Engineering strain for increasing supply of cofactors and application of engineering strain in improving production of epsilon-polylysine

Abstract

The invention discloses an engineering strain for increasing supply of cofactors and application of the engineering strain in improving production of epsilon-polylysine, and belongs to the technical field of microbial genetic engineering. The method comprises the following steps: by taking streptomyces albus for producing epsilon-polylysine as an original strain, simultaneously connecting a nicotinic acid ribose phosphate transferase gene pncB, a phosphoenolpyruvate carboxylase gene pck and a pyruvate dehydrogenase gene pdh in the strain with a pIB139 vector by utilizing a homologous recombination method to obtain a recombinant plasmid vector pIB139-pncB-pck-pdh, and introducing the recombinant plasmid vector into a competent cell to obtain the recombinant plasmid vector pIB139-pncB-pck-pdh. The engineering bacterium BP3 is obtained. The shake flask epsilon-PL yield of the engineering bacterium BP3 reaches 1.61 g / L and is improved by 30.89% compared with that of an original strain; the yield of fed batch fermentation reaches 37.21 g / L, the yield reaches 4.85 g / (L.d), and the maximum thallus concentration DCW reaches 43.47 g / L.

Description

Technical Field

[0001] This invention relates to an engineered strain that increases the supply of cofactors and its application in improving the production of ε-polylysine, belonging to the field of microbial genetic engineering technology. Background Technology

[0002] ε-Poly-L-lysine (ε-PL) is a novel homopolymer of amino acids formed by the condensation of α-COOH and ε-NH2, typically containing 25-35 L-lysine residues. ε-PL is a pale yellow powder with a slightly bitter taste, exhibiting strong hygroscopicity, easy solubility in water, good thermal stability, and a wide pH range (pH < 9). Due to its unique molecular structure, ε-PL possesses many excellent properties, such as good antibacterial properties, thermal stability, edibility, water solubility, biodegradability, and non-toxicity. ε-PL has a broad antibacterial spectrum and strong antibacterial effect; therefore, it has been approved for use as a food preservative additive in many countries and regions, including Japan, South Korea, the United States, and China. Furthermore, ε-PL can also be applied in antibacterial materials, cosmetics, medicine, and agriculture. Therefore, ε-PL has broad market application prospects.

[0003] Currently, ε-PL is mainly transmitted through Streptomyces albopictus (Streptomyces simulans) Streptomyces albulus ε-PL is produced through aerobic fermentation. To improve ε-PL production, researchers have employed various methods and strategies, such as culture medium optimization and fermentation process control (e.g., pH and dissolved oxygen (DO) control strategies, cell immobilization, and the addition of exogenous substances). Simultaneously, to obtain high-yielding ε-PL strains, various mutagenesis methods, such as physicochemical mutagenesis, ribosome engineering, genome rearrangement, and ambient-pressure room-temperature plasma, have also been applied to the selection of ε-PL strains. For example, Zhu et al. (2024) used a hybrid selection strategy consisting of diethyl sulfate (DES) mutagenesis, protoplast fusion, and ambient-pressure room-temperature plasma mutagenesis to obtain a genetically stable mutant strain GS114 with a shake-flask yield of 2.8–2.9 g / L. Li et al. (2024) used a combination of interspecific hybridization and multiple antibiotic resistance methods to obtain a mutant strain GX6 with a shake-flask ε-PL yield of 2.23 g / L, increasing the yield by 95.6%–136.8% in fed-batch fermentation.

[0004] With the rapid development of biotechnology and molecular biology, in recent years, an increasing number of researchers have attempted to use genetic engineering methods to modify ε-PL production strains in order to further improve their production performance. For example, Hamano et al. (2007) were among the first to conduct related research in this area, initially targeting the gene encoding aspartate kinase. Ask, the inhibition of aspartokinase activity by high intracellular concentrations of L-lysine and L-glycine can be effectively removed, and the production of ε-PL can be increased to a certain extent. This lays a certain foundation for the subsequent genetic engineering of ε-PL production strains.

[0005] With the progress of genetic technology and the in-depth analysis of the ε-PL synthesis pathway, in recent years, a series of progress has been made in this regard. For example, Wang Ze et al. (2020) in S. albulus M-Z18 homologous overexpression of ε-PL synthetase gene pls Gene, obtained an engineering strain M-Z18 / pIB139 -pls, Its pls up-regulated by 16.8 times at the transcriptional level, and the yield reached 2.74 g / L. Purev et al. (2020) in E. festucae E437 overexpression of ε-PL synthetase gene from fungi Epls Gene, the yield of ε-PL was increased by 6.72 times compared with the wild type. Overexpression of ε-PL synthetase gene ( pls ), which has a direct and important influence on ε-PL synthesis, indeed can increase the yield of ε-PL, and the effect is significant. However, due to various factors, such as the starting strain, experimental method, etc., the same overexpression of pls , there is a big difference in the range of product improvement.

[0006] Some researchers consider to study other genes in the ε-PL synthesis pathway. For example, Wang et al. (2015) found that the pH-sensitive sigma factor-HrdD factor can specifically bind to the upstream promoter of pls Gene, revealing that the factor affects the transcriptional regulation of pls by regulating the promoter region. Although these methods effectively increase the yield of ε-PL, there are still problems such as plasmid instability, gene expression imbalance, and increased metabolic burden.

