Ammonium salt-tolerant l-lactic acid engineering strain and construction method and application thereof
By integrating the ldhL gene and knocking out the ldhA gene in Escherichia coli, and combining this with ammonia domestication, an L-lactic acid engineered strain tolerant to ammonium salts was constructed. This solved the problem of poor tolerance of lactic acid engineered strains to ammonium salts, and enabled efficient and environmentally friendly lactic acid production, meeting industrial needs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
- Filing Date
- 2026-04-22
- Publication Date
- 2026-05-29
AI Technical Summary
Lactic acid engineered strains have poor tolerance to ammonium salts, resulting in unsatisfactory fermentation indicators during the ammonium salt method of lactic acid production, making it difficult to meet industrialization requirements.
An L-lactic acid engineered strain was constructed through adaptive domestication and genetic modification. The specific steps included integrating the ldhL gene driven by the M1-93 promoter and knocking out the ldhA gene into Escherichia coli, performing homologous recombination using CPEC technology, and then acclimatizing and culturing in ammonia water to gradually improve its tolerance to ammonium salts.
It significantly improved the strain's tolerance to ammonium salts, kept the fermentation broth homogeneous and clear during fermentation, reduced stirring energy consumption, produced no gypsum solid waste, and achieved an L-lactic acid yield of 72.96 g/L after 24 hours of fermentation, increasing biomass by 34% and significantly improving production efficiency.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of industrial microbiology technology, specifically relating to an ammonium salt-tolerant L-lactic acid engineered strain, its construction method, and its application. Background Technology
[0002] Lactic acid, as an important organic acid, is widely used in the food, chemical, and pharmaceutical industries. As a monomer, lactic acid can be used to produce polylactic acid (PLA), a biodegradable material (poly-L-lactic acid or poly-D-lactic acid). PLA is completely degradable in nature, aligning with the concept of green and sustainable development, and market demand is increasing year by year. With the growth of the PLA market, the market potential of lactic acid is also increasing annually.
[0003] Traditional lactic acid fermentation requires pH adjustment using neutralizing agents, commonly calcium hydroxide or calcium carbonate, which produce calcium lactate after fermentation. Due to the low solubility of calcium lactate, crystals easily precipitate during fermentation, making stirring difficult and increasing fermentation energy consumption. In subsequent lactic acid separation and extraction processes, sulfuric acid is added for acidolysis, generating calcium sulfate dihydrate (gypsum) solid waste (Komesu, A., Maciel, MRW, & Maciel Filho, R. Separation and Purification Technologies for Lactic Acid - A Brief Review. BioResources, 2017, 12(3): 6885-6901). Producing 1 ton of lactic acid generates 0.96 tons of gypsum solid waste, causing significant environmental harm. Compared with the calcium salt method, the ammonium salt method (using ammonia water as a neutralizing agent during fermentation) has significant advantages: (1) The product ammonium lactate has high solubility, and the fermentation liquid always maintains a clear homogeneous system, which is beneficial to mass and heat transfer and can greatly reduce the energy consumption of stirring; (2) Ammonium sulfate can be obtained during downstream extraction. This product can be used as fertilizer or directly as a raw material for amino acid production, with no gypsum solid waste discharge.
[0004] Although the ammonium salt method for producing lactic acid has obvious advantages, the lactic acid engineered strains have poor tolerance to ammonium salts, resulting in unsatisfactory fermentation indicators during the production of lactic acid using the ammonium salt method, which makes it difficult to meet the requirements for industrialization.
[0005] Therefore, improving the ammonium salt tolerance of lactic acid engineered strains is of great significance for the industrial production of lactic acid. Summary of the Invention
[0006] To address the problems existing in the prior art, this invention provides an ammonium salt-tolerant L-lactic acid engineered strain, its construction method, and its application. Through adaptive domestication and by using ammonia water as a neutralizing agent, the tolerance to ammonium salts is gradually improved.
[0007] On one hand, the present invention provides a method for constructing L-lactic acid engineered strains, comprising the following steps:
[0008] Starting with Escherichia coli, ldhL, driven by the M1-93 promoter, was integrated into the frdABCD gene locus. Gene knockout of the ldhA gene yielded a recombinant E. coli strain.
[0009] Specifically, the frdABCD gene locus is sequence 4214758-4218069 of GenBank No. NC_010468.1, and the ldhA gene locus is sequence 2508048-2509037 of GenBank No. NC_010468.1.
