Characterization of an Evolved Carotenoids Hyper-Producer of Saccharomyces Cerevisiae through Bioreactor Parameter Optimization and Raman Spectroscopy

We have previously developed an evolutionary engineering approach for enhancing heterologous carotenoids production in an engineered Saccharomyces cerevisiae strain, and isolated several carotenoids hyper-producers from the evolved populations. In this work, we optimized β-carotene production of the parental and one of the evolved carotenoids hyper-producers (SM14) in bench-top bioreactors. We found that maintaining a low pH and increasing the carbon-to-nitrogen ratio (C:N) from 8.8 to 50 in standard YNB medium resulted in a higher β-carotene production level at 25.52 ± 2.15 mg β-carotene g^-1 [dry cell weight] in the carotenoids hyper-producer. We hypothesized that the increased carotenoids production in the evolved hyper-producer and the increase in beta-carotene production at a high C:N ratio are due to increased cellular lipid contents. In this study, we demonstrate that Raman spectroscopy is capable of monitoring β-carotene production in these bioreactor cultures. Raman spectroscopy is a powerful tool because it is adaptable to large-scale fermentations and can return results in near real-time. Raman spectroscopy was successfully used to return the relative lipid compositions and protein content of the parental and SM14 strains at two different C:N ratios in the bioreactor. The data showed a higher total lipid content in SM14 compared with the parental strain and that an increased C:N ratio resulted in significant increase in total fatty acid content of both strains, strongly supporting our hypothesis. The data suggests a positive correlation between the yield of β-carotene per biomass with total lipid content of the cell, suggesting one mechanism for the hyper-production of β-carotene in SM14 to be related with increased total lipid content.