Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation

Abstract

Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE) was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS) was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress.

Keywords: Phaeodactylum tricornutum; UV mutagenesis; adaptive laboratory evolution (ALE); fucoxanthin; neutral lipids.

Figure 1

Schematic process for improving diatoms for value-added products.

Figure 2

(a) The lethality curve of P. tricornutum under different UV exposure times; (b) The growth of screened UVC treated strains. Each well of strain had been sub-cultured in biological triplicates and each triplicate had been technically measured twice. The seed culture of wild type for comparison was taken from an Erlenmeyer flask culture under logarithmic growth.

Figure 3

Analysis of neutral lipids in UVC treated strains using Nile red based fluorescence. Strains were incubated with Nile Red (Details in Experimental Design and Methods) in a dark room for 20 min and fluorescence intensities were measured. The intensity differences between stained samples and unstained samples correlate with the content of neutral lipid.

Figure 4

Comparison of fucoxanthin, β-carotene and chlorophyll a contents between wild type cells and UV-mutants. (a) Fucoxanthin; (b) β-carotene; (c) chlorophyll a. Data were averaged from biological triplicates; error bars represent standard deviation.

Figure 5

P. tricornutum growth rate and specific growth rate over cycles during adaptive laboratory evolution (ALE). The average growth rates correspond to biomass produced per day in one cycle. The results were averaged from three biological replicates and error bars represent standard deviation. Asterisk represents statistically significant difference between C11 and C1 (p < 0.05).

Figure 6

Changes of fucoxanthin, β-carotene and chlorophyll a contents during adaptive laboratory evolution. (a) fucoxanthin; (b) β-carotene; (c) chlorophyll a. Data were averaged from three biological replicate and error bars represented standard deviation. Asterisk means significant difference (p < 0.05) and “ns” indicates no significant difference between C11 and C1.