[0010] Specifically, the starting bacterium is *Escherichia coli* QJL008; the integration involves integrating ldhL containing the M1-93 promoter. The gene, bleomycin resistance gene, and upstream and downstream homologous arm sequences of the knockout gene were integrated by homologous recombination after CPEC technology to assemble the fragment. The nucleotide sequence of the assembled fragment is shown in SEQ ID NO.11. The knockout was achieved by one-step homologous recombination.
[0011] Specifically, the homologous recombination uses pKD46 plasmid to provide the recombinase.
[0012] Furthermore, it also includes;
[0013] The recombinant Escherichia coli strain was subjected to ammonium salt adaptation to obtain the L-lactic acid engineered strain described above.
[0014] Specifically, the adaptive acclimatization uses ammonia as a neutralizing agent, the pH is controlled at 7.0, and the acclimatization is carried out continuously for at least 50 generations.
[0015] Specifically, the acclimatization culture medium contains glucose, xylose, NH4H2PO4, (NH4)2HPO4, MgSO4·7H2O, betaine-HCl, KCl, FeSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, ZnSO4·7H2O, Na2MoO4·2H2O, MnCl2·4H2O, and EDTA.
[0016] On the one hand, the present invention provides an L-lactic acid engineered strain prepared using the construction method described above.
[0017] Specifically, the L-lactic acid engineered strain is Escherichia coli CC085, with the accession number CGMCCNo.38146.
[0018] On the one hand, the present invention provides the application of the L-lactic acid engineered strain in the fermentation production of L-lactic acid ammonium.
[0019] On the one hand, the present invention provides the application of the L-lactic acid engineered strain in the production of polylactic acid raw materials.
[0020] On the other hand, the present invention provides a method for producing L-lactic acid, wherein the L-lactic acid engineered strain is used for anaerobic fermentation to produce L-lactic acid, preferably, the pH is adjusted to 7.0 using ammonia water as a neutralizing agent during the fermentation process.
[0021] Compared with existing technologies, the present invention has the following beneficial effects: the CC085 strain constructed by the present invention has significantly improved ammonium salt tolerance, is adapted to the ammonium salt fermentation process neutralized by ammonia water, and overcomes the ammonium salt sensitivity defect of the original strains; its fermentation process uses ammonia water as a neutralizing agent, the fermentation broth remains homogeneous and clear, reduces stirring energy consumption, and produces no gypsum solid waste, making it green and environmentally friendly; the L-lactic acid yield reaches 72.96 g / L after 24 hours of fermentation, which is 3.68 times higher than the starting strain FY103, the biomass is increased by 34%, and the production efficiency is greatly improved; and downstream, ammonium sulfate can be produced as a by-product, which can be used as fertilizer or amino acid raw material, improving the economic efficiency of the process and meeting the needs of industrial production. Attached Figure Description
[0022] Figure 1 This is to verify ldhL Electrophoresis diagram of integration into the frdABCD site, where each lane represents M: marker, 1: FY103b integrated strain, and 2: control ATCC 8739 strain.
[0023] Figure 2 This is a bar chart showing the ammonium salt tolerance analysis of the FY103 engineered strain.
[0024] Figure 3 This is a schematic diagram of the engineered strain CC085 obtained by domestication from the FY103 engineered strain.
[0025] Figure 4 This is a bar chart showing the ammonium salt tolerance analysis of engineered strain FY103 and domesticated engineered strain CC085.
[0026] Instructions for the preservation of biological materials:
[0027] Escherichia coli CC085 was deposited on March 25, 2026, at the China General Microbiological Culture Collection Center (CGMCC, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 38146, and classified as Escherichia coli. Detailed Implementation
[0028] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.
[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0031] The QJL008 strain in the following examples is described in: Qiao J et al., Experimentalevolution reveals an effective avenue for d-lactic acid production from glucose-xylose mixtures via enhanced Glk activity and a cAMP-independent CRPmutation. Biotechnology Bioengineering, 2024 Nov, 121(11), 3514-3526. This biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and shall not be used for any other purpose.
[0032] The Slac007 strain in the following examples is described in patent: CN112852693A (publication date: 2021.05.28). The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.
