Genetic element for enhancing lactate metabolism flux of actinomyces

Abstract

The application discloses a genetic element for enhancing lactic acid metabolic flux of actinomycetes, and belongs to the field of synthetic biology and microbial metabolic engineering. sucC The application successfully constructs a single expression element and a co-expression element by cloning genes from Streptomyces coelicolor sucD , and verifies that the SucC protein and the SucD protein both have lactic acid coenzyme A synthetase activity through in-vitro protein purification and enzyme activity determination, and the enzyme activity of a protein complex (SucC-SucD, SucCD) expressed by the co-expression element is significantly higher than that of the single protein, indicating that the two proteins have a synergistic effect; the genetic element can efficiently catalyze the combination reaction of lactic acid and coenzyme A in vitro, accelerates the lactic acid metabolic speed, provides a core genetic element for the modification of the lactic acid metabolic pathway of actinomycetes, enriches a synthetic biology tool library, and has important industrial application prospects and academic value.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and microbial metabolic engineering, and in particular to a genetic element that enhances the lactic acid metabolic flux of actinomycetes. Background Technology

[0002] As important industrial production strains for secondary metabolites such as antibiotics, the optimization of the metabolic network of actinomycetes is crucial for improving the synthesis efficiency of target products. In actual production scenarios such as industrial fermentation, hypoxic or anaerobic environments are prone to occur in local fermentation systems, and lactic acid is a product that easily accumulates during the metabolism of actinomycetes under such conditions. Lactic acid metabolism is a key branch of the carbon metabolism pathway in actinomycetes. Efficient lactic acid degradation and conversion can not only provide sufficient energy and key precursors (such as lactyl-CoA) for cell growth and secondary metabolism, but also alleviate the adverse effects of lactic acid accumulation on cells under hypoxic conditions, thereby improving the growth status of actinomycetes and the synthesis efficiency of secondary metabolites under hypoxic levels. However, the lactic acid metabolism rate of natural actinomycetes is generally limited, resulting in low carbon source utilization efficiency. They are unable to effectively decompose lactic acid accumulated in hypoxic environments, nor can they fully convert lactic acid into the energy and precursors required for growth and product synthesis, indirectly restricting the synthesis level of secondary metabolites.

[0003] Streptomyces cerevisiae, as a model actinomycete, has its genome annotated with... sucC Genes and sucD The gene, originally thought to be related to the synthesis of succinyl-CoA, encodes corresponding subunits involved in its synthesis. Previous studies have found that the SucC / D protein in eukaryotes has the ability to metabolize lactate, but in prokaryotes... sucC / sucD Whether these genes are involved in lactate metabolism and whether they can catalyze the production of lactyl-CoA is currently unclear. Existing research only suggests that the prokaryotic SucC and SucD proteins, alone or in synergy, are involved in the synthesis of acyl-CoA, but there is no research on whether these two genes function as genetic elements in Streptomyces to enhance lactate metabolic flux.

[0004] Synthetic biology genetic elements are core tools for metabolic engineering, and developing highly efficient lactate metabolism genetic elements is of great significance for optimizing the carbon metabolism network of actinomycetes. Current technologies lack specific, highly efficient elements for lactate metabolism in actinomycetes, and those derived from *Streptomyces azurite*... sucC and sucD Whether these genes are involved in lactic acid metabolism, and whether co-expression of these two genes may produce a synergistic effect related to lactic acid metabolism, remains unclear. Therefore, verifying the lactic acid metabolism-related activities of co-expressed these two genes and clarifying their potential to improve lactic acid metabolism efficiency could provide efficient genetic elements for the targeted modification of lactic acid metabolism pathways in actinomycetes, and has significant application value. Summary of the Invention

[0005] The purpose of this invention is to provide a genetic element that enhances the lactic acid metabolic flux of actinomycetes, thereby solving the problems existing in the prior art. This is achieved by cloning a genetic element derived from *Streptomyces cerevisiae*. sucC Genes and sucD Genes were constructed and recombinant vectors for single and co-expression were developed. After in vitro protein purification, their lactyl-CoA synthase activity was verified, thereby enhancing the flux of lactic acid metabolism in vitro and providing efficient genetic elements for actinomycete metabolic engineering.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a genetic element that enhances the lactic acid metabolic flux of actinomycetes. The genetic element is a SucC-SucD co-expression element, which is integrated... sucC Genes and sucD Genes enable the simultaneous expression of the expression products of two genes and their assembly to form a functional complex; The sucC The nucleotide sequence of the gene is shown in SEQ ID NO.1. sucD The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0007] Optionally, the sucC Genes and the sucD The genes are linked by an RBS sequence, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0008] Optionally, the sucC Genes and the sucD The gene originates from Streptomyces azureense ( Streptomyces coelicolor ).

