Rapid Sorting of Fucoxanthin-Producing Phaeodactylum tricornutum Mutants by Flow Cytometry

Abstract

Fucoxanthin, which is widely found in seaweeds and diatoms, has many benefits to human health, such as anti-diabetes, anti-obesity, and anti-inflammatory physiological activities. However, the low content of fucoxanthin in brown algae and diatoms limits the commercialization of this product. In this study, we introduced an excitation light at 488 nm to analyze the emitted fluorescence of Phaeodactylum tricornutum, a diatom model organism rich in fucoxanthin. We observed a unique spectrum peak at 710 nm and found a linear correlation between fucoxanthin content and the mean fluorescence intensity. We subsequently used flow cytometry to screen high-fucoxanthin-content mutants created by heavy ion irradiation. After 20 days of cultivation, the fucoxanthin content of sorted cells was 25.5% higher than in the wild type. This method provides an efficient, rapid, and high-throughput approach to screen fucoxanthin-overproducing mutants.

Comparative Study Highlights the Potential of Spectral Deconvolution for Fucoxanthin Screening in Live Phaeodactylum tricornutum Cultures

“…Because the flow cytometry methods are measuring live cells rather than pigments in extracts, it is appropriate to assess fucoxanthin absorbance characteristics when bound to FCP. Also, an extra emission peak at 710 nm was observed by Fan et al (2021) in P. tricornutum, who confirmed a relationship between emission intensity at 710 nm and fucoxanthin content measured using HPLC [25]. Using these wavelengths has the likely benefit of being able to measure fucoxanthin content by its relationship to cellular FCP rather than by chlorophyll content as a proxy, like when measuring extracts using equations from Ritchie et al (2008) [18].…”

“…Measuring autofluorescence with flow cytometry is not only accurate for measuring fucoxanthin, it does not require extraction, dilution or dyeing, and this greatly improves screening time. Relevant to many projects that generate large mutant or transformant libraries, FACS can also be attached to this method to further improve screening and selection times, as also found in Gao et al [17] and Fan et al [25].…”

Random Mutagenesis as a Promising Tool for Microalgal Strain Improvement towards Industrial Production

“…Their higher frequency enables stronger cell penetration, which, by interacting with molecules such as water, gives origin to free radicals, which are able to disrupt macromolecules, namely DNA, causing high mutation rates [78,83]. Both methods, along with others, such as laser mutagenesis, involve the application of electromagnetic fields to me- Gamma and heavy-ion beam irradiation are both forms of high-frequency radiation that cause double-stranded DNA breakage by ionization, which often leads to the deletion of nucleotides as well as chromosome breaks and exchanges, respectively (Figure 2) [84,87,88]. Their higher frequency enables stronger cell penetration, which, by interacting with molecules such as water, gives origin to free radicals, which are able to disrupt macromolecules, namely DNA, causing high mutation rates [78,83].…”

“…Gamma and heavy-ion beam irradiation are both forms of high-frequency radiation that cause double-stranded DNA breakage by ionization, which often leads to the deletion of nucleotides as well as chromosome breaks and exchanges, respectively ( Figure 2 ) [ 84 , 87 , 88 ]. Their higher frequency enables stronger cell penetration, which, by interacting with molecules such as water, gives origin to free radicals, which are able to disrupt macromolecules, namely DNA, causing high mutation rates [ 78 , 83 ].…”

“…Therefore, the correlation of fluorescence to certain compounds needs to be established. For example, in a study on Phaeodactylum tricornutum , the correlation between fucoxanthin and chlorophyll autofluorescence was used to isolate high-fucoxanthin-producing mutants ( Table 1 ) [ 88 ].…”