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.
TIAN Mingke
,
ZOU Yu
,
WU Xiaoyue
,
BAI Zhonghu
,
LI Kang
,
LIU Xiuxia
. Secretion of recombinant lysozyme in Corynebacterium glutamicum[J]. Food and Fermentation Industries, 2025
, 51(24)
: 1
-8
.
DOI: 10.13995/j.cnki.11-1802/ts.042318
[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.
