贻贝足丝蛋白因其卓越的黏附能力、良好的生物相容性和生物降解性,在医药、食品和特种材料制造等领域具有广泛而独特的应用前景。通过合成生物学技术重组微生物表达贻贝足丝蛋白具有很大潜力。然而,工程菌表达条件不适合、表达元件不匹配是造成生产强度低的重要原因。该研究采用多种策略组合优化重组厚壳贻贝(Mytilus coruscus)足丝蛋白Mcofp-3在大肠杆菌中的表达,旨在实现Mcofp-3的高效发酵制备。首先,确定了重组Mcofp-3以包涵体形式表达时产量较高,初始产量为158.6 mg/L,且通过差速离心后蛋白占比超85%;接着,通过正交优化重组菌株的培养条件,确定在诱导时间为1 h,诱导剂浓度0.4 mmol/L,诱导后培养温度37 ℃ 时,利用TB培养基发酵培养,Mcofp-3表达量达到303.3 mg/L;在此基础上,结合载体选择、启动子及核糖体结合位点(ribosome binding site,RBS)工程多策略组合优化,在使用pACYCDuet为载体,T7启动子以及更换为翻译速率更高的RBS6,Mcofp-3的表达量进一步提高至484.2 mg/L。最终在5-L发酵罐中放大优化结果表明,发酵37 h后,Mcofp-3的表达水平达到1 720 mg/L,产率达46.49 mg/(L·h),结果为现有贻贝足丝蛋白重组表达报道中最高水平。以上研究为Mcofp-3的规模化工业生产奠定了基础,同时也为其他贻贝足丝蛋白的优化表达提供了重要参考。
Mussel foot protein is renowned for its remarkable adhesive properties, outstanding biocompatibility, and biodegradability, presenting broad and unique application prospects in fields such as medicine, food, and speciality materials manufacturing.The synthesis of mussel foot protein via synthetic biology techniques, specifically through recombinant microbial expression, holds significant potential.However, inappropriate expression conditions and a mismatched expression cassette in the engineered strain are major factors contributing to the low production intensity.Therefore, this study implemented a combination of strategies to optimize the expression of the recombinant mussel foot protein Mcofp-3 in Escherichia coli, with the aim of achieving efficient fermentation preparation of Mcofp-3.Initially, it was found that recombinant Mcofp-3 expressed in inclusion bodies resulted in a higher production, with an initial yield of 158.6 mg/L, and the protein proportion exceeded 85% by centrifugation.Subsequently, through orthogonal optimization of the culture conditions for the recombinant strain, it was determined that an induction time of 1 hour, an inducer concentration of 0.4 mmol/L, and a post-induction culture temperature of 37 ℃ in TB medium resulted in an Mcofp-3 expression level of 303.3 mg/L.Building on this, by integrating strategies, such as vector selection, promoter, and RBS engineering for further optimization, the expression level of Mcofp-3 was further increased to 484.2 mg/L when employing pACYCDuet as the vector, the T7 promoter, and substituting with the higher translation rate RBS6.The final optimization results in a 5-L fermenter indicated that after 37 hours of fermentation, the expression level of Mcofp-3 reached 1 720 mg/L, with a yield of 46.49 mg/(L·h), marking the highest level reported in the recombinant expression of mussel foot proteins to date.The above study lays the foundation for the large-scale industrial production of Mcofp-3, and also provides an important reference for the optimized expression of other mussel foot proteins.
[1] XU S C, KANG M Z, XIN X L, et al.Design principles and application research of mussel-inspired materials:A review[J].Journal of Environmental Chemical Engineering, 2024, 12(1):111655.
[2] GUO Q, CHEN J S, WANG J L, et al.Recent progress in synthesis and application of mussel-inspired adhesives[J].Nanoscale, 2020, 12(3):1307-1324.
[3] ZWIES C, VARGAS RODRíGUEZ Á M, NAUMANN M, et al.Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins-A case study for Mefp-3 from Mytilus edulis[J].Protein Expression and Purification, 2024, 219:106483.
[4] PRIEMEL T, PALIA R, BABYCH M, et al.Compartmentalized processing of catechols during mussel Byssus fabrication determines the destiny of DOPA[J].Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(14):7613-7621.
[5] WANG X E, LIAO Z, YANG Q M, et al.Characterization of a novel antioxidant byssal protein from Mytilus coruscus foot[J].International Journal of Biological Macromolecules, 2024, 273:133095.
[6] WANG Z, FANG Y, ZENG Y, et al.Immunomodulatory peptides from thick-shelled mussel (Mytilus coruscus):Isolation, identification, molecular docking and immunomodulatory effects on RAW264.7 cells[J].Food Bioscience, 2024, 59:103874.
[7] LI K, XU Z T, LIU X X, et al.Mussel foot inspired bionic adhesive material enhanced by a reconstructed in vitro system for interfacial adhesion[J].Chemical Engineering Journal, 2023, 452:139580.
[8] CASTILLO J J, SHANBHAG B K, HE L.Food Bioactives:Extraction and Biotechnology Applications [M].Cham:Springer International Publishing, 2017:111-135.
[9] WANG J, SCHEIBEL T.Recombinant production of mussel Byssus inspired proteins[J].Biotechnology Journal, 2018, 13(12):1800146.
[10] ESKANDARI A, NEZHAD N G, LEOW T C, et al.Essential factors, advanced strategies, challenges, and approaches involved for efficient expression of recombinant proteins in Escherichia coli[J].Archives of Microbiology, 2024, 206(4):152.
[11] WANG X Y, FENG X X, XUE R, et al.Promoting soluble expression of hybrid mussel foot proteins by SUMO-TrxA tags for production of mussel glue[J].International Journal of Biological Macromolecules, 2023, 225:840-847.
