Process for producing l-lactic acid monomer and production strain

Abstract

The application belongs to the technical field of microbial fermentation engineering, and particularly relates to a production method of L-lactic acid monomer and a production strain thereof. The application realizes dynamic reconfiguration of a metabolic pathway by precisely deleting an acetolactate synthase coding gene of a target lactic acid monomer high-yield strain and using a temperature-sensitive plasmid system, effectively realizes separation of a cell proliferation and a metabolic stage of target product synthesis, and significantly improves unit volume and unit time yield and purity of lactic acid monomer synthesis, thereby adapting to modern fermentation intelligent manufacturing and industrial demand of efficient preparation of a large amount of bio-based raw materials. Through metabolic regulation and innovative fermentation process of the strain, chemical purity and fermentation efficiency of L-lactic acid are significantly improved, and an effective solution is provided for industrial scale production of high-quality poly-L-lactic acid materials.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation engineering technology, specifically relating to a method for producing L-lactic acid monomer and its production strain. Background Technology

[0002] L-lactic acid is an important chiral carboxylic acid, widely used as a monomer raw material for poly-L-lactic acid (PLLA) in biodegradable materials, medical materials, and green packaging. Producing high-quality PLLA materials requires extremely high purity of the L-lactic acid monomer. Not only is high optical purity (≥99.5%) necessary to ensure the degree of polymerization, crystallinity, and thermal stability of the material, but high chemical purity (≥98%) is also required to guarantee the efficiency of the polymerization reaction and product quality. The purity of the L-lactic acid monomer is also a key factor determining the quality and production cost of lactide.

[0003] Currently, the industrial production of L-lactic acid monomers mainly employs microbial fermentation. In existing technologies, researchers have conducted extensive work to improve the yield and purity of L-lactic acid. For example: 1) Through metabolic engineering, deleting certain components from the acetic acid synthesis pathway in *E. coli*. pta and ackA 1) Genes were used to reduce the generation of the byproduct acetic acid, thereby increasing the total yield of L-lactic acid; 2) By optimizing the fermentation process and improving the culture conditions and feeding strategy, the yield and optical purity of L-lactic acid were significantly improved; 3) By using innovative separation and purification technologies, such as crystallization and membrane separation, the purity of L-lactic acid was further improved.

[0004] However, existing technologies for the efficient preparation of L-lactic acid monomers still face two key challenges: firstly, the chemical purity needs further improvement; and secondly, there is still room for improvement in fermentation efficiency. Previous studies, while screening and genetic improvement of fermentation strains, as well as separation and purification techniques during fermentation, have significantly reduced the content of common impurities in lactic acid monomers such as ethanol, acetic acid, formic acid, and succinic acid, in practical L-lactic acid fermentation production, we discovered a previously overlooked organic molecule—ketocarboxylic acid intermediates derived from the biosynthesis and degradation pathways of branched-chain amino acids—that competes with L-lactic acid dehydrogenase to generate hydroxycarboxylic acid impurities such as isopropyl malic acid and 3-methyl-2-hydroxybutyric acid. These impurities have similar chemical structures to L-lactic acid and are difficult to remove using conventional separation and purification methods, severely affecting the final chemical purity of the L-lactic acid monomer and the subsequent preparation and purification of lactide. More importantly, the branched-chain amino acid synthesis pathway and the lactate synthesis pathway compete for substrates, leading to an irrational distribution of metabolic flux. This not only affects the chemical purity of L-lactic acid but also significantly limits the synthesis rate and fermentation efficiency of L-lactic acid, thereby increasing production costs.

[0005] Therefore, how to solve the problem of L-lactic acid monomer chemical purity at the level of microbial metabolic network, while simultaneously improving L-lactic acid synthesis rate and fermentation efficiency, is a technical challenge that urgently needs to be addressed in this field. This invention aims to achieve high-purity and high-efficiency production of L-lactic acid without the addition of additional nutrients through ingenious strain design and precise temperature-controlled fermentation processes. Summary of the Invention

[0006] The purpose of this invention is to provide an efficient method and strain for producing high-purity L-lactic acid monomers, aiming to solve the problems of insufficient chemical purity and low fermentation efficiency of L-lactic acid monomers in existing technologies. This method involves precisely deleting the acetolactate synthase encoding gene in a high-yield strain of the target lactic acid monomer and dynamically reconstructing the metabolic pathway using a temperature-sensitive plasmid system. This not only effectively separates the metabolic stages of cell proliferation and target product synthesis but also significantly improves the yield (production intensity) and purity of lactic acid monomers per unit volume per unit time, meeting the industrial demands of modern intelligent fermentation manufacturing and the efficient preparation of large-scale bio-based raw materials. Through metabolic regulation of the strain and innovative fermentation technology, the chemical purity and fermentation efficiency of L-lactic acid are significantly improved, providing an effective solution for the industrial-scale production of high-quality poly-L-lactic acid materials.

