Lactic acid monomer production strain using temperature to regulate strain nutritional pattern and application thereof

Abstract

This invention relates to the field of microbial fermentation engineering technology, and particularly to lactic acid monomer production strains and their applications utilizing temperature-controlled strain trophic modes. This invention, through precise deletion of L-alanine synthesis-related genes in high-yield lactic acid monomer strains and dynamic reconstruction of metabolic pathways using a temperature-sensitive plasmid system, 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. This meets the industrial demands of modern intelligent fermentation manufacturing and the efficient preparation of bulk bio-based raw materials, and can be widely applied to the production of high-purity lactic acid monomers in the polylactic acid (PLA) industry.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation engineering technology, and in particular to lactic acid monomer production strains that utilize temperature-controlled strain nutrient modes and their applications. Background Technology

[0002] Lactic acid (2-hydroxypropionic acid) is an important raw material for the production of polylactic acid (PLA). As a biodegradable green polymer material, PLA occupies a core position in the development of sustainable materials. The industrial demand for L-lactic acid or D-lactic acid is increasing, therefore improving the efficiency, yield per unit time, and purity of lactic acid production has become a key research focus.

[0003] In the industrial production of lactic acid, the competition for metabolic flux between cell proliferation and lactic acid synthesis is one of the main bottlenecks limiting lactic acid yield and production intensity. During the rapid proliferation phase, cells dedicate most of their carbon source to building biomolecules. Even when entering the lactic acid production phase, cell proliferation still consumes some key metabolites, leading to a decrease in lactic acid synthesis efficiency. Effectively separating the cell proliferation and lactic acid synthesis phases is crucial for improving lactic acid production intensity.

[0004] L-alanine, an important amino acid, not only participates in protein synthesis but is also a crucial component of cell wall peptidoglycan, and its metabolic dynamics significantly influence cell growth and lactate synthesis. In *E. coli*, L-alanine is primarily produced through three synthetic pathways: valine pyruvate aminotransferase (VAT) avtA ), glutamate pyruvate aminotransferase ( alaA ) and glutamate pyruvate aminotransferase ( alaC These genes catalyze reactions. They have a significant impact on the growth and metabolic state of the strain.

[0005] In the field of metabolic regulation, existing research has explored methods to modulate the metabolic state of bacterial strains by controlling the availability of specific nutrients, achieving specific regulation of strain metabolism. Furthermore, temperature-sensitive plasmid systems have unique value in bacterial metabolic regulation research. Related studies have shown that temperature-sensitive plasmids can replicate and maintain at specific temperatures, while ceasing replication and being lost from the cell at other temperatures. This temperature-responsive characteristic provides a technological basis for constructing temperature-controlled metabolic regulation systems. In lactic acid bacteria metabolic engineering, existing research has used genetic engineering to regulate specific metabolic pathways, such as the expression level of lactate dehydrogenase (LDH), but a systematic method for effectively separating cellular metabolic state from growth stage by utilizing temperature to control the nutrient pattern of the strain is still lacking.

[0006] The application of gene deletion technology allows for more precise modification of metabolic networks and controllable impact on the genetic stability of strains. Combining temperature-sensitive plasmid systems with precise gene deletion technology can create strains with temperature-responsive characteristics, and with optimized fermentation process design, further improve the production intensity of lactic acid monomers.

[0007] In summary, based on previous research, this invention proposes a method for efficient production of lactic acid monomers by utilizing temperature-controlled strain trophic modes. By mediating the expression of the L-alanine synthesis gene through a temperature-sensitive plasmid, the strain can be controlled to transition from a growth state to a non-growth state while maintaining metabolic activity. This effectively separates the cell proliferation and lactic acid synthesis stages, significantly increasing the yield of lactic acid monomers and the production intensity per unit time. Summary of the Invention

[0008] This invention aims to solve the optimization problem of cellular metabolic flux distribution in existing microbial fermentation processes for lactic acid monomers, further enhance lactic acid monomer production intensity, and improve the level of process intelligence. It provides a high-efficiency lactic acid monomer production method and a dedicated engineered strain based on temperature-controlled regulation of the L-alanine metabolic pathway. By precisely deleting L-alanine synthesis-related genes in the target high-yield lactic acid monomer strain and using a temperature-sensitive plasmid system to dynamically reconstruct the metabolic pathway, it 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 bulk bio-based raw materials.

[0009] To achieve the above objectives, the technical approach adopted by the present invention is as follows:

[0010] One of the technical solutions provided by this invention is a temperature-sensitive expression plasmid, specifically the pRepTS plasmid. The pRepTS plasmid is obtained by removing non-essential sequences from the temperature-sensitive replication plasmid pKD46, retaining only the core sequences such as the temperature-sensitive replicon, the selective marker, and the necessary selective restriction enzyme sites. The nucleotide sequence of the pRepTS plasmid is shown in SEQ ID NO.1.