[0007] The above method mainly provides a large amount of precursor supply to improve the yield of ε-PL. However, in the synthesis process of ε-PL, in addition to the supply of a large amount of precursor, the supply of various coenzymes such as ATP and NADH is also needed to provide energy substances and reducing substances. At present, although a few studies have attempted to increase the yield by regulating the supply of a single coenzyme (such as ATP or NADH) in Streptomyces albus, such as the introduction of PPK-mediated ATP regeneration system by Yang et al. (2023), overexpression of ATP-related genes, which increased the yield of ε-PL by 21.24%, indicating that the increase of ATP supply is beneficial to biosynthesis. However, there are still few reports on the simultaneous improvement of coenzymes ATP and NADH levels by genetic engineering methods. This research gap is due to the challenges faced in the practical application of multi-gene co-expression technology, including plasmid instability, gene expression imbalance, and increased metabolic burden, which may lead to growth inhibition of the engineering strain or decrease of the yield of the target product. Therefore, how to improve the yield of ε-PL in Streptomyces albus based on coenzyme regulation is one of the problems to be solved at present. Therefore, this study attempts to co-overexpress the nicotinic acid phosphoribosyltransferase gene pncB , the phosphoenolpyruvate carboxylase gene pck and the pyruvate dehydrogenase gene pdh to construct an engineering strain to improve the supply of ATP and NADH, in order to improve the fermentation production of ε-PL. SUMMARY

[0008] To solve the above problems, the present application strengthens the supply of coenzymes ATP and NADH by homologous overexpression of related genes to construct a high-yield genetic engineering strain. Medium and fermentation process optimization strategies are used to further improve the yield of ε-PL; and the physiological characteristics of the engineering strain are analyzed and determined.

[0009] The first object of the present application is to provide a recombinant Streptomyces albus for improving the yield of ε-polylysine, which co-overexpresses the nicotinic acid phosphoribosyltransferase gene pncB , the phosphoenolpyruvate carboxylase gene pck and the pyruvate dehydrogenase gene pdh .

[0010] In an embodiment of the present application, the nucleotide sequence of the nicotinic acid phosphoribosyltransferase gene pncB is shown in SEQ ID NO. 1, the nucleotide sequence of the phosphoenolpyruvate carboxylase gene pck is shown in SEQ ID NO. 2, and the nucleotide sequence of the pyruvate dehydrogenase gene pdh is shown in SEQ ID NO. 3.

[0011] In an embodiment of the present application, the host cell of the recombinant Streptomyces albus is Streptomyces albus 。

[0012] In an embodiment of the present application, the recombinant Streptomyces albus expresses the nicotinic acid phosphoribosyl transferase gene, the phosphoenolpyruvate carboxylase gene and the pyruvate dehydrogenase gene with the pIB139 vector pncB pck pdh.

[0013] A second object of the present application is to provide a method for constructing the recombinant Streptomyces albus as described above, comprising the steps of: (1) cloning and amplifying the gene fragments pncB, pck, pdh using corresponding primers to obtain the desired gene fragments; (2) using the method of homologous recombination to link the gene fragments pncB, pck, pdh with the pIB139 vector to obtain the recombinant plasmid pIB139- pncB-pck-pdh; (3) transforming the recombinant plasmid pIB139- pncB-pck-pdh into E. coli ET12567, and then performing conjugation transfer with Streptomyces albus as the receptor to select the correct conjugant to obtain the recombinant Streptomyces albus.

[0014] In an embodiment of the present application, the genome of S. albulus is used as the template, and the primers F- PncB , R- PncB , F-2- PncB , R-2- PncB , F-2- Pck , R-2- Pck , F-3- PncB , R-3- PncB , F-3 -Pck , R-3- Pck , F-3- Pdh , R-3- Pdh are used to amplify the pncB , pck and pdh genes.

[0015] In an embodiment of the present application, the purified pncB , pck and pdh fragments obtained by recovery are linked to the pIB139 vector by homologous recombination, and transformed into the competent cell E.coli DH5α.

[0016] ​​In one embodiment of the present invention, the minimum inhibitory concentration (MIC) of the antibiotic used in conjugation transfer showed differences in the MICs of nalidixinone acid for *E. coli* containing different plasmids, while the MIC value of apramycin for *Streptomyces albopictus* was 50 mg / L. This was used as a reference concentration for subsequent drug addition in conjugation transfer experiments.

[0017] In one embodiment of the present invention, well-developed zygotes are selected for seed culture, genomic DNA is extracted, and zygote colony PCR identification is performed. One strain containing pIB139 was identified. -pncB Recombinant bacteria (named) S.albulus BP1) identified one strain containing pIB139. -pncB-pck Recombinant bacteria (named) S.albulus BP2) identified one strain containing pIB139 -pncB- pck-pdh Recombinant bacteria (named) S.albulus BP3).

[0018] A third objective of this invention is to provide a method for producing ε-polylysine, comprising the step of fermentation using the aforementioned recombinant Streptomyces albopictus.

[0019] In one embodiment of the present invention, the fermentation process involves inoculating the strain into the fermentation medium at an inoculation rate of 5-13% (v / v) and fermenting under conditions of a temperature of 27-36°C, a rotation speed of 150-700 rpm, and a dissolved oxygen content of 18-45%.

[0020] In one embodiment of the present invention, the fermentation medium comprises 40-60 g / L glucose, 3-8 g / L yeast extract, 7-12 g / L (NH4)2SO4, 0.3-0.8 g / L magnesium sulfate, 0.5-1.0 g / L dipotassium hydrogen phosphate, 1.20-1.60 g / L potassium dihydrogen phosphate, 0.02-0.05 g / L ferrous sulfate, and 0.03-0.06 g / L zinc sulfate.

[0021] In one embodiment of the present invention, the fermentation medium comprises 39.95 g / L glucose, 4.32 g / L yeast extract, 6.87 g / L (NH4)2SO4, 1.36 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O, 0.04 g / L ZnSO4·7H2O, 0.03 g / L FeSO4·7H2O, and pH 6.8.

[0022] In one embodiment of the present invention, the fermentation is either batch fermentation or fed-batch fermentation.

[0023] In one embodiment of the present invention, the batch fermentation includes the following steps: when the pH naturally decreases from the initial 6.5~7.6 to 3.7~4.2, the pH is maintained at 3.7~4.2 by adding a pH adjuster until the batch fermentation is completed.

[0024] In one embodiment of the present invention, during fed-batch fermentation, pH control includes the following steps: when the pH naturally decreases from the initial 6.5-7.6 to 4.5-5.6, a pH adjuster is fed in to maintain the pH at 4.5-5.6 for 6-12 hours; then, when the pH naturally decreases to 3.7-4.2, a pH adjuster is fed in to maintain the pH at 3.7-4.2 until the fed-batch fermentation is completed.