[12] XUE R, WANG J, HUANG W W, et al.Efficient production of recombinant hybrid mussel proteins with improved adhesion[J].International Journal of Biological Macromolecules, 2025, 289:139937.
[13] BOLGHARI N, SHAHSAVARANI H, ANVARI M, et al.A novel recombinant chimeric bio-adhesive protein consisting of mussel foot protein 3, 5, gas vesicle protein A, and CsgA curli protein expressed in Pichia pastoris[J].AMB Express, 2022, 12(1):23.
[14] CASTRO J M, BIANCHI V A, PASCUAL M M, et al.Immune and biochemical responses in hemolymph and gills of the Patagonian freshwater mussel Diplodon chilensis, against two microbiological challenges:Saccharomyces cerevisiae and Escherichia coli[J].Journal of Invertebrate Pathology, 2018, 157:36-44.
[15] SEONG C,GYUN K, LIM S, et al.Recombinant mussel adhesive protein fp-5 (MAP fp-5) as a bulk bioadhesive and surface coating material[J].Biofouling, 2011, 27(7):729-737.
[16] YANG B, GYUN K,HYUN S, et al.A comparative study on the bulk adhesive strength of the recombinant mussel adhesive protein fp-3[J].Biofouling, 2013, 29(5):483-490.
[17] LEE S J, HAN Y H, NAM B H, et al.A novel expression system for recombinant marine mussel adhesive protein Mefp1 using a truncated OmpA signal peptide[J].Molecules and Cells, 2008, 26(1):34-40.
[18] 袁升. 贻贝足丝蛋白Mgfp-5及其融合蛋白在大肠杆菌中的表达和粘附性能研究 [D].武汉:华中科技大学, 2021.
YUAN S.Expression and adhesion of mussel foot silk protein Mgfp-5 and its fusion protein in Escherichia coli[D].Wuhan:Huazhong University of Science and Technology, 2021.
[19] 吕玉伟, 吕亚维, 杨泽熵, 等.贻贝蛋白Mgfp-5的原核表达与纯化[J].西北大学学报(自然科学版), 2016, 46(3):390-396.
LYU Y W,LV Y W,YANG Z S,et al.Prokaryotic expression and purification of Mytilus galloprovincialis foot protein-5[J].Journal of Northwest University (Natural Science Edition), 2016, 46(3):390-396.
[20] 李楠楠. 厚壳贻贝足丝蛋白分子多样性及重组表达分析 [D].舟山:浙江海洋大学, 2012.
LI N N.Molecular diversity and recombinant expression analysis of Mytilus coruscus foot protein[D].Zhoushan:Zhejiang Ocean University, 2012.
[21] CHOI B H, CHEONG H, KEE J, et al.Highly purified mussel adhesive protein to secure biosafety for in vivo applications[J].Microbial Cell Factories, 2014, 13(1):52.
[22] GEHRKE C W, WALL L L Sr, ABSHEER J S, et al.Sample preparation for chromatography of amino acids:Acid hydrolysis of proteins[J].Journal of Association of Official Analytical Chemists, 1985, 68(5):811-821.
[23] ZHOU H Y, WU W J, NIU K, et al.Enhanced L-methionine production by genetically engineered Escherichia coli through fermentation optimization[J].3 Biotech, 2019, 9(3):96.
[24] CHEN Z, ZHOU P, ZHAO Z J, et al.Enhanced production of L-serine in Escherichia coli by fermentation process optimization and transcriptome sequencing[J].Biochemical Engineering Journal, 2023, 200:109109.
[25] 陈佳敏, 王阳, 堵国成, 等.优化前体供给与细胞膜通透性强化大肠杆菌合成麦角硫因[J].食品与生物技术学报, 2022, 41(8):43-52.
CHEN J M, WANG Y, DU G C, et al.Enhancement of ergothioneine synthesis in Escherichia coli via optimization of precursor supply and cell membrane permeability[J].Journal of Food Science and Biotechnology, 2022, 41(8):43-52.
[26] ZHOU Y L, LU Z H, WANG X, et al.Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli[J].Applied Microbiology and Biotechnology, 2018, 102(4):1545-1556.
[27] ZHANG Y, GUO M L, ZHANG X Y, et al.Multiple optimizations of recombinant plasmid for improving expression of Hepatitis B core antigen in Escherichia coli[J].Protein Expression and Purification, 2022, 198:106127.
[28] SANTOS S P O, GARCÉS L F S, SILVA F S R, et al.Engineering an optimized expression operating unit for improved recombinant protein production in Escherichia coli[J].Protein Expression and Purification, 2022, 199:106150.
[29] WANG Y, LIU Q T, WENG H J, et al.Construction of synthetic promoters by assembling the sigma factor binding -35 and -10 boxes[J].Biotechnology Journal, 2019, 14(1):1800298.
[30] HUANG Y K, YU C H, NG I S.Precise strength prediction of endogenous promoters from Escherichia coli and J-series promoters by artificial intelligence[J].Journal of the Taiwan Institute of Chemical Engineers, 2024, 160:105211.
[31] WANG D A, CHEN J M, WANG Y, et al.Engineering Escherichia coli for high-yield production of ectoine[J].Green Chemical Engineering, 2023, 4(2):217-223.
[32] 郑依琳, 胥睿睿, 王阳, 等.细菌酪氨酸酶高效异源表达及其在生物染发和丝素多肽多巴修饰中的应用[J].生物工程学报, 2024, 40(9):3083-3102.
ZHENG Y L, XU R R, WANG Y, et al.Heterologous expression of bacterial tyrosinase and its applications in biological hair dyeing and DOPA modification of hydrolyzed silk fibroin[J].Chinese Journal of Biotechnology, 2024, 40(9):3083-3102.
