Construction method and application of engineered strain of streptomyces mobaraensis producing phospholipase a1

Abstract

The application discloses a construction method and application of a streptomyces mobaraensis engineering strain for producing phospholipase A1, and belongs to the technical field of enzyme engineering and food processing. The application provides a streptomyces mobaraensis engineering strain for efficiently synthesizing phospholipase A1, and heterogeneously expresses a codon-optimized phospholipase A1 coding gene on the basis of streptomyces mobaraensis smYS1-ΔG11-ΔG8-Δ tg The application also discloses fermentation for producing enzymes by using the engineering strain, and enzymatic properties of the purified saPLA1 and application of the saPLA1 in degumming of soybean oil.The recombinant saPLA1 can reduce the phosphorus content of soybean oil from 113.01 mg / kg to below 7.36 mg / kg.The food-grade expression system constructed by the application has the advantages of high safety, high expression amount and low production cost, the saPLA1 has high degumming efficiency, and the application provides a new scheme for industrial production of food-grade PLA1 and realization of efficient oil degumming.

Description

Technical Field

[0001] This invention relates to the construction method and application of an engineered strain of Streptomyces morihara that produces phospholipase A1, belonging to the fields of enzyme engineering and food processing technology. Background Technology

[0002] Phospholipase A1 (EC 3.1.1.32, PLA1 for short) is a class of enzymes that specifically catalyze the breakdown of phospholipid molecules. Sn-1 Esterases that hydrolyze acyl bonds produce free fatty acids and lysophospholipids. This enzyme has significant applications in enzymatic degumming of oils and fats and is widely used in phospholipid modification, preparation of natural emulsifiers (lysophospholipids), and biosensing detection. Currently, phospholipase A1 (PLA1), which has important industrial applications, is distributed in various organisms in nature, with its main industrial production sources concentrated in [unclear - likely referring to specific regions or regions]. Serratia and Aspergillus However, the fermentation levels of wild-type strains are generally low, with yields often below 5 U / mL, while fungal expression systems face risks of glycosylation heterogeneity and enzyme activity instability. To improve yield, methods based on... Escherichia coli , Pichia pastoris and Bacillus subtilis Heterologous expression systems for multiple hosts, etc. However, all of the above systems have significant drawbacks: E. coli It easily produces endotoxins and forms inclusion bodies; P. pastoris Relying on methanol induction, which poses safety risks; while in food-grade hosts B. subtilis When expressed in [a specific location], enzyme activity is low. Although some studies have shown that... Streptomyces lividans While this bacterium achieves high enzyme activity, its fermentation requires the addition of antibiotics, resulting in high costs and making it unsuitable for food applications.

[0003] Despite the presence of Streptomyces mobara ( Streptomyces mobaraensis As a GRAS-certified food-grade host, it possesses mature fermentation technology and efficient secretion capabilities, and has been successfully applied to the production of transglutaminase (TGase). However, directly utilizing it for efficient expression of PLA1 still faces challenges. Previous studies have explored mutant strains obtained through mutagenesis and chassis strains smYS1-Δ constructed through gene editing. tg This lays the foundation for PLA1 expression; however, its expression efficiency, process stability, and final fermentation level still need further optimization and improvement. Therefore, how to achieve efficient, stable, and low-cost industrial production of PLA1 in this safe host is a core issue that urgently needs to be addressed to break through technological bottlenecks and meet the needs of the food industry. Summary of the Invention

[0004] This invention provides a *Streptomyces mogulata* chassis cell, wherein the chassis cell is obtained by knocking out the genomic island GIs gene, endogenous CRISPR / Cas system gene, and NHEJ repair pathway gene of *Streptomyces mogulata* smY2019 as the starting strain. ΔG5 C3 (described in the text of Chinese invention patent application with publication number CN120230690A); in smY2019 ΔG5 Based on C3, knock out the GI-8 and GI-11 genes in the genome;

[0005] The GI-8 gene is numbered in NCBI as follows: K7I03_23670, K7I03_23675, K7I03_23680, K7I03_23685, K7I03_23690, K7I03_23695, K7I03_23700. The GI-11 gene is numbered in NCBI as follows: K7I03_25135, K7I03_25140, K7I03_25145, K7I03_25150, K7I03_25155, K7I03_25160, K7I03_25165.

[0006] The NCBI numbers of the genes in the genomic island GIs are K7I03_28245, K7I03_28250, K7I03_28255, K7I03_28260, K7I03_28265, K7I03_28270, K7I03_28275, K7I03_28280, K7I03_28285, K7I03_28290, K7I03_28295, K7I03_28300, K7I03_28305, K7I03_28310, K7I03_28315, K7I03_28320, K7I03_28325, K7I03_28330, K7I03_28335, K7I03_28340, K7I03_28345, K7I03_28350, K7I03_28355, K7I03_28360, K7I03_28365, K7I03_28370, K7I03_28375, K7I03_28380, K7I03_28385, K7I03_28390, K7I03_28395, K7I03_28400, K7I03_28405, K7I03_28410, K7I03_28415, K7I03_28420, K7I03_28425, K7I03_28430, K7I03_28435, K7I03_28835, K7I03_28840, K7I03_28845, K7I03_28850, K7I03_28855, K7I03_28860, K7I03_28865, K7I03_28870, K7I03_28875, K7I03_28880, K7I03_28885, K7I03_28890, K7I03_28895, K7I03_28900, K7I03_28905, K7I03_28910, K7I03_28915, K7I03_28920, K7I03_28925, K7I03_28930, K7I03_28935, K7I03_28940, K7I03_28945, K7I03_28950, K7I03_28955, K7I03_28960, K7I03_28965, K7I03_28970, K7I03_28975, K7I03_28980, K7I03_28985, K7I03_28990, K7I03_28995, K7I03_29000, K7I03_29005, K7I03_18425, K7I03_18430, K7I03_18435, K7I03_18440, K7I03_18445, K7I03_18450, K7I03_18455,K7I03_18460, K7I03_18465, K7I03_18470, K7I03_18475, K7I03_18480, K7I03_18485, K7I03_18490, K7I03_18495, K7I03_18500, K7I03_1 8505, K7I03_18510, K7I03_18515, K7I03_18520, K7I03_18525, K7I03_18530, K7I03_18535, K7I03_18540, K7I03_18545, K7I03_18550, K7I 03_18555、K7I03_18560、K7I03_18565、K7I03_18570、K7I03_18575、K7I03_18580、K7I03_18585、K7I03_18590、K7I03_18595、K7I03_1860 0, K7I03_18605, K7I03_18610, K7I03_03350, K7I03_03355, K7I03_03360, K7I03_03365, K7I03_03370, K7I03_03375, K7I03_03380, K7I03_ 03385, K7I03_03390, K7I03_03395, K7I03_03400, K7I03_03405, K7I03_03410, K7I03_03415, K7I03_03420, K7I03_03425, K7I03_03430, K 7I03_03435, K7I03_03440, K7I03_03445, K7I03_03450, K7I03_03455, K7I03_03460, K7I03_03465, K7I03_01620, K7I03_01625, K7I03_016 30, K7I03_01635, K7I03_01640, K7I03_01645, K7I03_01650, K7I03_01655, K7I03_01660, K7I03_01665, K7I03_01670, K7I03_01675, K7I0 3_01680, K7I03_01685, K7I03_01690, K7I03_01695, K7I03_01700, K7I03_01705, K7I03_01710, K7I03_01715, K7I03_01720, K7I03_01725K7I03_01730; the endogenous CRISPR / Cas system genes are the CRISPR1 and CRISPR2 genes, wherein the CRISPR1 gene is located at positions 1358697 to 1368646 on the genome of *Streptomyces mobara* smY2019, and the CRISPR2 gene is located at positions 1437389 to 1442520 on the genome of *Streptomyces mobara* smY2019; the NCBI numbers of the NHEJ repair pathway genes are K7I03_29600, K7I03_29605, and K7I03_29610, respectively.

