溶菌酶又称细胞壁水解酶或肽聚糖水解酶,是一种天然抗菌酶,具有广谱抑菌活性,已被广泛应用于食品防腐领域,谷氨酸棒杆菌(Corynebacterium glutamicum)作为食品安全菌株生产溶菌酶具有很大的应用潜力。该研究旨在实现重组溶菌酶在C.glutamicum CGMCC1.15647中的分泌表达。首先,分析了不同来源溶菌酶在C.glutamicum中的表达情况,并选择表达效果最佳的溶菌酶作为目标蛋白,利用载体pGX19进行分泌表达,接着在表达元件上进行了信号肽筛、启动子和核糖体结合位点(ribosome binding site,RBS)组合优化,又从转运水平上提高重组溶菌酶的分泌。结果表明Tat途径的CgR0949信号肽明显提高重组溶菌酶表达量;经过启动子和RBS组合优化,其表达量提高了2.3倍;过表达不同来源的Tat转运系统,重组溶菌酶表达量提高了1.5倍,经多水平优化后,重组溶菌酶的摇瓶发酵水平较初始菌株提高8.3倍,达到0.2 g/L。最后,对表达的重组蛋白进行了纯化,并对其酶学性质进行了初步研究,最适pH值为7.5,最适反应温度为35 ℃。该研究首次在C.glutamicum中实现重组溶菌酶的分泌表达,并通过信号肽筛选、转录翻译元件优化及转运系统强化,为溶菌酶生产提供了新策略。
Lysozyme, also known as cell wall hydrolase or peptidoglycan hydrolase, is a natural antimicrobial enzyme with broad-spectrum antibacterial activity and has been widely used in food preservation.Corynebacterium glutamicum, a food-safe strain, shows significant potential for lysozyme production.This study aimed to achieve the secretion and expression of recombinant lysozyme in C.glutamicum CGMCC1.15647.First, the expression of lysozymes from different sources in C.glutamicum was analyzed, and the lysozyme with optimal expression efficiency was selected as the target protein.The pGX19 vector was employed for secretory expression.Subsequently, multi-level optimizations were performed, including signal peptide screening, promoter and ribosome binding site (RBS) combinations, and enhancement of the transport system.Results showed that the Tat pathway signal peptide CgR0949 significantly improved recombinant lysozyme expression.After promoter and RBS optimization, the expression level increased by 2.3-fold.Overexpression of the Tat transport system from diverse sources further enhanced recombinant lysozyme production by 1.5-fold.Through these optimizations, the shake-flask fermentation yield of recombinant lysozyme reached 0.2 g/L, representing an 8.3-fold improvement over the initial strain.Finally, the recombinant protein was purified, and its enzymatic properties were characterized, revealing optimal activity at pH 7.5 and 35 ℃.This study is the first to achieve secretory expression of recombinant lysozyme in C.glutamicum.By integrating signal peptide screening, transcription-translation element optimization, and transport system reinforcement, it provides a novel strategy for efficient lysozyme production.
[1] WEI Z X, WU S H, XIA J J, et al.Enhanced antibacterial activity of hen egg-white lysozyme against Staphylococcus aureus and Escherichia coli due to protein fibrillation[J].Biomacromolecules, 2021, 22(2):890-897.
[2] WANG S W, WANG T Y.Study on antibacterial activity and structure of chemically modified lysozyme[J].Molecules, 2023, 28(1):95.
[3] ZHANG W L, RHIM J W.Functional edible films/coatings integrated with lactoperoxidase and lysozyme and their application in food preservation[J].Food Control, 2022, 133:108670.
[4] FERRABOSCHI P, CICERI S, GRISENTI P.Applications of lysozyme, an innate immune defense factor, as an alternative antibiotic[J].Antibiotics, 2021, 10(12):1534.
[5] SHAHMOHAMMADI A.Lysozyme separation from chicken egg white:A review[J].European Food Research and Technology, 2018, 244(4):577-593.
