title: Secretory expression of Lentinula edodes intracellular laccase by yeast high-cell-density system: sub-milligram production of difficult-to-express secretory protein.
abstract: While a number of heterologous expression systems have been reported for extracellular laccases, there are few for the intracellular counterparts. The Lentinula edodes intracellular laccase Lcc4 is an industrially potential enzyme with its unique substrate specificity. The heterologous production of the intracellular laccase, however, had been difficult because of its expression-dependent toxicity. We previously demonstrated that recombinant yeast cells synthesized and, interestingly, secreted Lcc4 only when they were suspended to an inducing medium in a high cell-density (J. Biosci. Bioeng., 113, 154-159, 2012). The high cell-density system was versatile and applicable to other difficult-to-express secretory proteins. Nevertheless, the system's great dependence on aeration, which was a practical obstacle to scale-up production of the enzyme and some other proteins, left the secretion pathway and enzymatic properties of the Lcc4 uncharacterized. In this report, we demonstrate a successful production of Lcc4 by applying a jar-fermentor to the high cell-density system. The elevated yield (0.6 mg L(-1)) due to the sufficient aeration allowed us to prepare and purify the enzyme to homogeneity. The enzyme had been secreted as a hyper-glycosylated protein, resulting in smear band-formations in SDS-PAGE. The amino acid sequencing analysis suggested that the N-terminal 17 residues had been recognized as a secretion signal. The recombinant enzyme showed similar enzymatic properties to the naturally occurring Lcc4. The characteristics of the scale-upped expression system, which includes helpful information for the potential users, have also been described.
authors.affiliation: Biomolecular Engineering Lab, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kurose
authors.affiliation: School of Food and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Saito
authors.affiliation: School of Food and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kimata
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nakagawa
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Yano
authors.affiliation: Biomolecular Engineering Lab, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan; School of Food and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ito
authors.affiliation: Biomolecular Engineering Lab, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan; School of Food and Nutritional Sciences, University of Shizuoka, Yada 52-1, Suruga-ku, Shizuoka 422-8526, Japan. Electronic address: kawarsky@u-shizuoka-ken.ac.jp.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kawarasaki
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D020942:Shiitake Mushrooms / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D042845:Laccase
keywords: Difficult-to-express protein; High cell-density expression; Lentinula edodes laccase; Recombinant protein; Yeast extracellular production