[0007] In one embodiment, the chassis cell further includes the following improvement: integration of PkasO* at the attb site on the genome. bldD gene overexpressed by the Sp44 promoter;

[0008] The nucleotide sequence of the attb site is shown in SEQ ID NO.1, and the PkasO* The nucleotide sequence of the Sp44 promoter is shown in SEQ ID NO.2, and the nucleotide sequence of the bldD gene is shown in SEQ ID NO.3.

[0009] The present invention also provides a method for preparing TGase enzyme, wherein the enzyme is prepared by fermentation of the above-mentioned Streptomyces molybdenum chassis cells.

[0010] The present invention also provides a method for expressing a target protein, wherein the method comprises using the above-mentioned *Streptomyces molybdenum* chassis cells as host cells to express the target protein.

[0011] The present invention also provides a recombinant Streptomyces mogularia, wherein the recombinant Streptomyces mogularia uses the aforementioned Streptomyces mogularia chassis cells as host cells, knocks out the TGase enzyme on the genome, its GenBank number is UBI36310.1, and integrates the saPLA1 gene expression frame shown in SEQ ID NO.4 at the attb site on the genome.

[0012] This invention also provides a recombinant *Streptomyces mobara* strain, wherein the recombinant *Streptomyces mobara* strain uses the aforementioned *Streptomyces mobara* chassis cells as host cells, knocks out the TGase enzyme in its genome (GenBank ID: UBI36310.1), and sequentially integrates the saPLA1 gene expression cassette (SEQ ID NO.4) and PkasO* gene expression cassette (SEQ ID NO.4) at the attb site in its genome. bldD gene overexpressed by the Sp44 promoter.

[0013] The nucleotide sequence of the attb site is shown in SEQ ID NO.1, and the PkasO* The nucleotide sequence of the Sp44 promoter is shown in SEQ ID NO.2, and the nucleotide sequence of the bldD gene is shown in SEQ ID NO.3.

[0014] This invention provides a strain for the efficient synthesis of phospholipase A1. S. mobaraensis Engineered bacteria, in *Streptomyces mobara* smYS1-ΔG11-ΔG8-Δ tg Based on this, the codon-optimized phospholipase A1 encoding gene was heterologously expressed, and the properties of recombinant saPLA1 were determined. Finally, the efficient application of phospholipase A1 was achieved by optimizing the lipid degumming parameters.

[0015] In one embodiment, the S. mobaraensis The engineered bacteria also have at least one of the following improvements:

[0016] (1) Using promoter P gapdh The expression cassette of the saPLA1 gene was constructed using TGase signal peptide and TGase terminator for heterologous expression.

[0017] (2) The saPLA1 gene expression cassette was integrated into the chromosome of the chassis strain via φC31 integrase. attB Sites are used to achieve stable expression;

[0018] In one embodiment, the S. mobaraensis The engineered bacteria were strains smYS1-ΔG11-ΔG8-Δ from the chassis. tg Based on this, using the strong promoter P gapdh We constructed an efficient expression cassette for the saPLA1 gene using TGase signal peptide.

[0019] In one embodiment, the S. mobaraensis The engineered bacteria were strains smYS1-ΔG11-ΔG8-Δ from the chassis. tg Based on this, the saPLA1 gene expression cassette was integrated into the chromosome via the integration vector pSET152. attB Sites that enable stable expression without antibiotic selection.

[0020] In one embodiment, the nucleotide sequence of the saPLA1 gene is shown below (SEQ ID NO.5):

[0021] gccgccggcggctacgtcgcgctgggcgactcctactcctccggcgtgggcgccggctcgtacgactccgggtccggcgactgccggcggaccccgaaggcctacccggccctgtgggccgccgcgaactccccggcctccttcgacttcgtggcctgcagcggcgcggtcacctcggacgtcctgaacaagcagatggggcccctgaactcgtcgacgtccctggtctccctgaccatcggggggaacgacgccggcttcgccgacgtgatgaccacctgcgtcctgcagagcgaggccaactgcatcgcccgcgtcaacaccgccaaggccttcgtcgagtccaccctccccggccgcctggactccgtgtactcccaggtgcgcgccaaggccccctcggccaacgtcgtcgtgctgggctacccccggttctacaagctgaacggcacctgcgtcgccggcctcaccgagggggagcgcaccgccatcaacggcgcggccgacctgctcaactcggtgatctccaagcgcgccgccgaccacggctacgcctacggcgacatcgccgcggccttcaccggccacgagatctgctccggcgactcgtggctgcactccgtcaagtggaccggcatcaacgactcgtaccacccgaccgccgccggccagagcggcggctacctgccggtgctgaactccaaggcc

[0022] Promoter P gapdh The nucleotide sequence of the gene is shown below (SEQ ID NO.6):

[0023] gctgctccttcggtcggacgtgcgtctacgggcaccttaccgcagccgtcggctgtgcgacggacggatcgggcgaactggccgatgctgggagaagcgcgctgctgtacggcgcgcaccgggtgcggagcccctcggcg agcggtgtgaaacttctgtgaatggcctgttcggttgctttttttatacggctgccagataaggcttgcagcatctgggcggctaccgctatgatcggggcgttcctgcaattcttagtgcgagtatctgaaaggggatacgc

[0024] The nucleotide sequence of the TGase signal peptide gene is shown below (SEQ ID NO.7):

[0025] atgtcccaacgcgggagaactctcgtcttcgccgctctcggtgcggtcatgtgcaccaccgcgttaatgccgtccgcaggcgcggccaccggcagtggcagtggcagcggcaccgggg aagagaagaggtcctacgccgaaacgcaccgcctgacggcggatgacgtcgacgacatcaacgcgctgaacgaaagcgctccggccgcttcgagcgccggtccgtccttccgggccccc

[0026] The nucleotide sequence of the TGase terminator gene is shown below (SEQ ID NO.8):

[0027] ggaggggaggggaggcggagcatccggctcccctccccacc

[0028] In one embodiment, the recombinant vector pSET152-P gapdh -saPLA1 contains P gapdh Promoter, TGase signal peptide coding sequence, saPLA1 gene coding region, His tag sequence, and TGase terminator sequence.

[0029] In one embodiment, the S. mobaraensis The engineered bacteria were strains smYS1-ΔG11-ΔG8-Δ from the chassis. tgBased on this, the codon-optimized saPLA1 gene, derived from Streptomyces albopictus, was expressed. Streptomyces albidoflavus NA297.

[0030] In one embodiment, a fermentation medium is used for the above-mentioned... S. mobaraensis The engineered bacteria were cultured in a fishmeal fermentation medium, the components of which were 2% glycerol, 7.5% fishmeal peptone, 0.5% yeast extract, 0.55% corn steep liquor powder, 0.55% ammonium sulfate, 0.2% MgSO4, and 0.2% K2HPO4.

[0031] In one embodiment, the engineered strain of *Streptomyces molybdenum* was fermented in fishmeal fermentation medium at 30°C and 220 rpm for 48 h, and the extracellular phospholipase A1 activity reached 54.7 U / mL.

[0032] In one embodiment, the culture medium includes, but is not limited to, fishmeal fermentation medium and basal fermentation medium.

[0033] In one embodiment, the gene integration site is S. mobaraensis On chromosomes attB Site.

[0034] In one embodiment, the microorganisms include, but are not limited to, those mentioned above. S. mobaraensis .

[0035] The present invention also provides the above. S. mobaraensis The application of engineered bacteria in the production of phospholipase A1 requires protection of the bacteria. S. mobaraensis Application of recombinant phospholipase A1 expressed by engineered bacteria in the field of oil degumming.

[0036] In one embodiment, the optimized conditions for the recombinant phospholipase A1 to degumme soybean crude oil are: temperature 45℃, pH 5.5, enzyme addition amount 300U / 100g oil, reaction time 2.5 h, and stirring speed 250rpm.

[0037] In one embodiment, the recombinant phospholipase A1 can reduce the phosphorus content in crude soybean oil from 113.01 mg / kg to below 7.36 mg / kg, meeting the requirements of physical refining.

[0038] The present invention also provides a method for degumming soybean crude oil, wherein the method comprises adding the above-mentioned recombinant Streptomyces mogulata, or the recombinant enzyme obtained by fermentation of the above-mentioned recombinant Streptomyces mogulata, to soybean crude oil for reaction and degumming.