[0009] Optionally, the protein expressed by the genetic element has lactyl-CoA synthase activity, which can catalyze the combination of lactic acid and coenzyme A to generate lactyl-CoA, thereby enhancing the lactic acid metabolic flux of actinomycetes.

[0010] The present invention also provides the application of the genetic element in the modification of the lactic acid metabolism pathway in actinomycetes.

[0011] The present invention also provides the application of the genetic element in enhancing the lactic acid metabolic flux of actinomycetes.

[0012] The present invention also provides the application of the genetic element in constructing an in vitro lactate metabolism system.

[0013] The present invention also provides a method for enhancing the lactic acid metabolic flux of actinomycetes, comprising the step of introducing the genetic element into the actinomycetes.

[0014] The present invention discloses the following technical effects: This invention utilizes clones of Streptomyces cerevisiae. sucC Genes and sucD The gene was successfully constructed into individual expression elements and co-expression elements. In vitro protein purification and enzyme activity assays confirmed that both SucC and SucD proteins possess lactyl-CoA synthase activity. Furthermore, the enzyme activity of the protein complex expressed by the co-expression element (SucC-SucD, SucCD) was significantly higher than that of the individual proteins, indicating a synergistic effect between the two proteins. This genetic element can efficiently catalyze the binding reaction of lactate and CoA in vitro, enhancing lactate metabolic flux. It provides a core genetic element for the modification of lactate metabolic pathways in actinomycetes, enriches the synthetic biology tool library, and has significant industrial application prospects and academic value. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 pET-28a- sucC Structural diagrams of plasmids; Figure 2 pET-28a- sucD Structural diagrams of plasmids; Figure 3 pET-28a- sucC - sucD Structural diagrams of plasmids; Figure 4 target gene fragment sucC Gene (with homologous arm) (A) sucD PCR amplification results of gene (with homologous arm) (B) and pET-28a vector (C); Figure 5 Validation results for seamless cloning recombinant vectors (A) and sucC Gene (B) sucD Gene (C) and s ucC -s ucD Sequencing results alignment of the fusion gene (D); In A, the first lane on the left is the marker, and the first red box indicates... sucC Genes, indicated by the second red box sucD Gene, the third red box represents s ucC -s ucD Fusion genes; Figure 6 SDS-PAGE electrophoresis images of the purified recombinant protein; A: Purified SucC protein, the first lane on the left is the marker, and the other two lanes are purified SucC protein; B: Purified SucD protein, the first lane on the left is the marker, and the other two lanes are purified SucD protein; C: Purified SucC-SucD co-expressed protein, the first lane on the left is the marker, and the third lane is the purified SucC-SucD co-expressed protein; the second lane contains incidental protein samples from other experiments conducted concurrently, which are unrelated to the content of this invention; Figure 7 L-lactic acid consumption (A) and ATP consumption (B) for proteins expressed by different genetic elements. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] The invention used sucC The gene originates from Streptomyces azureense ( Streptomyces coelicolor Its nucleotide sequence is shown in SEQ ID NO.1, encoding the SucC protein, whose amino acid sequence is shown in SEQ ID NO.2; the used sucD The gene originates from Streptomyces azureense ( Streptomyces coelicolor The nucleotide sequence of the SucC-SucD protein is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4. The SucC-SucD co-expression element is linked by an RBS sequence. sucC Genes and sucD The gene, with the RBS sequence TCTAGAGATAAAGAGGAGAATACTAG (SEQ ID NO.5), is located at... sucC Gene stop codons and sucD Between gene start codons, used to mediate sucD Efficient gene translation leads to a convergence in the expression levels of SucC and SucD proteins, achieving co-expression of SucC and SucD proteins. The nucleotide sequence of the SucC-SucD co-expression element is the sequence shown in SEQ ID NO.1-SEQ ID NO.5-SEQ ID NO.3 linked together.