[0011] The second technical solution provided by the present invention is a lactic acid monomer producing strain. The strain is a strain with lactic acid production capacity as the starting strain. After the expression of genes related to L-alanine synthesis is deleted, the synthesis of L-alanine is controlled by a temperature-sensitive expression plasmid. Furthermore, the L-alanine synthesis-related genes include: valine pyruvate aminotransferase gene. avtA (GenBank No. 948087), glutamate-pyruvate aminotransferase gene alaA(GenBank ID 946772) and glutamate pyruvate aminotransferase gene alaC (GenBank No. 946850); Furthermore, the ways in which the deletion affects the expression of L-alanine synthesis-related genes include, but are not limited to: knocking out L-alanine synthesis-related genes, inserting, substituting, or deleting bases in L-alanine synthesis-related genes, etc., so that the protein encoded by the gene is not expressed at all, or even if it is expressed, it is not active. Furthermore, the control of L-alanine synthesis by thermosensitive expression plasmid involves expressing L-alanine synthesis-related genes using a thermosensitive plasmid. Furthermore, the temperature-sensitive expression plasmid includes, but is not limited to, the pRepTS plasmid; Furthermore, control via pRepTS plasmid avtA The expression of [the substance] allows for the control of L-alanine synthesis; Furthermore, the strains capable of producing lactic acid include: L-lactic acid monomer producing strain CGMCC No. 11060, or D-lactic acid monomer producing strain CGMCC No. 11059; Preferably, the lactic acid monomer producing strain is a starting strain of CGMCC No. 11060 or CGMCC No. 11059, which targets the genome... avtA , alaA and alaC After knockout, the pRepTS plasmid was introduced and avtA Recombinant gene expression vector pRep- avtA The result.

[0012] The third technical solution provided by the present invention is the application of the production strain described in the second 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 lactic acid monomer production stage carried out under increased temperature (38-44℃) conditions in the later stage. Furthermore, the method for producing lactic acid monomers using the aforementioned production strain through fermentation is as follows: (1) Bacterial proliferation stage Using M9 basic medium supplemented with glucose (concentration range 25-35 g / L) as the fermentation medium, the production strain was inoculated at an inoculum rate of 5%-10% (v / v). The culture was carried out at a temperature of 30-35℃ and a pH of 6.8-7.2, with dissolved oxygen controlled at 20%-80% by adjusting the rotation speed and airflow until the cell density reached OD500. 600 Reach 2-8; increase the culture temperature to 38-44℃ and continue culture until the bacterial density OD reaches 2-8.600 Reaching 20-35; (2) Lactic acid monomer production stage Maintain the temperature at 38-44℃, adjust the rotation speed and ventilation to enter the anaerobic fermentation stage, promote lactic acid monomer synthesis, maintain the pH value at 6.8-7.2, and continuously or in batches add glucose solution to maintain sufficient carbon source; The fermentation process is terminated when the concentration of lactic acid monomers no longer increases significantly or reaches the target concentration.

[0013] Furthermore, in step (1), the temperature is preferably 30℃, 31℃, 32℃, 33℃, etc.; the pH value is preferably 7.0; Furthermore, in step (1), the dissolved oxygen is controlled at 20%-80% by adjusting the rotation speed to 200-800 r / min and the air volume to 3-7 L / min; Furthermore, in step (1), the culture temperature is preferably increased to 40°C; the pH value is preferably 7.0; Further, in step (2), the rotation speed is adjusted to 200 r / min, ventilation is stopped, and the anaerobic fermentation stage begins; Furthermore, in step (2), the pH value is adjusted to 6.8-7.2 by adding 25 wt.% calcium hydroxide suspension; Furthermore, in step (2), 60 wt.% glucose solution is continuously or added in batches to maintain the glucose concentration at 5-40 g / L.

[0014] Beneficial effects This invention employs a gene deletion method to remove multiple enzyme genes closely related to L-alanine synthesis from a high-yield lactate monomer strain, constructing an L-alanine auxotrophic chassis strain. A temperature-sensitive engineered plasmid capable of stably expressing key genes for L-alanine metabolism is constructed, and a recombinant strain with temperature-controlled metabolic regulation is obtained through transformation. For this strain, a staged temperature-controlled process of "low-temperature proliferation-high-temperature fermentation" is developed, enabling the opening of metabolic pathways during cell proliferation and the closing of pathways during lactate monomer production, significantly improving metabolic flux direction and fermentation efficiency. Based on the characteristics of the novel engineered strain, a fermentation process suitable for temperature-controlled metabolic switching is created, improving the final concentration, chemical purity, and optical purity of lactate monomers.

[0015] 1. This invention achieves precise regulation of the L-alanine pathway in temperature-controlled Bacillus subtilis, optimizes fermentation process conditions, and completely decouples cell proliferation from the efficient synthesis of lactic acid monomers. This increases production intensity by more than 30% compared to existing technologies, shortens the fermentation cycle, reduces energy consumption and labor costs, and improves production efficiency. 2. The yield of industrial-grade lactic acid monomer can reach over 160 g / L, with a chemical purity of over 98% and an optical purity of over 99.5%, meeting the application requirements of the polylactic acid industry chain and its products. 3. The innovative use of a temperature-controlled plasmid regulation system eliminates the need for external chemical inducers, enabling intelligent process control and facilitating industrial scale-up and the construction of unmanned factories; 4. The process and strain of this invention are applicable to the production of both L-lactic acid monomers and D-lactic acid monomers, and have the versatility of a large-scale biomanufacturing platform and industrial promotion value.

[0016] 5. This invention provides a novel temperature-sensitive plasmid, pRepTS. This plasmid exhibits good genetic stability under low-temperature culture conditions, with 99% of cells retaining the plasmid after 4-8 hours of culture. Increasing the culture temperature significantly improves the plasmid loss efficiency to 75-83%, which is more conducive to the rapid entry of the strain into the product accumulation stage. Strict control of plasmid loss also further reduces the content of miscellaneous acids and increases the production intensity of lactate monomers. Furthermore, the addition of multiple cloning sites significantly improves the universality and convenience of exogenous gene cloning. Attached Figure Description

[0017] Figure 1 Electrophoresis pattern for diagnostic PCR verification of target gene deletion.