[0039] In one embodiment, the reaction conditions are: 250 rpm, pH 4.5-7.0, and reaction time: 2.5-3 h.

[0040] In one embodiment, the amount of recombinant enzyme added is 200 U / 100g-350 U / 100g.

[0041] The present invention also provides the application of the above-mentioned *Streptomyces molybdenum* chassis cells, or the above-mentioned recombinant *Streptomyces molybdenum*, in the degumming of soybean crude oil or in the preparation of products degummed from soybean crude oil.

[0042] Beneficial effects

[0043] This invention uses the food-grade GRAS strain *Streptomyces morihara* smYS1-Δ tg Using a host cell as the primary host, a safe and efficient heterologous expression system was constructed without antibiotic selection or inducer induction. This system successfully achieved highly efficient secretory expression of phospholipase A1 (saPLA1), with extracellular enzyme activity reaching 64.3 U / mL after 48 hours of fermentation, 42.6 times the yield of the original host cell. Furthermore, the fermentation medium used inexpensive fish peptone as the main nitrogen source, significantly reducing production costs. The obtained recombinant saPLA1 exhibits excellent enzymatic properties; its optimal operating conditions (50℃, pH 6.0) and broad pH stability (pH 5-8) are suitable for industrial environments, and it is Ca-independent. 2+ Furthermore, its citric acid-activated property fundamentally solves the problem of activity inhibition of traditional calcium-dependent PLA1 in the degumming process. Ultimately, under optimized conditions, this enzyme alone can reduce the phosphorus content of crude soybean oil from 113.01 mg / kg to below 7.27 mg / kg within 2.5 hours, meeting the physical refining standard, thus establishing a new, efficient, economical, and simplified enzymatic degumming process. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the expression vector for the saPLA1 gene and a schematic diagram of the construction process of the engineered strain.

[0045] Figure 2 For the shake-flask fermentation process smYS1-Δ tg ,smYS1-Δ tg -saPLA1、smYS1-ΔG11-ΔG8-Δ tg -saPLA1、smYS1-ΔG11-ΔG8-Δ tg -saPLA1-bldD strain saPLA1 enzyme activity change over time curve.

[0046] Figure 3 smYS1-Δ tgSDS-PAGE analysis of the fermentation supernatant of -saPLA1, where positions 1-6 in the figure are fermentation culture supernatant samples extracted at 12, 24, 36, 48, 60 and 72 hours, respectively.

[0047] Figure 4 SDS-PAGE analysis of purified saPLA1.

[0048] Figure 5 Enzymatic properties analysis of saPLA1.

[0049] Figure 6 This is a schematic diagram of the saPLA1 grease degumming process.

[0050] Figure 7 This is a schematic diagram showing the parameter optimization results of the saPLA1 grease degumming process. Detailed Implementation

[0051] The culture media involved in the following examples:

[0052] GYM solid medium: 1% glucose, 0.4% yeast extract, 0.3% malt extract, 2% agar powder;

[0053] Seed culture medium: 2% glycerol, 2% peptone, 0.5% yeast extract, 0.2% K2HPO4, 0.2% MgSO4, pH 7.2;

[0054] Fishmeal fermentation medium: 2% glycerol, 7.5% fishmeal peptone, 0.5% yeast extract, 0.55% corn steep liquor powder, 0.55% ammonium sulfate, 0.2% MgSO4, 0.2% K2HPO4, pH 7.0-7.2;

[0055] The fermentation conditions of *Streptomyces mobara* involved in the following examples:

[0056] Will S. mobaraensis The strain was cultured on GYM solid plates at 30°C for 4-5 days. Spores and mycelia were scraped off using an inoculation loop and transferred to a shake flask containing 30 mL of seed culture medium. The flask was then incubated at 30°C and 220 rpm for 24 h to obtain the desired results. S. mobaraensis Seed culture: Inoculate the seed culture into a shake flask containing 30 mL of fishmeal fermentation medium at an inoculation rate of 8% (v / v) and ferment at 30°C and 220 rpm.

[0057] The following examples illustrate the TGase enzyme activity assay method:

[0058] Every 12 hours, take 1 mL of fermentation broth, centrifuge at 10,000 rpm for 10 min at 4℃, collect the supernatant and keep it on ice. Add 60 μL of TGase sample solution to 150 μL of enzyme activity test substrate and mix well. Incubate in a metal bath at 37℃ for 10 min, then immediately add 60 μL of TGase stop solution. Centrifuge the reaction solution at 10,000 rpm for 1 min, and measure the absorbance of 200 μL of supernatant at 525 nm. For the blank control, first add 60 μL of enzyme activity test stop solution to 60 μL of enzyme sample solution, incubate at 37℃ for 10 min, and then add 150 μL of enzyme activity test substrate.

[0059] The following examples illustrate the methods for determining mycelial growth:

[0060] This study used the diphenylamine colorimetric method to determine the total DNA content to characterize bacterial biomass. Samples were taken every 12 hours, and bacterial cells were collected by centrifugation at 10000 r / min for 10 minutes at 4°C. After discarding 500 μL of supernatant, the bacterial pellet was washed twice with PBS buffer. The bacterial cells were resuspended in diphenylamine chromogenic solution (containing 1.5 g diphenylamine, 100 mL glacial acetic acid, 1.5 mL concentrated sulfuric acid, and 1 mL 1.6% acetaldehyde), vortexed for 1 minute, and then incubated in a 60°C water bath in the dark for 1 hour for color development. After the reaction was complete, the sample was centrifuged at 12000×g for 1 minute, and the absorbance of the supernatant was measured at a wavelength of 595 nm. All operations required protection from light, and the color development time had to be strictly controlled.

[0061] The following examples illustrate the method for determining the hydrolytic activity of saPLA1:

[0062] Add 200 μL of 5% (v / v) lecithin substrate to a test tube and preheat at 37°C for 5 minutes. Then add 50 μL of enzyme solution, mix well, and incubate at 37°C for 10 minutes. After the reaction is complete, immediately add 50 μL of 6 mol / L HCl and 300 μL of 95% ethanol to terminate the reaction. Mix thoroughly, then add 150 μL of isooctane and shake vigorously for 90 seconds. Let the mixture stand at 65°C until it separates into layers. After cooling to room temperature for 5 minutes, take 100 μL of the supernatant and mix it with 400 μL of isooctane and 100 μL of pyridine-copper salt colorimetric reagent, and shake for 90 seconds. After the solution is clear, take the upper organic phase and measure the absorbance at 715 nm. The blank control is set by adding the reaction termination solution first, followed by the enzyme solution, and the remaining steps are exactly the same as the experimental group.

[0063] Under certain conditions (constant temperature reaction at 37℃ for 10 minutes), the amount of phospholipase A1 required to generate 1 μmol of palmitic acid within 1 minute is defined as 1 enzyme activity unit.

[0064] The calculation formula is:

[0065] .

[0066] In the formula: U is the phospholipase A1 enzyme activity (U / mL); v is the volume of palmitic acid isooctane solution taken when measuring the standard curve (mL); V1 is the total volume of the colorimetric system (mL); V2 is the volume of supernatant taken from the reaction system (mL); C is the palmitic acid concentration (μmol / mL); n is the enzyme solution dilution factor; t is the enzyme reaction time (min); m is the enzyme dosage (mL).

[0067] The purification and desalting methods of saPLA1 involved in the following examples:

[0068] The supernatant was collected after 48 h of fermentation of the recombinant strain and purified by affinity chromatography using PrePack Ni-NTAPurose 6 FF from Jiaxing Qianchun Biotechnology Co., Ltd. Fractions with PLA1 activity were eluted using a buffer consisting of 50 mM Tris-HCl, 0.2 M NaCl, and 500 mM imidazole (pH 7.4). Subsequently, the eluted fractions were desalted using a 10 kDa ultrafiltration tube.