[0023] SEQ ID NO.1: SEQ ID NO.2: VDLFEYQARDLFAKHDVPVLAGEVIDTPEAAREITERLGGKSVVKAQVKVGGRGKAGGVKLAASADEAVARATDILGMDIKGHTVHKVMIAETAPEIVEEYYVSFLLDRANRTFLSIASVEGGVEIEEVAATRPEAVAKTPIDAIDGVTPEKAREIVEAAKFPAEVADKVADILVKLWDTFIKEDALLVEVNPLAKVVSGDVIALDGKVSLDDNAEFRHPDFEALHDKAAANPLEAAAKEKNLNYVKLDGEVGIIGNGAGLVMSTLDVVAYAGEAHGNVKPANFLDIGGASAQVMANGLEIILGDPDVRSVFVNVFGGITACDEVANGIVQALKLLEDRGEKVEKPLVVRLDGNNAELGRKILTDANHPLVQRVDTMDGAADKAAELAHAAAK; SEQ ID NO.3: ATGGCTATCTGGCTCAACAAGGACAGCAAGGTCATCGTCCAGGGCATGACCGGTGCCACCGGCATGAAGCACACCAAGCTCATGCTGGGTGACGGCACCGAGGTCGTGGGCGGCGTGAACCCGCGCAAGGCGGGCACCTCCGTGGACTTCGACGGCAACGAGGTACCGGTCTTCGGCACCGTCAAGGAGGCCATCGAGAAGACCGGCGCCAACGTCTCCGTCATCTTCGTGCCGGAGAAGTTCACCAAGGACGCCGTCGTCGAGGCCATCGACGCCGAGATCCCGCTGGCCGTCGTCATCACCGAGGGCATCGCCGTGCACGACTCGGCCGCCTTCTGGTCGTACGCCGGCAAGAAGGGCAACAAGACCCGGATCATCGGCCCGAACTGCCCCGGCATCATCACGCCGGGCCAGTCCAACGTCGGCATCATCCCGGGCGACATCACCAAGCCGGGCCGCATCGGCCTGGTCTCGAAGTCCGGCACGCTGACGTACCAGATGATGTACGAGCTGCGTGACATCGGCTTCTCGACCGCCGTCGGCATCGGTGGCGACCCGATCATCGGCACCACGCACATCGACGCGCTCGCCGCGTTCGAGGCCGACCCCGAGACCGACCTGATCGTCATGATCGGCGAGATCGGCGGCGACGCCGAGGAGCGTGCGGCCGAGTACATCTCGAAGAACGTGACGAAGCCGGTCGTCGGCTACGTCGCGGGCTTCACCGCGCCCGAGGGCAAGACCATGGGCCACGCCGGCGCCATCGTCTCCGGTTCGTCCGGCACCGCCCAGGCGAAGAAGGAGGCCCTGGAGGCCGCCGGCGTCAAGGTCGGCAAGACGCCGACCGAGACGGCGAAGCTCGCCCGCGCCATCCTGGCCGGCTGA; SEQ ID NO.4: MAIWLNKDSKVIVQGMTGATGMKHTKLMLGDGTEVVGGVNPRKAGTSVDFDGNEVPVFGTVKEAIEKTGANVSVIFVPEKFTKDAVVEAIDAEIPLAVVITEGIAVHDSAAFWSYAGKKGNKTRIIGPNCPGIITPGQSNVGIIPGD ITKPGRIGLVSKSGTLTYQMMYELRDIGFSTAVGIGGDPIIGTTHIDALAAFEADPETDLIVMIGEIGGDAEERAAEYISKNVTKPVVGYVAGFTAPEGKTMGHAGAIVSGSSGTAQAKKEALEAAGVKVGKTPTETAKLARAILAG.

[0024] Example 1 1. Plasmid construction The homologous arms were obtained by PCR amplification using primers SucC-F / R. sucC The gene fragment was cloned into the PCR-linearized pET-28a plasmid backbone using seamless cloning technology to construct the pET-28a plasmid for in vitro expression and purification of SucC. sucC (pET-28a(+)-sucC(SCO4808)-his, plasmid map as follows) Figure 1 (As shown).

