Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 91, Issue 3, Pages 677-687Publisher
SPRINGER
DOI: 10.1007/s00253-011-3281-8
Keywords
Protein secretion; Corynebacterium glutamicum; Tat-type signal sequence; PS2; Calcium
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Funding
- New Energy and industrial Technology Development Organization (NEDO)
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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 Ca2+ 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, alpha-amylase accumulation in C. glutamicum cultures was also enhanced by Ca2+ addition, suggesting that Ca2+ may affect general protein secretion in C. glutamicum.
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