[0069] The following examples involve the determination of the enzymatic properties of saPLA1:

[0070] Purified saPLA1 was used for catalytic characterization. Optimal temperature determination was performed within the range of 30-70℃, with reaction points set at 5℃ intervals. Relative enzyme activity at each temperature was calculated with the highest enzyme activity as 100%. For thermostability assessment, the enzyme solution was incubated for 30 minutes within the range of 25-60℃ (each 5℃ representing an experimental group), followed by measurement of residual enzyme activity. Relative activity was again calculated with the enzyme activity of the untreated sample as 100%.

[0071] To determine the optimal reaction pH, three buffer systems were used to adjust the reaction environment: 50 mM acetate-sodium acetate buffer (pH 4.0-5.5), 50 mM potassium phosphate buffer (pH 6.0-7.5), and 50 mM Tris-HCl buffer (pH 8.0-9.0). Enzyme activity was measured at 50°C, and the highest value was used to calculate the relative enzyme activity as 100%. pH stability was determined by incubating the enzyme solution in different pH buffers at 4°C for 1 hour, and then calculating the percentage of residual activity using the same method.

[0072] The effects of various metal ions (CaCl2, CoCl2, MgCl2, MnCl2, CuCl2, FeCl2, FeCl3, and AlCl3) at a final concentration of 10 mM, as well as 10 mM EDTA, 0.1% (w / v) Triton X-100, 0.1% (w / v) SDS, and 100 mM citric acid (to adjust the substrate pH to 7.2) on the activity of saPLA1 enzyme were investigated. The reaction system without any added reagents served as a control (set as 100%), and the relative enzyme activities under each condition were calculated.

[0073] The following embodiments involve optimization of the grease degumming process:

[0074] Based on typical vegetable oil enzymatic degumming process ( Figure 6 The catalytic conditions for saPLA1 were optimized. 100.0 g of crude soybean oil was accurately weighed and heated to 80°C in a constant-temperature water bath. 150 μL of 45% (w / w) citric acid solution was added, and the mixture was stirred at 80°C for 25 minutes. After cooling to the preset temperature, the pH of the system was adjusted with 4% (w / w) NaOH solution. Then, 5 mL of distilled water and an appropriate amount of saPLA1 enzyme solution (enzyme activity calculated as U / 100 g oil) were added, and the mixture was stirred at 500 r / min for 1 minute. Enzymatic degumming was then carried out at the set temperature. After the reaction, the sample was heated in a 100°C water bath for 10 minutes to inactivate the enzyme. The enzyme-inactivated oil sample was centrifuged at 10000 r / min for 15 minutes, and the upper oil phase was collected and dried in a 103°C oven for 1 hour. After cooling, the sample was used for phosphorus content determination.

[0075] Oil phase pH determination method: Mix 2 mL of oil-water emulsion with 2 mL of deionized water, centrifuge at 10000 r / min for 10 minutes, collect the lower aqueous phase and determine the pH. The phosphorus content of the oil sample was determined using the AOCS Official Method Ca12-55.

[0076] S. mobaraensis protoplasmic electroconversion

[0077] Take 70 μL S. mobaraensisThe protoplast solution was mixed with 5-10 μg of the constructed integrative expression plasmid and transferred into a 0.1 cm electroporation cuvette (Bio-Rad, USA). The mixture was electroporated once at 2500 V and 25 Ω. Immediately after electroporation, 1 mL of pre-chilled 1 M sorbitol was added, and the mixture was then transferred to sterile 1.5 mL EP tubes and incubated at 30 °C and 220 rpm for 2 h. The culture was then spread onto GYM plates containing apramycin and incubated for 5-6 days. Positive transformants were screened by PCR verification and gene sequencing analysis.

[0078] The primer sequences involved in the following examples are shown in Table 1:

[0079] Table 1 Primer Sequences

[0080]

[0081] Note: The underlined sequence indicates a one-step cloning of homologous sequences.

[0082] The knockout genomic island GI genes involved in the following examples are shown in Table 2:

[0083] Table 2: Genome Island GIs Genes

[0084]

[0085] The knockout primers used in the following examples are shown in Table 3:

[0086] Table 3: Primers

[0087]

[0088] The specific genes involved, from GI-6 to GI-18, are shown in Table 4 below:

[0089] Table 4: Genes

[0090]

[0091] Example 1: Construction of Chassis Cells

[0092] The method for constructing smYS1 is described in publication number CN120230690A, where smYS1 is smY2019 prepared in Example 2. Δ G5 C3, named smY2019 in this application. Δ G5 C3.

[0093] 1. Knockout of GI-6 to GI-18 individually

[0094] The specific steps are as follows:

[0095] (1) Construction of gene knockout plasmid:

[0096] First, sgRNAs targeting the target genes (GI-6-GI-18, TGase, and bldD) were evaluated and designed on the CRISPOR website. Highly specific and efficient sgRNAs were selected for primer design and synthesis. The relevant primer sequences are shown in Table 3. The original plasmid pCRISPomyces-2-P6 was digested with restriction endonuclease BpiⅠ at 37℃ for 3-4 hours. After successful nucleic acid electrophoresis, the plasmid was purified and recovered. The synthesized sgRNA was cloned and ligated into the enzyme-digested vector in a one-step process to obtain plasmids containing the corresponding sgRNAs, namely pCRISPR-sg6, pCRISPR-sg7, pCRISPR-sg8, pCRISPR-sg9, pCRISPR-sg10, pCRISPR-sg11, pCRISPR-sg12, pCRISPR-sg13, pCRISPR-sg14, pCRISPR-sg15, pCRISPR-sg16, pCRISPR-sg17, pCRISPR-sg18, pCRISPR-sg1, pCRISPR-sg3, and pCRISPR-sgbldD. The above plasmids were further digested with restriction endonuclease XbaI and then purified and recovered. Using smYS1 as a template, primer pairs 6-U-1000-F / R, 6-D-1000-F / R, 7-U-1000-F / R, 7-D-1000-F / R, 8-U-1000-F / R, 8-D-1000-F / R, 9-U-1000-F / R, 9-D-1000-F / R, 10-U-1000-F / R, 10-D-1000-F / R, 11-U-1000-F / R, 11-D-1000-F / R, 12-U-1000-F / R, 12-D-1000-F / R, 13-U-1000-F / R were used. / R, 13-D-1000-F / R, 14-U-1000-F / R, 14-D-1000-F / R, 15-U-1000-F / R, 15-D-1000-F / R, 16-U-1000-F / R, 16-D-1000-F / R, 17-U-1000-F / R, 17-D-1000-F / R, 18-U-1000-F / R, 18-D-1000-F / R, dTG-UF / R, and dTG-DF / R amplified 1000bp of each of the upstream and downstream homologous arms of the target gene (GI-6-GI-18, TGase).The upstream and downstream homologous arms were ligated to the pCRISPR vector fragment carrying sgRNA in a one-step cloning process to obtain the target gene knockout plasmids: pCRISPR-ΔG6, pCRISPR-ΔG7, pCRISPR-ΔG8, pCRISPR-ΔG9, pCRISPR-ΔG10, pCRISPR-ΔG11, pCRISPR-ΔG12, pCRISPR-ΔG13, pCRISPR-ΔG14, pCRISPR-ΔG15, pCRISPR-ΔG16, pCRISPR-ΔG17, pCRISPR-ΔG18, pCRISPR-sg1-Δtg, and pCRISPR-sg3-Δtg.

[0097] (2) Transform all of the above recombinant plasmids. E. coli JM109 was used and verified by PCR and sequencing.

[0098] (3) Take 70 μL of smYS1 protoplast solution and mix it with 5-10 μg of the integrated expression plasmid constructed in (2). Transfer the mixture into a 0.1 cm electroporation cuvette and electroporate once at 2500 V and 25 Ω. Immediately after electroporation, add 1 mL of pre-cooled 1 M sorbitol and finally transfer it to a sterile 1.5 mL EP tube. Incubate at 30℃ and 220 rpm for 2 h. Spread the culture medium to a container containing 50 μg·mL⁻¹ of sorbitol. -1 Positive transformants were screened by PCR verification and gene sequencing analysis after culturing on GYM plates containing aspirin for 5-6 days.

