Comparison of mCherry fluorescence in the supernatant of different constructs.

<p>mCherry fluorescence was measured in supernatant. mCherry fluorescence was measured from 96 individual colonies, grown in a deep-well plate in TAP media for 7 d under constant illumination and agitation. Data presented in the boxplot were collected from colonies where the total fluorescence signal was higher than the auto-fluorescence of the parental wild-type cc1690, above three standard deviations. pAH04 –construct without SP; pJP22 –construct with arylsulfatase 1 SP; pJP26 –construct with binding protein 1 SP; pJP28 –construct with carbonic anhydrase 1 SP; pJP29 –construct with ice-binding protein 1 SP; pJP30-35 –construct with <i>in silico</i> identified SP (iSP). n–number of positive results obtained for each construct. * represents the average result. ○ represents the outliers. No normalization was conducted for mCherry fluorescence.To confirm this hypothesis, a positive colony of each construct with the highest fluorescence was cultivated in 50 mL of TAP media for 7 d, and mCherry fluorescence was determined in both the total cultures and in the supernatant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192433#pone.0192433.s002" target="_blank">S2 Fig</a>). The supernatant percentage of mCherry fluorescence in the pAH04 strain cultivated in the flask was lower relative to that of the pAH0A strain cultivated on the plate (from 42% on the plate to ~8.5% in the flask), which was consistent with the culture condition hypothesis. Although the test in the flask presented a lower noise, it lacks the throughput to test several colonies, an important feature when comparing different construct designs. Since transformation is based mainly on a random insertion by non-homologous end joining (NHEJ) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192433#pone.0192433.ref039" target="_blank">39</a>], colonies presented a wide range of expression efficiency, from a relative standard deviation of 42.7% to 102.7% (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192433#pone.0192433.s008" target="_blank">S1 Table</a>). Therefore, we chose the 96 well plate assay to compare constructs efficiency since it could prevent sampling bias.</p

mCherry compartmentalization in immunoblotting analysis.

<p>Black arrow–mCherry bands, 27 KDa for unmodified and 29 KDa for post-translational modified; Grey arrow–mCherry band still fused with sh-ble and 2A autocleavage peptide, 46 KDa; Sup.–supernatant; Lys.–lysate, cc1690 –parental wild-type strain; pAH04 –construct without SP; pJP22 –construct with arylsulfatase 1 SP; pJP26 –construct with binding protein 1 SP; pJP28 –construct with carbonic anhydrase 1 SP; pJP29 –construct with ice-binding protein 1 SP; pJP30-35 –construct with <i>in silico</i> identified SP.</p

Comparison of mCherry fluorescence among expression vector constructs in the total culture.

<p>mCherry fluorescence was measured in the total culture sample. mCherry fluorescence was measured from 96 individual colonies, grown in a deep-well plate in TAP media for 7 d under constant illumination and agitation. Data presented in the boxplot were collected from colonies in which the total fluorescence signal was higher than that of the auto fluorescence signal of the parental wild-type cc1690, above three standard deviations. pAH04 –construct without SP; pJP22 –construct with arylsulfatase 1 SP; pJP26 –construct with binding protein 1 SP; pJP28 –construct with carbonic anhydrase 1 SP; pJP29 –construct with ice-binding protein 1 SP; pJP30-35 –construct with <i>in silico</i> identified SP (iSP). n–number of positive results obtained for each construct. * represents the average result. ○ represents the outliers. No normalization was conducted for mCherry fluorescence.</p

Characteristics of the signal peptide tested.

<p>Characteristics of the signal peptide tested.</p

Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for <i>Chlamydomonas reinhardtii</i>

<div><p>Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga <i>Chlamydomonas reinhardtii</i>, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP) for heterologous protein secretion in <i>C</i>. <i>reinhardtii</i> by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified <i>in silico</i>. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified <i>in silico</i> secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the <i>C</i>. <i>reinhardtii</i> genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications.</p></div

Live-cell fluorescence microscopy of the mCherry-expressing strains.

<p>The mCherry signal from the non-secreting pAH04 construct is distributed in the cytosol, while the secreting pJP transformants present a mCherry signal in vesicles. Live cells were plated on agar pads and images were acquired 0.4 -μm apart in each channel in the z-axis. Then, images were stacked using the Fiji software Z projects function, generating the final images. An argon laser at 543 nm was used to excite mCherry, and a spectral detector set at approximately 610–650 nm was used to detect emitted fluorescence. For chlorophyll, we used a laser at 405 nm for excitation, and a spectral detector set at 680 nm. cc1690 –parental wild-type strain; pAH04 –construct without SP; pJP22 –construct with arylsulfatase 1 SP; pJP26 –construct with binding protein 1 SP; pJP28 –construct with carbonic anhydrase 1 SP; pJP29 –construct with ice-binding protein 1 SP; pJP30-35 –construct with <i>in silico</i> identified SP. All images were processed identically. Scale bar = 5 μm.</p