[0007] To achieve the above objectives, the technical approach adopted by the present invention is as follows:

[0008] One of the technical solutions provided by the present invention is a lactate monomer production strain. The strain is a strain with lactate production capacity as the starting strain. After the expression of acetolactate synthase is deleted, the presence of the acetolactate synthase encoding gene is controlled by a temperature-sensitive replication plasmid, thereby controlling its functional expression. Furthermore, the acetolactate synthase encoding gene includes: ilvG / M, ilvI / H and ilvB / N ; The ilvG / M express ilvG and ilvM Genes; the stated ilvI / H represents ilvI and ilvH Genes; the stated ilvB / N express ilvB and ilvN Gene; in, ilvG The Gene ID in NCBI is: 2847699. ilvM The Gene ID is: 948279 ilvI The Gene ID is: 948793. ilvH The Gene ID is: 947267 ilvB The Gene ID is: 948182. ilvN The GeneID is: 948183; Furthermore, the methods for deleting acetolactate synthase expression include, but are not limited to: knocking out the acetolactate synthase encoding gene, inserting, substituting, or deleting bases in the acetolactate synthase encoding gene, etc., which can completely prevent the expression of the protein encoded by the gene, or even if it is expressed, it will not be active. Furthermore, control is achieved through temperature-sensitive replication plasmids. ilvG / M The expression; Going further, is to ilvG / M Genes are controlled by temperature-sensitive replication plasmids, thereby regulating their existence. ilvG / M The functional expression of acetolactate synthase is thus controlled. Furthermore, the wild-type low-copy thermosensitive plasmid pKD46, i.e., the smallest unit of low-copy and thermosensitive replication, is retained in the subsequent construction of recombinant expression plasmids. Furthermore, the backbone of the thermosensitive replication plasmid is the pRT gene fragment obtained by amplification using pKD46 plasmid as a template and pRT-F and pRT-R as primers. pRT-F: 5'-GTATGGACAGTTTTCCTTTGA -3'; pRT-R: 5'-GATGCAGGTGGCACTTTTCGGGGAA-3'; Furthermore, the strain capable of producing lactic acid includes: L-lactic acid monomer producing strain CGMCC No. 11060; Preferably, the lactic acid monomer producing strain is a CGMCC No. 11060 starting strain that produces lactic acid monomers from the genome. ilvG / M , ilvI / H and ilvB / N After knockout, the clone is transferred. ilvG / M Thermosensitive recombinant expression plasmid pRT- ilvG / M The obtained LLA619 strain; the recombinant expression plasmid pRT- ilvG / M It is by ilvG / M The gene was obtained by recombination and ligation of the pRT gene fragment.

[0009] The aforementioned lactic acid monomer-producing strain exhibits specific metabolic characteristics. Under low-temperature conditions (30-35℃), this strain can synthesize branched-chain amino acids, promoting cell proliferation. However, under high-temperature conditions (38-44℃), it ceases the synthesis of branched-chain amino acids, concentrating metabolic flux on the L-lactic acid synthesis pathway. This strain also exhibits the following temperature-dependent growth characteristics: at culture temperatures below or equal to 35℃, the strain can rapidly multiply; at culture temperatures above 37℃, the strain rapidly ceases proliferation.

[0010] The second technical solution provided by this invention is the application of the production strain described in the first technical solution in the production of lactic acid monomer; Furthermore, the method for producing lactic acid monomers by fermentation using the aforementioned production strain includes a cell proliferation stage carried out under low temperature (30-35℃) conditions in the early stage and a cell enrichment and lactic acid monomer fermentation production stage carried out under increased temperature (38-44℃, especially 40-42℃) conditions in the later stage. Furthermore, the method for producing lactic acid monomers using the aforementioned production strain through fermentation is as follows: (1) Cell proliferation stage: The production strain is inoculated into a carbon source medium at an inoculation rate of 5-10% (v / v) and cultured aerobically at 30-35℃ with dissolved oxygen controlled at 20%-80% until the cell density OD reaches 100%. 600 Reaching 2-8; Furthermore, the aerobic culture conditions are: rotation speed 200 r / min-800 r / min, ventilation 3 L / min-7 L / min; Furthermore, the carbon source includes glucose, sucrose, xylose, and glycerol; (2) Cell enrichment stage: Increase the culture temperature to 38-44℃ and continue aerobic culture until the cell density OD reaches 38-44℃. 600 Reaching 20-40; (3) Fermentation and acid production stage: Maintain the culture temperature at 38-44℃ and switch to anaerobic fermentation. During this period, continuously or in batches, add carbon source to maintain sufficient carbon source. Add pH neutralizer to maintain pH value at 6.8-7.2. When the concentration of lactic acid monomer no longer increases significantly or reaches the target concentration, terminate the fermentation process. Furthermore, the fermentation process is terminated when the concentration of L-lactic acid monomer is not less than 170 g / L; Furthermore, the anaerobic fermentation conditions are as follows: reduce the stirring speed to below 200 r / min, and simultaneously shut off the aeration.