[0099] Following the above method, recombinant strains with single-gene knockout were obtained respectively:

[0100] smYS1-ΔG6, smYS1-ΔG7, smYS1-ΔG8, smYS1-ΔG9, smYS1-ΔG10, smYS1-ΔG11, smYS1-Δ G12, smYS1-ΔG13, smYS1-ΔG14, smYS1-ΔG15, smYS1-ΔG16, smYS1-ΔG17, smYS1-ΔG18.

[0101] 2. The single-gene knockout recombinant strains (smYS1-ΔG6~smYS1-ΔG18) obtained in step 1 were fermented separately, as follows:

[0102] The recombinant bacterial transformants obtained from GYM plates were streaked onto the plates and incubated at 30°C for 4-5 days. Subsequently, a suitable amount of mycelium was scraped from the GYM solid plates using a sterile spreader, and the spore suspension was diluted to a final concentration of 10. 6Inoculate the seed culture medium with 8% (v / v) of fish meal at 30℃ and 220 rpm for 24-36 h, then transfer the inoculum to 250 mL Erlenmeyer flasks containing 30 mL of fish meal fermentation medium and continue culturing at 30℃ and 220 rpm for 84 h. The activity of TGase (transglutaminase, EC 2.3.2.13) in the fermentation broth was measured during fermentation. The NCBI ID for TGase is (GenBank: UBI36310.1). The results are shown in Table 5.

[0103] Table 5: TGase activity of recombinant strains obtained by knocking out different genes at different fermentation times

[0104]

[0105] The experimental results showed that, except for the recombinant strains with GI-10 and GI-17 knockouts, whose TGase activity did not change significantly, the enzyme activities of the other single-gene knockout strains were all increased to varying degrees. Among them, the GI-11 knockout strain smYS1-ΔG11 showed the most significant increase, with an enzyme activity reaching a maximum of 42.1 U / mL after 60 h of fermentation, which was 34.5% higher than the original strain smYS1 (31.3 U / mL). In addition, the biomass of the strains with GI-6, GI-7, GI-10, GI-13, GI-15, GI-16, and GI-18 knockouts increased significantly, with smYS1-ΔG15 showing the highest biomass.

[0106] 3. Based on the results of increased enzyme activity and biomass of the strains obtained in step 2, we screened out GI-6, GI-7, GI-8, GI-9, GI-13, GI-15, GI-16 and GI-18. Then, based on smYS1-ΔG11, we knocked out GI-6, GI-7, GI-8, GI-9, GI-13, GI-15, GI-16 or GI-18 to perform double gene knockout (the knockout method is the same as described in step 2).

[0107] The TGase activity of the strains obtained by double gene knockout was detected according to the method in step 2.

[0108] The results showed that among the double knockout strains, the strains that further knocked out GI-8 based on smYS1-ΔG11 had significantly increased TGase activity, specifically: smYS1-ΔG11-ΔG8 had the highest activity, reaching 50.13 U / mL, which was 19.1% higher than smYS1-ΔG11 and 60.2% higher than smYS1; the enzyme activities of the other double knockout strains did not change significantly.

[0109] The final chassis cell was: smYS1-ΔG11-ΔG8.

[0110] 4. Metabolic and physiological characterization of strain smYS1-ΔG11-ΔG8

[0111] The high-yielding TGase chassis strain smYS1-ΔG11-ΔG8 was evaluated in terms of gene and protein expression levels, cell metabolic status, and cell morphology.

[0112] The recombinant strains smYS1-ΔG11-ΔG8, smYS1, and smYS1-ΔG11 transformed from GYM plates were streaked onto the plates and incubated at 30°C for 4-5 days. Subsequently, a suitable amount of mycelium was scraped from the GYM solid plates using a sterile spreader, and the spore suspension was diluted to a final concentration of 10... 6 Inoculate the seed culture medium with 8% (v / v) of the fermentation supernatant and culture at 30°C and 220 rpm for 24-36 h. Then, transfer the supernatant to a 250 mL Erlenmeyer flask containing 30 mL of fishmeal fermentation medium and continue to culture at 30°C and 220 rpm for 84 h. The SDS-PAGE analysis of the fermentation supernatant provides direct evidence.

[0113] At the transcriptional level, qPCR analysis showed that the gene transcriptional activity of TGase gradually increased with gene knockout. Compared with the original strain smYS1, the transcriptional level of the single knockout strain smYS1-ΔG11 was significantly improved, while the transcriptional level of the double knockout strain smYS1-ΔG11-ΔG8 reached the highest level.

[0114] Electrophoresis patterns showed that, compared to smYS1, the smYS1-ΔG11 strain exhibited darker band staining near the corresponding TGase protein size (approximately 38 kDa); while the corresponding band was the most concentrated in the smYS1-ΔG11-ΔG8 strain. This indicates that gene knockout not only enhanced TGase transcription but also effectively promoted its protein translation and extracellular secretion efficiency.

[0115] 5. smYS1 ΔG11 ΔG8 PkasO* Sp44 Construction of bldD strain

[0116] (1) PkasO* respectively The Sp44 promoter and the bldD gene were sequentially ligated into the XbaI site of the pSET152 vector, resulting in pSET152-PkasO*. Sp44 bldD.

[0117] PkasO* Sp44 promoter (SEQ ID NO.2) sequence:

[0118] tgttcacattcgaaccgtctctgctTTGACAacatgctgtgcggtgttgTAAAGTctggtgtAggagaatacgacagcgtgcaggactgggggagtt

[0119] bldD gene (SEQ ID NO.3) sequence:

[0120] atgtccagcgaatacgccaaacagctcggggccaagctccgcgccatccgcacccagcagggcctctcactccatggtgtcgaggagaagtcccagggccgctggaaggccgtggtggcgggctc gtacgagcgcggcgaccgtgccgtgaccgtgcagcgccttgccgagctggccgacttctacggggtgcccgtccaggagctgctgcccggcaccacgcccggtggcgcggccgagcccccgccga agctggtcctggacctggagcgcctcgcccacgtcccggccgagaaggcgggcccgctccagcgttacgcggcgaccatccagagccagcgcggcgactacaacggcaaggtgctgtcgatccgc caggacgacctgcgcaccctcgccgtgatctacgaccagtcgccctcggtgctcaccgagcagctgatcagctggggcgtgctcgacgcggatgcgcgccgcgccgtgcagcacgaggacgcgtag

[0121] (2) Plasmid pSET152-PkasO* was transferred using a binding transfer method. Sp44 bldD was transformed into the attb site in smYS1-ΔG11-ΔG8 to obtain the recombinant strain: smYS1 ΔG11 ΔG8 PkasO* Sp44 bldD, the TGase enzyme activity of the obtained recombinant strain was detected according to the method in step 2.

[0122] The results show: smYS1 ΔG11 ΔG8 PkasO* Sp44 The enzyme activity of bldD reached 60.32 U / mL, compared to the chassis strain (smYS1). ΔG11 ΔG8) increased by 20.3%, while compared to the original starting strain smYS1, it increased significantly by 92.7%.

[0123] (3) Comparison of different promoters

[0124] The specific experiment is the same as steps (1) to (2), the difference is that different promoters are adjusted: according to the strength of the promoters, from high to low, they are: rpsL, tg-2, tg, ermE*, kasO*-Sp44, TipAL, kasO* and TipALS;

[0125] The promoter sequences obtained are rpsL, tg-2, tg, ermE*, kasO*-Sp44, TipAL, kasO*, and TipALS. Following steps (1) to (2), these sequences are applied to the constructed chassis strain smYS1. ΔG11 Overexpression of bldD in ΔG8 successfully yielded the following recombinant strain: smYS1 ΔG11 ΔG8 PrpsL bldD、smYS1 ΔG11 ΔG8 Ptg bldD、smYS1 ΔG11 ΔG8 Ptg 2 bldD、smYS1 ΔG11 ΔG8 PkasO* Sp44 bldD、smYS1 ΔG11 ΔG8 PkasO* bldD、smYS1 ΔG11 ΔG8 PermE* bldD、smYS1 ΔG11 ΔG8 PTipAL bldD and smYS1 ΔG11 ΔG8 PTipALS bldD. And detect TGase enzyme activity according to the method in step 2.

