Biotechnological approaches to enhance fucoxanthin production in a model diatom Phaeodactylum tricornutum

Abstract

Diatoms are a major group of algae in the phytoplankton community and can be found in waters worldwide wherever sufficient nutrients and lights are present. Marine diatoms, such as Phaeodactylum tricornutum, can accumulate high levels of lipids and have the potential to be engineered into cell factories for sustainable bio-based industries. They are expected to be a promising resource for future clean energy supply and sustainable production of bioactive compounds such as value-added unsaturated fatty acids and carotenoids. The aim of this study was to explore rational biotechnological approaches to increase the yield of valuable products, especially fucoxanthin. Following a summary of diatoms’ morphology, ecology and reproduction methods, a review of bioactive compounds of microalgae, especially marine diatoms, is given. Firstly, we described the potential of microalgae as a large-scale industrial production source of bioactive compounds including carotenoids such as fucoxanthin, astaxanthin and valuable lipids. Ultraviolet C (UVC) light radiation was used as a physical mutagen to induce mutations in the model diatom species Phaeodactylum tricornutum and selected positive mutants with enhanced lipids and carotenoid accumulation. Adaptive laboratory evolution (ALE) was also applied to improve phenotypical performance in P. tricornutum. Liquid chromatography-mass spectrometry (LC-MS) was applied to quantify the major pigments in the wild type, UVC mutants and ALE strains. It was reported for the first time that ALE was successfully applied to diatoms, increasing both the productivity of valuable carotenoids and biomass production. The final fucoxanthin content was doubled compared with the wild type and the growth rate in the final cycle was approximately two-fold higher than the growth at the beginning of ALE. For the next study, P. tricornutum was mutated with chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine (NTG). EMS exhibited higher efficiency in creating positive mutants with high carotenoid content than did NTG with a similar lethality rate. Furthermore, as we found that both chlorophyll a and the lipids had significant correlations with total carotenoids, we established a high-throughput screening method for iii selecting high carotenoid accumulation strains: five mutants were selected using this screening method. These five mutants were then cultivated in repeated batch cultures over two months for strain stability validation: four out of five mutants remained stable while one strain faded. In general, four mutants out of approximately 1,000 isolated strains exhibited at least 33% enhancement of fucoxanthin production and the high-throughput screening method significantly increased the screening efficiency.

Stór hluti svifþörunga eru kísilþörungar og finnast þeir í vötnum um allan heim þar sem næg næringarefni og ljós eru til staðar. Sjávarþörungar, eins og Phaeodactylum tricornutum, geta innihaldið mikið magn fituefna og eru því möguleikar á að nýta þá til framleiðslu á verðmætum lífefnum. Vonir eru bundnar við þá til framleiðslu á lífeldsneyti og ýmsum lífvirkum efnum eins og ómettuðum fitusýrum og karótenóíðum. Markmiðið þessa verkefnis var að kanna mögulegar aðferðir í líftækni til að auka framleiðslu verðmætra efna í þörungnum, sérstaklega fucoxanthin. Fyrst var framkvæmd ítarlega heimildarvinna um form, vistfræði og æxlun kísilþörunga, síðan er yfirlit yfir lífvirk efni í smáþörungum, sérstaklega saltvatns-þörungum. Að lokum var þörungurinn Phaeodactylum tricornutum rannsakaður nánar. Honum var stökkbreytt með útfjólubláu ljósi og ræktaður í margar kynslóðir (þróaður) með það fyrir augum að fá fram afbrigði með aukinn vaxtarhraða og meiri myndun karótenóíðum. Útfjólublátt ljós (UVC) var notað til að valda stökkbreytingum og afbrigði af P. tricornutum voru síðan valin til áframhaldandi ræktunar. Afbrigðin voru ræktuð í margar kynslóðir til að þróa nýjar svipgerðir af P. tricornutum. Við þetta náðist aukin vöxtur í P. tricornutum ásamt aukinni myndun verðmætra kartenóíða. Að lokum var P. tricornutum stökkbreytt með EMS (ethyl methanesulfonate) og afbrigði með auknu fucoxanthin innihaldi valin með nýrri afastamikilli aðferð sem gerði mögulegt að velja jákvæð afbrigði meðal þúsunda afbrigða. Fimm afbrigði voru valin með þessari aðferð og ræktuð áfram í tvo mánuði til að kanna stöðugleika þeirra. Fjögur þeirra sýndu stöðugleika og jókst fucoxanthin innihald þeirra um 33%.