[0018] Figure 2 Nutritional characterization of mutant strains of lactic acid monomer producing strains in, Figure 2 a represents the nutritional characterization of strain CGMCC No.11060 and mutant strain LLA88; Figure 2 b represents the nutritional characterization of strain CGMCC No. 11059 and mutant strain DLA88.

[0019] Figure 3 Physical and restriction maps of plasmid pRepTS in, Figure 3 a represents the physical map and restriction enzyme digestion map of plasmid pRepTS; Figure 3 b is plasmid pRep- avtA Restriction enzyme digestion map.

[0020] Figure 4 Thermosensitive recombinant expression plasmid pRep- avtA The results of enzyme digestion verification.

[0021] Figure 5 Growth characteristics of the strain in, Figure 5 a represents industrial strains CGMCC No. 11060, strain LLA88, and strain LLA818; Figure 5b represents industrial strains CGMCC No. 11059, strain DLA88, and strain DLA818.

[0022] Figure 6 The acid production performance of industrial and optimized strains during fermentation. in, Figure 6 a represents industrial strains CGMCC No. 11060 and LLA818; Figure 6 b consists of industrial strains CGMCC No. 11059 and DLA818. Detailed Implementation

[0023] 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.

[0024] This invention provides a highly efficient method for producing lactic acid monomers based on temperature-controlled regulation of the L-alanine metabolic pathway, comprising the following key steps: 1. Select a suitable high-yield lactic acid monomer strain as the starting strain; 2. Precisely target key genes in the L-alanine synthesis pathway using gene deletion technology without leaving a trace; 3. Construct a thermosensitive recombinant expression plasmid to achieve temperature-controlled regulation of the L-alanine metabolic pathway; 4. Adopt a staged fermentation strategy of "low-temperature proliferation-high-temperature fermentation" to optimize production conditions and parameters.

[0025] Compared with existing technologies, this invention has the following significant advantages: First, it achieves automatic switching of metabolic flux for engineered lactic acid monomer strains, overcoming the technical challenge of coordinating cell proliferation and product accumulation; second, through a novel metabolic regulation mechanism, it effectively improves the final yield and production intensity of lactic acid monomers, with optical and chemical purity meeting the requirements of downstream polymer and pharmaceutical grades; third, it requires no exogenous inducers throughout the process, making it easy to automate and scale up industrial applications, with low energy consumption and high economic efficiency. Preliminary verification shows that the production intensity of lactic acid monomers is increased by more than 30% compared with existing mainstream production processes, and the final product purity reaches over 98%, with an optical purity of over 99.5%, significantly improving the economic benefits and industrial value of existing industrial applications.

[0026] 1. Strain construction 1.1 Selection of starting strain This invention uses CGMCC No. 11060 as the L-lactic acid monomer production strain and CGMCC No. 11059 as the D-lactic acid monomer production strain. Both strains are characterized by high yield, high conversion rate, and low byproduct production, and have been deposited at the China General Microbiological Culture Collection Center; they are existing technologies.

[0027] 1.2 Primers used in gene manipulation The key primer sequences used in the embodiments of this invention are shown in Table 1. These primers are used for gene deletion, gene cloning, mutation verification, and gene expression.

[0028] Table 1. Main primer sequences used in this invention

[0029] Note: The underlined part in the primer sequence indicates the added restriction endonuclease site.

[0030] 1.3 Construction of L-alanine auxotrophic strains This invention utilizes a scarless gene deletion technique to sequentially delete three key genes related to L-alanine synthesis: avtA (Valine pyruvate aminotransferase gene, GenBank accession number 948087) alaA (Glutamate-pyruvate aminotransferase gene, GenBank accession number 946772) and alaC (Glutamate pyruvate aminotransferase gene, GenBank accession number 946850).

[0031] The gene deletion operation employed a traceless gene deletion technique based on the Red recombination system (Zhou Li et al., Multi-gene Deletion Method for Escherichia coli Based on Red and Xer Recombination Systems, Bulletin of Microbiology, 2010, 37(6): 923-928), specifically including the following steps: (1) Amplify using primers P1-P4, P7-P10 and P13-P16 in Table 1 respectively. avtA , alaA and alaC Upstream and downstream homologous arm sequences of the gene were used to construct a gene containing gentamicin resistance and site-specific recombinant sequences. difference Homologous recombination fragments at (sites); (2) The target gene and the recombination fragment are precisely replaced by homologous recombination mediated by the Red recombination system; (3) Recognition using endogenous Xer / dif recombinase in cells difference two sites difference Homologous recombination occurs at the site, and circularization removes the resistance gene, achieving traceless deletion; (4) The accuracy of gene deletion was verified by PCR using the verification primers P5-P6, P11-P12 and P17-P18 in Table 1.

[0032] Using the above method, the present invention constructed L-lactic acid monomer strain LLA88 and D-lactic acid monomer strain DLA88. Both strains exhibit L-alanine auxotrophy and require exogenous supplementation of L-alanine to maintain normal growth.

[0033] It is understood that the methods provided by this invention are merely exemplary, and those skilled in the art can employ any gene editing method (such as gene knockout, base insertion, substitution, deletion, etc.) to ultimately achieve [the desired result]. avtA , alaA and alaC The protein encoded by the gene is not expressed at all, or even if it is expressed but has no activity, and is therefore within the scope of protection of this invention.

