(614c) Investigating Overflow Metabolism during Heterotrophic Growth in the Green Alga, Chromochloris Zofingiensis

Chromochloris zofingiensis is an emerging model system for the production of biofuels and bioproducts that is attractive due to its lipid and astaxanthin production capability. Astaxanthin is a high value product (~47k per kg) with uses in the pharmaceutical, nutraceutical, and cosmetic industries. This organism demonstrates high levels of triacylglycerol accumulation when additional organic carbon sources are provided, making it ideal for metabolic or genetic engineering focused on increasing algal lipid production. A genome scale metabolic model, iCre1925 [1], was constructed for this organism, and used to conduct flux balance analysis (FBA) studies of growth in different media conditions. The results of FBA simulations conducted using this model predicted the excretion of overflow metabolism products when C. zofingiensis is grown heterotrophically on glucose. This prediction was later confirmed with experimental data on extracellular metabolites in C. zofingiensis cultures. Overflow metabolism is not observed when cultures are grown in a nutrient dense media with high levels of iron. To further investigate this behavior, an isotopically assisted metabolic flux analysis experiment was performed. In this experiment, isotopically labeled glucose substrate was provided and cultures were harvested at metabolic and isotopic steady state. Mass isotopomer distributions of central metabolites were measured via GC-MS, and this data was used along with external flux measurements to calculate fluxes through the central metabolic network of the cell. Results of this analysis indicate that glucose is transported to the plastid after uptake and is either directed into starch biosynthesis and the pentose phosphate pathway in nutrient replete cultures, whereas the nutrient deplete cultures display more flux through glycolytic pathways with the majority of the carbon flux ultimately ending up in the citric acid cycle, product secretion, and some gluconeogenesis in the cytosol. Despite both cultures being grown in the presence of continuous light, and both network models including carbon fixation reactions, flux through these pathways was only observed in nutrient replete media, confirming prior observations that photosynthetic activity is lost during this overflow metabolism. The lack of overflow product secretion and ability to maintain photosynthetic activity in the nutrient dense media seems to indicate that sufficient iron concentration allows for true mixotrophic growth and a more efficient utilization of glucose.

References

1. Meagher, M., et al., Genome-Scale Metabolic Model Accurately Predicts Fermentation of Glucose by Chromochloris zofingiensis. bioRxiv, 2021: p. 2021.06.22.449518.