[0025] SucC-F: 5'-GTGCCGCGGCAGCCATATGGTGGACCTGTTCGAGTACCAGGC-3', SEQ IDNO.6; SucC-R: 5'-CAGTGATGATGATGATGATGCTTGGCGGCGGCGTGAGCC-3', SEQ ID NO. 7.

[0026] The homologous arms were obtained by PCR amplification using primers SucD-F / R. sucD The gene fragment was cloned into the linearized pET-28a plasmid using the same seamless cloning method to construct the in vitro expression and purification plasmid pET-28a- sucD (pET-28a(+)-sucD(SCO4809)-his-1, plasmid map as follows) Figure 2 (As shown).

[0027] SucD-F: 5'-CGCGGCAGCCATATGatggctatctggctcaacaaggaca-3', SEQ ID NO.8; SucD-R: 5'-ATCAGTGATGATGATGATGATGgccggccaggatggC-3', SEQ ID NO. 9.

[0028] Using primers SucC-RBS-F / SucC-RBS-R / SucD-RBS-F / SucD-RBS-R, streptococcal amplification with Streptomyces cerevisiae genomic DNA as a template was performed to obtain si with homologous arms. ucC - SucD The fusion gene fragment was cloned into the linearized pET-28a plasmid backbone using seamless cloning technology to construct the co-expression plasmid pET-28a- sucC - SucD (pET-28a(+)-4808-rbs-4809(1), plasmid map as follows) Figure 3 (As shown).

[0029] SucC-RBS-F: 5'-CGCGGCAGCCATATGGTGGACCTGTTCGAGTACCAGGC-3', SEQ ID NO.10; SucC-RBS-R: 5'-TATTCTCCTCTTTAATCTCTAGATTACTTGGCGGCGGCGTGA-3', SEQ IDNO.11; SucD-RBS-F: 5'-GAGATTAAAGAGGAGAATACTAGatggctatctggctcaacaaggacagC-3', SEQ ID NO.12; SucD-RBS-R: 5'-ATCAGTGATGATGATGATGATGgccggccaggatggcgc-3', SEQ ID NO. 13.

[0030] Linearization of the pET-28a vector was achieved by PCR. The primers used were pET-28a linearization-F / R (pET-28a linearization-F: 5'-CATCATCATCATCATCACTGATGAGATCCGG-3', SEQ ID NO.14; pET-28a linearization-R: 5'-CATATGGCTGCCGCGCG-3', SEQ ID NO.15). The PCR reaction system consisted of 25 μL buffer, 1 μL dNTP, 1 μL upstream primer, 1 μL downstream primer, 1 μL pET-28a plasmid template, 1 μL high-fidelity DNA polymerase, and 20 μL ddH2O.

[0031] The PCR amplification reaction system for the target gene and fusion fragment was as follows: 25 μL buffer, 1 μL dNTP, 1 μL upstream primer, 1 μL downstream primer, 1 μL Streptomyces cerevisiae genomic DNA template, 1 μL high-fidelity DNA polymerase, and 20 μL ddH2O.

[0032] All the above PCR reaction procedures were as follows: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 15 s, 58℃ annealing for 30 s, 72℃ extension for 30 s / 1000 bp, the denaturation to extension process was repeated 35 times, after which the extension was performed at 72℃ for 10 min, and finally cooled to 12℃.

[0033] After PCR products were subjected to 1% agarose gel electrophoresis, the target gene fragment, fusion fragment, and linearized vector were compared with the marker in size. Once the size was correct, the gel extraction kit was used for purification and recovery.

[0034] The purified target fragment was ligated into the linearized pET-28a vector using a seamless cloning enzyme. The recombination reaction conditions were as follows: 6 μL of seamless cloning enzyme, 4 μL of target fragment (or fusion fragment), and 2 μL of linearized vector were mixed and incubated at 50°C for 30 min. After incubation on ice for 5 min, the entire mixture was added to DH5α competent cells and gently mixed. After incubation on ice for 30 min, the mixture was heat-shocked at 42°C for 90 s and incubated on ice for 3 min. Then, 750 μL of LB medium was added and the mixture was activated at 37°C and 220 rpm for 45 min. The activated bacterial culture was then plated onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C for 12–18 h. Single clones were picked and sequenced. After alignment with the designed sequence, the clones with correct sequencing were retained to obtain the pET-28a-s vector. ucC pET-28a- sucD and pET-28a- sucC - sucD Transformants of plasmids.

