Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus

Microbial Cell Factories

Research

Rinji Akada¹  Hisashi Hoshida¹  Tohru Yarimizu²  Mikiko Nakamura³ 

[1] Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, 2-16-1 Tokiwadai, 755-8611, Ube, Japan;Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, 2-16-1 Tokiwadai, 755-8611, Ube, Japan;Present address: Environmental Biofunction Division, National Institute for Agro-Environmental Sciences, 3-1-3 Kan-nondai, 305-8604, Tsukuba, Ibaraki, Japan;Innovation Center, Yamaguchi University, 2-16-1 Tokiwadai, 755-8611, Ube, Japan;

关键词: Synthetic signal sequence;    Hydrophobic core;    Secretion;    Thermotolerant yeast;    Heterologous protein production;   

DOI  :  10.1186/s12934-015-0203-y

 received in 2014-09-19, accepted in 2015-01-27,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundTargeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences.ResultsMutational analysis of the GLuc signal sequence revealed that the GLuc hydrophobic peptide length was lower limit for effective secretion and that the N-terminal basic residue was indispensable. Deletion of the 16th Glu caused enhanced levels of secreted protein, suggesting that this hydrophilic residue defined the boundary of a hydrophobic peptide stretch. Consequently, we redesigned this domain as a repeat of a single hydrophobic amino acid between the N-terminal Lys and C-terminal Glu. Stretches consisting of Phe, Leu, Ile, or Met were effective for secretion but the number of residues affected secretory activity. A stretch containing sixteen consecutive methionine residues (M16) showed the highest activity; the M16 sequence was therefore utilized for the secretory production of human leukemia inhibitory factor protein in yeast, resulting in enhanced secreted protein yield.ConclusionsWe present a new concept for the provision of secretory signal sequence ability in the yeast K. marxianus, determined by the number of residues of a single hydrophobic residue located between N-terminal basic and C-terminal acidic amino acid boundaries.

【 授权许可】

Unknown   
© Yarimizu et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.