Proteomic and phosphoproteomic analysis of green and red stage Haematococcus pluvialis metabolism

Abstract

The green microalga, Haematococcus pluvialis, is desirable for industrial cultivation because it produces high amounts of the powerful antioxidant, astaxanthin. Currently, the primary bottlenecks for the algae astaxanthin industry are the low biomass yield and high production cost of H. pluvialis. With rapid development of genetic engineering toolboxes in microalgae, targeting specific genes, proteins, or pathways directly through strain engineering is an attractive alternative strategy to improve biomass and astaxanthin yield. Omics technologies, including proteomics and phosphoproteomics, have enabled the global characterization of genes, proteins, metabolites, etc. under different experimental conditions and pathway analysis to reveal promising targets for strain engineering. However, as a non-model species, interpretation of data from omics analyses in H. pluvialis remains challenging. This work aims to 1) develop a method for analyzing complex proteomics and phosphoproteomics data from H. pluvialis that can also be used in other non-model species, 2) investigate the heterotrophic (green stage) metabolism of H. pluvialis, and 3) investigate the high light (red stage) metabolism of H. pluvialis. Two mutant strains, KREMS 23D-3 and JWHIB 27-38, were generated via chemical mutagenesis (ethyl methane sulfonate; EMS) and physical radiation (heavy-ion beams), respectively. Both demonstrate desirable phenotypes under small-scale laboratory conditions, such as a high cell division rate and high astaxanthin content per cell. A Tandem Mass Tag-based proteomics and phosphoproteomics approach was used along with physiological and biochemical characterization to understand the dynamic metabolism of H. pluvialis. Comparison of protein expression and phosphorylation levels between the mutant strains and the wild type revealed proteins and phosphoproteins that were potentially responsible for the observed phenotypes. This work established a proteomics and phosphoproteomics analysis pipeline for H. pluvialis that might also be useful to analyze omics data from other non-model algal species and identified promising targets for strain engineering to improve biomass and astaxanthin production from H. pluvialis. Ultimately, these efforts may promote widespread industrial cultivation of H. pluvialis for astaxanthin production.