Abstract
BackgroundProtein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation enzymes derived from Thermobifida fusca YX and S. lividans.ResultsThree candidate signal peptides were isolated and evaluated for their protein secretion ability using β-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the β-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the β-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the β-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the β-glucosidase fused to S. cinnamoneus phospholipase D signal peptide.ConclusionIn the present study, we focused on signal peptide sequences derived from biomass degradation enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic β-glucosidase.
Highlights
Protein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure
We reported the secretory production of two cellulases and transglutaminase using a secretory system that consisted of a putative promoter and terminator regions, and the signal peptide sequence (Plds) derived from the phospholipase D gene of Streptomyces cinnamoneus [15]
We focused on the copy number of BGL genes in order to achieve further protein secretion, and introduced a gene encoding BGL fused to the xylanase C signal peptide (XCs) into S. lividans using a multi-copy vector and an integrative vector, pUC702 and pTYM18 [15,23]
Summary
Protein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. We focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomassdegradation enzymes derived from Thermobifida fusca YX and S. lividans. The secretory production of proteins has three major potential advantages: secreted target proteins are usually natively folded, the degradation of target proteins by intracellular proteases can be suppressed, and there is a reduced requirement for expensive extraction and purification procedures [1,2,3,4]. The secretory production of valuable proteins is an industrially effective method to simplify purification procedures and avoid refolding processes and extraction from cells. With E. coli, produced proteins are usually obtained in the intracellular fraction and need to be extracted from cells
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