[0011] Furthermore, in step (1), the temperature is preferably 35°C; Furthermore, in steps (2) and (3), the culture temperature is preferably increased to 40-42℃; Furthermore, in step (3), the pH value is adjusted to 6.8-7.2 by adding 25 wt.% calcium hydroxide suspension; the preferred pH value is 7.0. Furthermore, in step (3), 60 wt.% glucose solution is continuously or added in batches to maintain the glucose concentration at 5-40 g / L.

[0012] Using the above method, the fermentation intensity can reach 15.6 g / (L·h) or higher, and the chemical purity of the obtained L-lactic acid monomer is ≥98.5% and the optical purity is ≥99.7%.

[0013] The present invention also relates to the application of the production strain described in one of the technical solutions in the industrial-scale fermentation production of L-lactic acid monomer, wherein the L-lactic acid monomer is used to produce poly-L-lactic acid materials.

[0014] Beneficial effects: 1. This invention innovatively constructs a metabolically regulated L-lactic acid-producing strain, which ingeniously solves the metabolic conflict between the branched-chain amino acid synthesis pathway and the lactic acid synthesis pathway, and provides a novel metabolic flux regulation strategy. This strain can synthesize branched-chain amino acids to promote cell proliferation under low temperature (≤35℃) conditions, and stops synthesizing branched-chain amino acids under conditions of increased culture temperature (>37℃), thus eliminating the interference of ketocarboxylic acids on L-lactic acid synthesis. 2. Through a three-stage fermentation process, the spatiotemporal separation of cell proliferation and product synthesis is achieved, which significantly improves fermentation efficiency. The fermentation intensity can reach more than 15.6 g / (L·h), which is more than 35% higher than the existing technology. 3. This method effectively inhibits the accumulation and transformation of metabolic intermediates such as ketocarboxylic acids, reducing the generation of difficult-to-remove impurities such as isopropyl malic acid and 3-methyl-2-hydroxybutyric acid from the source. This application is the first to achieve a product in which L-lactic acid fermentation products are free of impurities such as isopropyl malic acid, 3-methyl-2-hydroxybutyric acid, and acetic acid, with a chemical purity of over 98.5% and an optical purity of over 99.7%. The elimination of L-lactic acid chemically similar compounds such as isopropyl malic acid and 3-methyl-2-hydroxybutyric acid greatly reduces the difficulty of separation and purification, which is beneficial for improving chemical purity and subsequent preparation and purification of lactide. Attached Figure Description

[0015] Figure 1 To knock out ilvG / M , ilvI / H and ilvB / N Electrophoresis patterns of PCR identification of strains after three gene sets in, Figure 1 a: Gene knockout ilvG / M PCR identification of electrophoretic patterns; Figure 1 b: Gene knockout ilvI / H PCR identification of electrophoretic patterns; Figure 1 c: Gene knockout ilvB / N PCR identification of electrophoretic patterns.

[0016] Figure 2 For recombinant plasmid pRT- ilvG / M Physical spectrum and enzyme digestion verification in, Figure 2 a: Recombinant plasmid pRT- ilvG / M Physical map; Figure 2 b: Recombinant plasmid pRT- ilvG / M Enzyme digestion verification via agarose gel electrophoresis; Lane M: DNA molecule size reference; Lane 1: pRT- ilvG / M A single 5.4 kb band was released after digestion with BamHI.

[0017] Figure 3 Temperature-dependent validation curves for recombinant strain LLA619.

[0018] Figure 4 The fermentation process curve of recombinant strain LLA619 in the upper tank is shown.

[0019] Figure 5 HPLC detection chromatograms were generated for the detection of relevant impurities in the fermentation broth. Detailed Implementation

[0020] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various improvements and modifications to the present invention without departing from the spirit and scope of the present invention.