[0126] The sequences involved are shown in Table 6 below:

[0127] Table 6: Starter Sequence

[0128]

[0129] The results showed that among all recombinant strains, smYS1... ΔG11 ΔG8 PkasO* Sp44 bldD exhibited the highest enzyme activity; therefore, PkasO* was subsequently used. Sp44 promoter overexpresses the bldD gene.

[0130] Example 2: Construction of a recombinant strain overexpressing saPLA1 enzyme

[0131] The specific steps are as follows:

[0132] 1. smYS1-Δ tg ,smYS1-ΔG11-ΔG8-Δ tg Construction

[0133] Streptomyces mogulata ( S .mobaraensis Using the entire genome of DSM40587 as a template, primer dtg was used. FF / FR(dtg FF:gccggggcgttttttatctagCCGGTTGTGGGAGAATCCGGCCGC, SEQ ID NO.142; dtg FR:TCGGCTCTACAGCTCGTGGCCCGTC, SEQ ID NO.143) and dtg RF / RR(dtg RF:cacgagctgtagagccgaGGCTCCAAAACCGGCCACTCCGTCA,, SEQ ID NO.144; dtg RR:ttacggttcctggcctctagCATCGGCACCACGACCCTCACCGTC (SEQ ID NO.145) were used to amplify approximately 1000 bp of homologous sequences upstream and downstream of the tg gene using PCR. These fragments were then ligated into the pCRISPR vector using a one-step cloning method to obtain the plasmid pCRISPR. Δtg. The plasmid pCRISPR was transferred using a binding transfer method. Δtg was transformed into smYS1 and smYS1-ΔG11-ΔG8 respectively, and the primer Dver was used. F / R (Dver) F:GGTCTCCGGGATTTGTTCGCATGCC, SEQ ID NO.146; Dver The recombinant strain smYS1 was obtained by PCR verification using R:CATCCTCATGTTCGAACCCACGGGC (SEQ ID NO. 147). Δtg、smYS1-ΔG11-ΔG8-Δ tg .

[0134] 2. Construction of recombinant strains

[0135] (1) Construction of pET28a(+)-saPLA1 recombinant plasmid ( Figure 1 )

[0136] The saPLA1 enzyme was ligated to the pET28a(+) plasmid to prepare the recombinant plasmid: pET28a(+)-saPLA1.

[0137] (2) Construction of recombinant strains

[0138] Using the plasmid pET28a(+)-saPLA1, which was synthesized in its entirety by Sangon Biotech (Shanghai) Co., Ltd. and carries the coding sequence of saPLA1, as a template, the saPLA1 gene was amplified using primers saPLA1-F / R; pET28a(+)-P gapdh Using the template, primer gapdhp-F / R was used to amplify promoter P. gapdh Using genomic DNA of smY2019 (described in publication number CN120230690A) as a template, the complete TGase signal peptide sequence was amplified using primers TG signal-F / R; using pSET152 plasmid as a template, the linearized vector backbone was amplified using primers pSET152-His-F / pSET152-R.

[0139] saPLA1 gene expression cassette sequence (SEQ ID NO.4):

[0140] gctgctccttcggtcggacgtgcgtctacgggcaccttaccgcagccgtcggctgtgcgacacggacg gatcgggcgaactggccgatgctgggagaagcgcgctgctgtacggcgcgcaccgggtgcggagcccctcggcgag cggtgtgaaacttctgtgaatggcctgttcggttgctttttttatacggctgccagataaggcttgcagcatctgg gcggctaccgctatgatcggggcgttcctgcaattcttagtgcgagtatctgaaaggggatacgcATGTCCCAACG CGGGAGAACTCTCGTCTTCGCCGCTCTCGGTGCGGTCATGTGCACCACCGCGTTAATGCCGTCCGCAGGCGCGGCC ACCGGCAGTGGCAGTGGCAGCGGCACCGGGGAAGAGAAGAGGTCCTACGCCGAAACGCACCGCCTGACGGCGGATG ACGTCGACGACATCAACGCGCTGAACGAAAGCGCTCCGGCCGCTTCGAGCGCCGGTCCGTCCTTCCGGGCCCCCgc cgccggcggctacgtcgcgctgggcgactcctactcctccggcgtgggcgccggctcgtacgactccgggtccggc gactgccggcggaccccgaaggcctacccggccctgtgggccgccgcgaactccccggcctccttcgacttcgtgg cctgcagcggcgcggtcacctcggacgtcctgaacaagcagatggggcccctgaactcgtcgacgtccctggtctc cctgaccatcggggggaacgacgccggcttcgccgacgtgatgaccacctgcgtcctgcagagcgaggccaactgc atcgcccgcgtcaacaccgccaaggccttcgtcgagtccaccctccccggccgcctggactccgtgtactcccagg tgcgcgccaaggccccctcggccaacgtcgtcgtgctgggctacccccggttctacaagctgaacggcacctgcgt cgccggcctcaccgagggggagcgcaccgccatcaacggcgcggccgacctgctcaactcggtgatctccaagcgc gccgccgaccacggctacgcctacggcgacatcgccgcggccttcaccggccacgagatctgctccggcgactcgt ggctgcactccgtcaagtggaccggcatcaacgactcgtaccacccgaccgccgccggccagagcggcggctacct gccggtgctgaactccaaggccCACCATCACCATCACCAT TGATGTAAGCGG GGAGGGGAGGGGAGGCGGAGCATC CGGCTCCCCTCCCCACC

[0141] Note: The underscore is P. gapdh Promoter sequence; wavy underline indicates signal peptide sequence; dashed underline indicates PLA1 sequence; double wavy underline indicates His tag; double underline indicates terminator sequence.

[0142] The gene expression cassette described above was ligated to a vector using a one-step cloning method to obtain the plasmid pSET152-P. gapdh -saPLA1. The plasmid pSET152-P was transferred using a binding transfer method. gapdh -saPLA1 is converted to smYS1-Δ tg ,smYS1-ΔG11-ΔG8-Δ tg The attb site was identified, and PCR verification was performed using primers very-attb-F / R to obtain the recombinant strain smYS1-Δ. tg -saPLA1、smYS1-ΔG11-ΔG8-Δ tg -saPLA1.

[0143] The sequence of the attb site is as follows (SEQ ID NO.1):

[0144] CGGTGCGGGTGCCAGGGGGTGCCCTTCGGCTCCCCGGCCGCGTAGTCCACC

[0145] 3. Fermentation

[0146] smYS1- tg、smYS1-Δ tg -saPLA1、smYS1-ΔG11-ΔG8-Δ tg-saPLA1 transformants were streaked onto GYM agar medium and cultured at 30°C for 4-5 days to obtain spores. An appropriate amount of spores was inoculated into an Erlenmeyer flask containing 30 mL of seed culture medium and cultured at 30°C and 220 r / min with shaking for 24 hours. Subsequently, 2.4 mL of the seed culture was transferred to 30 mL of fishmeal fermentation medium and cultured under the same conditions for another 3 days. Samples were taken every 12 hours during fermentation, and the samples were centrifuged at 12000×g for 10 minutes at 4°C. The supernatant was collected to determine the saPLA1 hydrolytic activity and to perform SDS-PAGE analysis of the fermentation supernatant.

[0147] The results show that ( Figures 2 to 3 ):

[0148] (1) smYS1- No phospholipase A1 hydrolytic activity was detected during TG fermentation;

[0149] (2) smYS1- The extracellular saPLA1 enzyme activity of tg-saPLA1 gradually increased with fermentation time, reaching a maximum value of 39.4 U / mL at 48 h; the cell biomass reached a peak value of 0.6064 (A595) at 60 h.

[0150] SDS-PAGE results further showed that a specific band corresponding to the theoretical molecular weight of saPLA1 (approximately 25.0 kDa) could be detected in the supernatant from 12 h of fermentation. The intensity of this band gradually increased with fermentation time, decreasing after 48 h. These results indicate that the combination of the strong Pgapdh promoter with the TGase signal peptide and terminator can effectively drive the secretory expression of saPLA1 in *Streptomyces malnourished*.

