title: High yield secretion of heterologous proteins in Corynebacterium glutamicum using its own Tat-type signal sequence.
abstract: Efficient protein secretion, the basis of large-scale production of many compounds central to the biotechnology industry, is achieved by signal peptide and propeptide optimization in addition to optimizing host factors affecting heterologous protein production. Here, we fused green fluorescent protein (GFP) to the recently identified Tat-type secretory signal peptide of CgR0949 to demonstrate a high-yield protein secretion system of Corynebacterium glutamicum. The resultant secretion vector facilitated effective secretion of active-form GFP (20 mg l(-1)) into C. glutamicum culture medium. The expression of GFP was enhanced 2.9-fold using the Shine-Dalgarno sequence of triosephosphate isomerase in the secretion vector. Moreover, GFP drastically accumulated in the culture supernatant upon addition of calcium chloride even though Ca(2+) addition did neither enhanced the transcription of gfp nor resulted in the accumulation of cytosolic GFP. Active-form GFP concentration reached 1.8 g l(-1) after 48-h incubation in a jar fermentor. Likewise, α-amylase accumulation in C. glutamicum cultures was also enhanced by Ca(2+) addition, suggesting that Ca(2+) may affect general protein secretion in C. glutamicum.
authors.affiliation: Molecular Microbiology and Biotechnology Group, Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizugawa, Kyoto 619-0292, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Teramoto
authors.forename: Keiro
authors.initials: K
authors.lastname: Watanabe
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Suzuki
authors.forename: Masayuki
authors.initials: M
authors.lastname: Inui
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yukawa
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001709:Biotechnology; D002118:Calcium; D048230:Corynebacterium glutamicum / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics*; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014305:Triose-Phosphate Isomerase / Q000235:genetics; D000516:alpha-Amylases / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D049452:Green Fluorescent Proteins; D000516:alpha-Amylases; D014305:Triose-Phosphate Isomerase; D002118:Calcium