[0025] In one embodiment of the present invention, the feeding batch fermentation includes the following steps: when the residual glucose concentration in the fermentation broth drops to below 10 g / L, a glucose solution is added to control the glucose concentration in the fermentation broth at 7~13 g / L, and a nitrogen source solution is added to maintain the amino nitrogen concentration in the fermentation broth at 0.7~1.3 g / L.

[0026] In one embodiment of the present invention, the concentration of the glucose solution is 380~870 g / L; the nitrogen source solution is a mixed nitrogen source solution of 60~130 g / L ammonium sulfate and 70~130 g / L yeast powder.

[0027] In an embodiment of the present invention, the batch fermentation includes the following steps: The sterilized fermenter is placed on a base, sterile air is immediately introduced and condensate is added, the agitator is turned on, and the aeration rate is set to 1.0 vvm and the rotation speed to 200 rpm. When the temperature drops to 30 ℃, the pH is adjusted to 6.8 with 12.5% ​​ammonia water, and then 300 mL of seed culture is added to begin fermentation. Samples are taken every 8 hours, and dissolved oxygen (DO) is controlled above 15% by adjusting the agitator speed and aeration rate. Fermentation ends after 72 hours.

[0028] Beneficial effects (1) This invention confirms that the engineered strains BP1, BP2, and BP3 can significantly improve the production level of ε-polylysine, with shake-flask yields of 1.43 g / L, 1.49 g / L, and 1.61 g / L, respectively, and concentration-to-weight ratios (DCW) of 9.83 g / L, 11.34 g / L, and 13.17 g / L, respectively. Compared with the starting strain, BP3 showed a 30.89% increase in ε-polylysine production and a 47.48% increase in cell concentration (DCW).

[0029] (2) SEM, respiration rate, and enzyme activity assays revealed significant physiological and biochemical changes in the engineered strain BP3. SEM results showed that the hyphae of the engineered strain were thicker and denser, and the respiration rate was also higher, indicating that the engineered strain's activity was significantly higher than the original strain. This result is consistent with the higher DCW of the engineered strain. Enzyme activity results showed that the POD, ATP, and PEPCK contents of the engineered strain were significantly higher than those of the original strain, while the MDA content was significantly lower. This indicates that the engineered strain experienced less oxidative damage during fermentation than the original strain. These results suggest that the engineered strain BP3, by increasing the supply of cofactors ATP and NADH and reducing intracellular PA concentration, provides higher energy and reducing power for cell metabolism, enhancing the strain's resistance to oxidative damage, thereby promoting cell growth and ε-PL synthesis.

[0030] (3) Batch and fed-batch fermentation experiments were conducted on engineered strain BP3 in a 5 L fermenter. In batch fermentation, the yield of BP3 reached 9.46 g / L, with a yield rate of 3.15 g / L / d. In fed-batch fermentation, the maximum yield of engineered strain BP3 reached 37.21 g / L, with a yield rate of 4.85 g / L / d, and its maximum DCW reached 43.47 g / L. Attached Figure Description

[0031] Figure 1 This is a PCR verification diagram of the engineered strain; Figure 2 Roadmap for plasmid construction; Figure 3 The shake flask yields of the starting strain, engineered strains BP1, BP2, and BP3 are shown in the graph. Figure 4 The colony and hyphal morphology and respiratory activity of the starting strain and the engineered strain BP3 are as follows: (A) Colony morphology of the starting strain; (B) Hyphae morphology of the starting strain; (C) Respiratory activity of the starting strain; (D) Colony morphology of the engineered strain BP3; (E) Hyphae morphology of BP3; (F) Respiratory activity of BP3. Figure 5 Volcano plot for differentially expressed genes; Figure 6 This is a diagram showing the transcriptional levels of genes related to cell metabolism and signal transduction systems. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0033] The strain used in this invention is *Streptomyces albopictus* (…Streptomyces albulus ) ， This strain has been published in (Fu J,Li C, Ju X, Bai J, Zhou Y, Zhang Y, Wang Y, Sun Z, Hu C, Li L, Ji L. Efficient production of ε-poly-L-lysine from cassava bagasse hydrolysate used as carbon source by Streptomyces albulus US3-18. Bioprocess Biosyst Eng. 2022Aug;45(8):1407-1419. doi: 10.1007 / s00449-022-02755-3. Epub 2022 Jul 24. PMID:35871697.).

[0034] The culture media involved in the following examples are as follows: M3G medium (g / L): glucose 50, yeast extract 5, (NH4)2SO4 10, K2HPO4·3H2O 1.36, MgSO4·7H2O 0.5, ZnSO4·7H2O 0.04, FeSO4·7H2O 0.03, pH 6.8.

[0035] BTN (g / L): glucose 10, fish meal peptone 2, yeast powder 1, pH 7.5.

[0036] MS (g / L): Mannitol 20, soybean flour 20, agar powder 20.

[0037] 2×YT (g / L): tryptone 16, yeast extract 10, NaCl 5, pH 7.0.

[0038] The primers involved in the following examples: Table 1 PCR Primers

[0039] The detection methods involved in the following embodiments are as follows: 1. Determination of the ε-PL standard curve Nine clean test tubes were used to add 0, 1.5, 3.0, 4.5, 6.0, 7.5, and 9.0 mL of ε-PL stock solution sequentially, and then diluted to 10 mL with sterile water to obtain a series of standard solutions with concentrations of 0, 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 g / L. Each gradient solution was diluted 20-fold by volume and vortexed thoroughly. An equal volume of the diluted sample was mixed with an equal volume of methyl orange indicator. The mixture was transferred to a 30°C water bath shaker and vortexed at 200 rpm for 30 min in a dark environment. After the reaction was terminated, the mixture was centrifuged at 5000×g for 15 min, and the supernatant was collected. The supernatant was diluted a second time by 20-fold, and the absorbance was measured at 465 nm using a UV spectrophotometer. A standard curve was plotted based on the obtained absorbance, and the linear equation y = -3.2078x + 2.1107 was fitted, with a correlation coefficient R² = 0.999, showing a good linear relationship.

