Evaluation of secretion reporters to microalgae biotechnology: Blue to red fluorescent proteins

Efficient protein secretion could potentially be exploited for improving the performance of heterologous protein expression system. However, efficient protein secretion depends on numerous factors that are not always known, and rapid development of protein secretion depends on efficient reporters. This study aimed to compare the secretion efficiency of seven fluorescent proteins (FPs)-mTagBFP, mCerulean, Emerald, CrGFP, cOFP, tdTomato, and mCherry-which are photoactive across the visible light spectrum in the microalgal expression system, Chlamydomonas reinhardtii. To fully compare the efficiency of FPs, three expression vectors-a non-secreting vector used as a control and two secreting vectors employing arylsulfatase 1 (ARS1) signal peptide-were used. Up to 94 transformants for each construct were evaluated to better determine the secretion detection efficiency and variability of each FP construct. Fluorescence measurements of all FPs in the whole culture and supernatant were conducted using a microplate reader. Furthermore, we observed fluorescence of six FPs, among the seven tested, in the secretory pathway by confocal fluorescence microscopy. The results indicated that orange and red FPs are the most suitable for this photosynthetic pigment-rich organism, but the FP reporter for secretion should be selected carefully, since some may be deleteriously modified, as observed in the case of tdTomato, in our study. Overall, cOFP and mCherry performed admirably with a high fluorescence signal to noise ratio (S/N > 6) for all constructs as compared to that of the wild type auto-fluorescence, and showed a high positive rate of transformants (> 75%). This FP may play an important role in protein secretion studies, supporting the development of this potential expression system based on microalgae.