[0034] 1.4 Construction of thermosensitive recombinant expression plasmid To achieve temperature-controlled regulation of the L-alanine synthesis pathway, this invention constructs a temperature-sensitive recombinant expression plasmid pRep- avtA Its construction steps include: (1) Construction of the temperature-sensitive plasmid pRepTS: Using primers P21 and P22 from Table 1, the plasmid pKD46 (Datsenko, KA and Wanner, BL, One-step inactivation of chromosomal genes in Escherichia coli Based on the study of K-12 using PCR products. Proc Natl Acad Sci USA, 2000, 97(12): 6640-6645), the temperature-sensitive expression vector pRepTS was constructed by inverse PCR amplification, removal of non-essential sequences, and retention of only core sequences such as temperature-sensitive replicons, selective markers, and necessary selective restriction enzyme sites. After self-circularization, this plasmid was used to construct the plasmid. This plasmid can stably replicate at culture temperatures below 37°C (especially 30-35°C, such as 30°C, 31°C, 32°C, 33°C, etc.), but its replication ability is limited at culture temperatures above 37°C (especially 38-44°C, preferably 40°C), leading to plasmid loss from the cells.

[0035] (2) avtA Gene cloning: Using the genome of the starting strain as a template, primers P19 and P20 from Table 1 were used to amplify the sample via PCR. avtAThe full-length gene sequence (including its natural promoter and terminator) was cloned into the thermosensitive expression vector pRepTS to construct the thermosensitive recombinant expression plasmid pRep- avtA .

[0036] (3) Construction of recombinant strains: Thermosensitive recombinant expression plasmid pRep- was converted using electroporation and other methods. avtA The temperature-controlled strains LLA818 and DLA818 were constructed by transferring them into L-alanine auxotrophic strains LLA88 and DLA88, respectively.

[0037] The strain constructed using the above method, under low-temperature conditions (30-35℃), exhibits thermosensitive recombinant expression plasmid pRep- avtA Stable existence and expression avtA The gene enables cells to synthesize L-alanine, exhibiting a prototrophic phenotype that allows for rapid growth and proliferation; however, under elevated culture temperatures (38-44℃), the temperature-sensitive recombinant expression plasmid pRep- avtA Lost due to loss of self-replication ability. avtA Loss of expression results in cells losing the ability to synthesize L-alanine, exhibiting an L-alanine auxotrophic phenotype. Cell division and proliferation cease, and metabolic flow shifts to lactate monomer synthesis.

[0038] 2. Fermentation process 2.1 Bacterial proliferation stage (1) Culture medium composition: M9 basal medium was used, supplemented with glucose (concentration range 5-40 g / L, preferably 25-35 g / L), pH 6.8-7.2.

[0039] (2) Cultivation conditions: Temperature: Controlled within the range of 25-35℃, preferably 33℃; pH value: Maintained at 6.8-7.2, preferably 7.0; Dissolved oxygen: Maintain at 20%-80% to ensure sufficient oxygen supply (speed 200-800 r / min, air volume 3-7 L / min). Cultured to cell density OD 600 Reaching 2-8.

[0040] At this stage, the thermosensitive recombinant expression plasmid pRep- avtA Stable existence and expression avtA The gene and strain exhibit an L-alanine protrophic form, enabling rapid proliferation.

[0041] Increase the culture temperature to 38-44℃, preferably 40℃. Continue culturing until the bacterial cell density OD reaches 100%. 600 Achieving 20-35, with optimal OD 600 =28.

[0042] As the culture temperature increases, the temperature-sensitive recombinant expression plasmid pRep- avtA Losing its ability to self-replicate, the strain gradually transforms into an L-alanine auxotroph.

[0043] 2.2 Lactic acid monomer production stage (1) Fermentation conditions: Maintain the temperature between 38-44℃, preferably 40℃; pH value: Maintained at 6.8-7.2, adjusted by adding 25 wt.% calcium hydroxide suspension; Gas environment: Adjust the rotation speed and shut off the ventilation to enter the anaerobic fermentation stage and promote the synthesis of lactic acid monomers; Carbon source supply: Continuously or in batches, add 60 wt.% glucose solution to maintain sufficient carbon source.

[0044] (2) Fermentation process control: At this stage, since the temperature-sensitive recombinant expression plasmid has been lost, cell proliferation has essentially ceased, and metabolic flux is efficiently directed to lactate monomer synthesis. By controlling the glucose supplementation rate and pH value, the yield and conversion rate of lactate monomers can be effectively improved.

[0045] (3) Fermentation terminated: The fermentation process is terminated when the concentration of lactic acid monomers no longer increases significantly or reaches the target concentration (usually exceeding 160 g / L).

[0046] 3. Analytical Methods 3.1 Biomass determination The OD of the culture was determined using a spectrophotometer. 600 Values ​​are used to assess bacterial growth.

[0047] 3.2 Determination of lactic acid monomer concentration The concentration of lactic acid monomers was determined by high performance liquid chromatography (HPLC). The chromatographic column was an Aminex HPX-87H ion-exchange column; the mobile phase was 5 mM H₂SO₄ solution; the flow rate was 0.6 mL / min; the column temperature was 65℃; and the detector was a differential refractive index detector.

[0048] 3.3 Determination of optical purity of lactic acid monomer L-lactic acid and D-lactic acid were separated using a chiral column, their ratio was determined, and their optical purity was calculated. Chromatographic conditions: chiral column CHIRALPAK MA(+); mobile phase: 1 mM CuSO4 solution; flow rate: 1.0 mL / min; detection wavelength: 254 nm.

[0049] 3.4 Determination of chemical purity of lactic acid monomer Chemical purity is calculated by analyzing the content of impurity components in lactic acid monomers using HPLC or gas chromatography.

