Macular pigment-enriched oil production from genome-edited microalgae

Abstract

Background The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. Results Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g−1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g−1 DCW), and lipids (450.09 ± 25.48 mg g−1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g−1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g−1 lutein of oil and 7.69 ± 1.03 mg g−1 zeaxanthin of oil was produced. Conclusion Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

Microalgal lutein biosynthesis: Recent trends and challenges to enhance the lutein content in microalgal cell factories

“…Heterologous expression of this gene in C. reinhardtii boosted lutein content up to 8.9 mg/g which was 60% higher than the wild type under low light intensities of 75 mmol photons m −2 s −1 (Rathod et al, 2020). Recently, Song et al (2022), used CRISPR-Cas9 RNP mediated knock out strategies to delete the zeaxanthin epoxidase (ZEP) gene along with the ADP−glucose pyrophosphorylase (AGP) gene in the C. reinhardtii that is responsible for xanthophyll and starch biosynthesis in this microalga (Song et al, 2022). This double knock-out mutant enabled to accumulate 2.93 mg/g of lutein under optimal cultivation conditions (Song et al, 2022).…”

“…Elucidation of carotenoids production in bacteria, algae, protist (Patel et al, 2022) and higher plants has progressed significantly, and it is expected that primary carotenoids biosynthesis in these species follows a similar process. Figure 1 depicts the lutein synthesis route in green algae (Chlorophyta), which is subdivided into the different stages (Huang et al, 2021;Kato et al, 2021;Lou et al, 2021;Xie et al, 2021;Song et al, 2022;Velmurugan and Kodiveri Muthukaliannan, 2022). The major metabolic pathway includes the synthesis of isopentenyl diphosphate (IPP), geranylgeranyl pyrophosphate, phytoene, and desaturation of phytoene into lycopene, followed by cyclisation and hydroxylation of lycopene to lutein.…”

“…Some examples of genetically modified microalgal strains for overproduction of lutein are listed in Table 1. In a study, the lutein content in C. reinhardtii was boosted by improving the biomass Biosynthetic pathway of lutein in microalgae; source references (Huang et al, 2021;Kato et al, 2021;Lou et al, 2021;Xie et al, 2021;Song et al, 2022;Velmurugan and Kodiveri Muthukaliannan, 2022).…”

“…Recently, Song et al (2022), used CRISPR-Cas9 RNP mediated knock out strategies to delete the zeaxanthin epoxidase (ZEP) gene along with the ADP−glucose pyrophosphorylase (AGP) gene in the C. reinhardtii that is responsible for xanthophyll and starch biosynthesis in this microalga (Song et al, 2022). This double knock-out mutant enabled to accumulate 2.93 mg/g of lutein under optimal cultivation conditions (Song et al, 2022). Baek et al (2018) suggested that genetically modified C. reinhardtii CC-4349 through knocking out the zeaxanthin epoxidase gene by preassembled DNA-free CRISPR-Cas9 ribonucleoproteins, showed a 56-fold increment in zeaxanthin content without affecting the lutein content significantly, which increased from 2.71 ± 0.04 mg/g to 3.08 ± 0.07 (Baek et al, 2018).…”

Emerging Trends in Genetic Engineering of Microalgae for Commercial Applications

“…There is a growing interest in the new research areas such as integrated biosystems to produce H 2 in mixed-cultures and direct utilization of residual algal biomass as a primary input for hydrogen production by a consecutive anaerobic digestion stage. Interdisciplinary approaches, such as nanotechnology and electrochemistry, immobilization of cells on novel materials and utilization of wastewater resources as a convenient and renewable source of nutrients for H 2 production [ 65 ], were also reported [ 107 ]. Recently, Khanna et al reported a novel methodology regarding apo-hydrogenase activation via insertion of a synthetic cofactor in vivo, and the potential to create catalytically active “semi-synthetic” hydrogenases in living cells was demonstrated [ 108 ].…”