[0021] Example 1. Construction of a dedicated microbial strain for L-lactic acid monomer production 1.1 Strains and Plasmids The starting strain used in this invention is Escherichia coli (E. coli). Escherichia coli CGMCC No. 11060 (published in ZL201580000781.7) is an L-lactic acid monomer producing bacterium. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 7, 2015.

[0022] The pKD46 plasmid (Datsenko, KA and Wanner, BL, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA, 2000, 97(12): 6640-6645) contains the λ phage Red recombinase system and is used as a gene knockout helper plasmid (Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000;97(12):6640-6645.). pSK- dif The GmR plasmid contains a gentamicin resistance gene cassette, which has flanking sections... dif Sequences were used as knockout markers (Liu Dawei, Niu Dandan, Zhang Liang, Shi Guiyang, Wang Zhengxiang. Construction of a series of plasmids to facilitate the isolation and molecular recombination of resistance genes [J]. Bulletin of Microbiology, 2007, 34(5): 0926-0928).

[0023] 1.2 Gene Knockout Primer Design According to GenBank login number ilvG : 2847699、 ilvM : 948279、 ilvI : 948793、 ilvH :947267、 ilvB : 948182 and ilvN Based on the sequence information of 948183, the following primers (Table 1) were designed for gene knockout and verification.

[0024] Table 1. Gene deletion primer sequences used in this invention

[0025] 1.3 Gene Knockout Procedure 1.3.1 Sequential knockout ilvG / M , ilvI / H and ilvB / N Gene It should be noted that the construction methods provided in the embodiments of the present invention are exemplary and not restrictive. Those skilled in the art can use any technical means to achieve the final gene editing purpose.

[0026] by ilvG / M Taking gene knockout as an example, the specific operation is as follows: (1) Primers ilvGM-KO-F and ilvGM-KO-R were used as templates to amplify and prepare the gene fragment to be deleted from Escherichia coli CGMCC No.11060. ilvG / M The obtained PCR product was cloned into plasmid pSKsym. Sma I restriction site, to obtain the corresponding recombinant plasmid pSK- ilvGM After that Eco The RV enzyme was used to remove a portion of the middle sequence of the gene to be deleted, and then cloned into... dif Gm fragment, to obtain recombinant plasmid pSK- ilvGM :: difGm .

[0027] (2) Primers ilvGM-KO-F and ilvGM-KO-R were used with pSK- ilvGM :: dif Using Gm plasmid as a template, DNA fragments containing gentamicin resistance gene cassettes were amplified by PCR.

[0028] PCR reaction system (50 μL): 5 μL 10×PCR buffer, 4 μL 2.5 mM dNTPs, 0.5 μL 10 mM forward primer, 0.5 μL 10 mM reverse primer, pSK- ilvGM::difGm Add 0.5 μL template DNA, 0.5 μL DNA polymerase, and ddH2O to a final volume of 50 μL.

[0029] PCR program: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 15 s, 58℃ annealing for 30 s, 72℃ extension for 2.5 min, for a total of 30 cycles; 72℃ final extension for 10 min.

[0030] (3) The PCR products were separated by agarose gel electrophoresis, the target DNA fragments were recovered by gel excision, and purified using a DNA recovery kit (Zhuangmeng Biotechnology).

[0031] (4) CGMCC No.11060 strain containing pKD46 plasmid was inoculated into LB medium containing 100 μg / mL ampicillin and 10 mM L-arabinose and cultured at 30℃ until OD600 was about 0.3-0.4 to express the Red recombinase system.

[0032] (5) Collect bacterial cells and prepare electroporation competent cells. The specific steps of electroporation are as follows: CGMCC No.11060 strain was inoculated into 50 mL LB medium and cultured overnight at 30°C. Take 1 mL of culture medium, centrifuge to collect bacterial cells, wash twice each with ice-cold ddH2O and 10% glycerol solution, and finally resuspend in 100 μL 10% glycerol solution.

[0033] Add 300-500 ng of the purified PCR product from step (3), mix well, and transfer to a pre-cooled 2 mm electroporation cuvette. Set the electroporator parameters to 1.8 kV and perform electroporation. Immediately add 900 μL of recovery solution, incubate at 30°C for 2 h, spread on LB agar plates containing 20 μg / mL gentamicin, and incubate at 30°C for 16-18 h. Pick single colonies for subsequent operations.

[0034] (6) Select candidate colonies and design identification primers ilvGM-check-F and ilvGM-check-R that cross the knockout region, and verify them by PCR. ilvG / M Gene knockout status (e.g.) Figure 1 (As shown).

