5-羟基色氨酸(5-hydroxytryptophan, 5-HTP)是哺乳动物中重要的神经递质前体,已被证明在治疗多种神经疾病方面具有重要的医用价值,市场前景广阔。酶催化法具有高转化率和可控的反应条件等优点,是一种有前景的生产方法。然而,酶法催化也存在蛋白纯化过程复杂且成本高昂等问题。该研究旨在提升色氨酸羟化酶的表达、催化和分泌效率,无需复杂蛋白纯化过程,利用分泌的酶蛋白粗酶液催化实现5-HTP的高效生物转化。通过基因工程对表达宿主进行遗传改造,有效阻断了底物和产物的降解途径。通过蛋白截短和引入促融标签,获得了表达效率最佳且热稳定性较高的酶突变体M11。进一步通过优化催化体系参数,确立了最佳催化条件,提高了酶的催化效率,催化2 h获得了0.17 g/L的5-HTP,转化率达85%。结合绿色荧光蛋白实变体与促释放化学试剂,建立了一种高效胞外分泌酶蛋白的策略,蛋白分泌效率高达90%,节省了传统纯酶催化的操作成本,为5-HTP的工业化生产提供了潜在的替代方案。
5-Hydroxytryptophan (5-HTP) is a vital precursor to significant neurotransmitters in mammals.Its medicinal value in treating neurological diseases gives it a broad market prospect.Enzymatic catalysis emerges as a promising production approach due to its high conversion rates, controllable reaction conditions, and eco-friendly nature.But challenges still exist, such as the costly and time-consuming purification of enzyme proteins.This study intend to improve 5-HTP expression, catalysis, and secretion to enhance the biotransformation of 5-HTP from tryptophan utilizing crude enzyme extract.The expression host was genetically modified to effectively block the degradation pathway of substrate and product.The introduction of truncation and solubility-enhancing fusion tags yielded the M11 enzyme mutant with optimal expression efficiency and improved thermal stability.Optimization of catalysis system parameters established the best catalytic conditions, enhancing enzyme catalysis efficiency.The M11 mutant catalyzed in the optimal reaction system produced 0.17 g/L of 5-HTP within 2 h, achieving an 85% conversion ratio.An efficient strategy for the extracellular release of enzyme proteins was developed by combining a green fluorescent protein variant with chemical release agents.The secretion efficiency achieved up to 90%, reducing the operational costs compared with traditional pure enzyme catalysis and offering a potential alternative for the industrial production of 5-HTP.
[1] LIU X X, ZHANG B, AI L Z.Advances in the microbial synthesis of 5-hydroxytryptophan[J].Frontiers in Bioengineering and Biotechnology, 2021, 9:624503.
[2] BONO G, CRISCUOLI M, MARTIGNONI E, et al.Serotonin precursors in migraine prophylaxis[J].Advances in Neurology, 1982, 33:357-363.
[3] WYATT R J, ZARCONE V, ENGELMAN K, et al.Effects of 5-hydroxytryptophan on the sleep of normal human subjects[J].Electroencephalography and Clinical Neurophysiology, 1971, 30(6):505-509.
[4] CECI F, CANGIANO C, CAIRELLA M, et al.The effects of oral 5-hydroxytryptophan administration on feeding behavior in obese adult female subjects[J].Journal of Neural Transmission, 1989, 76(2):109-117.
[5] ZMILACHER K, BATTEGAY R, GASTPAR M.L-5-hydroxytryptophan alone and in combination with a peripheral decarboxylase inhibitor in the treatment of depression[J].Neuropsychobiology, 1988, 20(1):28-35.
[6] MAFFEI M E.5-Hydroxytryptophan (5-HTP):Natural occurrence, analysis, biosynthesis, biotechnology, physiology and toxicology[J].International Journal of Molecular Sciences, 2020, 22(1):181.