[0033] The pORF1 plasmid used in the following examples is a bleomycin-containing anti-plasmid. pORF1 is described in: Zheng F, Kawabe Y, Murakami M, Takahashi M, Nishihata K, Yoshida S, Ito A, Kamihira M. LINE-1 vectors mediate recombinant antibody gene transfer by retrotransposition in Chinese hamster ovary cells. Biotechnol J. 2021 Jul;16(7):e2000620. doi: 10.1002 / biot.202000620. Epub 2021 May 20. PMID: 33938150. This biological material is available to the public from Addgene #206025. This biological material is only for repeating the experiments of this invention and should not be used for other purposes. The enzymes in this invention are all obtained by prokaryotic expression according to genetic engineering methods.
[0034] The pKD46 plasmid used in the following examples is a recombinase-containing plasmid. pKD46 is described in: Datsenko, KA, Wanner, BL, 2000. One-step inactivation of chromosomal genes in Escherichiacoli K-12 using PCR products. Proc. Nat. Acad. Sci. USA 97, 6640-6645. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.
[0035] The promoters M1-93 used in the following examples are described in: Lu, J., Tang, JL, Liu, Y., Zhu, XN, Zhang, TC, Zhang, XL (2012). Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization. Appl Microbiol Biotechnol, 93(6), 2455-2462. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of the present invention and shall not be used for any other purpose.
[0036] The pXZ-CS plasmid used in the following examples is described in: Tan, Z., Zhu, X., Chen, J., Li, Q., & Zhang, X. (2013). Activating phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in combination for improvement of succinate production. Applied and environmental microbiology, 79(16), 4838-4844. This biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and may not be used for any other purpose.
[0037] Table 1. Strains used in this invention
[0038] ;
[0039] Table 2. Primer information used in this invention
[0040] ;
[0041] Example 1: Construction of L-lactic acid producing strains
[0042] Starting with recombinant Escherichia coli QJL008, ldhL was synthesized using a one-step method. The gene (its nucleotide sequence is shown in SEQ ID NO. 11) was integrated into the knockout frdABCD gene site (its nucleotide sequence is positions 4214758-4218069 of the sequence shown in GenBank No. NC_010468.1; the nucleotide sequence containing the frdA gene is positions 4214758-4218069 of the sequence shown in GenBank No. NC_010468.1, and the nucleotide sequence containing the frdB gene is GenBank No. NC_010468.1). The sequence shown in NC_010468.1 was used to obtain recombinant E. coli FY103b (positions 4216559-4217293 of which contain the frdC gene nucleotide sequence; positions 4217304-4217699 of which contain the frdD gene nucleotide sequence; and positions 4217710-4218069 of which contain the frdD gene nucleotide sequence). The specific experimental steps are as follows:
[0043] (1) Using the genome of Slac007 strain (CN112852693A, publication date: 2021.05.28, biological accession number: CGMCCNo.19459) as a template, PCR amplification was performed using primers frd-up / ldhL-R-CPEC (SEQ ID NO.1-SEQ ID NO.2). The product size was 1497bp, named fragment A. This fragment contains the upstream homologous arm of the frd knockout gene and ldhL. Encoding genes and the M1-93 promoter. Using pORF1 plasmid as a template, PCR amplification was performed using primers BleR-up-CPEC / BleR-down-CPEC (SEQ ID NO.3-SEQ ID NO.4), yielding a 513 bp product named fragment B, which contains the bleomycin resistance gene BleR. Using the genome of Slac007 strain as a template, PCR amplification was performed using primers frd-F1-CPEC / frd-down (SEQ ID NO.5-SEQ ID NO.6), yielding a 417 bp product named fragment C, which contains the downstream homologous arm of the frd knockout gene.
[0044] (2) Fragments A, B and C were seamlessly connected sequentially in the order of A, B and C using CPEC technology (Quan J, Tian J, Ho PL. Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways[J]. PlosOne, 2009, 4(7):e6441.DOI:10.1371 / journal.pone.0006441.) to obtain a fragment of size 2383bp (as shown in SEQ ID NO.11).
[0045] (3) Preparation of electrocompetent cells of QJL008: (i) Pick single clones from LB plates, inoculate into 30 mL of LB liquid medium, and culture at 37 °C and 250 r / min until OD. 550Approximately 0.5 μL, incubate on ice for 10 min. (ii) Transfer to a 50 mL sterile centrifuge tube, centrifuge at 5000 rpm for 5 min at 4 °C; (iii) Discard the supernatant, resuspend the precipitate in 20 mL sterile water, centrifuge at 5000 rpm for 5 min at 4 °C; (iv) Discard the supernatant, resuspend the precipitate in 15 mL sterile water, centrifuge at 5000 rpm for 5 min at 4 °C; (v) Discard the supernatant, resuspend the precipitate in 10 mL 10% glycerol, centrifuge at 5000 rpm for 5 min at 4 °C; (vi) After discarding the supernatant, resuspend the bacterial cells in the residual liquid, and transfer 50 µL to a new 1.5 mL centrifuge tube; it can be used directly for electroporation or stored in a -80 °C refrigerator. The pKD46 plasmid containing recombinase was transformed and integrated into the electroporation competent cells of QJL008, and grown on ampicillin plates to obtain the intermediate strain QJL008pKD46.