[0050] The following embodiments are used to further illustrate the technical solutions of the present invention, but do not constitute a limitation thereof. Those skilled in the art can make various improvements and modifications based on the content disclosed herein, and all such improvements and modifications are within the protection scope of the present invention.

[0051] Example 1: Construction of L-alanine auxotrophic strains LLA88 and DLA88 This embodiment, based on the technical route described in the foregoing specific implementation, elaborates in detail the construction process and verification results of L-alanine auxotrophic strains.

[0052] 1.1 Gene Deletion Operation Procedure Starting with either strain CGMCC No.11060 (a high-yield strain of L-lactate monomer) or CGMCC No.11059 (a high-yield strain of D-lactate monomer), the following alanine metabolism-related genes were sequentially knocked out using gene deletion techniques based on the Red recombination system and the Xer recombination system: (1) Valine pyruvate aminotransferase gene ( avtA )delete: Amplification was performed using primers P1-P4 from Table 1. avtA The upstream and downstream homologous arm sequences of the gene were edited and recombined using overlap PCR technology to form... avtA Upstream and downstream homologous arms of a gene are simultaneously introduced at the splicing point of the upstream and downstream sequences during the overlap process. EcoR V restriction enzyme cleavage site; With both sides difference The gentamicin resistance gene fragment at the locus (see reference: Liu Dawei et al., "Construction of a series of plasmids for easy isolation and molecular recombination of resistance genes," Microbiology Bulletin, 2007) was cloned into... avtA Upstream and downstream homologous arms of genes Eco R⁵ site, forming avtA Gene mutation cassette; after recovering the mutation cassette by PCR, it was transformed into the target strain by electroporation, and transitional recombinant strains whose genomes were replaced by homologous recombination by this mutation cassette were screened on gentamicin-resistant plates. Using the Xer / dif recombination system, bacterial Xer recombinase specifically recognizes difference two sites difference Homologous recombination occurs at the site, and cyclization removes the gentamicin resistance gene, achieving... avtA Seamless deletion of genes; PCR verification was performed using primers P5-P6 from Table 1 to confirm... avtA The gene has been deleted.

[0053] (2) Glutamate pyruvate aminotransferase gene ( alaA )delete: exist avtA Based on the gene-deleted strain, the same method was used to delete the gene using primers P7-P10 from Table 1. alaA Gene; PCR verification was performed using primers P11-P12 from Table 1 to confirm... alaA The gene has been deleted.

[0054] (3) Glutamate pyruvate aminotransferase gene ( alaC )delete: exist avtA , alaA Based on the double-gene deletion strain, the same method was used to delete the gene using primers P13-P16 from Table 1. alaC Gene; PCR verification was performed using primers P17-P18 from Table 1 to confirm... alaC The gene has been precisely deleted.

[0055] 1.2 Gene Deletion Validation Results After each gene knockout, PCR verification was performed using specifically designed primers to confirm the deletion of the target gene. The PCR products were analyzed by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown. Figure 1 a showed avtA The gene knockout results were confirmed, including in the originating bacteria. avtA The band size was approximately 1.2 kb (lane 2) when the gene was not knocked out, while the band size decreased to approximately 0.9 kb (lane 1) when the gene was successfully knocked out. Figure 1 b showed alaA The gene knockout verification results showed that the band size in the original strain was approximately 1.4 kb (lane 4), and the band size after knockout was reduced to approximately 1.1 kb (lane 3). Figure 1 c showed alaC The gene knockout validation results showed that the band size in the originating strain was approximately 1.2 kb (lane 6), while the knockout band size decreased to approximately 0.6 kb (lane 5). PCR validation results confirmed that the target gene had been precisely deleted and contained no exogenous sequences or resistance markers.

[0056] Through the above operations, we have constructed the following based on CGMCC No. 11060. avtA , alaA and alaCThe three-gene deletion strain LLA88 is an L-alanine auxotrophic strain used for L-lactic acid monomer production; obtained based on CGMCC No. 11059. avtA , alaA and alaC The three-gene deletion strain DLA88 is an L-alanine auxotroph strain used for the production of D-lactic acid monomers.

[0057] 1.3 Phenotypic Verification of L-Alanine Auxiliary Strains Both strains LLA88 and DLA88 were inoculated into M9 medium with or without L-alanine and cultured on plates. The results are as follows: Figure 2 As shown. The culture results indicate that: Both LLA88 and DLA88 were able to grow normally in M9 medium supplemented with L-alanine (100 mg / L); In M9 medium without the addition of L-alanine, LLA88 and DLA88 cannot grow, exhibiting a typical L-alanine auxotrophic phenotype.

[0058] This phenotypic validation result further confirms the existence of three L-alanine synthesis-related genes ( avtA , alaA and alaC The successful deletion of ) resulted in the mutant strain exhibiting an L-alanine auxotrophic behavior.

[0059] Example 2: Thermosensitive recombinant expression plasmid pRep- avtA Construction This embodiment describes in detail the thermosensitive recombinant expression plasmid pRep- avtA The construction process and verification results.

[0060] 2.1 Thermosensitive carrier pRepTS 2.1.1 Construction of the temperature-sensitive carrier pRepTS 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.

