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Background: Canthaxanthin is a ketocarotenoid with high antioxidant activity, and it is primarily produced by microalgae, among which Nannochloropsis oceanica, a marine alga widely used for aquaculture. In the last decade, N. oceanica has become a model organism for oleaginous microalgae to develop sustainable processes to produce biomolecules of interest by exploiting its photosynthetic activity and carbon assimilation properties. N. oceanica can accumulate lipids up to 70% of total dry weight and contains the omega-3 fatty acid eicosapentaenoic acid (EPA) required for both food and feed applications. The genome sequence, other omics data, and synthetic biology tools are available for this species, including an engineered strain called LP-tdTomato, which allows homologous recombination to insert the heterologous genes in a highly transcribed locus in the nucleolus region. Here, N. oceanica was engineered to induce high ketocarotenoid and canthaxanthin production. Results: We used N. oceanica LP-tdTomato strain as a background to express the key enzyme for ketocarotenoid production, a β-carotene ketolase (CrBKT) from Chlamydomonas reinhardtii. Through the LP-tdTomato strain, the transgene insertion by homologous recombination in a highly transcribed genomic locus can be screened by negative fluorescence. The overexpression of CrBKT in bkt transformants increased the content of carotenoids and ketocarotenoids per cell, respectively, 1.5 and 10-fold, inducing an orange/red color in the bkt cell cultures. Background (LP) and bkt lines productivity were compared at different light intensities from 150 to 1200 µmol m-2 s-1: at lower irradiances, the growth kinetics of bkt lines were slower compared to LP, while higher productivity was measured for bkt lines at 1200 µmol m-2 s-1. Despite these results, the highest canthaxanthin and ketocarotenoids productivity were obtained upon cultivation at 150 µmol m-2 s-1. Conclusions: Through targeted gene redesign and heterologous transformation, ketocarotenoids and canthaxanthin content were significantly increased, achieving 0.3% and 0.2% dry weight. Canthaxanthin could be produced using CO2 as the only carbon source at 1.5 mg/L titer. These bkt-engineered lines hold potential for industrial applications in fish or poultry feed sectors, where canthaxanthin and ketocarotenoids are required as pigmentation agents. [ABSTRACT FROM AUTHOR]

Background: 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. Results: 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−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 different 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 ketocarotenoids accumulation. S4 strain can thus be considered as novel platform for ketocarotenoids and EPA production for different industrial applications. [ABSTRACT FROM AUTHOR]

Abstract: The genetic engineering of plants to facilitate the reclamation of soils and waters contaminated with inorganic pollutants is a relatively new and evolving field, benefiting from the heterologous expression of genes that increase the capacity of plants to mobilize, stabilize and/or accumulate metals. The efficiency of phytoremediation relies on the mechanisms underlying metal accumulation and tolerance, such as metal uptake, translocation and detoxification. The transfer of genes involved in any of these processes into fast‐growing, high‐biomass crops may improve their reclamation potential. The successful phytoextraction of metals/metalloids and their accumulation in aerial organs have been achieved by expressing metal ligands or transporters, enzymes involved in sulfur metabolism, enzymes that alter the chemical form or redox state of metals/metalloids and even the components of primary metabolism. This review article considers the potential of genetic engineering as a strategy to improve the phytoremediation capacity of plants in the context of heavy metals and metalloids, using recent case studies to demonstrate the practical application of this approach in the field. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, the non-proteinogenic amino acid ornithine is the precursor of arginine and polyamines (PAs). The final step of ornithine biosynthesis occurs in plants via a cyclic pathway catalyzed by N2-acetylornithine:N-acetylglutamate acetyltransferase (NAOGAcT). An alternative route for ornithine formation, the linear pathway, has been reported for enteric bacteria and a few other organisms; the acetyl group of N2-acetylornithine is released as acetate by N2-acetylornithine deacetylase (NAOD). NAOD activity has never been demonstrated in plants, although many putative NAOD-like genes have been identified. In this investigation, we examined the effect of down-regulation of the putative Arabidopsis thaliana NAOD gene by using AtNAOD-silenced (sil#17) and T-DNA insertional mutant (atnaod) plants. The ornithine content was consistently reduced in sil#17 and atnaod plants compared with wild-type plants, suggesting that in addition to NAOGAcT action, AtNAOD contributes to the regulation of ornithine levels in plant cells. Ornithine depletion was associated with altered levels of putrescine and spermine. Reduced AtNAOD expression resulted in alterations at the reproductive level, causing early flowering and impaired fruit setting. In this regard, the highest level of AtNAOD expression was observed in unfertilized ovules. Our findings suggest that AtNAOD acts as a positive regulator of fruit setting and agree with those obtained in tomato auxin-synthesizing parthenocarpic plants, where induction of SlNAOD was associated with the onset of ovary growth. Thus, here we have uncovered the first hints of the functions of AtNAOD by connecting its role in flower and fruit development with the regulation of ornithine and PA levels. [ABSTRACT FROM AUTHOR]