Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga Coccomyxa subellipsoidea C169

Abstract

Edited by George M. CarmanDeriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO 2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U- The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions.

The assimilation of glycerol into lipid acyl chains and associated carbon backbones of Nannochloropsis salina varies under nitrogen replete and deplete conditions

“…synthesis before acyl editing reactions (Allen et al, 2017). This is consistent with the highest extent of 13 C incorporation in the C18:1 acyl chains of the PL fraction (Figure 4c), which is one of the primary precursors for the acyl editing cycle reactions (Bates & Browse, 2012).…”

“…Under nitrate replete and deplete conditions, the relative proportion of 13 C was generally higher in de novo synthesized monounsaturated (e.g., C16:1 and C18:1) than the saturated C16:0 or polyunsaturated C20:5 (Figure 4) acyl chains of TAG. This reflects the flux of carbon from glycerol going first to de novo fatty acid synthesis before acyl editing reactions (Allen et al, 2017). This is consistent with the highest extent of 13 C incorporation in the C18:1 acyl chains of the PL fraction (Figure 4c), which is one of the primary precursors for the acyl editing cycle reactions (Bates & Browse, 2012).…”

“…The use of isotope labeled carbon sources has provided a means of investigating carbon metabolism in algae. Previous studies have analyzed the carbon flux using radioactive or stable isotope labeling techniques and by following the kinetics of a labeled substrate at various stages of metabolism (Allen, DiRusso, & Black, 2017; Bates & Browse, 2012; Bates, Durrett, Ohlrogge, & Pollard, 2009; Villanova et al, 2017). Stable isotope labeling enables detailed investigations into the metabolism of carbon using mass spectrometry‐based analytical techniques (Elahee Doomun, Loke, O'Callaghan, & Callahan, 2016).…”

“…As such, label enrichment on polyunsaturated fatty acid chains such as C20:5 can be used to detect membrane lipid turnover (Ecker & Liebisch, 2014; Janssen et al, 2019; Zhang, Misra, Nargund, Coleman, & Sriram, 2018). A study of the growth of Coccomyxa subellipsoidea on 13 C 3 ‐glucose, 13 CO 2 , or D 2 O under nitrogen‐limited conditions revealed that 80% of TAG was produced by de novo synthesis via the Kennedy pathway, while acyl chain transfer from membrane turnover only contributed approximately 20% of TAG (Allen et al, 2017). Meanwhile, 13 C‐sodium bicarbonate was used to demonstrate the translocation of 23% of EPA from polar lipids to TAG during nitrogen starvation in photoautotrophically grown N. gaditana (Janssen et al, 2019).…”

Induction of oil accumulation by heat stress is metabolically distinct from N stress in the green microalgae Coccomyxa subellipsoidea C169

“…Total lipid extractions were performed on freeze dried, pre-weighed algae using a modified version of the Bligh & Dyer [ 23 ] method as described in [ 24 ]. TAGs were isolated for GC-MS analysis by first re-suspending the total lipid extract in 1 mL of methylene chloride.…”