[0061] In plasmid pKD46 ( Figure 3 Based on a), all relevant coding genes of the λ-Red recombination system, which guides DNA homologous recombination by this plasmid, were further deleted, including the gene... exo , bet , gameAt the same time, genes controlling transcription of the λ-Red recombination system and L-arabinose-specific induction were deleted. araC The plasmid pRepTS was constructed by using its associated promoter and terminator sequences. Figure 3 b). The specific construction method is as follows: Using the temperature-sensitive replication plasmid pKD46 as a template, reverse PCR amplification was performed using primers P21 and P22 from Table 1. The amplified region contained the temperature-sensitive replicon, the selective marker gene, and the necessary multiple cloning site. The specific steps are as follows: PCR amplification conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 3 min, 30 cycles; 72℃ extension for 10 min. The PCR product was detected by agarose gel electrophoresis, and the target band was approximately 3.0 kb. The PCR products were recovered, self-ligation was performed, and the cells were transformed into E. coli DH5α competent cells. Plasmids were extracted from transformants and confirmed to be thermosensitive expression vector pRepTS by restriction endonuclease digestion and sequencing. The nucleotide sequence of plasmid pRepTS is shown in SEQ ID NO.1.

[0062] 2.2.2 Comparison of the effects of the temperature-sensitive vector pRepTS and the original plasmid pKD46 Using the LLA88 strain constructed above as the experimental strain, pRepTS and pKD46 were transformed into the LLA88 strain, respectively, to obtain LLA88(pKD46) and LLA88(pRepTS) strains. The LLA88(pKD46) and LLA88(pRepTS) strains were inoculated into LB medium and cultured at 32℃, followed by subculture at 37℃ and 40℃. Samples were taken periodically for viable cell counting, seeding on resistant and non-resistant plates, and calculations were made for plasmid retention rate at 32℃ and plasmid loss per division per subculture at 37℃ and 40℃.

[0063] Plasmid possession rate (%) = Number of cells containing plasmid (CPU / mL) / Total number of cells per unit (CPU / mL) × 100% Plasmid loss rate per division generation (%) = (1 - plasmid retention rate) 1 / N ) × 100% (N: bacterial cell division number) The results are shown in Table 2.

[0064] Table 2 Comparison of genetic stability of different plasmids at different culture temperatures

[0065] The percentage of cells that retain plasmids after 6 hours of culture (%) The percentage of plasmid loss per division after subculture (%) Table 2 shows that under low-temperature culture conditions (32℃), the pRepTS plasmid exhibits good genetic stability, with 99% of cells retaining the plasmid after 6 hours of culture. Increasing the culture temperature (37℃ and 40℃) significantly improves the pRepTS plasmid loss efficiency to 75-83%, which is more conducive to the strain rapidly entering the product accumulation stage and increasing production intensity. Furthermore, the addition of multiple cloning sites significantly improves the universality and convenience of exogenous gene cloning.

[0066] 2.2 avtA Gene cloning and construction of recombinant expression plasmids Using genomic DNA from CGMCC No. 11060 or CGMCC No. 11059 as a template, PCR amplification was performed using primers P19 and P20 in Table 1 to obtain... avtA Full-length gene sequence (including promoter and terminator regions); The PCR product was detected by agarose gel electrophoresis, and the target band was approximately 1.3 kb. The expression vector pRepTS constructed above was subjected to... Sma Ⅰ and Bam HⅠ restriction enzyme linearization; linearized pRepTS and avtA Gene fragments were ligated, and the ligation product was transformed into E. coli DH5α competent cells. Plasmids were extracted from transformants, and the thermosensitive recombinant expression plasmid pRep- was confirmed to be obtained through restriction enzyme digestion. avtA . Figure 4 The thermosensitive recombinant expression plasmid pRep- was shown. avtA The enzyme digestion verification results confirmed... avtA The gene has been successfully inserted into the pRepTS vector. Restriction enzyme digestion verification results indicate that the temperature-sensitive recombinant expression plasmid pRep- avtA Successfully constructed and ready for subsequent transformation experiments.

[0067] Example 3: Construction and phenotypic verification of temperature-controlled strains LLA818 and DLA818 This embodiment details the construction process of the temperature-controlled strain and the verification of its temperature-dependent growth characteristics.

[0068] 3.1 Construction of temperature-controlled strains The thermosensitive recombinant expression plasmid pRep- constructed in Example 2 was converted by electroporation. avtA The strains were introduced into the L-alanine auxotrophic strains LLA88 and DLA88 constructed in Example 1, as follows: Prepare LLA88 and DLA88 electrocompetent cells; pRep- avtA Plasmid DNA (approximately 100 ng) was mixed with competent cells and electroporated. The transformed cells were plated on LB plates containing the corresponding antibiotics and cultured at 33°C for 16-20 h. Single colonies were selected for recombinant plasmid DNA extraction and restriction enzyme digestion verification to confirm that the plasmid had been successfully introduced into the host strain.

[0069] Using the methods described above, temperature-controlled strains LLA818 and DLA818 were successfully constructed. LLA818 is a temperature-controlled strain for the efficient production of L-lactic acid monomers; DLA818 is a temperature-controlled strain for the efficient production of D-lactic acid monomers.

[0070] 3.2 Verification of the growth characteristics of temperature-controlled strains To verify the temperature-dependent growth characteristics of the temperature-controlled strain, the following experiments were conducted: The strain was inoculated into M9 medium containing 100 mg / L L-alanine or without L-alanine; it was cultured at 33℃ and 40℃ respectively, and OD was monitored. 600 Value changes. Experimental results for growth characteristics are as follows: Figure 5 As shown.

[0071] Figure 5 The growth curves of LLA88 (L-alanine auxotrophic strain), LLA818 (temperature-controlled strain), and industrial strain CGMCC No.11060 (control strain) under different conditions are shown: 33℃ without L-alanine: LLA818 can grow normally, while LLA88 cannot grow; CGMCC No.11060, as a control, can grow normally. 40℃ without L-alanine: LLA818 and LLA88 cannot grow, while CGMCC No.11060 can grow normally. 40℃ with L-alanine: LLA818, LLA88, and CGMCC No.11060 can all grow.

