Abstract: Collagen is the most abundant structural protein in human body and a considerable component of cytoplasmic matrix. Collagen, attributed to its unique triple helix structure, has good mechanical properties and high biocompatibility, thereby being widely used in many applications, such as biomedicine, tissue engineering and cosmetics. The use of microorganisms to express recombinant collagen has the advantages of short fermentation cycle, low cost, and convenient genetic manipulation, which has been developed rapidly in recent years. However, the secretion of recombinant collagen with a triple helix structure remains a challenge.In this study, a platform for the secretion of recombinant collagen by Corynebacterium glutamicum was established. Six different signal peptides (Cg1514, Cg2052, CgR0949, CspB, PorB or TorA) were fused to the N-terminal of V-B derived from Streptococcus pyogenes and ligated into pXMJ19 to construct a recombinant expression vector. The recombinant plasmid was transferred into the food-grade expression host C. glutamicum ATCC 13032 to construct six recombinant strains. The secretion efficiency of these signal peptides was compared, and the expression conditions were optimized. The results showed that PorB was the most efficient signal peptide for V-B secretion. At the same time, the yields of collagen mediated by the Sec-dependent signal peptides Cg1514, CspB and PorB were higher than those of CgR0949 and TorA, which were the Tat-dependent signal peptides. This is consistent with the latest research result that C. glutamicum mainly secretes spider silk protein in a Sec-dependent manner. Therefore, it is speculated that the Sec-dependent secretion pathway is conducive to the secretion of fibrous protein such as collagen and spider silk protein.In addition, the culture medium, initial induction OD600, induction duration, IPTG concentration, and dissolved oxygen were optimized for the secretory expression mediated by PorB signal peptide. The optimal expression conditions were as follows: cultured in 500 mL baffled shake flask with 50 mL culture medium 1(20 g/L glucose, 20 g/L corn steep powder, 20 g/L (NH4)2SO4, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/L MgSO4, and 42 g/L MOPS [3-(N-Morpholinyl) propanesulfonic acid sodium salt]), with an initial induction OD600 of 6 and induced by 0.5 mmol/L IPTG for 40 h. It was found that dissolved oxygen was the key factor affecting collagen production at the flask level, which provided guidance for further scale-up test at industrial production level. Subsequently, the supernatant was taken for affinity purification, followed by trypsin digestion. The V domain was cut into small peptides, while the collagen domain B was not digested due to its rigid triple helix structure when it’s correctly folded. After desalting, lyophilization and weighing, the final secretion yield of collagen domain B at the shake flask level reached 30 mg/L, which was 3 times that before optimization. The secondary structure was characterized by circular dichroism showing that it was correctly folded into a typical triple helix structure of collagen. The melting curve was scanned at 225 nm from 10 ℃ to 70 ℃ and the melting temperature (Tm) value of collagen domain B was calculated to be 25.82 ℃, which was close to that reported previously.The Corynebacterium glutamicum expression and secretion system established in this study is of food safety grade, and can efficiently secrete collagen with a triple helix structure, which provides a new host choice for the recombinant expression of collagen. Further, this development can provide solutions and theoretical basis for the production of recombinant collagen as well as other functional biomaterial proteins for the application in biomedicine and tissue engineering.
IRASTORZA A, ZARANDONA I, ANDONEGI M, et al.The versatility of collagen and chitosan:From food to biomedical applications[J].Food Hydrocolloids, 2021, 116:106633.
[4]
BÁEZ J, OLSEN D, POLAREK J W.Recombinant microbial systems for the production of human collagen and gelatin[J].Applied Microbiology and Biotechnology, 2005, 69(3):245-252.
[5]
XU Y, KEENE D R, BUJNICKI J M, et al.Streptococcal Scl1 and Scl2 proteins form collagen-like triple helices[J].Journal of Biological Chemistry, 2002, 277(30):27 312-27 318.
[6]
YU Z X, BRODSKY B, INOUYE M.Dissecting a bacterial collagen domain from Streptococcus pyogenes:Sequence and length-dependent variations in triple helix stability and folding[J].Journal of Biological Chemistry, 2011, 286(21):18 960-18 968.
[7]
PENG Y Y, HOWELL L, STOICHEVSKA V, et al.Towards scalable production of a collagen-like protein from Streptococcus pyogenes for biomedical applications[J].Microbial Cell Factories, 2012, 11(1):146.
[8]
GUO J Q, LUO Y E, FAN D D, et al.Analysis of metabolic products by response surface methodology for production of human-like collagen II[J].Chinese Journal of Chemical Engineering, 2010, 18(5):830-836.
[9]
JIA Q L, LUO Y E, FAN D D, et al.The different roles of chaperone teams on over-expression of human-like collagen in recombinant Escherichia coli[J].Journal of the Taiwan Institute of Chemical Engineers, 2014, 45(6):2 843-2 850.