[0046] (4) Preparation of electrocompetent cells of QJL008pDK46: (i) Pick single clones from LB plates containing 50 µg / ml ampicillin antibiotic, inoculate into 30 mL LB liquid medium, and culture at 37 °C and 250 r / min containing 2.5% L+ arabinose until OD. 550 Approximately 0.5 μL, incubate on ice for 10 min. (ii) Transfer to a 50 mL sterile centrifuge tube, centrifuge at 5000 rpm for 5 min at 4 °C; (iii) Discard the supernatant, resuspend the precipitate in 20 mL sterile water, centrifuge at 5000 rpm for 5 min at 4 °C; (iv) Discard the supernatant, resuspend the precipitate in 15 mL sterile water, centrifuge at 5000 rpm for 5 min at 4 °C; (v) Discard the supernatant, resuspend the precipitate in 10 mL 10% glycerol, centrifuge at 5000 rpm for 5 min at 4 °C; (vi) After discarding the supernatant, resuspend the bacterial cells in the residual liquid, and transfer 50 µL to a new 1.5 mL centrifuge tube; it can be used directly for electroporation or stored in a -80 °C refrigerator.
[0047] (5) The 2383bp fragment assembled in (2) (as shown in SEQ ID NO.11) was electroporated into competent cells electroporated with QJL008pDK46, plated on LB plates containing bleomycin (50ug / ml), and the resulting clone was verified and sequenced using frdB-up / frdD-down. The size was 2383bp (results are shown in SEQ ID NO.11). Figure 1 The clone that was verified to be correct was named FY103b.
[0048] Starting with recombinant E. coli FY103b, the ldhA gene (its nucleotide sequence is positions 2508048-2509037 of the sequence shown in GenBank number NC_010468.1) was knocked out in one step (the knockout portion is positions 2508074-2508996 of the sequence). The specific experimental steps are as follows:
[0049] Using pXZ-CS plasmid DNA as a template, a 2719bp DNA fragment (containing a 50bp upstream homologous arm of the ldhA gene, a 2619bp cat-sacB expression cassette, and a 50bp downstream homologous arm of the ldhA gene, where cat is the chloramphenicol gene and sacB is the sucrose lethal gene) was amplified using primers ldhA-cat-up / ldhA-sacB-down (SEQ ID NO.7-SEQ ID NO.8). This fragment was used for one-step homologous recombination to integrate the resistance gene and achieve insertional inactivation of ldhA: First, the pKD46 plasmid was transformed into FY103b by electroporation to obtain recombinant E. coli FY103bpKD46 containing pKD46; then, the DNA fragment was electroporated into FY103bpKD46. The competent cells were prepared and the electroporation conditions were the same as those described in Example 1 for ldhL. Gene integration method: 200 μl of bacterial culture was plated on LB agar plates containing chloramphenicol (final concentration 34 μg / ml), incubated overnight at 37°C, and single colonies were selected for PCR verification. The primers were cat-up / XZ-ldhA-down (SEQ ID NO.9-SEQ ID NO.10), 844 bp in size. The correct clone was named FY103.
[0050] Example 2: Fermentation of recombinant Escherichia coli FY103
[0051] Seed culture medium: NaCl: 10 g / L, Tryptone: 10 g / L, yeast extract: 5 g / L, solvent: water.
[0052] The fermentation medium consists of the following components:
[0053] Macroelements: Glucose 50g / L, Xylose 50g / L, NH4H2PO4 0.1756g / L, (NH4)2HPO4 0.526g / L, MgSO4·7H2O 0.37g / L, Betaine-HCl 0.15361g / L, KCl 0.1875g / L.
[0054] Trace elements: FeSO4·7H2O 0.00768g / L, CoCl2·6H2O 0.00129g / L, CuSO4·5H2O 0.0011727g / L, ZnSO4·7H2O 0.0153g / L, Na2MoO4·2H2O 0.00084g / L, MnCl2·4H2O 0.00075g / L, EDTA 0.02865g / L.