[0072] Figure 5 b shows the growth curves of DLA88 (L-alanine auxotrophic strain), DLA818 (temperature-controlled strain), and industrial strain CGMCC No.11059 (control strain) under the same conditions, exhibiting similar growth characteristics to the LLA series strains. Among them, CGMCC No.11059 can grow normally under all conditions.

[0073] Experimental results confirm: (1) Under 33℃ conditions, the thermosensitive recombinant expression plasmid pRep- avtA Stable existence and expression avtA The gene enables LLA818 and DLA818 to exhibit prototrophic behavior, allowing them to grow normally in media without added L-alanine. (2) Under 40℃ conditions, the thermosensitive recombinant expression plasmid pRep- avtA Losing their ability to self-replicate, LLA818 and DLA818 become L-alanine auxotrophs and cannot grow in media without added L-alanine. (3) All strains can grow normally in the culture medium with added L-alanine, regardless of the temperature. (4) Industrial control strains CGMCC No.11060 and CGMCC No.11059 were able to grow normally under all conditions, verifying the reliability of the experiment.

[0074] These results fully demonstrate that the temperature-controlled strain constructed in this invention has the expected temperature-dependent growth characteristics, laying the foundation for the efficient production of lactic acid monomers in the future.

[0075] Example 4: Fermentation production and product analysis of lactic acid monomers This embodiment describes in detail the process flow and results analysis of lactic acid monomer production using temperature-controlled strains LLA818 and DLA818.

[0076] 4.1 Fermentation process flow The lactic acid monomer production performance of strains LLA818 and DLA818 was evaluated in a 5 L fermenter. The specific fermentation process is as follows: (1) Cell proliferation stage: Culture medium: M9 basal medium, glucose concentration 30 g / L, pH 7.0; Inoculation amount: 5% (v / v) logarithmic growth stage seed solution; Temperature control: 33℃, at which temperature the temperature-sensitive recombinant expression plasmid pRep- avtA They exist stably and can proliferate rapidly. Speed ​​control: 200 r / min - 800 r / min; Ventilation rate control: 3-7 L / min; Dissolved oxygen control: 20%-80%; When the bacterial cell density reaches OD 600 When the value is approximately 4.9, the incubation temperature is increased to 40℃; Continue culturing until the bacterial cell density reaches OD. 600The value is approximately 27.

[0077] (2) Lactic acid monomer production stage: Maintain the temperature at 40℃; Turn off the ventilation and control the stirring speed at 200 r / min; The glucose concentration in the culture medium was maintained at 5-40 g / L by adding 60 wt.% glucose solution. The pH was maintained at 6.5-7.2 by adding a 25 wt.% calcium hydroxide suspension. The fermentation process continues until the rate of glucose consumption decreases significantly.

[0078] The above fermentation process achieves an organic combination of rapid cell growth and efficient lactic acid monomer production through temperature control in two key stages: the 33℃ stage promotes cell proliferation, and the 40℃ stage induces cells to stop growing but maintain high metabolic activity, efficiently converting the carbon source into the target product, lactic acid monomer.

[0079] 4.2 Evaluation of fermentation performance Lactic acid monomer fermentation experiments were conducted using industrial strains CGMCC No.11060 and CGMCC No.11059, as well as the temperature-controlled strains LLA818 and DLA818 constructed in this invention, according to the fermentation process flow in 4.1. The acid production performance of each strain was monitored by tracking the fermentation curves.

[0080] Fermentation results as follows Figure 6 As shown in Table 3.

[0081] Figure 6 The figure shows a comparison curve of lactic acid monomer production between LLA818 and CGMCC No.11060. LLA818 reached a lactic acid monomer concentration of 162 g / L after 35 h of fermentation, while CGMCC No.11060 only reached 116 g / L in the same time period and required 45 h to reach a similar lactic acid monomer concentration.

[0082] Figure 6 b shows the comparison curves of lactic acid monomer production between DLA818 and CGMCC No.11059. DLA818 reached a lactic acid monomer concentration of 160 g / L after 36 h of fermentation, while CGMCC No.11059 only reached 113 g / L in the same time period, and also required 47 h to reach a similar lactic acid monomer concentration.

[0083] By comparing the fermentation curves, the key fermentation parameters of the temperature-controlled strain and the industrial strain were calculated as follows: 1. Shorter fermentation time: Temperature-controlled strains have a fermentation time that is more than 34% shorter than industrial strains; 2. Increased acid production rate: The average acid production rate of the temperature-controlled strain is about 30% higher than that of the industrial strain; 3. Improved glucose conversion rate: The glucose-lactic acid conversion rate of the temperature-controlled strain reached 0.95 g / g, which is about 10% higher than that of the industrial strain.

[0084] 4.3 Product Purity Analysis After fermentation, the chemical and optical purity of the lactic acid monomers were analyzed. The chemical purity reached 98%, and the optical purity was above 99.5%.

[0085] Table 3 Summary of data on lactic acid monomers in fermentation broth

[0086] By constructing new strains LLA818 and DLA818 and optimizing the two-stage fermentation process, this invention significantly improves the efficiency and purity of lactic acid production.

[0087] Example 5: Fermentation production of lactic acid monomers This embodiment describes in detail the process flow for lactic acid monomer production using temperature-controlled strains LLA818 and DLA818.