[10]
常海燕, 范代娣, 骆艳娥, 等.重组大肠杆菌高密度发酵生产类人胶原蛋白Ⅱ条件优化[J].微生物学通报, 2009, 36(6):870-874.CHANG H Y, FAN D D, LUO Y E, et al.Optimization of recombinant E.coli high-density fermentation for expressing human-like collagen Ⅱ[J].Microbiology, 2009, 36(6):870-874.
[11]
刘斌. 巴氏毕赤酵母基因工程菌高密度发酵表达重组人源胶原蛋白[D].南京:南京理工大学, 2012.LIU B.High-density fermentation of genetically engineered Pichia pastoris expressing recombinant human-source collagen[D].Nanjing:Nanjing University of Science and Technology, 2012.
[12]
LI L B, FAN D D, MA X X, et al.High-level secretory expression and purification of unhydroxylated human collagen α1(III) chain in Pichia pastoris GS115[J].Biotechnology and Applied Biochemistry, 2015, 62(4):467-475.
[13]
PAKKANEN O, HÄMÄLÄINEN E R, KIVIRIKKO K I, et al.Assembly of stable human type i and iii collagen molecules from hydroxylated recombinant chains in the yeast Pichia pastoris:Effect of an engineered c-terminal oligomerization domain foldon[J].Journal of Biological Chemistry, 2003, 278(34):32 478-32 483.
[14]
KEIZER-GUNNINK I, VUORELA A, MYLLYHARJU J, et al.Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris[J].Matrix Biology, 2000, 19(1):29-36.
[15]
PAKKANEN O, PIRSKANEN A, MYLLYHARJU J.Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris[J].Journal of Biotechnology, 2006, 123(2):248-256.
[16]
HANSMEIER N, CHAO T C, PÜHLER A, et al.The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032[J].Proteomics, 2006, 6(1):233-250.
[17]
YIM S S, CHOI J W, LEE R J, et al.Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum[J].Biotechnology and Bioengineering, 2016, 113(1):163-172.
[18]
YIM S S, AN S J, CHOI J W, et al.High-level secretory production of recombinant single-chain variable fragment (scFv) in Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology, 2014, 98(1):273-284.
[19]
KIKUCHI Y, DATE M, YOKOYAMA K I, et al.Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum:Processing of the pro-transglutaminase by a cosecreted subtilisin-like protease from Streptomyces albogriseolus[J].Applied and Environmental Microbiology, 2003, 69(1):358-366.
[20]
WATANABE K, TSUCHIDA Y, OKIBE N, et al.Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences[J].Microbiology (Reading, England), 2009, 155(Pt 3):741-750.
[21]
KIKUCHI Y, DATE M, ITAYA H, et al.Functional analysis of the twin-arginine translocation pathway in Corynebacterium glutamicum ATCC 13869[J].Applied and Environmental Microbiology, 2006, 72(11):7 183-7 192.
[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]
胡立涛, 王阳, 李佳莲, 等.代谢工程改造谷氨酸棒杆菌合成透明质酸[J].食品与发酵工业, 2020, 46(18):1-7.HU L T, WANG Y, LI J L, et al.Metabolic engineering of Corynebacterium glutamicum for hyaluronic acid production[J].Food and Fermentation Industries, 2020, 46(18):1-7.
[24]
BUTKOWSKI R J, NOELKEN M E, HUDSON B G.Estimation of the size of collagenous proteins by electrophoresis and gel chromatography[J].Methods in Enzymology, 1982, 82:410-423.
[25]
ANNE J, VRANCKEN K, VAN MELLAERT L, et al.Protein secretion biotechnology in Gram-positive bacteria with special emphasis on Streptomyces lividans[J].Biochimica et Biophysica Acta, 2014, 1843(8):1 750-1 761.
[26]
HEMMERICH J, ROHE P, KLEINE B, et al.Use of a sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum[J].Microbial Cell Factories, 2016, 15(4):323-324.
[27]
JIN Q, PAN F, HU C F, et al.Secretory production of spider silk proteins in metabolically engineered Corynebacterium glutamicum for spinning into tough fibers[J].Metabolic Engineering, 2022, 70:102-114.
[28]
MALAKAR P, VENKATESH K V.Effect of substrate and IPTG concentrations on the burden to growth of Escherichia coli on glycerol due to the expression of Lac proteins[J].Applied Microbiology and Biotechnology, 2012, 93(6):2 543-2 549.
[29]
YANG Z F, PEI X L, XU G, et al.Efficient inducible expression of nitrile hydratase in Corynebacterium glutamicum[J].Process Biochemistry, 2019, 76:77-84.
[30]
侯亚茹, 张萌, 许菲.引入非天然氨基酸胶原蛋白表达及交联成键的优化[J].生物工程学报, 2021, 37(9):3 231-3 241.HOU Y R, ZHANG M, XU F.Optimization of unnatural amino acid incorporation in collagen and the cross-linking through thioether bond[J].Chinese Journal of Biotechnology, 2021, 37(9):3 231-3 241.