[0035] (7) The verified strain was cultured overnight, diluted 100-fold, and inoculated into fresh LB medium. It was then cultured at 30°C until OD500. 600 Approximately 0.6 μg / mL gentamicin was then plated onto LB agar plates without antibiotics and incubated overnight at 30°C. Single colonies were picked and plated separately onto LB agar plates containing 20 μg / mL gentamicin and those without antibiotics, respectively, for comparative culture to confirm the removal of the antibiotic marker. This is because endogenous Xer / Red recombinases can recognize... dif The sequence is mediated and recombination is carried out, thereby enabling the automatic removal of resistance markers.

[0036] Knock them out one by one using the same method. ilvI / H and ilvB / N Genes (corresponding primers are shown in Table 1), ultimately yielding three gene groups in CGMCC No. 11060 ( ilvG / M, ilvI / H and ilvB / N The basic strain LLA69, which was knocked out in all cases (PCR verification as shown), was also eliminated. Figure 1 (As shown).

[0037] 1.4 Construction of Temperature-Regulated Recombinant Expression Plasmid 1.4.1 ilvG / M Cloning of genes and their promoters Design primers for amplification ilvG / M Genes and their natural promoters: ilvGM-clone-F: 5'-TTCAGGACGGGGAACTAACT-3' ilvGM-clone-R: 5'-GCGCAAAAGGAATATAAAAA-3' Using CGMCC No. 11060 genomic DNA as a template and ilvGM-clone-F / R as primers, PCR amplification was performed. The PCR reaction system and procedure were the same as above, except that the annealing temperature was adjusted to 58℃ and the extension time was extended to 3 min. The PCR products were separated by agarose gel electrophoresis, the target fragment was recovered by gel excision, and purified using a DNA recovery kit.

[0038] 1.4.2 Construction of Recombinant Plasmids Because pKD46 possesses DNA recombination capabilities, its plasmid DNA molecules lose their self-replication ability and undergo a certain degree of random chromosome integration when culture conditions are unsuitable (such as increased culture temperature). This delays the clearance of its DNA molecules by the host cell, resulting in low loss efficiency and leading to problems such as increased impurities and low yield in subsequent lactate production stages. Therefore, the existing pKD46 plasmid needs to be modified.

[0039] Design primers to amplify plasmid backbone fragments: pRT-F: 5'-GTATGGACAGTTTTCCTTTGA-3' pRT-R: 5'-GATGCAGGTGGCACTTTTCGGGGAA-3' (1) The DNA fragment of the temperature-sensitive replication minimum element in the backbone of the plasmid obtained by reverse amplification using the temperature-sensitive replication plasmid pKD46 as a template was named pRT fragment. After purification, it was combined with the amplified gene. ilvG / M Connect the fragments.

[0040] (2) Ligation reaction system (10 μL): Vector DNA (pRT fragment) 2-4 μL, insert fragment DNA 4-6 μL, 10× ligation buffer 1 μL, PEG4000 1 μL, T4 DNA ligase 1 μL. Ligate overnight at 16℃.

[0041] (3) The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 25 μg / mL ampicillin. The cells were incubated overnight at 30°C. Candidate colonies were picked, plasmid DNA was extracted, and the correct recombinant plasmid was confirmed by enzyme digestion and named pRT- ilvG / M (Physical diagrams and enzyme digestion verification, as shown) Figure 2 ).

[0042] 1.5 Construction of recombinant strain LLA619 The pRT-ilvG / M plasmid was electrotransformed into LLA69 competent cells using the same transformation parameters as above. The cells were plated on LB agar plates containing 25 μg / mL ampicillin and incubated overnight at 30°C. Candidate colonies were picked and confirmed to contain pRT-ilvG / M plasmid by enzyme digestion. ilvG / M The strain for which the plasmid was derived was named LLA619.

[0043] 1.6 Temperature-dependent verification of recombinant strain LLA619 1.6.1 Growth curve of recombinant bacteria The LLA619 strain was cultured at 30℃, 33℃, 35℃, 37℃, 39℃ and 42℃, and the OD was measured every 2 h. 600 Values, plot growth curves ( Figure 3 The results showed that the strain could grow normally at 30℃, 33℃, and 35℃, with the fastest growth rate at 35℃, a significant slowdown in growth at 37℃, and near-complete cessation of growth at 39℃ and 42℃, consistent with the expected temperature-dependent characteristics. That is, the strain can reproduce rapidly at cultivation temperatures below or equal to 35℃; and rapidly ceases proliferation at cultivation temperatures above 37℃, such as 38-44℃ (especially 38-42℃).

