Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum

Abstract

Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom Phaeodactylum tricornutum, a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). We found that both chlorophyll a and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied P. tricornutum common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.

Combined artificial high-silicate medium and LED illumination promote carotenoid accumulation in the marine diatom Phaeodactylum tricornutum

“…The optical density at 625 nm wavelength (OD625) was also applied to estimate the biomass production. The detailed correlation between biomass production and OD625 was demonstrated in previous publication [6]. The unit of the pigment yields that we apply in this study was the dry biomass percentage (mg/g DCW).…”

“…The i LB1025 genome-scale computational model was applied in this study to predict the linearly correlated reactions and enzymes that associated with fucoxanthin accumulation. Detailed method was described in our previous publication [6].…”

“…Commercial applications of fucoxanthin have been explored in last decades [5], and diatoms have been recognized as a preferred source for their higher content of fucoxanthin and capability to grow in controlled bioreactors to avoid outdoor contaminations in comparison with sea weeds. Therefore, rational biotechnological approaches should be developed in order to make fucoxanthin production in diatoms feasible [3, 6].…”

Bioengineering of Microalgae: Recent Advances, Perspectives, and Regulatory Challenges for Industrial Application

“…These forward genetic screening in C. reinhardtii and some other model microalgae have been performed mostly to identify the genetic factors responsible for desirable traits, such as higher biomass and cell culture density (Thung et al, 2018 ), enhanced lipid content (Cagnon et al, 2013 ; Lee et al, 2014 ), or to understand the basic cellular processes such as photosynthesis (Dent et al, 2015 ; Li et al, 2019 ), non-photochemical quenching (Schierenbeck et al, 2015 ), lipid metabolism (Li et al, 2016b ; Schulz-Raffelt et al, 2016 ; Cheng et al, 2017 ), and flagellar responses (Hilton et al, 2016 ; Cheng et al, 2017 ). In an integrative approach, the P. tricornutum mutants with enhanced carotenoid biosynthesis were subjected to genome-scale metabolic network simulation to identify the metabolic reactions that are highly correlated with the carotenoid biosynthesis (Yi et al, 2018 ). This study exemplified the use of system-biology approach to target the key pathway(s) that should be considered during bioengineering in diatoms.…”

“…For the screening of biopigments accumulation, a 96-well microplate-based high-throughput assay was developed to identify P. tricornutum mutants with high carotenoid content (Yi et al, 2018 ). The assay was based on the fact that fluorescence intensity of chlorophyll a and neutral lipids (stained with fluorescence dye) has a significant correlation with the carotenoid content during exponential growth phase of P. tricornutum .…”