[0055] Anaerobic fermentation of recombinant Escherichia coli FY103 includes the following steps:
[0056] (1) Seed culture: 30 mL of seed culture medium was prepared in a 250 mL Erlenmeyer flask and sterilized at 115 °C for 15 min. After cooling, single clones of recombinant Escherichia coli FY103 were picked from the plate and inoculated into the seed culture medium. The culture was carried out at 37 °C and 250 rpm for 12 hours to obtain the seed liquid, which was used for inoculation of the fermentation medium.
[0057] (2) Fermentation culture: The fermentation medium volume in the 500mL anaerobic tank is 250mL. The seed culture from step (1) is added according to the final concentration OD. 550 Inoculate the fermentation medium with an inoculum size of 0.1 nm, ferment at 37°C for 1 day to obtain the fermentation broth.
[0058] To maintain the pH of the fermenter at 7.0, a neutralizing agent of 20% Ca(OH)₂ or 7.8M ammonia solution can be used. When using Ca(OH)₂ as the neutralizing agent, the rotation speed should be 350 rpm; when using ammonia solution, the rotation speed should be 150 rpm. The fermentation broth consists of all substances within the fermenter. No gas is introduced during the cultivation process.
[0059] Analytical methods: Components in the fermentation broth after 96 hours were determined using an Agilent-1200 high-performance liquid chromatograph. The concentrations of glucose, xylose, and organic acids in the fermentation broth were determined using a Biorad Aminex HPX-87H organic acid analytical column. The optical purity of lactic acid was analyzed using a Sumika Chemical Analysis Service (Japan) SUMICHIRALOA-6000 chiral column.
[0060] Glucose, xylose, and lactic acid standards were purchased from Merck Life Sciences, catalog numbers 50-99-7, 58-86-6, and 50-21-5, respectively. Specific detection procedures followed the methods described in the literature (Zhu X, Tan Z, Xu H, et al. Metabolicevolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli[J]. Metabolic Engineering, 2014, 24:87-96.DOI:10.1016 / j.ymben.2014.05.003.). The lactic acid production of recombinant Escherichia coli FY103 fermentation in different neutralizing agents is as follows: Figure 2 As shown, when Ca(OH)2 is used as a neutralizing agent, the 24h OD 550The OD value was 4.6, and the lactic acid yield was 25.8 g / L; while when ammonia was used as a neutralizing agent, the OD value after 24 hours was... 550 The concentration was 4.1, and the lactic acid yield was 19.8 g / L; the yield was 23% lower, indicating that strain FY103 had low tolerance to ammonium salts.
[0061] Example 3: Adaptive domestication to obtain ammonium salt tolerant engineered strain CC085
[0062] To further improve the ammonium salt tolerance and L-lactic acid production capacity of engineered strains, we started with recombinant Escherichia coli FY103, using ammonia as a neutralizing agent, and improved cell growth, ammonium salt tolerance, and L-lactic acid production capacity through adaptive domestication.
[0063] The fermentation medium used for adaptation and domestication consists of the following components (water as the solvent):
[0064] Macroelements: Glucose 75g / L, Xylose 55g / L, NH4H2PO4 0.1756g / L, (NH4)2HPO4 0.526g / L, MgSO4·7H2O 0.37g / L, Betaine-HCl 0.15361g / L, KCl 0.1875g / L.
[0065] Trace elements: FeSO4·7H2O 0.00768g / L, CoCl2·6H2O 0.00129g / L, CuSO4·5H2O 0.0011727g / L, ZnSO4·7H2O 0.0153g / L, Na2MoO4·2H2O 0.00084g / L, MnCl2·4H2O 0.00075g / L, EDTA 0.02865g / L.
[0066] Adaptation and acclimatization process: A 500mL fermenter was used with 250mL of fermentation medium. 7.8M ammonia was used as a neutralizing agent, and the pH of the fermenter was maintained at 7.0. No gas was introduced during the cultivation process. Initial OD 550 The nm value was 0.1, and after 96 hours of growth, the fermentation broth was transferred to a new fermenter. 550 nm reached 0.05, 37℃, 150 rpm, and acclimatization was carried out for 85 generations according to this scheme (e.g. Figure 3 (As shown).