[0035] 2. In vitro expression and purification of proteins Plasmid extraction: 50 μL of DH5α Escherichia coli bacterial culture containing the overexpression plasmid was added to a 5 mL LB liquid medium test tube and cultured at 37℃ and 220 rpm for 24 h. The cultured bacterial culture was then used to extract and purify the plasmid using a plasmid extraction kit.

[0036] After plasmid extraction, 1 μL of each of the three plasmids was added to 50 μL of BL21 competent cells. The cells were incubated on ice for 30 min, then heat-shocked at 42°C for 90 s. After 3 min on ice, 750 μL of LB medium was added, and the cells were activated at 37°C and 220 rpm for 45 min using a shaker. The activated bacterial cultures were then spread onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C for 12–18 h. Single clones were picked to obtain the recombinant expression strain BL21 / pET-28a-s. ucC BL21 / pET-28a- sucD and BL21 / pET-28a-s ucC -s ucD .

[0037] Purified bacterial culture: Take 50 μL of the above three recombinant bacterial cultures and add them to 5 mL LB liquid medium containing 50 μg / mL kanamycin in a test tube. Incubate at 37℃ and 220 rpm for 18 h to obtain seed cultures. Then, add each seed culture to 100 mL LB liquid medium containing 50 μg / mL kanamycin and incubate at 37℃ and 220 rpm until OD (dose retardation). 600 Add IPTG to a concentration of 96 μg / mL, set the pH to 0.4-0.6, and transfer to a shaker at 18℃ and 220 rpm for overnight incubation.

[0038] Protein in vitro purification: After culture, the three types of bacterial cells were centrifuged separately (4℃, 8000 rpm, 10 min) and washed twice with PBS buffer (pH 7.4); the bacterial cells were then disrupted using an ultrasonic disruptor at low temperature (400 W, on for 3 seconds, off for 5 seconds) to dissolve the protein in PBS buffer, and centrifuged at 4℃, 8000 rpm for 20 min to remove impurities after disruption. Protein purification was performed using a nickel column. The nickel column was washed and equilibrated with 10 mL of imidazole 10 buffer (20 mM NaH2PO4, 500 mM NaCl, and 10 mM imidazole). The supernatant after centrifugation was added to the nickel column through a 0.45 μm filter at a flow rate of 15 mL / h. Unbound contaminating proteins in the nickel column were washed with 10 mL of imidazole 20 buffer (20 mM NaH2PO4, 500 mM NaCl, and 20 mM imidazole). The target protein was eluted with 5 mL of imidazole 250 buffer (20 mM NaH2PO4, 500 mM NaCl, and 250 mM imidazole). Finally, the nickel column was washed with 5 mL of 0.5 M NaOH and stored in 20 mL of imidazole 10 buffer. The three target proteins eluted were verified by SDS-PAGE electrophoresis to confirm that their sizes were consistent with expectations (SucC approximately 43 kDa and SucD approximately 33 kDa). The target proteins were then stored in PBS buffer by ultrafiltration for subsequent use.

[0039] Protein ultrafiltration: Use an ultrafiltration tube with a pore size approximately 1 / 3 the size of the target protein for ultrafiltration (based on SucC 43 kDa and SucD 33 kDa, a 10 kDa ultrafiltration tube can be used). Replace the protein buffer with PBS. First, fill the ultrafiltration tube with deionized water and centrifuge at 4°C and 3000 g for 10-15 min. While rinsing the ultrafiltration tube, observe whether the ultrafiltration tube is intact, and then discard the deionized water. Fill the ultrafiltration tube with PBS buffer and centrifuge at 4°C and 3000 g for 10-15 min to further rinse the ultrafiltration tube. Add the three purified protein solutions respectively, then fill with PBS buffer, centrifuge at 4°C and 3000 g until the remaining liquid volume is about 1 mL, then fill with PBS buffer again, centrifuge at 4°C and 3000 g until the remaining liquid volume is 500-1000 μL. Take out the protein solution at this time for subsequent experiments.

[0040] The purity of the ultrafiltered protein was verified by SDS-PAGE electrophoresis, and the protein was destained with Coomassie Brilliant Blue and photographed using a gel imaging system.