[6] NAVEED M, WEN S, CHAN M W H, et al.Expression of BSN314 lysozyme genes in Escherichia coli BL21:A study to demonstrate microbicidal and disintegarting potential of the cloned lysozyme[J].Brazilian Journal of Microbiology, 2024, 55(1):215-233.
[7] LAMPPA J W, TANYOS S A, GRISWOLD K E.Engineering Escherichia coli for soluble expression and single step purification of active human lysozyme[J].Journal of Biotechnology, 2013, 164(1):1-8.
[8] CALLEWAERT L, MASSCHALCK B, DECKERS D, et al.Purification of Ivy, a lysozyme inhibitor from Escherichia coli, and characterisation of its specificity for various lysozymes[J].Enzyme and Microbial Technology, 2005, 37(2):205-211.
[9] 王亚森. 人源溶菌酶在毕赤酵母中的表达优化[D].无锡:江南大学, 2021.
Wang Y S.Optimization of human lysozyme expression in Pichia pastoris[D].Wuxi:Jiangnan University, 2021.
[10] 于政权, 樊宝良, 戴蕴平, 等. 转基因小鼠乳腺表达重组人溶菌酶. 科学通报, 2003, 48(20):2149-2153.
YU Z Q, FAN B L, DAI Y P, et al. Expression of recombinant human lysozyme in mammary gland of transgenic mice. Chinese Science Bulletin, 2003, 48(20):2149-2153.
[11] WANG M M, SHI Z, GAO N, et al.Sustainable and high-level microbial production of plant hemoglobin in Corynebacterium glutamicum[J].Biotechnology for Biofuels and Bioproducts, 2023, 16(1):80.
[12] LIU X X, ZHANG W, ZHAO Z H, et al.Protein secretion in Corynebacterium glutamicum[J].Critical Reviews in Biotechnology, 2017, 37(4):541-551.
[13] CALABRETTA P, HODGES H L, KRAFT M B, et al.Bacterial cell wall modification with a glycolipid substrate[J].Journal of the American Chemical Society, 2019, 141(23):9262-9272.
[14] HO T D, HASTIE J L, INTILE P J, et al.The Bacillus subtilis extracytoplasmic function σ factor σ(V) is induced by lysozyme and provides resistance to lysozyme[J].Journal of Bacteriology, 2011, 193(22):6215-6222.
[15] GREEN D S.Effects of microplastics on European flat oysters, Ostrea edulis and their associated benthic communities[J].Environmental Pollution, 2016, 216:95-103.
[16] MADANI A, KRAUSE B, GREENE E R, et al.Large language models generate functional protein sequences across diverse families[J].Nature Biotechnology, 2023, 41(8):1099-1106.
[17] SUN M M, GAO A X, LI A, et al.Bicistronic design as recombinant expression enhancer:Characteristics, applications, and structural optimization[J].Applied Microbiology and Biotechnology, 2021, 105(20):7709-7720.
[18] JIANG Y, QIAN F H, YANG J J, et al.CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum [J].Nature Communications, 2017, 8:15179.
[19] 余心宇. 基于CRISPR—微流控筛选系统鉴定谷氨酸棒杆菌高效分泌蛋白的关联基因 [D], 无锡:江南大学,2022.
YU X Y.Identification of genes associated with efficient protein secretion in C.glutamicum using a CRISPR-microfluidic screening system[D].Wuxi:Jiangnan University,2022.
[20] 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.
[21] DUAN Y T, ZHAI W J, LIU W J, et al.Fine-tuning multi-gene clusters via well-characterized gene expression regulatory elements:Case study of the arginine synthesis pathway in C.glutamicum[J].ACS Synthetic Biology, 2021, 10(1):38-48.
[22] KIKUCHI Y, ITAYA H, DATE M, et al.TatABC overexpression improves Corynebacterium glutamicum Tat-dependent protein secretion[J].Applied and Environmental Microbiology, 2009, 75(3):603-607.
[23] YU X Y, LI S, FENG H B, et al.CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion[J].Metabolic Engineering, 2023, 75:192-204.
