硫酸软骨素A(chondroitin sulfate A, CSA)是关节软骨修复中不可或缺的重要成分。CSA由软骨素4-O-磺基转移酶-1(chondroitin 4-O-sulfotransferase-1, C4ST-1)催化软骨素中N-乙酰氨基半乳糖胺(N-acetylgalactosamine, GalNAc)4号位羟基磺酸化合成。然而,由于C4ST-1酶活性较低,其催化能力受限,进而阻碍了CSA的工业化生产进程。为此,该研究旨在通过结合重组菌构建和培养基优化提升C4ST-1酶活性。首先在筛选确定了以毕赤酵母GS115为底盘细胞的基础上,进一步优化了以OST1-α分泌信号肽和SUMO Pro 3促溶标签的组合方式进行C4ST-1分泌表达,经摇瓶发酵C4ST-1最高酶活性为1 889.2 U/L。鉴于前期研究发现无机盐对C4ST-1酶活性的抑制以及现有培养基成本较高问题,该研究通过优化培养基组分,发现不添加昂贵成分酵母无氨基氮源(yeast nitrogen base without amino acids, YNB)时,C4ST-1酶活性较原培养基提高68.4%。此外,结合碳源、其他氮源以及生物素的筛选与优化使C4ST-1酶活性进一步提高。最终,在5L发酵罐补料分批发酵72 h时,获得最高酶活性为5 040.7 U/L。该研究不仅为CSA规模化生产奠定了基础,也将为其他糖胺聚糖(如肝素、硫酸皮肤素)合成所需的磺基转移酶发酵生产提供借鉴。
Chondroitin sulfate A (CSA) is an essential component in articular cartilage repair.CSA is synthesized by the hydroxyl sulfonation of N-acetylgalactosamine (GalNAc) at position 4 in chondroitin catalyzed by chondroitin 4-O-sulfotransferase-1 (C4ST-1).However, the low enzyme activity of C4ST-1 limits its catalytic efficiency, thereby hindering the industrial production of CSA.For this reason, this study aimed to enhance C4ST-1 enzyme activity by combining recombinant strain construction and medium optimization.Initially, based on the screening and determination of Komagataella phaffii GS115 as the chassis cell, the combination of OST1-α secretion signal peptide and SUMO Pro 3 solubility-enhancing tag was further optimized for the secretion and expression of C4ST-1, and the highest enzyme activity of C4ST-1 was 1,889.2 U/L after shake flask fermentation.Given that previous studies found inorganic salts to inhibit C4ST-1 enzyme activity and the high cost of the existing medium, this study optimized the medium components and found that omitting the expensive component, yeast nitrogen base without amino acids (YNB), resulted in a 68.4% increase in C4ST-1 enzyme activity compared to the original medium.Furthermore, the screening and optimization of carbon sources, other nitrogen sources, and biotin further increased the C4ST-1 enzyme activity.Finally, fed-batch fermentation in a 5 L fermenter for 72 hours achieved a maximum enzyme activity of 5 040.7 U/L.This study not only lays the foundation for the large-scale production of CSA, but will also provide a reference for the fermentation and production of sulfotransferases required for the synthesis of other glycosaminoglycans (e.g., heparin, dermatan sulfate).
[1] 赵琳, 云振宇, 吴希, 等.硫酸软骨素钠的制备及抗骨关节炎活性研究进展[J].医药导报, 2024, 43(6):916-925.
ZHAO L, YUN Z Y, WU X, et al.Research progress on preparation and anti-osteoarthritis activity of chondroitin sulfate sodium[J].Herald of Medicine, 2024, 43(6):916-925.
[2] MARTEL-PELLETIER J, FARRAN A, MONTELL E, et al.Discrepancies in composition and biological effects of different formulations of chondroitin sulfate[J].Molecules, 2015, 20(3):4277-4289.
[3] VOLPI N.Quality of different chondroitin sulfate preparations in relation to their therapeutic activity[J].Journal of Pharmacy and Pharmacology, 2009, 61(10):1271-1280.
[4] HE W Q, ZHU Y Y, SHIRKE A, et al.Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris[J].Applied Microbiology and Biotechnology, 2017, 101(18):6919-6928.
[5] TAKASHIMA M, SUZUKI K, MOCHIZUKI H, et al.Expression of highly active chondroitin 4-O-sulfotransferase-1 in Escherichia coli by a trigger factor fusion protein expression system[J].Process Biochemistry, 2022, 115:146-151.
[6] BADRI A, WILLIAMS A, AWOFIRANYE A, et al.Complete biosynthesis of a sulfated chondroitin in Escherichia coli[J].Nature Communications, 2021, 12:1389.
[7] LIU H Y, WEI W Q, PANG Z X, et al.Protein engineering, cofactor engineering, and surface display engineering to achieve whole-cell catalytic production of chondroitin sulfate A[J].Biotechnology and Bioengineering, 2023, 120(7):1784-1796.