[0044] 1.6.2 Plasmid stability analysis Plasmid stability tests were performed on bacterial cells cultured at different temperatures. The constructed LLA619 strain was used as the experimental strain, and pKD46 was transformed into the LLA69 strain to obtain the LLA69(pKD46) strain as a control. The cells were inoculated into LB medium and cultured at 30℃, followed by subculture at 35℃, 39℃, and 42℃. Samples were taken periodically for viable cell counting, and for seeding on resistant and non-resistant plates. The plasmid retention rate at 30℃ and the plasmid loss rate per division per subculture at 35℃, 39℃, and 42℃ were calculated.

[0045] Plasmid holding rate (%) = Number of cells containing plasmid (CPU / mL) / Total number of cells per unit (CPU / mL) × 100%.

[0046] Plasmid loss rate per cell division (%) = (1 - plasmid retention rate 1 / N) × 100% (N: bacterial cell division number) The results showed that as the culture temperature increased, pRT- ilvG / M The plasmid loss rate was significantly higher than that of the pKD46 plasmid (Table 2). Under culture conditions at 30℃, the plasmid retention rate was close to 100%; however, under culture conditions at 39℃ and 42℃, the plasmid loss rate reached 87% and 95% after 24 h, respectively. This confirms that increasing the fermentation temperature can eliminate the recombinant plasmid pRT- ilvG / M Furthermore, by controlling the temperature of the simple fermentation process, the branched-chain amino acid synthesis pathway can be efficiently eliminated. This effectively solves the metabolic conflict between branched-chain amino acid synthesis and lactic acid synthesis, providing a new metabolic basis for the efficient biosynthesis of lactic acid monomers and creating a new metabolic scenario for the fermentation preparation of lactic acid monomers, which is conducive to achieving efficient and high-quality biosynthesis of lactic acid.

[0047] Table 2 Comparison of genetic stability of different plasmids at different culture temperatures

[0048] Example 2. Efficient Production Method of L-Lactic Acid Monomer 2.1 Culture medium and fermentation conditions M9 culture medium composition (g / L): glucose 30.0, Na2HPO4 6.0, KH2PO4 3.0, NaCl 0.5, NH4Cl 1.0, MgSO4·7H2O 0.5, CaCl2·2H2O 0.01, 0.1% trace element stock solution.

[0049] Trace element solution composition (g / L): FeSO4·7H2O 5.0, MnSO4·H2O 3.0, ZnSO4·7H2O 3.0, CoCl2·6H2O 1.0, CuSO4·5H2O 0.2, H3BO3 0.1, Na2MoO4·2H2O 0.1.

[0050] The composition of the feed solution (g / L) is: 600.0 g / L glucose.

[0051] pH neutralizer: 25% (w / v) calcium hydroxide suspension.

[0052] 2.2 Three-stage fermentation process 2.2.1 Bacterial proliferation stage (1) Inoculate LLA619 strain (and control CGMCC No.11060) into 50 mL M9 medium and culture at 35℃ for 12 h to obtain seed culture.

[0053] (2) Take 2% (v / v) seed culture and inoculate it into a 1 L shake flask containing 200 mL M9 medium. Incubate at 35℃ and 200 r / min for 8-10 h.

[0054] (3) Take 10% (v / v) of the above culture solution and inoculate it into a 50 L fermenter containing 25 L of M9 medium. Fermentation conditions: temperature 35℃, stirring speed 200 r / min-800 r / min, aeration rate 1.0 vvm, pH automatically controlled at 7.0±0.1.

[0055] 2.2.2 Bacterial cell enrichment stage (1) When the bacterial cell concentration OD 600 When the value is 5, the fermentation temperature is gradually increased from 35℃ to 40℃ at a rate of 1℃ / min.

[0056] (2) Maintain stirring speed of 200 r / min-800 r / min, aeration rate of 1.0 vvm, and pH of 7.0±0.1.

[0057] (3) Based on the glucose consumption, add feed solution by feed in a fed-batch manner, and control the glucose concentration at 5-10 g / L.

[0058] 2.2.3 Fermentation and Acid Production Stage (1) Keep the fermentation temperature constant at 40℃, and when the cell concentration OD 600 When the temperature reaches 28°C, reduce the stirring speed to 150 r / min, turn off the aeration, and switch to anaerobic fermentation.

[0059] (2) The pH value was controlled at 7.0±0.1, and 25% calcium hydroxide suspension was automatically added to neutralize the lactic acid produced.

[0060] (3) Add feed solution according to glucose consumption, and control glucose concentration at 10-20 g / L.

[0061] (4) Samples were taken every 4 hours to measure the concentration of L-lactic acid, glucose and byproducts.

[0062] (5) Continue fermentation until the L-lactic acid concentration reaches 170 g / L or higher, then stop fermentation.

