Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.

Abstract

The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels.

The dilemma for lipid productivity in green microalgae: importance of substrate provision in improving oil yield without sacrificing growth

“…Metabolic flux analysis performed in Chlorella protothecoides showed that carbon flow from acetyl-CoA to FA pools increased from 58 to 109% of glucose uptake during nitrogen-limited growth [71], indicating that algal cells substantially reorganize their metabolism to divert more acetyl-CoA towards lipid production. Increased NADPH pool in a Chlorella pyrenoidosa mutant overexpressing an A. thaliana NADH kinase (AtNADK3) has also been associated with enhanced lipid content of up to 110% [72] (Table 1). Oleaginicity is hence attributed to the presence and activity of enzymes responsible for the supply of acetyl-CoA and NADPH to FA synthesis.…”

Nitrogen-induced metabolic changes and molecular determinants of carbon allocation in Dunaliella tertiolecta

“…In addition, they can be cultivated in wastewater and saltwater systems which will not directly compete with resources necessary for agricultural food production56. When cultivated under stressful conditions such as nitrogen depletion (N-depletion), photosynthetic microalgae alter their metabolism to redirect energy towards the production and accumulation of energy-rich storage compounds such as starch and lipids789, but at the cost of diminished growth. This ability enables the microalgae to survive adverse environmental changes as the energy deposits can be easily mobilized when growth conditions are restored10.…”

“…Traditionally, Nannochloropsis oceanica strains are favored for studying lipid accumulation131415, and the freshwater microalgal species Chlamydomonas reinhardtii 1617 and Chlorella vulgaris 81819 are most commonly studied for starch accumulation. The latter two species have thus far been cultivated mixotrophically or heterotrophically with the addition of organic carbon such as acetate and glucose20.…”

Current advances in molecular, biochemical, and computational modeling analysis of microalgal triacylglycerol biosynthesis

“…By contrast, knockout of CrNRR1, a specific TAG regulator for nitrogen deprivation, resulted in 50% decrease in TAG content in C. reinhardtii under nitrogen deprivation conditions (Boyle et al, 2012). A recent whole genome and transcriptome study reconstructed the central carbon metabolism of Chlorella pyrenoidosa, including lipid biosynthesis pathways, and suggested NAD(H) kinase3 (NADK3) controlled NADPH supply and contributed to lipid production in this alga (Fan et al, 2015). Overexpression of an Arabidopsis NAD(H) kinase in C. pyrenoidosa enhanced cellular lipid content effectively by 110.4% without slowing down growth rate (Fan et al, 2015), representing a significant increase in lipid production in industrial-relevant algal strains.…”

“…A recent whole genome and transcriptome study reconstructed the central carbon metabolism of Chlorella pyrenoidosa, including lipid biosynthesis pathways, and suggested NAD(H) kinase3 (NADK3) controlled NADPH supply and contributed to lipid production in this alga (Fan et al, 2015). Overexpression of an Arabidopsis NAD(H) kinase in C. pyrenoidosa enhanced cellular lipid content effectively by 110.4% without slowing down growth rate (Fan et al, 2015), representing a significant increase in lipid production in industrial-relevant algal strains. These studies confirm the potential of omics studies and pathway modeling in identification of metabolic switches for maximizing TAG production in algae.…”