Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris
Tóm tắt
Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.
Tài liệu tham khảo
Vaaje-Kolstad G, et al. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science. 2010;330:219–23.
Forsberg Z, et al. Cleavage of cellulose by a cbm33 protein. Protein Sci. 2011;20(9):1479–83.
Agger JW, et al. Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation. Proc Natl Acad Sci U S A. 2014;111(17):6287–92.
Vu VV, Beeson WT, Span EA, Farquhar ER, Marletta MA. A family of starch-active polysaccharide monooxygenases. Proc Natl Acad Sci U S A. 2014;111(38):13822–7.
Frommhagen M, et al. Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase. Biotechnol Biofuels. 2015;8:101.
Vaaje-Kolstad G, Horn SJ, Van Aalten DMF, Synstad B, Eijsink VGH. The non-catalytic chitin-binding protein CBP21 from Serratia marcescens is essential for chitin degradation. J Biol Chem. 2005;280(31):28492–7.
Harris PV, et al. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: Structure and function of a large, enigmatic family. Biochemistry. 2010;49(15):3305–16.
Cannella D, Hsieh CWC, Felby C, Jørgensen H. Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content. Biotechnol Biofuels. 2012;5:26.
Hemsworth GR, Johnston EM, Davies GJ, Walton PH. Lytic polysaccharide monooxygenases in biomass conversion. Trends Biotechnol. 2015;33(12):747–61.
Johansen KS. Discovery and industrial applications of lytic polysaccharide mono-oxygenases. Biochem Soc Trans. 2016;44:143–9.
Müller G, Chylenski P, Bissaro B, Eijsink VGH, Horn SJ. The impact of hydrogen peroxide supply on LPMO activity and overall saccharification efficiency of a commercial cellulase cocktail. Biotechnol Biofuels. 2018;11:209.
Eijsink VGH, et al. On the functional characterization of lytic polysaccharide monooxygenases (LPMOs). Biotechnol Biofuels. 2019;12:58.
L. Rieder, N. Teuschler, K. Ebner, and A. Glieder, “Eukaryotic expression systems for industrial enzymes,” in Industrial Enzyme Applications, Wiley‐VCH Verlag, 2019, pp. 47–69.
Fischer JE, Glieder A. Current advances in engineering tools for Pichia pastoris. Curr Opin Biotechnol. 2019;59:175–81.
Vogl T, et al. A toolbox of diverse promoters related to methanol utilization: functionally verified parts for heterologous pathway expression in Pichia pastoris. ACS Synth Biol. 2015;5(2):172–86.
Quinlan RJ, et al. Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci U S A. 2011;108(37):15079–84.
Nakagawa YS, et al. A small lytic polysaccharide monooxygenase from Streptomyces griseus targeting α- And β-chitin. FEBS J. 2015;282(6):1065–79.
Tanghe M, et al. Recombinant expression of Trichoderma reesei Cel61A in Pichia pastoris: Optimizing yield and N-terminal processing. Mol Biotechnol. 2015;57:1010–7.
Labourel A, et al. A fungal family of lytic polysaccharide monooxygenase-like copper proteins. Nat Chem Biol. 2020;16:345–50.
Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev. 2000;24(1):45–66.
Courtade G, Le SB, Sætrom GI, Brautaset T, Aachmann FL. A novel expression system for lytic polysaccharide monooxygenases. Carbohydr Res. 2017;448:212–9.
Kittl R, Kracher D, Burgstaller D, Haltrich D, Ludwig R. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay. Biotechnol Biofuels. 2012;5:79.
Frandsen KEH, Haon M, Grisel S, Henrissat B, LoLeggio L, Berrin JG. Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs). J Biol Chem. 2020;296:100086.
Tolstorukov I, Cregg JM. Yeast promoters from Pichia pastoris, US2016/0097053A1, 2016.
Liang S, Zou C, Lin Y, Zhang X, Ye Y. Identification and characterization of PGCW14: A novel, strong constitutive promoter of Pichia pastoris. Biotechnol Lett. 2013;35(11):1865–71.
Petrović DM, et al. Methylation of the N-terminal histidine protects a lytic polysaccharide monooxygenase from auto-oxidative inactivation. Protein Sci. 2018;27(9):1636–50.
Kojima Y, et al. A lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus Gloeophyllum trabeum and its action on cellulose-xyloglucan complexes. Appl Environ Microbiol. 2016;82(22):6557–72.
Petrović DM, et al. Comparison of three seemingly similar lytic polysaccharide monooxygenases from Neurospora crassa suggests different roles in plant biomass degradation. J Biol Chem. 2019;294(41):15068–81.
Sygmund C, et al. Characterization of the two Neurospora crassa cellobiose dehydrogenases and their connection to oxidative cellulose degradation. Appl Environ Microbiol. 2012;78(17):6161–71.
Filiatrault-Chastel C, et al. AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes. Biotechnol Biofuels. 2019;12:55.
Kadowaki MAS, Magri S, de Godoy MO, Monclaro AV, Zarattini M, Cannella D. A fast and easy strategy for lytic polysaccharide monooxygenase-cleavable 6His-tag cloning, expression and purification. Enzyme Microb Technol. 2020;143:109704.
Isaksen T, et al. A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides. J Biol Chem. 2014;289(5):2632–42.
Frandsen KEH, et al. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases. Nat Chem Biol. 2016;12(4):298–303.
Fitzgerald I, Glick BS. Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting. Microb Cell Fact. 2014;13:125.
Barrero JJ, Casler JC, Valero F, Ferrer P, Glick BS. An improved secretion signal enhances the secretion of model proteins from Pichia pastoris. Microb Cell Fact. 2018;17:161.
Vogl T, Gebbie L, Palfreyman RW, Speight R. Effect of plasmid design and type of integration event on recombinant protein expression in Pichia pastoris. Appl Environ Microbiol. 2018;84(6):e02712-e2717.
Schwarzhans JP, Wibberg D, Winkler A, Luttermann T, Kalinowski J, Friehs K. Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microb Cell Fact. 2016;15:84.
PV Harris and M Wogulis, Polypetides having amylolytic enhancing activity and polynucleotides encoding same WO 2010/059413, 2010.
Leggio LL, et al. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase. Nat Commun. 2015;6:5961.
Barrero JJ, Pagazartaundua A, Glick BS, Valero F, Ferrer P. Bioreactor-scale cell performance and protein production can be substantially increased by using a secretion signal that drives co-translational translocation in Pichia pastoris. N Biotechnol. 2021;60(September):85–95.
Sturmberger L, et al. Refined Pichia pastoris reference genome sequence. J Biotechnol. 2016;235:121–31.
Weis R, Luiten R, Skranc W, Schwab H, Wubbolts M, Glieder A. Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena. FEMS Yeast Res. 2004;5(2):179–89.
Westereng B, et al. Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases. J Chromatogr A. 2013;1271(1):144–52.
Loose JSM, Forsberg Z, Fraaije MW, Eijsink VGH, Vaaje-Kolstad G. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase. FEBS Lett. 2014;588(18):3435–40.
Tuveng TR, Arntzen MØ, Bengtsson O, Gardner JG, Vaaje-Kolstad G, Eijsink VGH. Proteomic investigation of the secretome of Cellvibrio japonicus during growth on chitin. Proteomics. 2016;16(13):1904–14.