[0040] 2. ε-PL concentration determination Take 10 mL of the fermentation broth sample and transfer it to a 50 mL centrifuge tube. Centrifuge at 4500 × g for 15 min (follow Itzhaki's procedure). Dilute the clear supernatant and mix an equal volume of the diluted solution with 1 mmol / L methyl orange working solution. Place the mixture in a 30 ℃ constant temperature water bath shaker and react at 200 rpm in the dark for 30 min. After the reaction is terminated, centrifuge again using the same parameters (4500 × g, 15 min) and collect the supernatant. Dilute the obtained supernatant 20 times and measure the absorbance at 465 nm using a UV spectrophotometer. Calculate the actual concentration of ε-PL using the standard curve equation y = -3.2078x + 2.1107.

[0041] 3. Cell dry weight (DCW) determination Weigh the clean centrifuge tube empty and record the initial mass. Transfer 10 mL of fermentation broth sample to a labeled centrifuge tube, centrifuge at 4500×g for 15 min, and remove the supernatant. Add deionized water to the cell pellet, vortex to resuspend thoroughly, and centrifuge under the same conditions (4500×g, 1 min) to remove the supernatant. Wash the pellet twice. Transfer the washed mycelia to a 60 ℃ constant temperature drying oven and continue drying until constant weight. The dry weight of the cells can be obtained by subtracting the initial mass of the centrifuge tube from the total mass.

[0042] 4. ATP assay ATP content was measured using an ATP analysis kit (Jiangsu Keming, China). The precipitate from the centrifuged fermentation broth was mixed with the lysis buffer from the kit, and then sonicated at 4 °C. The mixture was centrifuged at 8000 × g for 10 min to prepare a supernatant for ATP detection. Buffer solutions were added sequentially as indicated in the instructions, followed by the sample. The absorbance was measured at 700 nm, and the ATP content was determined based on the measured protein content. A creatine kinase-mediated enzymatic reaction system was also used. This enzyme specifically catalyzes the reaction of creatine with adenosine triphosphate (ATP) to produce phosphocreatine. Phosphocreatine was analyzed colorimetrically at 700 nm; the absorbance change was linearly correlated with ATP concentration, thus determining the ATP content.

[0043] 5. MDA determination The determination of malondialdehyde (MDA) content in samples using a thiobarbituric acid colorimetric method employed an MDA detection kit (Jiangsu Keming Biotechnology Co., Ltd.). The principle is that in a high-temperature or acidic reaction system, thiobarbituric acid specifically binds to malondialdehyde to form a reddish-brown complex. This complex exhibits a peak absorbance at 532 nm, while the absorbance is lower at 600 nm. The concentration of malondialdehyde can be determined by calculating the difference between the two wavelengths.

[0044] 6. NADH Measurement The concentration of NADH was measured using an NADH assay kit (Jiangsu Keming, China). After centrifugation of the fermentation broth and removal of the supernatant, the extract was then subjected to ultrasonic cell disruption and centrifugation at 10,000 × g for 10 min. Buffer solutions were added sequentially, followed by the sample supernatant. After 5 min of incubation at a constant temperature, the absorbance was measured at 570 nm using a microplate reader to determine the NADH concentration. The mechanism of action is as follows: NADH, via a PMS-mediated hydrogen transfer reaction, reduces oxidized thiazolyl blue (MTT) to formazan crystals. This blue crystalline product exhibits an absorption peak at 570 nm, and its absorbance intensity is linearly correlated with the NADH concentration, thus allowing for the determination of NADH content.

[0045] 7. Determination of pyruvic acid content The detection of pyruvate content is based on the colorimetric principle of dinitrophenylhydrazine. In an acidic reaction system, pyruvate specifically binds with dinitrophenylhydrazine to form a phenylhydrazone complex, which turns brownish-red upon oxidation. The absorbance is measured at 520 nm using a UV-Vis spectrophotometer, and the concentration of pyruvate in the sample can be determined by combining the absorbance with a standard curve.

[0046] 8. PEPCK determination The activity of phosphoenolpyruvate carboxykinase (PEPCK) was determined using a PEPCK assay kit (Jiangsu Keming, China). This enzyme catalyzes the conversion of oxaloacetate to phosphoenolpyruvate (PEP) and releases CO2. Pyruvate kinase and lactate dehydrogenase can further mediate the oxidation of NADH to NAD. + The rate of decrease in NADH content can be obtained at 340 nm using a UV spectrophotometer. The rate of decrease in absorbance is positively correlated with PEPCK enzyme activity, thus enabling the determination of PEPCK enzyme activity.

[0047] 9. POD determination Peroxidase (POD) activity was determined using a POD assay kit (Jiangsu Keming, China). Guaiacin, sodium hydroxide, and POD buffer were mixed in a specific ratio. This mixture was then placed in a sonicated sample. In the reaction system, POD catalyzed the oxidation of guaiaacin to produce a brownish-yellow product. The absorbance of this complex was measured at 470 nm. The absorbance intensity was positively correlated with enzyme activity, thus allowing for the determination of POD content.

[0048] 10. Transcriptome sequencing The experimental procedure includes the following key steps: First, total RNA is isolated and its quality is assessed using electrophoresis. cDNA is then generated via reverse transcription, and a sequencing library is constructed. Next, the library size is assessed using an Agilent 2100 Bioanalyzer, and the total concentration of the library is checked using quantitative real-time sequencing. Finally, sequencing is performed.

[0049] Example 1 Construction of engineered bacteria 1. pncB , pck and pdh Cloning and construction of expression vectors.

[0050] Search for nicotinic acid phosphoribosyltransferase on NCBI pncB and phosphoenolpyruvate carboxykinase pck and pyruvate dehydrogenase pdh Based on the sequence information, primers were designed (Table 1) F- PncB R- PncB F-2- PncB R-2- PncB F-2- pck R-2- pck F-3- PncB R-3- PncB F-3- pck R-3- pck F-3- pdh R-3- pdh .

