Macular pigment-enriched oil production from genome-edited microalgae.

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.

The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii.

BACKGROUND: Zeaxanthin, a major xanthophyll pigment, has a significant role as a retinal pigment and antioxidant. Because zeaxanthin helps to prevent age-related macular degeneration, its commercial use in personalized nutritional and pharmaceutical applications has expanded. To meet the quantitative requirements for personalized treatment and pharmaceutical applications, it is necessary to produce highly purified zeaxanthin. RESULTS: In this study, to meet the quantitative requirements for industrial applications, we generated a double knockout mutant which is gene-edited by the CRISPR-Cas9 ribonucleoprotein-mediated knock-in system. The lycopene epsilon cyclase (LCYE) was edited to the elimination of α-branch of xanthophyll biosynthesis in a knockout mutant of the zeaxanthin epoxidase gene (ZEP). The double knockout mutant (dzl) had a 60% higher zeaxanthin yield (5.24 mg L- 1) and content (7.28 mg g- 1) than that of the parental line after 3 days of cultivation. Furthermore, medium optimization improved the 3-day yield of zeaxanthin from the dzl mutant to 6.84 mg L- 1. CONCLUSIONS: A Chlamydomonas strain with the elimination of lutein production by gene editing using CRISPR-Cas9 has been successfully developed. This research presents a solution to overcome the difficulties of the downstream-process for the production of high-purity zeaxanthin.

Digital panoramic radiographs are useful for diagnosis of osteoporosis in Korean postmenopausal women.

OBJECTIVE: The purpose of this study was to determine whether digital panoramic radiographs could be used for the diagnosis of osteoporosis through evaluation of the radiographs based on the correlation with bone mineral density (BMD). METHODS: One hundred and ninety-four post-menopausal women were selected from participants who had participated in the Dong-gu study. Panoramic radiographic indices measured are mental index (MI), mandibular cortical index (MCI) and simple visual estimation (SVE). BMD at the lumbar spine and proximal femur was measured by dual-energy X-ray absorptiometry (DXA). The Pearson's correlation test was performed to analyse the correlation between MI and age and BMD at the lumbar spine, femoral neck and total hip. Multiple linear regression analysis was performed to analyse the association of MI, MCI and SVE with BMD after adjusting for age, height and weight. To determine the optimal cut-off point of MI for the diagnosis of osteoporosis, the receiver operating characteristic analysis was performed. RESULTS: The MI was positively correlated with BMDs: lumbar spine: r = 0.36, femoral neck: r = 0.59 and total hip: r = 0.58 (p < 0.001). As age increased, MI decreased (r = -0.46). BMD at the lumbar spine and total hip were significantly lower in participants with reduction of mandibular width, thinning and resorption of mandibular cortex by the MI, SVE and MCI, respectively. The optimal cut-off value of MI for the diagnosis of spinal osteoporosis was 2.22 mm. CONCLUSION: Thickness and morphological changes of mandibular inferior cortical bone are associated with BMD, independent of age, height and weight. These results suggest that MI, MCI and SVE may be useful indices for the diagnosis of osteoporosis in a Korean population.