[0041] 3. Experiment to determine the activity of SucC, SucD, and SucCD lactyl-CoA synthases. The activity of lactyl-CoA synthase was measured based on an in vitro enzymatic reaction system. The enzyme activity was quantified by detecting the consumption of the substrate L-lactic acid during the reaction using an ELISA kit and the consumption of the substrate ATP during the reaction using a direct reaction ATP assay kit. The enzyme activity was positively correlated with the consumption of lactic acid and ATP.

[0042] Preparation of the enzyme-catalyzed reaction system (200 μL): The system contains 20 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 50 mM L-lactic acid, 0.6 mM coenzyme A (CoA), 0.8 mM ATP, and 30 μM purified target protein (SucC protein, SucD protein, and SucC-SucD complex (SucCD)). Make up the volume of the system with enzyme-free pure water.

[0043] Three groups were set up: the blank control group was replaced with an equal volume of enzyme-free pure water to replace the target protein, the heat-inactivated control group was added to the system after the target protein was inactivated by heating at 95℃ for 5 min, and the experimental groups were the reaction systems corresponding to the three purified target proteins mentioned above. Each group was set up with three biological replicates.

[0044] Reaction conditions: The prepared reaction system was placed in a dark environment and incubated at 30°C for 1 h. Immediately after incubation, the L-lactic acid and ATP consumption were measured using a kit.

[0045] Detection method: (1) L-Lactate consumption detection: The L-lactate ELISA kit was used according to the instructions: 1) Add the reacted sample and L-lactate standards of varying concentrations to the 96-well microplate provided with the kit, 100 μL per well, and set up blank wells; 2) Add 50 μL of enzyme-labeled reagent to each well, gently shake to mix, and incubate at 37℃ for 30 min; 3) After incubation, discard the liquid in the wells, wash each well thoroughly 5 times with washing buffer, and pat dry any remaining liquid; 4) Add 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B to each well, mix well, and incubate at 37℃ in the dark for 15 min; 5) Add 50 μL of stop solution to each well, mix well, and within 10 min, measure the absorbance (OD) of each well at a wavelength of 450 nm. 450 ).

[0046] (2) ATP consumption detection: A direct reaction ATP content detection kit was used, and the procedure was followed according to the instructions: 1) Add the reacted sample and gradient concentrations of ATP standards to each well of a 96-well plate, 50 μL per well; 2) Add 50 μL of the kit's reaction solution to each well and gently shake to mix; 3) Incubate at 30°C for 15 min in a dark environment; 4) Immediately after incubation, measure the absorbance (OD) of each well at a wavelength of 560 nm. 560 ).

[0047] Enzyme activity calculation: A standard curve was plotted based on the absorbance values ​​of L-lactic acid standards at gradient concentrations. The remaining amount of L-lactic acid in each reaction system was calculated by substituting the sample absorbance values ​​into the curve. The lactic acid consumption was then calculated by the difference between the initial addition and the remaining amount. Simultaneously, an ATP standard curve was plotted based on the absorbance values ​​of ATP standards at gradient concentrations. The remaining amount of ATP in each reaction system was calculated by substituting the corresponding sample absorbance values ​​into the curve. The ATP consumption was then calculated by the difference between the initial addition (0.8 mM) and the remaining amount (in the lactyl-CoA synthesis reaction, L-lactic acid and ATP consumption are in an equimolar ratio, allowing for cross-validation of the results). The specific activity of lactyl-CoA synthetase is defined as: the number of micromoles of L-lactic acid consumed per μmol of protein per minute under light-protected conditions at 30°C. - ¹・μmol - ¹); The specific activities of lactyl-CoA synthases of SucC protein, SucD protein and SucC-SucD complex were calculated respectively, and the differences in enzyme activities among the three were statistically analyzed.

[0048] 5. Results and Analysis This embodiment focuses on synthetic biological genetic elements that enhance the lactic acid metabolic flux of actinomycetes. Through experiments such as plasmid construction, protein expression and purification, and enzyme activity assays, it systematically verifies the... sucC Gene, sucD The functions of genes and co-expression elements, and the results of each step are as follows: (1) Results of construction of recombinant plasmids and recombinant strains like Figure 4 As shown, homologous arms were obtained by PCR amplification. sucC Gene fragment (approximately 1200 bp) sucD Gene fragment (approximately 900 bp) and linearized pET-28a vector (approximately 5000 bp) were obtained by agarose gel electrophoresis. The results showed that the size of each fragment was consistent with the expected size, with no interference from other bands. After gel recovery, high-purity target fragments and linear vectors were obtained.