[7] ZHANG Z, YU Z C, WANG J D, et al.Metabolic engineering of Escherichia coli for efficient production of L-5-hydroxytryptophan from glucose[J].Microbial Cell Factories, 2022, 21(1):198.
[8] XU D, FANG M J, WANG H J, et al.Enhanced production of 5-hydroxytryptophan through the regulation of L-tryptophan biosynthetic pathway[J].Applied Microbiology and Biotechnology, 2020, 104(6):2481-2488.
[9] FITZPATRICK P F.Tetrahydropterin-dependent amino acid hydroxylases[J].Annual Review of Biochemistry, 1999, 68:355-381.
[10] HARA R, KINO K.Enhanced synthesis of 5-hydroxy-l-tryptophan through tetrahydropterin regeneration[J].AMB Express, 2013, 3(1):70.
[11] MORA-VILLALOBOS J A, ZENG A P.Protein and pathway engineering for the biosynthesis of 5-hydroxytryptophan in Escherichia coli[J].Engineering in Life Sciences, 2017, 17(8):892-899.
[12] WANG B B, LIU S, WANG H D, et al.Efficient biosynthesis of high-value 5-hydroxytryptophan using a multienzyme cascade[J].Molecular Catalysis, 2023, 546:113274.
[13] FARID S S.Process economics of industrial monoclonal antibody manufacture[J].Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 2007, 848(1):8-18.
[14] MCKINNEY J, KNAPPSKOG P M, PEREIRA J, et al.Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris[J].Protein Expression and Purification, 2004, 33(2):185-194.
[15] CARKACI-SALLI N, FLANAGAN J M, MARTZ M K, et al.Functional domains of human tryptophan hydroxylase 2 (hTPH2)[J].The Journal of Biological Chemistry, 2006, 281(38):28105-28112.
[16] YANG J, HE B J, JANG R, et al.Accurate disulfide-bonding network predictions improve ab initio structure prediction of cysteine-rich proteins[J].Bioinformatics, 2015, 31(23):3773-3781.
[17] BULAJ G.Formation of disulfide bonds in proteins and peptides[J].Biotechnology Advances, 2005, 23(1):87-92.
[18] STEWART E J, ASLUND F, BECKWITH J.Disulfide bond formation in the Escherichia coli cytoplasm:An in vivo role reversal for the thioredoxins[J].The EMBO Journal, 1998, 17(19):5543-5550.
[19] SØRENSEN H P, MORTENSEN K K.Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli[J].Microbial Cell Factories, 2005, 4(1):1.
[20] FRANCIS D M, PAGE R.Strategies to optimize protein expression in E.coli[J].Current Protocols in Protein Science, 2010,5(1):5.24.1-5.24.5.24.29.
[21] KINO K, HARA R, NOZAWA A.Enhancement of L-tryptophan 5-hydroxylation activity by structure-based modification of L-phenylalanine 4-hydroxylase from Chromobacterium violaceum[J].Journal of Bioscience and Bioengineering, 2009, 108(3):184-189.
[22] ZHANG L, CHOU C P, MOO-YOUNG M.Disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by Escherichia coli expression system[J].Biotechnology Advances, 2011, 29(6):923-929.
[23] ZHANG Z, TANG R X, ZHU D W, et al.Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli[J].Scientific Reports, 2017, 7(1):6990.
[24] KIPRIYANOV S M, MOLDENHAUER G, LITTLE M.High level production of soluble single chain antibodies in small-scale Escherichia coli cultures[J].Journal of Immunological Methods, 1997, 200(1-2):69-77.
[25] ARISTIDOU A A, YU P, SAN K-Y.Effects of glycine supplement on protein production and release in recombinant Escherichia coli[J].Biotechnology Letters, 1993, 15(4):331-336.
[26] SIMMONS L C, YANSURA D G.Translational level is a critical factor for the secretion of heterologous proteins in Escherichia coli[J].Nature Biotechnology, 1996, 14(5):629-634.