[0051] PCR amplification using the genome of the originating strain as a template pncB , pck and pdh Genes, using electrophoresis, were analyzed separately. pncB , pck and pdh These three genes were verified. The electrophoresis results matched the theoretical values, allowing for a preliminary assessment. pncB , pck and pdh These three genes were successfully amplified by PCR, and were recovered after gel extraction for use in subsequent experiments. pncB , pck and pdh The gene and the double-digested plasmid pIB139 were ligated using homologous recombination to obtain the recombinant plasmid pIB139-. pncB pIB139- pncB - pck and pIB139- pncB - pck - pdh (The construction process is as follows) Figure 1 (As shown).

[0052] 2. Construction of recombinant cloning vector.

[0053] After introducing the recombinant product into *E. coli* DH5α competent cells, the bacterial culture was evenly spread on LB agar plates containing apramycin (50 μg / mL) for antibiotic selection. Single colonies obtained after culture were identified by colony PCR. Positive clones containing the target band were then amplified and sequenced for comparison. The size of the bacterial PCR bands and the target band were compared. pncB (1329), pncB - pck (3165), pncB - pck - pdh (4908) The size matches, which preliminarily confirms that the recombinant plasmid vector pIB139- has been obtained. pncB - pck - pdh .

[0054] 3. Bonding transfer.

[0055] The following sections detail the determination and verification of conjugation transfer concentrations, but they do not explain how the conjugation transfer process actually occurs. (1) Determination of the minimum concentration of antibiotics used for conjugation transfer The antibacterial effect of naphthylpyridinol against *Escherichia coli* was tested using a gradient dilution method: 200 μL of bacterial suspension was spread onto LB solid medium using the dilution plating method. After incubation at 30 °C for 12–18 h, 1 mL of sterile aqueous solution containing gradient concentrations of naphthylpyridinol was applied to the surface of MS medium, and the culture was continued for 3–5 days to observe the antibacterial effect. The apramycin tolerance test against *Streptomyces albopictus* was also conducted using the plating method: equal volumes of bacterial suspension were inoculated onto BTN plates containing gradient concentrations of apramycin (20–70 μg / mL), and incubated at 30 °C for 48–72 h to record growth inhibition.

[0056] As shown in Table 2, the MICs for nalidixic acid differed among E. coli containing different plasmids, with the MIC for apramycin in Streptomyces albus being 50 mg / L. This was used as a reference concentration for subsequent drug addition in binding and transfer experiments.

[0057] Table 2. Determination of minimum inhibitory concentration of antibiotics used in conjugation transfer. A

[0058] B

[0059] C

[0060] Note: A. Contains pIB139 -pncB The MIC of Escherichia coli and Streptomyces albus, B. containing pIB139- pncB- pck The MIC, C of Escherichia coli and Streptomyces albus. Contains pIB139- pncB-pck-pdh MIC of Escherichia coli and Streptomyces albus.

[0061] The specific procedure for the conjugation transfer experiment is as follows: Import the successfully sequenced vector into... E.coliIn ET12567 / pUZ8002 competent cells, the transformation products were evenly spread on LB agar plates containing three antibiotics at the following concentrations: kanamycin 25 μg / mL, chloramphenicol 25 μg / mL, and apramycin 50 μg / mL. Positive monoclonal selection was then performed. Positive colonies were cultured in LB liquid medium with the same antibiotic concentration at 37°C with shaking until the logarithmic growth phase (OD600≈0.6). After three centrifugations and washings on sterile LB medium, a concentrated bacterial suspension of 200 μL was prepared. Simultaneously, the Streptomyces spore suspension was mixed with an equal volume of 2×YT medium, heat-shocked at 50°C for 10 min, and pre-germinated at 37°C for 2.5 h. Finally, the donor bacteria and germinating spores were mixed 1:1, allowed to stand for 5 min, centrifuged to concentrate, spread on MS medium, and incubated at 30°C for 18 h.

[0062] After 18 h of conjugation culture, positive conjugates were screened using a specific antibiotic: Sterile water containing a specific concentration of nalidixic acid and apramycin was evenly distributed on the surface of MS plates, and incubated at 30 °C for 2-3 days until resistant conjugates formed. Single colonies of dominant spores were picked and transferred to BTN plates containing apramycin for secondary screening. After 5-8 days of culture, genomic verification was performed using the following procedure: Mature spores were inoculated into seed culture medium and cultured with shaking at 30 °C for 24 h. 10 mL of the seed culture was collected by centrifugation at 8000 × g for 5 min, and the DNA genome was extracted. PCR amplification was performed using this genome as a template, and agarose gel electrophoresis was used to confirm the target band and whether the gene was successfully overexpressed.

[0063] (2) Conjugation transfer verification Well-grown conjugates were selected and subjected to secondary screening on BTN plates containing apramycin. Successful single colonies were then transferred to M3G medium and cultured for 24 h. Genomic DNA was extracted and validated by PCR. Gel electrophoresis results are shown below. Figure 2 As shown. This indicates that BP1 (pIB139) -pncB ), BP2 (pIB139- pncB-pck ) and BP3 (pIB139- pncB-pck-pdh Successfully constructed.

[0064] Example 2: Shake-flask fermentation with engineered bacteria 1. Preparation of seed liquid Prepare bacterial suspensions from the starting strain preserved in glycerol and the engineered strain. Inoculate the suspensions into prepared solid culture media (slant / plate) using the serial dilution spread method or streak plate method, and incubate at 30 °C for 7-8 days. Using a sterile inoculation loop, scrape two loops of spores from the slant and transfer them to an Erlenmeyer flask containing M3G medium. Incubate at 30 °C for 24 h at 200 rpm.

