牛奶的蛋白质主要以酪蛋白为主,κ-酪蛋白是酪蛋白的一种,约占牛脱脂乳中总酪蛋白含量的13%。κ-酪蛋白具有乳蛋白的营养作用,可应用于未来牛奶的全合成,同时它水解产生的肽类物质还具有抑制血液凝固及抗血栓形成的作用。谷氨酸棒状杆菌(Corynebacterium glutamicum)作为一种安全生产菌株,是食用蛋白生产的优势细胞平台。该研究利用谷氨酸棒状杆菌作为受体菌株表达异源蛋白的优势实现κ-酪蛋白的外源表达。根据κ-酪蛋白的信号肽和前肽的分析设计并合成谷氨酸棒状杆菌密码子偏好性κ-酪蛋白基因,利用谷氨酸棒状杆菌ATCC 13032菌株用于表达κ-酪蛋白,并对该蛋白N端促溶标签、表达元件信号肽、表达宿主等进行优化,同时对发酵条件进一步优化。结果表明,对κ-酪蛋白进行二级结构及无序性分析,预测结果显示κ-酪蛋白具有固定的无序区域。采用SDS-PAGE和Western blotting技术检测κ-酪蛋白的表达情况,结果表明κ-酪蛋白能在谷氨酸棒状杆菌ATCC 13032菌株中成功表达,在其N端添加SUMO、FH8促溶标签能提高κ-酪蛋白可溶表达量,进行信号肽和宿主筛选能进一步提高蛋白产量。该研究在谷氨酸棒状杆菌中成功表达κ-酪蛋白,并通过优化手段进一步提高了产量,为未来牛奶全合成的实现奠定了基础。
Milk's primary protein component is casein, with κ-casein (κ-CN) comprising roughly 13% of the total casein content in bovine skim milk.κ-CN offers the nutritional benefits typical of milk proteins and holds potential for future applications in total milk synthesis.Moreover, peptides derived from κ-CN hydrolysis exhibit anticoagulant and antithrombotic properties. Corynebacterium glutamicum, a safely produced strain, served as an advantageous platform for edible protein production.This study leveraged C.glutamicum's capability to express heterologous proteins, focusing on the exogenous expression of κ-CN.The κ-CN gene was codon-optimized and synthesized, and the C.glutamicum ATCC13032 strain was utilized to express κ-CN-6×His protein.Optimization efforts targeted the N-terminal solubilization tag, signal peptide, expression host, and fermentation conditions.Results showed that structural analysis revealed fixed disordered regions within κ-CN.Expression of κ-CN was confirmed via SDS-PAGE and western blotting, indicating successful expression in C.glutamicum ATCC13032 strains.Soluble expression was enhanced by incorporating SUMO and FH8 solubilization tags at the N-terminus, with further improvements in protein yield achieved through signal peptide and host screening.This study demonstrates the successful expression of κ-CN in Corynebacterium glutamicum, with optimization strategies enhancing yield and laying the groundwork for future advancements in total milk synthesis.
[1] MALIK S, SIDHU N S, KUMAR S, et al.Kappa-casein alleles in Zebu and cross-bred (1/2 Friesian, 1/4 Jersey, 1/4 Hariana) cattle from India using polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSOP)[J].Genetic Analysis:Biomolecular Engineering, 1997, 14(2):61-63.
[2] CAPRIOTTI A L, CAVALIERE C, PIOVESANA S, et al.Recent trends in the analysis of bioactive peptides in milk and dairy products[J].Analytical and Bioanalytical Chemistry, 2016, 408(11):2677-2685.
[3] MIGLIORE-SAMOUR D, JOLLÈS P.Casein, a prohormone with an immunomodulating role for the newborn?[J].Experientia, 1988, 44(3):188-193.
[4] BERTHOU J, MIGLIORE-SAMOUR D, LIFCHITZ A, et al.Immunostimulating properties and three-dimensional structure of two tripeptides from human and cow caseins[J].FEBS Letters, 1987, 218(1):55-58.
[5] FOX P F.Milk proteins:General and historical aspects[M].Advanced Dairy Chemistry—1 Proteins.Boston, MA:Springer US, 2003:1-48.
[6] O'MAHONY J A, FOX P F.Milk proteins:Introduction and historical aspects[M].Advanced Dairy Chemistry.Boston, MA:Springer US, 2012:43-85.
