Heterologous expression of a fungal sterol esterase/lipase in different hosts: Effect on solubility, glycosylation and production.
Ophiostoma piceae secretes a versatile sterol-esterase (OPE) that shows high efficiency in both hydrolysis and synthesis of triglycerides and sterol esters. This enzyme produces aggregates in aqueous solutions, but the recombinant protein, expressed in Komagataella (synonym Pichia) pastoris, showed higher catalytic efficiency because of its higher solubility. This fact owes to a modification in the N-terminal sequence of the protein expressed in Pichia pastoris, which incorporated 4-8 additional amino acids, affecting its aggregation behavior. In this study we present a newly engineered P. pastoris strain with improved protein production. We also produced the recombinant protein in the yeast Saccharomyces cerevisiae and in the prokaryotic host Escherichia coli, corroborating that the presence of these N-terminal extra amino acids affected the protein's solubility. The OPE produced in the new P. pastoris strain presented the same physicochemical properties than the old one. An inactive form of the enzyme was produced by the bacterium, but the recombinant esterase from both yeasts was active even after its enzymatic deglycosylation, suggesting that the presence of N-linked carbohydrates in the mature protein is not essential for enzyme activity. Although the yield in S. cerevisiae was lower than that obtained in P. pastoris, this work demonstrates the importance of the choice of the heterologous host for successful production of soluble and active recombinant protein. In addition, S. cerevisiae constitutes a good engineering platform for improving the properties of this biocatalyst.
Centro de Investigaciones Biológicas, Department of Environmental Biology, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
María Eugenia
ME
Vaquero
Centro de Investigaciones Biológicas, Department of Environmental Biology, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Jorge
J
Barriuso
Centro de Investigaciones Biológicas, Department of Environmental Biology, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Francisco Javier
FJ
Medrano
Centro de Investigaciones Biológicas, Department of Environmental Biology, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Alicia
A
Prieto
Centro de Investigaciones Biológicas, Department of Environmental Biology, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain. Electronic address: mjmartinez@cib.csic.es.
María Jesús
MJ
Martínez
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D006868:Hydrolysis; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055156:Ophiostoma / Q000201:enzymology / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D002787:Sterol Esterase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D002787:Sterol Esterase; D008049:Lipase
Aggregation behavior; Escherichia coli; Glycosylation; Lipase; Pichia pastoris; Saccharomyces cerevisiae; Sterol esterase