[0063] 2.3 L-Lactic Acid Monomer Analysis Method 2.3.1 Determination of L-lactic acid concentration L-lactic acid concentration was determined by high-performance liquid chromatography (HPLC). Chromatographic conditions: Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad); mobile phase 5 mM H₂SO₄; flow rate 0.6 mL / min; column temperature 65℃; detector: refractive index detector (RID); injection volume 25 μL. L-lactic acid concentration was calculated using a standard curve.

[0064] 2.3.2 Determination of the optical purity of L-lactic acid The optical purity of L-lactic acid was determined by chiral high-performance liquid chromatography (HPLC). Chromatographic conditions: chiral column CHIRALPAKMA+ (50 mm × 4.6 mm, Daicel); mobile phase 1 mM CuSO4 solution; flow rate 1.0 mL / min; column temperature 25℃; detection wavelength 254 nm; injection volume 5 μL. The contents of L-lactic acid and D-lactic acid were determined separately using a standard curve, and the optical purity was calculated. Optical purity (%) = [L-lactic acid] / ([L-lactic acid] + [D-lactic acid]) × 100% 2.3.3 Determination of chemical purity of L-lactic acid The chemical purity of L-lactic acid was determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were the same as in Section 2.3.1. The contents of L-lactic acid and other organic acids were calculated using a standard curve to obtain the chemical purity. Chemical purity (%) = [L-lactic acid] / (total organic acid content) × 100% 2.4 Analysis of Fermentation Results Under the optimized three-stage fermentation process, after a total fermentation time of 52 h, the L-lactic acid concentration produced by strain LLA619 reached 173.3 g / L. Figure 4 (See Table 3) The glucose conversion rate reached 95.4%. The fermentation intensity (based on the highest L-lactic acid production rate) reached 15.6 g / (L·h), which was 35.5% higher than that of the control group (starting strain). It can be seen that the technology of this invention significantly improves the biosynthesis efficiency of L-lactic acid.

[0065] Table 3 Comparison of L-lactic acid production performance of strains

[0066] Further testing revealed that the L-lactic acid sample produced by strain LLA619 had an optical purity of 99.8% and a chemical purity of 98.7%. HPLC analysis showed no detection of impurities such as isopropyl malic acid, 3-methyl-2-hydroxybutyric acid, and acetic acid, indicating that the specific strain and fermentation method of this invention effectively block the formation of impurity molecules isopropyl malic acid and 3-methyl-2-hydroxybutyric acid, which are structurally similar to L-lactic acid. In contrast, isopropyl malic acid and 3-methyl-2-hydroxybutyric acid were visible in the fermentation product of the control strain. Figure 5 ).

[0067] Example 3. Fermentation performance test under different carbon source conditions To test the adaptability of the strain LLA619 of the present invention under different carbon source conditions, fermentation was carried out using glucose, sucrose, xylose, and glycerol as carbon sources, respectively, according to the method of Example 2. The initial concentration of each carbon source was 30 g / L (only the glucose in the M9 medium and feed solution in Example 2 was replaced with an equal amount of sucrose, xylose, or glycerol), and the carbon source concentration in the feed solution was 600 g / L.

[0068] Table 4 summarizes the highest fermentation performance under different raw materials. The LLA619 strain performed best under glucose and sucrose conditions, with similar L-lactic acid yield and purity. Under xylose conditions, the yield was slightly lower (130.5) g / L, but the purity remained at a high level (98.2% chemical purity, 99.7% optical purity). Under glycerol conditions, the yield reached 130.8 g / L, also exhibiting high purity. This indicates that the strain and method of this invention have good substrate adaptability and can utilize various renewable resources as carbon sources.

[0069] Table 4. Fermentation performance of strain LLA619 under different carbon source conditions

[0070]

[0071] When using sucrose as a raw material, add 0.8 U / g sucrase to the culture medium; when using glycerol as a raw material, maintain a microaerobic state during the fermentation stage.

[0072] Example 4. Optimization of different temperature conditions during the acid production stage To determine the optimal temperature conditions for the acid production stage of fermentation, comparative experiments were conducted at four temperature gradients: 38°C, 40°C, 42°C, and 45°C. Other conditions were performed as in Example 2.

[0073] The results are shown in Table 5. The highest L-lactic acid yield and fermentation intensity were observed at temperatures between 40℃ and 42℃. Furthermore, the highest L-lactic acid purity was achieved at 40℃ and 42℃, with chemical purities of 98.7% and 98.6%, respectively. Considering yield, fermentation intensity, and product purity, 40-42℃ was determined to be the optimal temperature range for the acid production stage.

[0074] Table 5. Fermentation performance under different temperature conditions during the acid production stage.