[0065] 2. Shake-flask fermentation broth culture Take the cultured seed liquid and inoculate it at a volume ratio of about 10% (i.e., 5 mL of seed liquid inoculated into 50 mL of fermentation medium), and set the same parameters (200 rpm, 30 ℃) for 72 h fermentation.

[0066] 3. Fermentation production of ε-polylysine by strains The production of ε-polylysine by the strain was determined, and the results are as follows: Figure 3 As shown, the ε-PL yield of the original strain was 1.23 g / L, and the cell biomass reached 8.93 g / L. The ε-PL yield of the genetically engineered strain BP1 was 1.43 g / L, which was 16.26% higher than that of the original strain. The ε-PL yield of the engineered strain BP2 was 1.49 g / L, which was 21.38% higher than that of the original strain.

[0067] The above results indicate that the ε-PL yield of engineered strain BP2 was only 4.20% higher than that of BP1, suggesting that... pck Overexpression of the gene did not significantly enhance the synthesis efficiency of ε-PL. This may be because... pncB Overexpression promotes NAD+ supply, accelerates glycolysis, and leads to increased pyruvate production. pck While overexpression of [specific compound] can theoretically optimize metabolic flux through PEP replenishment, its catalytic process of oxaloacetate to PEP conversion consumes ATP and may reduce oxaloacetate supply in the TCA cycle, thus affecting the generation of subsequent ε-PL precursors. Therefore, further overexpression is considered. pdh. pdh The gene encodes pyruvate dehydrogenase, which catalyzes the irreversible oxidation and decarboxylation of pyruvate to produce acetyl-CoA. Acetyl-CoA can then enter the tricarboxylic acid (TCA) cycle, providing energy and more precursor substances for cellular metabolism. Overexpression pdh After genes, on the one hand, the cause can be pncB Overexpression of pyruvate leads to its rapid conversion to acetyl-CoA, preventing excessive intracellular accumulation of pyruvate and metabolic flux blockage. Furthermore, sufficient acetyl-CoA entering the TCA cycle promotes the regeneration of oxaloacetate, alleviating the effects of overexpression. pck The insufficient supply of TCA cycle intermediates caused by gene overexpression depletes oxaloacetic acid, thereby ensuring the stable generation of ε-PL synthesis precursors.

[0068] The results showed that, in overexpression pdh After gene generation, the engineered bacterium BP3 (containing pIB139) -pncB-pck-pdh The yield significantly increased to 1.61 ± 0.13 g / L, an increase of 30.89%. Clearly, overexpression of ATP-related genes can effectively increase the yield of bacterial metabolites. This may be because nicotinic acid phosphoribosyltransferase (NAPRTase) is produced by… pncB Encoding this enzyme, it is the rate-limiting enzyme in the NAD(H) synthesis pathway. Overexpression of this enzyme significantly increases ε-PL and DCW. Furthermore, overexpression... pck The encoded phosphoenolpyruvate carboxylkinase can also increase ATP levels. Similarly, overexpression of pyruvate dehydrogenase (PDH) pdh It can reduce energy consumption during the generation of acetyl-CoA and does not produce harmful byproducts.

[0069] Example 3: Physiological Performance Testing of Engineered Strains (1) Plate colony morphology: The genetically engineered strain and the starting strain were prepared into bacterial suspensions, diluted and spread on BTN plates, and cultured at a constant temperature of 30 ℃ for 7-8 days. Then, single colonies were selected for morphological observation, photographed and compared.

[0070] Figure 4 (A) Colony morphology of the starting strain and the engineered strain on BTN plates. The results indicate that, morphologically, the starting strain... S. albulus The surface of the bacterial colonies showed a "wrinkled" appearance, and there was a "crack" in the middle, while the engineered bacteria... Figure 4 (D) shows a more obvious bulge in the middle; in terms of the number of strains, after the same dilution, the number of the starting strains is more concentrated but the diameter is smaller, while the engineered strains have a larger diameter but a smaller number; in terms of the color of the strains, the middle of the starting strain shows a part of light gray, while the entire single colony of the engineered strain BP3 is white.

[0071] (2) Observation of mycelial morphology: 2 mL of fermentation broth was transferred to a sterile centrifuge tube and centrifuged at 8000×g for 5 min. The supernatant was removed and the precipitate was retained. After washing with sterile water three times (centrifuged at 7500×g for 5 min each time), 2.5% glutaraldehyde fixative (v / v) was injected into the centrifuge tube containing the bacterial precipitate. The fixation was carried out at room temperature for 30 min and then transferred to a refrigerator for overnight fixation.

[0072] Figure 4 (B) and (E) are scanning electron microscope (SEM) images of the mycelia of the original strain and the engineered strain BP3, respectively. The results show that the original strain...S. albulus The hyphae of the engineered strain were sparser and thinner, while those of the original strain were thicker and more tightly intertwined. This is consistent with the previous finding that the DCW of the engineered strain was higher than that of the original strain. Furthermore, the hyphae of the original strain showed more significant breakage than those of the engineered strain. The results indicate that the hyphae of the engineered strain BP3 grew more rapidly, were thicker, and exhibited stronger strain activity, suggesting that the increased ε-PL yield of the engineered strain is closely related to the higher activity of the hyphae.

[0073] (3) Determination of the respiratory intensity of the strain According to the Bacstain-CTC kit instructions, 750 μL of deionized water was added to the CTC solution, and the mixture was thoroughly vortexed to prepare the working solution (final concentration 50 mM). The working solution was mixed with the bacterial sample at a ratio of 4:1, incubated in the dark, and then observed under a fluorescence microscope. After incubation at 37°C for 30 min, the samples were observed using a laser confocal microscope.

[0074] Comparison of differences in cellular respiration intensity under laser confocal microscopy, as follows: Figure 4 As shown in (C) and (F), the engineered BP3 cells exhibit significantly brighter red fluorescence than the original strain, indicating higher cellular respiration intensity and greater cell growth viability. This is likely due to the three overexpressed genes enhancing strain viability by increasing the supply of cofactors ATP and NADH, resulting in better cell growth and stronger survival. This phenomenon is consistent with previous SEM results. In conclusion, the engineered BP3 strain demonstrates stronger respiration intensity and a clear growth advantage.

