Abstract
BackgroundThe 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.ResultsUsing 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.ConclusionOur 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.
Highlights
The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recog‐ nized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids
Given that ADP-glucose pyrophosphorylase (AGP) is one of the key regulatory enzymes in the production of lipids in C. reinhardtii, the AGP gene was selected as a gene-editing target of the zep line by CRISPR-Cas9
Because a mutation in the AGP gene was created without the insertion of any antibiotic resistance gene, it was difficult to select the correct mutants from many transformants by PCR
Summary
The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recog‐ nized 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. The model green microalgae Chlamydomonas reinhardtii has recently been re-evaluated as an industrial strain, which can be made commercially available [10]. An attempted strategy for the commercialization of C. reinhardtii is to enable high lipid production or high value-added pigment production. This is because fatty acid and carotenoids biosynthesis are well defined [11]
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