant Kluyveromyces marxianus FMME LA04 and FMME LA05 producing lactic acid
[0067] The sequence SEQ ID NO.5 encoding the cytochrome oxidase gene CYB2 was retrieved from the database. Using the genome of K. marxianus NBRC1777 as a template, CYB2-up and CYB2-down were amplified by primers CYB2-U-F1 / R1 and CYB2-D-F1 / R1, respectively, for 500 bp. CYB2-up and CYB2-down were connected by fusion PCR to obtain a CYB2 knockout cassette. The recombinant plasmid PUCC01-CYB2 constructed in Example 3 and the CYB2 knockout cassette were introduced into FMME LA02, and sequenced to obtain FMME LA03.
[0068] Using pY26-01 plasmid as template, the fragments shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 were amplified by CYB2-LDH-F / R primers to obtain the LDH integration frame. CYB2-up, LDH integration frame, and CYB2-down were connected by fusion PCR to obtain the CYB2 knockout frame. The recombinant plasmid PUCC01-CYB2 and the CYB2 knockout frame were introduced into the strain FMME LA03 by chemical transformation to construct the ΔCYB2::Lp-LDH strain, named FMME LA04. Among them, the strain carries two copies of the L-lactate dehydrogenase gene.
[0069] The ΔPDH1::Lp-LDH strain was constructed according to the same method as above and named FMME LA05. The strain carries three copies of the L-lactate dehydrogenase gene.
[0070] Example 6: Shake flask fermentation of recombinant strains and result analysis
[0071] The strains FMME-LA01-LA05 constructed in Examples 4 and 5 were streaked on YNB plates and cultured at 37°C for 36 h. After a single colony grew out, a single colony was picked and inoculated into 50 mL YPD liquid medium. After culturing for 14 h, the seed culture medium was inoculated at an initial OD of 600 = 1 was inoculated into 100 mL of fermentation medium and fermented at 37°C and 200 rpm for 60 h. The fermentation broth was centrifuged and the supernatant was collected and the L-lactic acid content in the fermentation broth was determined by HPLC.
[0072] The results showed that L-lactic acid was not detected in the fermentation broth of K. marxianus NBRC1777. Figure 4 As shown, the L-lactic acid contents in the fermentation broths of the genetically engineered strains FMME LA01, FMME LA02, FMME LA03, FMME LA04 and FMME LA05 were 0 g / L, 72.55 g / L, 76.64 g / L, 121.14 g / L and 132.45 g / L, respectively. Therefore, FMME LA05 was selected as the starting strain for the subsequent fermentation tank.
[0073] Example 7: Fermentation in a fermenter to produce L-lactic acid
[0074] The fermentation performance of FMME LA05 strain was tested in a 5L fermenter. The cultured FMME LA05 bacterial solution was transferred to 50mL seed culture medium and cultured for 12h. The OD 600 = 1 was transferred to 500 mL of fermentation medium. After culturing for 12 h, 10% of the inoculum was inoculated into a 5 L fermentation tank with a liquid volume of 3 L. After inoculation, the OD 600=1, and use CaCO3 to adjust the pH to between 5 and 6. The temperature is 45°C, the ventilation volume is 1vvm, the rotation speed is 300rpm, and the fermentation period is 72h. Samples are taken every 12h to determine the contents of glucose, lactic acid, and pyruvic acid in the fermentation broth.
[0075] like Figure 5 As shown in the figure, the L-lactic acid production of strain FMME LA05 after 72h of fermentation reached 145.36 g / L. Although the L-lactic acid production of this strain was higher than that of other strains, it was only increased by 11.82% compared with FMME LA04. In addition, the maximum OD 600 The value of FMME LA04 was only 8.29, which was 32.1% lower than that of FMME LA04. The strain has good genetic stability and is suitable for industrial production.
[0076] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A recombinant Kluyveromyces marxianus for producing L-lactic acid, characterized in that: The gene PDC1 encoding pyruvate decarboxylase, the gene CYB2 encoding cytochrome oxidase, and the gene PDH1 encoding pyruvate dehydrogenase were knocked out, and 2 to 3 copies of the L-lactate dehydrogenase gene were integrated.
2. The recombinant Kluyveromyces marxianus according to claim 1, characterized in that The integration site of the L-lactate dehydrogenase gene is selected from: the PDC1 site encoding pyruvate decarboxylase, the CYB2 site encoding cytochrome oxidase, and the PDH1 site encoding pyruvate dehydrogenase.
3. The recombinant Kluyveromyces marxianus according to claim 1 or 2, characterized in that The nucleotide sequence of the lactate dehydrogenase gene LDH is shown in SEQ ID NO.
1.
4. The recombinant Kluyveromyces marxianus according to any one of claims 1 to 3, characterized in that Kluyveromyces marxianus NBRC1777 was used as the host.
5. The recombinant Kluyveromyces marxianus according to any one of claims 1 to 4, characterized in that The nucleotide sequence encoding the pyruvate decarboxylase gene PDC1 is shown in SEQ ID NO.4, the nucleotide sequence encoding the cytochrome oxidase gene CYB2 is shown in SEQ ID NO.5, and the nucleotide sequence encoding the cytochrome oxidase gene PDH1 is shown in SEQ ID NO.
6.
6. A method for constructing the recombinant Kluyveromyces marxianus according to claim 4, characterized in that: The following steps are involved: (1) constructing an integration frame containing the L-lactate dehydrogenase gene; the integration frame includes a DNA fragment with the structure of "integration site upstream homology arm-GPD promoter-LDH gene-CYC1 terminator-integration site downstream homology arm"; (2) The integration frame of the L-lactate dehydrogenase gene constructed in step (1) is transformed into Kluyveromyces marxianus NBRC1777.
7. A method for increasing the L-lactic acid production of Kluyveromyces marxianus, characterized in that: The improvement described in (1) and / or (2) is performed on Kluyveromyces marxianus: (1) Knockout the gene PDC1 encoding pyruvate decarboxylase, the gene CYB2 encoding cytochrome oxidase, and the gene PDH1 encoding pyruvate dehydrogenase; (2) Integration of 2 to 3 copies of the L-lactate dehydrogenase gene.
8. A method for preparing L-lactic acid, characterized in that: The recombinant Kluyveromyces marxianus according to any one of claims 1 to 5 is fermented in a culture medium, and L-lactic acid in the fermentation liquid is collected.
9. The use according to claim 8, characterized in that: The fermentation uses glucose as substrate.
10. Use of the recombinant Kluyveromyces marxianus according to any one of claims 1 to 5 in producing products containing L-lactic acid or its derivatives in the fields of biology, medicine, food or chemical industry.