[7] GUTIÉRREZ-ADÁN A, MAGA E A, MEADE H, et al.Alterations of the physical characteristics of milk from transgenic mice producing bovine κ-casein[J].Journal of Dairy Science, 1996, 79(5):791-799.
[8] 庞雨. 牛乳蛋白在大肠杆菌中的异源表达和组合合成[D].北京:中国农业科学院, 2021.
PANG Y.Heterologous expression and combinatorial synthesis of bovine milk proteins in Escherichia coli[D].Beijing:Chinese Academy of Agricultural Sciences, 2021.
[9] BECKER J, ROHLES C M, WITTMANN C.Metabolically engineered Corynebacterium glutamicum for bio-based production of chemicals, fuels, materials, and healthcare products[J].Metabolic Engineering, 2018, 50:122-141.
[10] CHENG F Y, YU H M, STEPHANOPOULOS G.Engineering Corynebacterium glutamicum for high-titer biosynthesis of hyaluronic acid[J].Metabolic Engineering, 2019, 55:276-289.
[11] XU G Q, ZHA J, CHENG H, et al.Engineering Corynebacterium glutamicum for the de novo biosynthesis of tailored poly-γ-glutamic acid[J].Metabolic Engineering, 2019, 56:39-49.
[12] YU X Y, LIU X X, GAO X, et al.Development of a novel platform for recombinant protein production in Corynebacterium glutamicum on ethanol[J].Synthetic and Systems Biotechnology, 2022, 7(2):765-774.
[13] PARK S H, KIM H U, KIM T Y, et al.Metabolic engineering of Corynebacterium glutamicum for L-arginine production[J].Nature Communications, 2014, 5:4618.
[14] LIU X X, YANG Y K, ZHANG W, et al.Expression of recombinant protein using Corynebacterium Glutamicum:Progress, challenges and applications[J].Critical Reviews in Biotechnology, 2016, 36(4):652-664.
[15] SATAKARNI M, CURTIS R.Production of recombinant peptides as fusions with SUMO[J].Protein Expression and Purification, 2011, 78(2):113-119.
[16] JOHANSSEN V A, BARNHAM K J, MASTERS C L, et al.Generating recombinant C-terminal prion protein fragments of exact native sequence[J].Neurochemistry International, 2012, 60(3):318-326.
[17] LU W G, CAI X T, GU Z H, et al.Production and characterization of hirudin variant-1 by SUMO fusion technology in E.coli[J].Molecular Biotechnology, 2013, 53(1):41-48.
[18] SINGH A, YADAV D, RAI K M, et al.Enhanced expression of rabies virus surface G-protein in Escherichia coli using SUMO fusion[J].The Protein Journal, 2012, 31(1):68-74.
[19] LI J F, ZHANG J, ZHANG Z, et al.SUMO mediating fusion expression of antimicrobial peptide CM4 from two joined genes in Escherichia coli[J].Current Microbiology, 2011, 62(1):296-300.
[20] WANG Z Y, LI N, WANG Y Y, et al.Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein production and purification[J].Protein Expression and Purification, 2012, 82(1):174-178.
[21] COSTA S J, COELHO E, FRANCO L, et al.The Fh8 tag:A fusion partner for simple and cost-effective protein purification in Escherichia coli[J].Protein Expression and Purification, 2013, 92(2):163-170.
[22] VAN DER REST M E, LANGE C, MOLENAAR D.A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA[J].Applied Microbiology and Biotechnology, 1999, 52(4):541-545.
[23] 李祥魁, 范翠英, 崔亚君, 等.重组蛋白可溶性表达促进标签的研究进展[J].生物技术, 2013, 23(2):93-97.
LI X K, FAN C Y, CUI Y J, et al.Recent advances on the solubility-enhancing fusion tags for recombinant protein expression[J].Biotechnology, 2013, 23(2):93-97.
[24] 彭枫, 刘秀霞, 陈静, 等.不同表达模式组合对谷氨酸棒状杆菌中脑钠肽蛋白表达的影响[J].食品与发酵工业, 2018, 44(8):1-7.
PENG F, LIU X X, CHEN J, et al.The effect of different expression patterns on B-type natriuretic peptide expression in Corynebacterium glutamicum[J].Food and Fermentation Industries, 2018, 44(8):1-7.
[25] SUN M M, GAO A X, LEDESMA-AMARO R, et al.Hypersecretion of OmlA antigen in Corynebacterium glutamicum through high-throughput based development process[J].Applied Microbiology and Biotechnology, 2022, 106(8):2953-2967.