[0049] like Figure 5 As shown, after seamless cloning ligation, single-clone sequencing results indicate that... sucC Gene, sucDAll genes were correctly inserted into the designated positions in the pET-28a vector, and the nucleotide sequences perfectly matched the designed sequences, with no base mutations, deletions, or frameshifts, confirming the correct insertion of pET-28a-s. ucC pET-28a- sucD and pET-28a- sucC - SucD Three recombinant plasmids were successfully constructed. After transforming the recombinant plasmids into BL21 competent cells, the cells were screened for kanamycin resistance to obtain a recombinant expression strain BL21 / pET-28a- with good growth. sucC BL21 / pET-28a- sucD and BL21 / pET-28a- sucC - sucD This lays the foundation for subsequent in vitro protein expression.

[0050] (2) The SucC monomer, SucD monomer and SucC-SucD complex (SucCD) were successfully expressed and purified in vitro. SDS-PAGE electrophoresis results showed that ( Figure 6 Following IPTG induction, nickel column affinity chromatography, and ultrafiltration purification, the SucC monomer, SucD monomer, and SucC-SucD complex were successfully expressed and purified in vitro. The SucC-SucD complex showed detectable specific bands corresponding to both monomers, consistent with the expected theoretical molecular weights. Coomassie brilliant blue staining and gel imaging analysis after destaining confirmed that the purity of the target products exceeded 90%, meeting the requirements for in vitro enzyme activity assays. Furthermore, ultrafiltration successfully replaced the protein buffer with PBS buffer, effectively removing residual imidazole and high concentrations of NaCl, providing a suitable environment for subsequent enzymatic reactions.

[0051] (3) The SucC-SucD complex of actinomycetes has the ability to metabolize lactate and synthesize lactyl-CoA. The activities of SucC monomer, SucD monomer, and SucC-SucD complex were quantified using a dual-indicator ELISA kit and a direct-reaction ATP assay kit. Figure 7 The experimental results showed that all three recombinant proteins could mediate the consumption of lactate and ATP, and the consumption level was directly related to the activity of lactyl-CoA synthesis from metabolized lactate. Among them, the lactate and ATP consumption of the SucC-SucD complex was significantly higher than that of the SucC monomer and the SucD monomer, and its specific activity was also significantly higher than that of the two monomers expressed alone. This confirms that the complex formed by co-expression of SucC and SucD has a significant synergistic catalytic effect, which can drive lactate metabolism-related reactions more efficiently and can be used as a genetic element to enhance the lactate metabolism capacity of actinomycetes.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A genetic element that enhances the lactic acid metabolic flux of actinomycetes, characterized in that, The genetic element is a SucC-SucD co-expression element, which is integrated... sucC Genes and sucD Genes enable the simultaneous expression of the expression products of two genes and their assembly to form a functional complex; The sucC The nucleotide sequence of the gene is shown in SEQ ID NO.

1. sucD The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. The genetic element as claimed in claim 1, characterized in that, The sucC Genes and the sucD The genes are linked by an RBS sequence, the nucleotide sequence of which is shown in SEQ ID NO.

5.

3. The genetic element as claimed in claim 1, characterized in that, The sucC Genes and the sucD The gene originates from Streptomyces azureense ( Streptomyces coelicolor ).

4. The genetic element as claimed in claim 1, characterized in that, The protein expressed by the genetic element has lactoyl-CoA synthase activity, which can catalyze the combination of lactic acid and coenzyme A to generate lactoyl-CoA, thereby enhancing the lactic acid metabolic flux of actinomycetes.

5. The application of the genetic element as described in any one of claims 1-4 in the modification of the lactic acid metabolic pathway in actinomycetes.

6. The use of the genetic element as described in any one of claims 1-4 in enhancing the lactic acid metabolic flux of actinomycetes.

7. The use of the genetic element as described in any one of claims 1-4 in constructing an in vitro lactate metabolism system.

8. A method for accelerating the lactic acid metabolism rate of actinomycetes, characterized in that, The step includes introducing the genetic element of any one of claims 1-4 into actinomycetes.

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