Dipartimento di Biotecnologie, Università degli Studi di Verona, Strada le Grazie 15, 37134, Verona, Italy.
Dipartimento di Scienze della Vita e Biologia dei Sistemi, Unità di Fisiologia Vegetale, Università di Torino, Via Quarello 15/a, 10135, Turin, Italy.
Microb Cell Fact. 2022 Jun 16;21(1):117. doi: 10.1186/s12934-022-01847-9.
Astaxanthin is a ketocarotenoid with high antioxidant power used in different fields as healthcare, food/feed supplementation and as pigmenting agent in aquaculture. Primary producers of astaxanthin are some species of microalgae, unicellular photosynthetic organisms, as Haematococcus lacustris. Astaxanthin production by cultivation of Haematococcus lacustris is costly due to low biomass productivity, high risk of contamination and the requirement of downstream extraction processes, causing an extremely high price on the market. Some microalgae species are also primary producers of omega-3 fatty acids, essential nutrients for humans, being related to cardiovascular wellness, and required for visual and cognitive development. One of the main well-known producers of omega-3 fatty eicosapentaenoic acid (EPA) is the marine microalga Nannochloropsis gaditana (named also Microchloropsis gaditana): this species has been already approved by the Food and Drug Administration (FDA) for human consumption and it is characterized by a fast grow phenotype.
Here we obtained by chemical mutagenesis a Nannochloropsis gaditana mutant strain, called S4, characterized by increased carotenoid to chlorophyll ratio. S4 strain showed improved photosynthetic activity, increased lipid productivity and increased ketocarotenoids accumulation, producing not only canthaxanthin but also astaxanthin, usually found only in traces in the WT strain. Ketocarotenoids produced in S4 strain were extractible in different organic solvents, with the highest efficiency observed upon microwaves pre-treatment followed by methanol extraction. By cultivation of S4 strain at different irradiances it was possible to produce up to 1.3 and 5.2 mgL day of ketocarotenoids and EPA respectively, in a single cultivation phase, even in absence of stressing conditions. Genome sequencing of S4 strain allowed to identify 199 single nucleotide polymorphisms (SNP): among the mutated genes, mutations in a carotenoid oxygenase gene and in a glutamate synthase gene could explain the different carotenoids content and the lower chlorophylls content, respectively.
By chemical mutagenesis and selection of strain with increased carotenoids to chlorophyll ratio it was possible to isolate a new Nannochloropsis gaditana strain, called S4 strain, characterized by increased lipids and ketocarotenoids accumulation. S4 strain can thus be considered as novel platform for ketocarotenoids and EPA production for different industrial applications.
虾青素是一种具有高抗氧化能力的酮类类胡萝卜素，在医疗保健、食品/饲料补充剂以及水产养殖中的着色剂等多个领域都有应用。虾青素的主要生产者是一些微藻物种，单细胞光合生物，如盐生杜氏藻（Haematococcus lacustris）。由于生物量生产率低、污染风险高以及下游提取工艺的要求，培养盐生杜氏藻生产虾青素的成本很高，这导致市场上虾青素的价格极高。一些微藻物种也是ω-3 脂肪酸的主要生产者，ω-3 脂肪酸是人体必需的营养物质，与心血管健康有关，也是视觉和认知发育所必需的。海洋微藻三角褐指藻（Nannochloropsis gaditana，也称为微拟球藻（Microchloropsis gaditana））是一种主要的 ω-3 脂肪酸二十碳五烯酸（EPA）生产者：该物种已被美国食品和药物管理局（FDA）批准可用于人类食用，其具有快速生长的表型。
在这里，我们通过化学诱变获得了一株盐生杜氏藻突变株，称为 S4，其类胡萝卜素与叶绿素的比例增加。S4 株系表现出增强的光合作用活性、提高的脂质生产力和增加的酮类胡萝卜素积累，不仅产生了角黄素，还产生了通常仅在 WT 株系中微量存在的虾青素。S4 株系产生的酮类胡萝卜素可以用不同的有机溶剂提取，在微波预处理后用甲醇提取的效率最高。通过在不同光照下培养 S4 株系，在单一培养阶段，甚至在没有胁迫条件下，分别可以生产高达 1.3 和 5.2 mgL-1 天的酮类胡萝卜素和 EPA。S4 株系的基因组测序鉴定出 199 个单核苷酸多态性（SNP）：在突变基因中，类胡萝卜素加氧酶基因和谷氨酸合酶基因的突变可以分别解释不同的类胡萝卜素含量和较低的叶绿素含量。
通过化学诱变和选择类胡萝卜素与叶绿素比例增加的菌株，我们成功分离出一株新的盐生杜氏藻菌株，称为 S4 株系，其特点是脂质和酮类胡萝卜素积累增加。因此，S4 株系可以被视为用于不同工业应用的酮类胡萝卜素和 EPA 生产的新型平台。