[0151] Pgapdh strong promoter (SEQ ID NO.6):

[0152] Gctgctccttcggtcggacgtgcgtctacgggcaccttaccgcagccgtcggctgtgcgacggacggatcgggcgaactggccgatgctgggagaagcgcgctgctgtacggcgcgcaccgggtgcggagcccctcggcg agcggtgtgaaacttctgtgaatggcctgttcggttgctttttttatacggctgccagataaggcttgcagcatctgggcggctaccgctatgatcggggcgttcctgcaattcttagtgcgagtatctgaaaggggatacgc

[0153] TGase signal peptide (SEQ ID NO.7):

[0154] atgtcccaacgcgggagaactctcgtcttcgccgctctcggtgcggtcatgtgcaccaccgcgttaatgccgtccgcaggcgcggccaccggcagtggcagtggcagcggcaccgggg aagagaagaggtcctacgccgaaacgcaccgcctgacggcggatgacgtcgacgacatcaacgcgctgaacgaaagcgctccggccgcttcgagcgccggtccgtccttccgggccccc

[0155] Terminator (SEQ ID NO.8):

[0156] ggaggggaggggaggcggagcatccggctcccctccccacc

[0157] (3) smYS1 ΔG11 ΔG8 After 48 h of shake-flask fermentation, Δtg-saPLA1 reached its maximum enzyme activity of 54.7 U / mL, compared to smYS1. The enzyme activity of the Δtg-saPLA1 strain was increased by 38.9%.

[0158] 4. Construction of recombinant strains co-expressing saPLA1 and bldD

[0159] (1) pSET152 saPLA1 Construction of bldD plasmid

[0160] PkasO* The Sp44 promoter and bldD gene were ligated to pSET152-P via one-step cloning. gapdh pSET152 was prepared from the XbaI site of the -saPLA1 plasmid. saPLA1 bldD plasmid.

[0161] (2) Using the binding transfer method to transfer pSET152 saPLA1 bldD plasmid was transformed into smYS1-ΔG11-ΔG8-Δ tgThe attb site was identified, and PCR verification was performed using primers very-attb-F / R to obtain the recombinant strain smYS1. ΔG11 ΔG8 Δtg-saPLA1-bldD;

[0162] (3) Following the method in step 3, process smYS1 ΔG11 ΔG8 Δtg-saPLA1-bldD was used for shake-flask fermentation. The results showed that after 48 h of shake-flask fermentation, saPLA1 reached its maximum enzyme activity of 64.3 U / mL, which was further increased by 17.6%.

[0163] Example 3: Purification and Enzymatic Properties Determination of Recombinant saPLA1

[0164] Using the method in step 3 of Example 2, smYS1 ΔG11 ΔG8 The recombinant strain Δtg-saPLA1-bldD was fermented. After 48 h of fermentation, the fermentation broth was centrifuged at 10000 r / min for 10 min, and the supernatant was collected. Purification was performed using nickel affinity chromatography, followed by desalting via ultrafiltration, and the enzymatic properties were determined. Figures 4 to 5 ).

[0165] (1) Electrophoretic analysis:

[0166] The purified PLA1 band was single, and the protein concentration was as expected (approximately 25 kDa).

[0167] The amino acid sequence of saPLA1 (SEQ ID NO.148)

[0168] AAGGYVALGDSYSSGVGAGSYDSGSGDCRRTPKAYPALWAAANSPASFDFVACSGAVTSDVLNKQMGPLNSSTSLVSLTIGGNDAGFADVMTTCVLQSEANCIARVNTAKAFVESTLPGRL DSVYSQVRAKAPSANVVVLGYPRFYKLNGTCVAGLTEGERTAINGAADLLNSVISKRAADHGYAYGDIAAAFTGHEICSGDSWLHSVKWTGINDSYHPTAAGQSGGYLPVLNSKAHHHHHH*

[0169] (2) Using soybean PC as a substrate, its kinetic properties and stability were systematically evaluated.

[0170] The specific method is as follows:

[0171] 1) Thermal stability testing:

[0172] Catalytic properties were analyzed using purified saPLA1. The optimal reaction temperature was determined within the range of 30-70℃, with reaction points set at 5℃ intervals. The highest enzyme activity was defined as 100%, and the relative enzyme activity at each temperature was calculated. Thermal stability was assessed by measuring residual enzyme activity after incubating the enzyme solution at 25-60℃ (at 5℃ intervals) for 30 minutes. Relative activity was calculated with the enzyme activity of the untreated sample as 100%.

[0173] 2) pH stability test:

[0174] Optimal pH was determined using three buffer systems: 50 mM acetate-sodium acetate buffer (pH 4.0-5.5), 50 mM potassium phosphate buffer (pH 6.0-7.5), and 50 mM Tris-HCl buffer (pH 8.0-9.0). Reactions were carried out at 50°C, with the highest enzyme activity considered as 100%. pH stability was determined by incubating the enzyme solution in different pH buffers at 4°C for 1 hour and measuring residual enzyme activity.

[0175] The results show:

[0176] The enzyme exhibited maximum activity at 50°C and pH 6.0. Thermostability tests showed that after incubation at 35°C-40°C for 4 hours, saPLA1 retained over 60% of its residual activity, and after incubation at 45°C for 2 hours, it retained approximately 40%. Above 50°C, its activity decreased sharply. Furthermore, storage stability tests showed that after storage at 4°C for 60 hours, the enzyme retained less than 50% of its activity. These properties are largely consistent with saPLA1 expressed in other host systems. Regarding pH stability, saPLA1 retained over 60% of its initial activity after exposure to pH 5-8 at 4°C for 8 hours. This performance is superior to PLA1 derived from *Serratia marcescens* and *Saccharomyces cerevisiae* KS58-2.

[0177] (3) We further evaluated the effects of metal ions, surfactants and metal chelators on the activity of saPLA1.

[0178] The specific method is as follows:

[0179] To investigate the effect of chemical reagents on enzyme activity, the reaction system was supplemented with 10 mM of metal ions (CaCl2, CoCl2, MgCl2, MnCl2, CuCl2, FeCl2, FeCl3, AlCl3), 10 mM EDTA, 0.1% (w / v) Triton X-100, 0.1% (w / v) SDS, or 100 mM citric acid (to adjust the substrate pH to 7.2). A reaction system without any added reagents was used as a control (set as 100%), and the relative enzyme activity under each condition was calculated.

[0180] The results showed that 10 mM Mn² + 10 mM Fe² + Or 100 mM citrate significantly stimulated activity, increasing it to 215%, 184%, and 195% of the control, respectively. Conversely, Co² + Fe³ + and Al³ + It strongly inhibits enzyme activity. Notably, 10 mMCa² + and Mg² + No significant effect was observed, confirming that the catalysis of saPLA1 is Ca² independent. + This indicates that the recombinase belongs to the non-Ca group. 2+ Phospholipase A1-dependent.

[0181] Example 4: Application of recombinant saPLA1 in soybean crude oil degumming

[0182] The specific steps are as follows:

[0183] 1. Preparation of recombinase

[0184] (1) Separately, smYS1- tg-saPLA1, smYS1 ΔG11 ΔG8 Δtg-saPLA1-bldD transformants were streaked onto GYM agar medium and cultured at 30℃ for 4-5 days to obtain spores. An appropriate amount of spores was inoculated into an Erlenmeyer flask containing 30 mL of seed culture medium and cultured at 30℃ and 220 r / min with shaking for 24 hours. Subsequently, 2.4 mL of the seed culture was transferred to 30 mL of fishmeal fermentation medium and cultured at 30℃ and 220 r / min for 48 h to prepare the fermentation broth.

[0185] (2) Treatment of fermentation broth:

[0186] The supernatant was collected after 48 h of fermentation of the recombinant strain and purified by affinity chromatography using PrePack Ni-NTAPurose 6 FF from Jiaxing Qianchun Biotechnology Co., Ltd. Fractions with PLA1 activity were eluted using a buffer consisting of 50 mM Tris-HCl, 0.2 M NaCl, and 500 mM imidazole (pH 7.4). Subsequently, the eluted fraction was desalted using a 10 kDa ultrafiltration tube, and the purified desalted protein was concentrated using a 30 kDa ultrafiltration tube.