[8] ZHOU Z X, LI Q, HUANG H, et al.A microbial-enzymatic strategy for producing chondroitin sulfate glycosaminoglycans[J].Biotechnology and Bioengineering, 2018, 115(6):1561-1570.
[9] ZHANG W J, ZHANG P, WANG H, et al.Enhancing the expression of chondroitin 4-O-sulfotransferase for one-pot enzymatic synthesis of chondroitin sulfate A[J].Carbohydrate Polymers, 2024, 337:122158.
[10] KLÜPPEL M, WIGHT T N, CHAN C, et al.Maintenance of chondroitin sulfation balance by chondroitin-4-sulfotransferase 1 is required for chondrocyte development and growth factor signaling during cartilage morphogenesis[J].Development, 2005, 132(17):3989-4003.
[11] YUSA A, KITAJIMA K, HABUCHI O.N-linked oligosaccharides are required to produce and stabilize the active form of chondroitin 4-sulphotransferase-1[J].Biochemical Journal, 2005, 388(Pt 1):115-121.
[12] 李成成, 黄义德.毕赤酵母表达系统及其发酵策略[J].福建轻纺, 2023(10):31-36.
LI C C, HUANG Y D.Pichia pastoris expression system and its fermentation strategy[J].The Light & Textile Industries of Fujian, 2023(10):31-36.
[13] LI J, SHAO Q N, XIANG Y L, et al.High-activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane[J].Biotechnology Journal, 2024, 19(5):2400098.
[14] GLEESON M A, WHITE C E, MEININGER D P, et al.Generation of protease-deficient strains and their use in heterologous protein expression[J].Methods in Molecular Biology, 1998, 103:81-94.
[15] DUAN G D, DING L M, WEI D S, et al.Screening endogenous signal peptides and protein folding factors to promote the secretory expression of heterologous proteins in Pichia pastoris[J].Journal of Biotechnology, 2019, 306:193-202.
[16] LIANG S L, LI C, YE Y R, et al.Endogenous signal peptides efficiently mediate the secretion of recombinant proteins in Pichia pastoris[J].Biotechnology Letters, 2013, 35(1):97-105.
[17] RUOHONEN L, HACKMAN P, LEHTOVAARA P, et al.Efficient secretion of Bacillus amyloliquefaciens α-amylase cells by its own signal peptide from Saccharomyces cerevisiae host[J].Gene, 1987, 59(2-3):161-170.
[18] CHUNG B H, NAM S W, KIM B M, et al.Highly efficient secretion of heterologous proteins from Saccharomyces cerevisiae using inulinase signal peptides[J].Biotechnology and Bioengineering, 1996, 49(4):473-479.
[19] MASSAHI A, ÇALıK P.Endogenous signal peptides in recombinant protein production by Pichia pastoris:From in-silico analysis to fermentation[J].Journal of Theoretical Biology, 2016, 408:22-33.
[20] AW R, MCKAY P F, SHATTOCK R J, et al.Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris[J].AMB Express, 2017, 7(1):70.
[21] BARRERO J J, CASLER J C, VALERO F, et al.An improved secretion signal enhances the secretion of model proteins from Pichia pastoris[J].Microbial Cell Factories, 2018, 17(1):161.
[22] GOVERDE C A, PACESA M, GOLDBACH N, et al.Computational design of soluble and functional membrane protein analogues[J].Nature, 2024, 631(8020):449-458.
[23] ESPOSITO D, CHATTERJEE D K.Enhancement of soluble protein expression through the use of fusion tags[J].Current Opinion in Biotechnology, 2006, 17(4):353-358.
[24] LIU L, SPURRIER J, BUTT T R, et al.Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions[J].Protein Expression and Purification, 2008, 62(1):21-28.
[25] KRONQVIST N, SARR M, LINDQVIST A, et al.Efficient protein production inspired by how spiders make silk[J].Nature Communications, 2017, 8:15504.
[26] ABELEIN A, CHEN G F, KITOKA K, et al.High-yield production of amyloid-β peptide enabled by a customized spider silk domain[J].Scientific Reports, 2020, 10(1):235.
[27] ZARZHITSKY S, JIANG A, E STANLEY E, et al.Harnessing synthetic biology to enhance heterologous protein expression[J].Protein Science, 2020, 29(8):1 698-1 706.
[28] INAN M, MEAGHER M M.Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris[J].Journal of Bioscience and Bioengineering, 2001, 92(6):585-589.
[29] 潘林, 孙建义.生物素的生理功能及其分子作用机制[J].中国饲料, 2005(6):21-24.
PAN L, SUN J Y.Physiological functions and molecular mechanisms of biotin[J].China Feed, 2005(6):21-24.