[0075] Example 4: Assay of cofactors and related enzyme activities When the engineered strain BP3 and the control group were fermented for 24, 48, and 72 h, samples were taken to measure the levels of intracellular cofactors, intermediate metabolites, and key enzymes.

[0076] The results showed that the ATP content of the engineered strain was significantly higher than that of the control group, with BP3 containing twice the ATP content of the original strain at 72 h. At 72 h, the NADH content of BP3 was 1.93 times that of the original strain. This indicates that a large amount of NADH is present in BP3, enabling the synthesis of more ATP. At 48 h, the peroxidase (POD) activity of BP3 reached its peak. At this point, the POD activity of the engineered strain BP3 was 18.75% higher than that of the original strain. This suggests that in the middle and late stages of shake-flask fermentation, the fermentation broth environment gradually becomes unsuitable for cell growth, and the strain chooses to cope with this environment by enhancing POD activity, more actively eliminating excessive free radicals, reducing the adverse effects of oxidative stress on cells, and thus improving its survival ability. Furthermore, the concentration of pyruvate (PA) in the engineered strain BP3 was lower than that in the original strain. This result indicates that overexpression of PA in the engineered strain... pdh The gene leads to increased PA utilization in the TCA cycle. Furthermore, enhancing the TCA cycle not only provides cells with more ATP but also provides more oxaloacetate during L-Lysine synthesis. However, at 72 h, PA levels begin to gradually increase as PA utilization shows a decreasing trend.

[0077] The PEPCK level of the engineered strain BP3 was significantly higher than that of the original strain. At 48 h, the PEPCK activity of the engineered strain BP3 was 1.67 times that of the original strain, peaking at 48 h and then decreasing after 72 h. The MDA level in strain BP3 was significantly lower than that in the control group. The decrease in MDA level was related to the increase in NADH concentration. The increased NADH concentration enhanced the interaction with free radicals, thereby inhibiting the peroxidation process.

[0078] The above results indicate that simultaneous overexpression of these three genes does indeed increase intracellular ATP and NADH supply levels, providing more energy and reducing agents for ε-PL synthesis. Simultaneously, PEPCK and POD enzyme activities are also increased, which is beneficial for increasing energy supply and enhancing antioxidant capacity. Furthermore, the decrease in PA levels enhances TCA cycle activity, providing more precursors for ε-PL synthesis.

[0079] Example 5: Determination of Differentially Expressed Genes in the Transcriptome 10 mL of bacterial culture from Example 2 (BP3) and the control group after 24 h of fermentation was taken respectively, and differentially expressed genes (DEGs) were measured in the transcriptome to reveal the molecular mechanism of simultaneous overexpression of three genes on ε-PL synthesis at the transcriptional level.

[0080] The results are as follows Figure 5As shown, this is a volcano diagram of differentially expressed genes, where CK is the blank control group and T is the experimental group (Example 2). A total of 1455 differentially expressed genes were found, of which 712 genes were upregulated in the experimental group compared to the control group, while 743 genes were downregulated.

[0081] Among the upregulated and downregulated genes, the GO enrichment analysis results showed that, under the biological process classification (BP), the most significant enrichment genes were those related to fatty acid metabolism, siderophore biosynthetic process, and siderophore biosynthetic process. Under the cell composition classification (CC), the most significant enrichment genes were those related to the ABC transporter complex, ATPase-dependent transmembrane transport complex, and transmembrane transporter complex. Furthermore, in terms of molecular function (MF), the most significant enrichment genes were those related to transmembrane transporter activity, transporter activity, and flavin adenine dinucleotide binding.

[0082] Among the upregulated genes, DEGs were most significantly enriched in metabolic pathways, including nitrogen metabolism, oxidative phosphorylation, and the synthesis of various antibiotics. Among the downregulated genes, the enrichment was most significant in the cellular process category of "ferroptosis." Ferroptosis primarily refers to the downregulation of genes in the Fe... 2+ Under the action of ester oxygenase, unsaturated fatty acids on the cell membrane are catalyzed, resulting in lipid peroxidation and thus cell death. Ferraphobia, as a unique form of cell death, integrates the different components of cellular metabolism into a tight network, including iron, amino acids, lipids, and redox reactions.

[0083] The distribution of DEGs in metabolic pathways, including proteasome, aminoacyl-tRNA biosynthesis, and endoplasmic reticulum protein processing, was further analyzed using the KEGG database. DEGs associated with ε-PL synthesis, such as... Figure 6 As shown, in the L-lysine biosynthesis pathway, genes asd Upregulated, genes gabD It was downgraded. (By...) asd Genetically regulated aspartate hemialdehyde dehydrogenase can convert aspartate phosphate into aspartate hemialdehyde, while... gabD Genetically regulated succinate semialdehyde dehydrogenase catalyzes the oxidation of succinate semialdehyde to succinate. Therefore, more L-aspartate can participate in this pathway, while fewer L-lysine can be transferred to other byproducts. In the TCA cycle, cs Upregulation of this gene leads to an increased supply of acetyl-CoA, which is involved in the TCA cycle and maintains cellular energy metabolism. Furthermore, citrate synthase, the only enzyme in the cycle responsible for forming carbon-carbon bonds, promotes the Claisen reaction between acetyl-CoA and oxaloacetate to produce citrate. Genes involved in the conversion of L-aspartic acid to fumarate are also included. aspA The regulation was downregulated, thus allowing more L-aspartic acid to participate in the synthesis of L-lysine. Furthermore, the gene... nadE Upregulation led to an increase in the NAD(P)H pool. Several genes related to energy and redox reactions were also upregulated, such as... nuoA and ndh These genes enhanced ATP synthesis, which is beneficial for ε-PL production. These results indicate that the up- and down-regulated genes are directed in a direction favorable to ε-PL accumulation, suggesting that this study represents an effective strategy for increasing ε-PL production.

