Cecchin et al. Microbial Cell Factories (2022) 21:117 https://doi.org/10.1186/s12934-022-01847-9 RESEARCH Astaxanthin and eicosapentaenoic acid production by S4, a new mutant strain of Nannochloropsis gaditana Michela Cecchin 1 , Stefano Cazzaniga 1 , Flavio Martini 1 , Stefania Paltrinieri 1 , Simone Bossi 2 , Massimo E. Mafei 2 and Matteo Ballottari 1* Abstract Background: Astaxanthin is a ketocarotenoid with high antioxidant power used in diferent felds 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 micro- algae 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 Micro- chloropsis 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. Results: Here we obtained by chemical mutagenesis a Nannochloropsis gaditana mutant strain, called S4, character- ized 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 diferent organic solvents, with the highest efciency observed upon microwaves pre-treatment followed by methanol extrac- tion. By cultivation of S4 strain at diferent irradiances it was possible to produce up to 1.3 and 5.2 mgL -1 day -1 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 diferent carotenoids content and the lower chlorophylls content, respectively. Conclusions: 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 keto- carotenoids accumulation. S4 strain can thus be considered as novel platform for ketocarotenoids and EPA production for diferent industrial applications. Keywords: Astaxanthin, Microalgae, Omega-3 fatty acids, Eicosapentaenoic acid, Carotenoids © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Nannochloropsis gaditana (named also Microchloropsis gaditana) is a microalgae species belonging to the Eus- tigmatophytes group which raised a high interest for Open Access Microbial Cell Factories *Correspondence: matteo.ballottari@univr.it 1 Dipartimento di Biotecnologie, Università degli Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy Full list of author information is available at the end of the article