[0088] Lactic acid monomers were produced by strains LLA818 and DLA818 in a 5 L fermenter. The specific fermentation process is as follows: (1) Cell proliferation stage: Culture medium: M9 basal medium, glucose concentration 31 g / L, pH 7.0; Inoculation amount: 5% (v / v) logarithmic growth stage seed solution; Temperature control: 32℃, at which temperature the temperature-sensitive recombinant expression plasmid pRep- avtA They exist stably and can proliferate rapidly. Speed ​​control: 200 r / min - 800 r / min; Ventilation rate control: 3-7 L / min; Dissolved oxygen control: 20%-80%; When the bacterial cell density reaches OD 600 When the value is approximately 3.0, the incubation temperature is increased to 42℃; Continue culturing until the bacterial cell density reaches OD. 600 The value is approximately 25.

[0089] (2) Lactic acid monomer production stage: Maintain the temperature at 42℃; Turn off the ventilation and control the stirring speed at 200 r / min; The glucose concentration in the culture medium was maintained at 5-40 g / L by adding 60 wt.% glucose solution. The pH was maintained at 6.5-7.2 by adding a 25 wt.% calcium hydroxide suspension. The fermentation process continues until the rate of glucose consumption decreases significantly.

[0090] Example 6: Fermentation production of lactic acid monomers This embodiment describes in detail the process flow for lactic acid monomer production using temperature-controlled strains LLA818 and DLA818.

[0091] Lactic acid monomers were produced by strains LLA818 and DLA818 in a 5 L fermenter. The specific fermentation process is as follows: (1) Cell proliferation stage: Culture medium: M9 basal medium, glucose concentration 35 g / L, pH 7.0; Inoculation amount: 10% (v / v) of logarithmic growth stage seed solution; Temperature control: 35℃, at this temperature the temperature-sensitive recombinant expression plasmid pRep- avtA They exist stably and can proliferate rapidly. Speed ​​control: 200 r / min - 800 r / min; Ventilation rate control: 3-7 L / min; Dissolved oxygen control: 20%-80%; When the bacterial cell density reaches OD 600 When the value is approximately 5.0, the incubation temperature is increased to 40℃; Continue culturing until the bacterial cell density reaches OD. 600 The value is approximately 35.

[0092] (2) Lactic acid monomer production stage: Increase the temperature to 44°C; Turn off the ventilation and control the stirring speed at 200 r / min; The glucose concentration in the culture medium was maintained at 5-40 g / L by adding 60 wt.% glucose solution. The pH was maintained at 6.5-7.2 by adding a 25 wt.% calcium hydroxide suspension. The fermentation process continues until the rate of glucose consumption decreases significantly.

[0093] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A temperature-sensitive expression plasmid, characterized in that, Specifically, it is the pRepTS plasmid, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. A lactic acid monomer-producing strain, characterized by, The strain was started by a strain capable of producing lactic acid. After the expression of genes related to L-alanine synthesis was deleted, the synthesis of L-alanine was controlled by a thermosensitive expression plasmid. The thermosensitive expression plasmid is the pRepTS plasmid.

3. The lactic acid monomer-producing strain as described in claim 2, characterized in that, The L-alanine synthesis-related genes include: a valine pyruvate aminotransferase gene avtA a glutamate pyruvate aminotransferase gene alaA and a glutamate pyruvate aminotransferase gene alaC .

4. The lactic acid monomer-producing strain as described in claim 2, characterized in that, The methods for deleting the expression of L-alanine synthesis-related genes include: knocking out L-alanine synthesis-related genes, or inserting, substituting, or deleting bases in L-alanine synthesis-related genes in a manner that ensures the protein encoded by the gene is not expressed at all, or even if expressed, it is inactive.

5. The lactic acid monomer producing strain as described in claim 2, characterized in that, The method of controlling L-alanine synthesis using a thermosensitive expression plasmid involves expressing L-alanine synthesis-related genes using a thermosensitive plasmid.

6. The lactic acid monomer-producing strain as described in claim 5, characterized in that, The thermosensitive expression plasmid is the pRepTS plasmid; the expression is controlled by the pRepTS plasmid. avtA The expression of [something] allows for control over the synthesis of L-alanine.

7. A lactic acid monomer-producing strain as described in claim 2, characterized in that, The strains capable of producing lactic acid include: L-lactic acid monomer producing strain CGMCC No.11060 or D-lactic acid monomer strain CGMCC No.11059.

8. A lactic acid monomer-producing strain as described in claim 2, characterized in that, Starting with CGMCC No. 11060 or CGMCC No. 11059, the genome was analyzed. avtA , alaA and alaC After knockout, the pRepTS plasmid was introduced and avtA Recombinant gene expression vector pRep- avtA The result.

9. The use of the strain according to any one of claims 2-8 in the production of lactic acid monomers.

10. The application as described in claim 9, characterized in that, The method for producing lactic acid monomers using the aforementioned strain through fermentation is as follows: (1) Bacterial proliferation stage The M9 basic medium with added glucose is used as the fermentation medium, the production strain is inoculated at an inoculation amount of 5%-10%, and the dissolved oxygen is controlled at 20%-80% by adjusting the rotation speed and air volume under the conditions of a temperature of 30-35°C and a pH value of 6.8-7.2, and the culture is carried out until the cell density OD 600 reaches 2-8; the culture temperature is increased to 38-44°C, and the culture is continued until the cell density OD 600 reaches 20-35; (2) Lactic acid monomer production stage Maintain the temperature at 38-44℃, adjust the rotation speed and ventilation to enter the anaerobic fermentation stage, promote the synthesis of lactic acid monomers, maintain the pH value at 6.8-7.2, and continuously or in batches add glucose solution to maintain sufficient carbon source.

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