[0084] Example 6: Batch and Feed-in Batch Fermentation in a Fermenter (1) Batch fermentation Approximately 3.0 L of initial fermentation medium was placed in a 5L fermenter. About 0.33 L of seed culture, which had been cultured for 24 hours, was then inoculated into the initial fermentation medium, and the pH was adjusted to 6.8. During fermentation, the fermentation temperature was controlled at 30℃, and dissolved oxygen was maintained above 15% by adjusting the stirring speed (150 rpm~700 rpm) and aeration rate (0.3 vvm~2.5 vvm). A three-stage pH control method was used: the pH was allowed to naturally decrease from the initial 6.8 to 5.0; then, the pH was maintained at 5.0 for approximately 8 hours; subsequently, as the pH naturally decreased to approximately 3.8, ammonia was automatically added to maintain the pH at 3.8 until fermentation was complete.

[0085] When the residual glucose concentration in the fermentation broth dropped below 10 g / L, a sterilized and concentrated solution containing 600 g / L glucose was automatically added to maintain the glucose concentration in the fermentation broth at around 10 g / L. Simultaneously, a sterilized mixed nitrogen source solution containing 80 g / L ammonium sulfate and 100 g / L yeast extract was automatically added to maintain the amino nitrogen concentration at around 1 g / L. Samples were taken at 8-hour intervals to determine residual sugar content, pH, DCW, and ε-PL yield.

[0086] The results showed that when the genetically engineered strain BP3 used glucose as a carbon source (M3G medium), after 72 h of batch fermentation, the ε-PL yield and DCW were 9.46 g / L and 16.23 g / L, respectively, which were 38.51% and 47.55% higher than those of the starting strain (6.83 g / L and 11.00 g / L, respectively).

[0087] (2) Feeding and fermentation in batches Following the batch fermentation procedure, the glucose content in the tank was measured after a certain fermentation period. Feeding was initiated when the glucose concentration fell below 10 g / L, and the glucose concentration in the fermentation broth was controlled at approximately 10 g / L. Before feeding, samples of the fermentation broth were collected every 8 hours to measure the residual glucose concentration, and feeding was adjusted according to the glucose consumption rate. The results showed that the maximum DCW of the engineered strain BP3 reached 43.47 g / L (176 h), the maximum ε-PL yield increased to 37.21 g / L (192 h), and the yield reached 4.85 g / (L·d).

[0088] The nucleotide sequences involved in the examples are as follows: Nicotinic acid phosphoribosyltransferase gene ( PncB The nucleotide sequence of ) is SEQ ID NO.1: Phosphoenolpyruvate carboxylase gene ( pck The nucleotide sequence of ) is SEQ ID NO.2: Pyruvate dehydrogenase gene ( pdh The nucleotide sequence of ) is SEQ ID NO.3: 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 recombinant Streptomyces albopictus strain for increasing ε-polylysine production, characterized in that, The recombinant Streptomyces co-overexpressed the nicotinic acid phosphoribosyltransferase gene. pncB Phosphoenolpyruvate carboxylase gene pck and pyruvate dehydrogenase gene pdh Three genes.

2. A method for constructing the recombinant Streptomyces albopictus according to claim 1, characterized in that, Includes the following steps: (1) Gene fragments pncB, pck, pdh The desired gene fragment was obtained by cloning and amplification using appropriate primers. (2) Using homologous recombination to separate gene fragments pncB, pck, pdh Ligated with the pIB139 vector, the recombinant plasmid pIB139- was obtained. pncB-pck-pdh; (3) The recombinant plasmid pIB139- pncB-pck-pdh Transfer E. coli ET12567 was then used as a recipient for conjugation transfer with Streptomyces leuciscus as the recipient. The correct conjugate was selected to obtain the recombinant Streptomyces leuciscus.

3. A method for producing ε-polylysine, characterized in that, The step includes the fermentation production of recombinant Streptomyces albopictus as described in claim 1.

4. The method according to claim 3, characterized in that, The fermentation process involves inoculating the bacterial strain into the fermentation medium at an inoculation rate of 5-13% v / v, and fermenting under conditions of 27-36℃, 150-700 rpm, and dissolved oxygen of 18-45%.

5. The method according to claim 4, characterized in that, The fermentation medium includes 40-60 g / L glucose, 3-8 g / L yeast extract, 7-12 g / L (NH4)2SO4, 0.3-0.8 g / L magnesium sulfate, 0.5-1.0 g / L dipotassium hydrogen phosphate, 1.20-1.60 g / L potassium dihydrogen phosphate, 0.02-0.05 g / L ferrous sulfate, and 0.03-0.06 g / L zinc sulfate.

6. The method according to claim 3, characterized in that, The fermentation is either batch fermentation or fed-batch fermentation.

7. The method according to claim 6, characterized in that, During batch fermentation, the following steps are included: when the pH naturally decreases from the initial 6.5~7.6 to 3.7~4.2, the pH is maintained at 3.7~4.2 by adding a pH adjuster until the batch fermentation is completed.

8. The method according to claim 6, characterized in that: During fed-batch fermentation, pH control is performed using the following steps: when the pH naturally decreases from the initial 6.5-7.6 to 4.5-5.6, a pH adjuster is fed in to maintain the pH at 4.5-5.6 for 6-12 hours; then, when the pH naturally decreases to 3.7-4.2, a pH adjuster is fed in to maintain the pH at 3.7-4.2 until the fed-batch fermentation is completed.

9. The method according to claim 6, characterized in that, The fed-batch fermentation process includes the following steps: when the residual glucose concentration in the fermentation broth drops below 10 g / L, a glucose solution is added to control the glucose concentration in the fermentation broth at 7~13 g / L, and a nitrogen source solution is added to maintain the amino nitrogen concentration in the fermentation broth at 0.7~1.3 g / L.

10. The method according to claim 9, characterized in that, The concentration of the glucose solution is 380~870 g / L; the nitrogen source solution is a mixed nitrogen source solution of 60~130 g / L ammonium sulfate and 70~130 g / L yeast powder.

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