[0187] Recombinase-1 (smYS1-) was prepared separately. tg-saPLA1), recombinase-2 (smYS1) ΔG11 ΔG8 Δtg-saPLA1-bldD).

[0188] In subsequent experiments on degumming of crude soybean oil, the enzyme dosage used was 200 U / 100 g soybean oil or 300 U / 100 g soybean oil.

[0189] When using recombinant enzyme-1, the required protein mass to achieve enzyme dosages of 200 U and 300 U is 6.23 mg and 9.33 mg, respectively.

[0190] When using recombinant enzyme-2, the required protein mass to achieve enzyme dosages of 200 U and 300 U is 3.82 mg and 5.72 mg, respectively.

[0191] 2. Single-factor experiment

[0192] The specific method is as follows: Accurately weigh 100.0 g of crude soybean oil and heat it to 80℃ in a constant temperature water bath. Add 150 μL of 45% (w / w) citric acid solution and stir at 80℃ for 25 minutes. After cooling to the preset temperature, adjust the pH of the system with 4% (w / w) NaOH solution. Then add 5 mL of distilled water and an appropriate amount of recombinant saPLA1 enzyme solution (enzyme activity calculated as U / 100 g oil), stir and mix at 500 r / min for 1 minute, and carry out enzymatic degumming reaction under the set temperature, speed, and pH conditions. After the reaction, heat the sample in a 100℃ water bath for 10 minutes to inactivate the enzyme. Centrifuge the enzyme-inactivated oil sample at 10000 r / min for 15 minutes, collect the upper oil phase, dry it in a 103℃ oven for 1 hour, and use it for phosphorus content determination after cooling.

[0193] (1) Add the purified recombinant enzyme-1 or recombinant enzyme-2 prepared in step 1 to crude soybean oil (purchased from Shandong Hongda Economic and Trade Co., Ltd., with a phosphorus content of 113.01 mg / kg). The amount of enzyme added is 200 U / 100 g soybean oil. Adjust the pH of the reaction system to 5.0. React for 3 h at a temperature of 35-65℃ and a rotation speed of 250 r / min. Detect the phosphorus content in the crude soybean oil.

[0194] The results showed that (since the amount of recombinase added was the same, either 200U or 300U, the results for the two groups of recombinases were identical):

[0195] Under initial reaction conditions (200 U / 100 g oil, 250 rpm, pH 5.0, 3 h), we first screened the temperature range of 35–65 °C. The phosphorus content (an indicator of residual phospholipids) decreased significantly with increasing temperature, reaching a minimum of 16.94 mg / kg at 45 °C, after which it rebounded.

[0196] (2) The specific method is the same as step (1), except that the temperature is fixed at 45℃ and the pH is adjusted from 4.5 to 7.0; the minimum phosphorus content is reached at pH 5.5.

[0197] (3) The specific method is the same as step (1). The difference is that, under the condition that the temperature (45℃) and pH (5.5) are fixed, we increased the amount of enzyme added: 300 U / 100 g oil, which significantly reduced the phosphorus content to below 10 mg / kg. After 2.5 hours of reaction, the phosphorus content was significantly reduced to 7.36 mg / kg.

[0198] Single-factor experiments showed that temperature and pH had the most significant effects on the efficiency of enzymatic degumming.

[0199] 3. Two-factor, three-level full factorial design

[0200] The specific method is the same as step (1), except that we fixed the enzyme addition amount (300 U / 100 g oil) and degumming time (2.5 hours), and used a two-factor, three-level full factorial design to systematically study the effects of temperature (40, 45, 50℃) and pH (5.0, 5.5, 6.0) on the phosphorus content of degummed oil.

[0201] The optimal degumming conditions were determined to be 45℃, pH 5.5, enzyme dosage of 300 U / 100 g oil, and reaction time of 2.5 hours. Under these conditions, the phosphorus content could be reduced to 7.27 mg / kg.

[0202] It is speculated that these conditions achieve an optimal balance between maintaining enzyme structural stability and maximizing its catalytic activity. This is consistent with the observed high enzyme activity of saPLA1 at 45°C and pH 5.5, which facilitates efficient degumming while minimizing the risk of phospholipid oxidation.

[0203] The results showed that ( Figure 7 ):

[0204] The optimal degumming conditions for recombinant saPLA1 are: temperature 45℃, pH 5.5, enzyme dosage 300 U / 100 g, reaction time 2.5 h, and stirring speed 250 r / min. Under these conditions, the phosphorus content in crude soybean oil can be reduced to 7.27 mg / kg, meeting the requirements of physical refining.

[0205] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Streptomyces molybdenum* basal cell, characterized in that, The chassis cells were obtained by knocking out the genomic island GIs gene, endogenous CRISPR / Cas system gene, and NHEJ repair pathway gene of *Streptomyces moharaja* smY2019 as the starting strain. ΔG5 C3, in smY2019 ΔG5 Based on C3, knock out the GI-8 and GI-11 genes in the genome; The GI-8 gene is numbered in NCBI as follows: K7I03_23670, K7I03_23675, K7I03_23680, K7I03_23685, K7I03_23690, K7I03_23695, K7I03_23700. The GI-11 gene is numbered in NCBI as follows: K7I03_25135, K7I03_25140, K7I03_25145, K7I03_25150, K7I03_25155, K7I03_25160, K7I03_25165.

2. The *Streptomyces mogulata* basal cell according to claim 1, characterized in that, The chassis cells also include the following improvements: integration of PkasO* at the attb site on the genome. bldD gene overexpressed by the Sp44 promoter; The nucleotide sequence of the attb site is shown in SEQ ID NO.1, and the PkasO* The nucleotide sequence of the Sp44 promoter is shown in SEQ ID NO.2, and the nucleotide sequence of the bldD gene is shown in SEQ ID NO.

3.

3. A method for preparing TGase enzyme, characterized in that, The method involves preparing the product using the *Streptomyces molybdenum* chassis cell fermentation method as described in claim 1 or 2.

4. A method for expressing a target protein, characterized in that, The method involves using the *Streptomyces molybdenum* chassis cells as described in claim 1 or 2 as host cells to express the target protein.

5. A recombinant Streptomyces mogularia, characterized in that, The recombinant Streptomyces mogulane uses the chassis cells of Streptomyces mogulane as described in claim 1 or 2 as the host cell, knocks out the TGase enzyme in the genome (GenBank number UBI36310.1), and integrates the saPLA1 gene expression cassette shown in SEQ ID NO.4 at the attb site in the genome. The nucleotide sequence of the attb site is shown in SEQ ID NO.

1.

6. A recombinant Streptomyces mogularia, characterized in that, The recombinant *Streptomyces mogularia* was prepared using the *Streptomyces mogularia* chassis cells as described in claim 1 or 2 as the host cell, with the TGase enzyme knocked out from the genome (GenBank ID: UBI36310.1), and integrating the saPLA1 gene expression cassette and PkasO* gene (as shown in SEQ ID NO.4) at the attb site in the genome. bldD gene overexpressed by the Sp44 promoter; The nucleotide sequence of the attb site is shown in SEQ ID NO.1, and the PkasO* The nucleotide sequence of the Sp44 promoter is shown in SEQ ID NO.2, and the nucleotide sequence of the bldD gene is shown in SEQ ID NO.

3.

7. A method for degumming crude soybean oil, characterized in that, The method involves adding the recombinant Streptomyces molybdenum as described in claim 5 or 6, or the recombinant enzyme obtained by fermentation of the recombinant Streptomyces molybdenum as described in claim 5 or 6, to soybean crude oil for reaction and then degumming.

8. The method according to claim 7, characterized in that, The reaction conditions were: 250 rpm, pH 4.5-7.0, and reaction time: 2.5-3 h.

9. The method according to claim 7 or 8, characterized in that, The amount of recombinant enzyme added is 200 U / 100g-350 U / 100g.

10. The use of the *Streptomyces mogulata* chassis cells as described in claim 1 or 2, or the recombinant *Streptomyces mogulata* as described in claim 5 or 6, in the degumming of crude soybean oil or in the preparation of degummed products of crude soybean oil.

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