[0075] Example 5. Validation of reproducibility in scaled-up batch fermentation To verify the reproducibility of the method of the present invention, the optimized conditions of Examples 2, 3, and 4 were used, with glucose as the carbon source and the strain culture temperature at 35°C. When the bacterial cell concentration OD... 600 When the temperature is raised to 42℃ at 5°C, the bacterial cell concentration OD600 At 28:00, the fermentation transitioned to the acid-producing stage, with a fermentation temperature of 42℃ (other conditions were the same as in Example 2). Three batches of fermentation experiments were conducted consecutively. The L-lactic acid concentrations of each batch were 175.6 g / L, 171.8 g / L, and 177.5 g / L, respectively; the fermentation intensities were 15.8 g / (L·h), 15.2 g / (L·h), and 16.1 g / (L·h), respectively; the chemical purities were 98.7%, 98.5%, and 98.6%, respectively; and the optical purity was above 99.7% for all batches. The results indicate that the method of this invention has good repeatability and stability, providing a reliable guarantee for industrial production.

[0076] The above embodiments demonstrate that the specialized microbial strains and efficient production methods provided by this invention can effectively improve the yield, fermentation intensity, and product purity of L-lactic acid monomers, especially chemical purity, providing a reliable monomer source for the production of high-quality poly-L-lactic acid materials. This method is simple to operate and has significant economic and social benefits.

Claims

1. A lactic acid monomer-producing strain, characterized in that, The strains used were lactic acid-producing strains as the starting strains. After the expression of acetolactate synthase was deleted, the expression of the acetolactate synthase encoding gene was controlled by a temperature-sensitive replication plasmid.

2. The lactic acid monomer-producing strain as described in claim 1, characterized in that, The acetyllactate synthase includes: ilvG / M, ilvI / H and ilvB / N ; The ways to prevent the expression of acetolactate synthase include: knocking out the acetolactate synthase encoding gene, inserting, substituting, or deleting bases in the acetolactate synthase encoding gene to achieve complete non-expression of the protein encoded by the gene, or even if expressed, it is inactive.

3. The lactic acid monomer-producing strain as described in claim 1, characterized in that, Control of acetyllactate synthase using temperature-sensitive replication plasmids ilvG / M Expression of encoding genes.

4. The lactic acid monomer-producing strain as described in claim 1, characterized in that, The backbone of the thermosensitive replication plasmid is the pRT gene fragment obtained by amplification using pKD46 plasmid as template and pRT-F and pRT-R as primers. pRT-F: 5'-GTATGGACAGTTTTCCTTTGA -3'; pRT-R: 5'-GATGCAGGTGGCACTTTTCGGGGAA-3'.

5. The lactic acid monomer producing strain as described in claim 1, characterized in that, The strains capable of producing lactic acid include: L-lactic acid monomer producing strain CGMCC No.11060.

6. The lactic acid monomer producing strain according to claim 1, characterized in that, The lactic acid monomer-producing strain is based on CGMCC No. 11060 as the starting strain, and it targets the genome... ilvG / M , ilvI / H and ilvB / N After knockout, the clone is transferred. ilvG / M Thermosensitive recombinant expression plasmid pRT- ilvG / M The obtained LLA619 strain; the recombinant expression plasmid pRT- ilvG / M It is by ilvG / M The gene was obtained by recombination and ligation of the pRT gene fragment as described in claim 4.

7. The use of the strain described in any one of claims 1-6 in the fermentation production of lactic acid monomer.

8. The application as described in claim 7, characterized in that, The method for producing lactic acid monomers using the strain is as follows: (1) Cell proliferation stage: The strain was inoculated into a culture medium containing a carbon source and cultured aerobically at 30-35℃, with dissolved oxygen controlled at 20%-80%, until the cell density OD reached the target value. 600 Reaching 2-8; (2) Cell enrichment stage: Increase the culture temperature to 38-44℃ and continue aerobic culture until the cell density OD reaches 38-44℃. 600 Reaching 20-40; (3) Fermentation acid production stage: Maintain the culture temperature at 38-44℃ and switch to anaerobic fermentation. During this period, continuously or in batches, add carbon source to maintain sufficient carbon source. Add pH neutralizer to maintain pH value at 6.8-7.

2. When the concentration of lactic acid monomer no longer increases significantly or reaches the target concentration, terminate the fermentation process.

9. The application as described in claim 8, characterized in that, The carbon sources include glucose, sucrose, xylose, and glycerol.

10. The application as described in claim 8, characterized in that, Furthermore, in step (1), the temperature is 35℃; in steps (2) and (3), the culture temperature is increased to 40-42℃.

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