Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway

Abstract

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Delta, vps22Delta, and vps34Delta, were determined on account of their hGH secretion efficiency. The mutant vps10Delta was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.

Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

“…(45). Although very recently the vps10 gene deletion was found to be effective for the production of recombinant human growth hormone (rhGH) in the fission yeast S. pombe (14), this is the first report of targeted disruption of a vacuolar protein sorting gene in a filamentous fungus which could dramatically enhance the production levels of heterologous proteins. Previously, we used DNA microarrays to monitor the expression of 134 protease genes for screening of the disruption target (18).…”

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe

“…The synthesized open reading frame of CpFAH12 was excised from E. coli plasmid pMK-CpFAH12 as a NcoI-SalI fragment by using artificially added restriction enzyme sites at the 5′ and 3′ ends of the gene, and it was cloned at the AarISalI multiple cloning site of pSL10, an integration type plasmid with a leu1 marker, to construct pL2428-9. pSL10 is a derivative of pSL6P3 (Idiris et al 2010a), and it has nmt1 promoter and inv1 terminator instead of hCMV1 promoter and human lipocortin 1 terminator of pSL6P3. Hence, CpFAH12 is expressed under the control of nmt1 promoter of S. pombe in pL2428-9.…”

“…S. pombe is particularly attractive, having served as a model organism for the development of metabolic engineering strategies to produce foreign gene products and certain metabolites (Giga-Hama et al 1994;Isoai et al 2002;Ikeda et al 2004;Giga-Hama et al 2007;Takegawa et al 2009;Idiris et al 2006Idiris et al , 2010a. Also, its fatty acid composition is simple.…”

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion

“…Yeasts have become a well utilized platform for recombinant protein production thanks to their ability to secrete proteins of interest (1)(2). This greatly facilitates product purification and thus helps to make a process economically competitive (3). As unicellular microorganisms they are easy to cultivate and high cell densities can be reached for industrial production processes.…”

“…Secretion of recombinant human transferrin has been reported, and single-chain antibody fragments have been produced with titers up to 5 mg/L (11)(12). In recent times a significant amount of effort has been focused toward engineering S. pombe as a competitive host system for recombinant protein secretion (3,11). Despite these efforts, S. pombe is still underdeveloped as an industrial cell factory and further research will be necessary to create a host system competitive to S. cerevisiae and P. pastoris.…”