[0067] After 85 transfers, recombinant Escherichia coli CC085 was obtained. Recombinant Escherichia coli CC085 is Escherichia coli, which is deposited at the China General Microbiological Culture Collection Center (CGMCC, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 38146.
[0068] Example 4: Fermentation of CC085 engineered strain
[0069] Anaerobic fermentation of recombinant Escherichia coli CC085 includes the following steps:
[0070] (1) Seed culture: 30 mL of seed culture medium was prepared in a 250 mL Erlenmeyer flask and sterilized at 115 °C for 15 min. After cooling, single clones of recombinant Escherichia coli CC085 were picked from the plate and inoculated into the seed culture medium. The culture was carried out at 37 °C and 250 rpm for 12 hours to obtain the seed liquid, which was used for inoculation of the fermentation medium.
[0071] (2) Fermentation culture: The fermentation medium volume in the 500mL anaerobic tank is 250mL. The seed culture from step (1) is added according to the final concentration OD. 550 Inoculate the culture medium with an inoculum size of 0.1 nm, ferment at 37°C and 150 rpm for 1 day to obtain the fermentation broth.
[0072] The neutralizing agent was 7.8M ammonia water, maintaining the pH of the fermenter at 7.0. The fermentation broth consisted of all substances within the fermenter. No gas was introduced during the cultivation process.
[0073] See Example 2 for the seed culture medium and fermentation culture medium.
[0074] The fermentation broth of recombinant Escherichia coli CC085 was tested, and the results are as follows: Figure 4 As shown, when ammonia is used as a neutralizing agent, the OD24h value is... 550 The OD value was 6.25, and the lactic acid production was 72.96 g / L, compared to the starting strain FY103 (24h OD value). 550 The biomass increased by 34%, and the lactic acid yield increased by 3.68 times. These results indicate that the ammonium salt tolerance of the CC085 engineered strain was significantly enhanced.
[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A method for constructing an L-lactic acid engineered strain, characterized in that, Includes the following steps: Starting with Escherichia coli, ldhL, driven by the M1-93 promoter, was integrated into the frdABCD gene locus. Gene knockout of the ldhA gene yielded a recombinant E. coli strain.
2. The construction method according to claim 1, characterized in that, The frdABCD gene locus is sequence position 4214758-4218069 of GenBank No. NC_010468.1, and the ldhA gene locus is sequence position 2508048-2509037 of GenBank No. NC_010468.
1.
3. The construction method according to claim 1, characterized in that, The starting bacterium is Escherichia coli QJL008, and the integration is the integration of ldhL containing the M1-93 promoter. After assembling the gene, bleomycin resistance gene, and upstream and downstream homologous arm sequences of the knockout gene using CPEC technology, homologous recombination was performed to achieve integration. The nucleotide sequence of the assembled fragment is shown in SEQ ID NO.
11. The knockout was achieved using one-step homologous recombination.
4. The construction method according to claim 3, characterized in that, The homologous recombination was performed using pKD46 plasmid to provide the recombinase.
5. The construction method according to claim 1, characterized in that, Also includes: Recombinant Escherichia coli strains were subjected to ammonium salt adaptation to obtain L-lactic acid engineered strains with enhanced ammonium salt tolerance.
6. The construction method according to claim 5, characterized in that, The adaptive acclimatization was carried out using ammonia as a neutralizing agent, with the pH controlled at 7.0, and continued for at least 50 generations.
7. The construction method according to claim 5, characterized in that, The acclimatization culture medium contains glucose, xylose, NH4H2PO4, (NH4)2HPO4, MgSO4·7H2O, betaine-HCl, KCl, FeSO4·7H2O, CoCl2·6H2O, CuSO4·5H2O, ZnSO4·7H2O, Na2MoO4·2H2O, MnCl2·4H2O, and EDTA.
8. L-lactic acid engineered strains prepared using the construction method according to any one of claims 1-7.
9. The L-lactic acid engineered strain as described in claim 8, characterized in that, Its accession number is CGMCC No.38146.
10. The use of the L-lactic acid engineered strain according to claim 8 or 9 in the fermentation production of L-lactic acid ammonium or in the production of polylactic acid raw materials.
11. A method for producing L-lactic acid, characterized in that, The L-lactic acid engineered strain described in claim 8 or 9 is used for anaerobic fermentation to produce L-lactic acid.
12. The production method as described in claim 11, characterized in that, The pH was adjusted to 7.0 using ammonia as a neutralizing agent during the fermentation process.