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.

Keywords: Phaeodactylum tricornutum; flow cytometry; fucoxanthin; heavy ion irradiation.

Figure 1

Fluorescence emission spectra of different microalgae strains (A) and P. tricornutum cultured with different concentrations of diphenylamine for 5 days (B). All the samples used the same OD₇₅₀ value; the excitation wavelength is 488 nm.

Figure 2

The fluorescence intensity and content of fucoxanthin in P. tricornutum cells treated with different concentrations of diphenylamine. (A) Scatter plot of the cells treated with different concentrations of diphenylamine by flow cytometry (SSC vs. FSC). (B) Histogram of the cells treated with different concentrations of diphenylamine (SSC vs. PerCP-Cy-5-5). Purple, blue, red, orange, and green points/lines in (A,B) represent cells incubated with 0, 25, 50, 75, and 100 µM diphenylamine, respectively. (C) TLC of the cells treated with different concentrations of diphenylamine in the culture period. (D) Correlation of fucoxanthin content and mean fluorescence intensity (MFI) in P. tricornutum cells. Each dot represents the cells treated by different concentrations of diphenylamine (25, 50, 75, and 100 µM). The fucoxanthin contents were determined using HPLC.

Figure 3

Schematic diagram of flow sorting of P. tricornutum wild type and mutant strains with different fucoxanthin content.

Figure 4

Cells were selected and sorted by flow cytometry. (A) R0 gate selected the cells with higher fluorescence (8%). R1 gate selected the cells with lower fluorescence (8%). R2 represents all the events. (B) The histogram shows the different mean fluorescence intensity (MFI) at the channel of PerCP-Cy5-5. (C) The negative mutations (left) and positive mutations (right) of P. tricornutum cells after 2 weeks’ culture. (D) Fucoxanthin content in the different samples. The positive group shows higher fucoxanthin content than the wild-type and negative groups. Error bars represent standard deviations, n = 3 (* p-value < 0.05).