Detailed Identification of Fatty Acid Isomers Sheds Light on the Probable Precursors of Triacylglycerol Accumulation in Photoautotrophically Grown Chlamydomonas reinhardtii

Abstract

Chlamydomonas reinhardtii is a model alga for studying triacylglycerol (TAG) accumulation in the photosynthetic production of biofuel. Previous studies were conducted under photoheterotrophic growth conditions in medium supplemented with acetate and/or ammonium. We wanted to demonstrate TAG accumulation under truly photoautotrophic conditions without reduced elements. We first reidentified all lipid components and fatty acids by mass spectrometry, because the currently used identification knowledge relies on data obtained in the 1980s. Accordingly, various isomers of fatty acids, which are potentially useful in tracing the flow of fatty acids leading to the accumulation of TAG, were detected. In strain CC1010 grown under photoautotrophic conditions, TAG accumulated to about 57.5 mol% of total lipids on a mole fatty acid basis after the transfer to nitrogen-deficient conditions. The content of monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol decreased drastically. The accumulated TAG contained 16:0 as the major acid and 16:4(4,7,10,13), 18:2(9,12), and 18: 3(9,12,15), which are typically found in chloroplast lipids. Additionally, 18:1(11) and 18:3(5,9,12), which are specific to extrachloroplast lipids, were also abundant in the accumulated TAG. Photosynthesis and respiration slowed markedly after the shift to nitrogen-deficient conditions. These results suggest that fatty acids for the production of TAG were supplied not only from chloroplast lipids but also from other membranes within the cells, although the possibility of de novo synthesis cannot be excluded. Under nitrogen-replete conditions, supplementation with a high concentration of CO 2 promoted TAG production in the cells grown photoautotrophically, opening up the possibility to the continuous production of TAG using CO 2 produced by industry. Photosynthetic production of various forms of reduced carbon is the ultimate source of biological free energy, which enables biological, social, and industrial human activities. Agricultural production has been the major source of human nutrition, but biofuel is now attracting social interest as an alternative to fossil resources. Algal production of bioresources is a new perspective as a means to supply both biofuel and chemical materials, avoiding competition with nutritional resources (1). Among various algal products, triacylglycerol (TAG), hydrocarbons, and carbohydrates are the major targets of bioengineering. In many algae, TAG is known to accumulate under conditions of nutrient deficiency (2). A unicellular green alga, Chlamydomonas reinhardtii, is currently studied as a model organism for elucidating the metabolic pathway and signaling pathway of TAG accumulation. This alga has been used in studies of photosynthesis, reproduction, and motility, mainly because the methodology for genetic analysis is applicable (3, 4). In addition to classical crossing, molecular genetic manipulation (5, 6) is becoming useful, especially on the basis of the genomic information (7). Vari...

Revisiting the Algal “Chloroplast Lipid Droplet”: The Absence of an Entity That Is Unlikely to Exist

“…EM, Electron micrograph. chloroplast membrane lipids to the TAG pool was not important under this condition, in contrast to the nitrogen-deficient conditions reported previously (Li et al, 2012;Sakurai et al, 2014).…”

“…To confirm that the wild-type cells accumulate neutral lipids in response to high-light intensity, as reported in strain CC-124 (Goold et al, 2016), we used strain CC-1010 (Sakurai et al, 2014) and examined lipid droplets stained with BODIPY after a shift from low light (40 mmol m -2 s -1 ) to high light (200 mmol m -2 s -1 ; Fig. 2A).…”

“…It was noted that the fatty acids specific to chloroplast lipids, namely 16:4 and 18:3(9,12,15), did not appreciably accumulate in TAG. As reported by Sakurai et al (2014), 18:4 was supposed to be a mixture of 18:4(5,9,12,15) and 18:4(6,9,12,15), but, as reported by Sato et al (2016), 18:4(6,9,12,15) was not detected in Chlamydomonas CC-1010. (7) 2.0 6 0.4 2.5 6 0.6 1.7 6 0.1 2.0 6 0.5 16:2(7,10) 4.8 6 1.0 3.3 6 0.7 1.1 6 0.3 2.1 6 0.8 16:3(4,7,10) 0.9 6 0.2 0.9 6 0.0 0.3 6 0.3 0.4 6 0.1 16:3(7,10,13) 7.8 6 0.3 7.7 6 0.8 6.7 6 0.8 6.9 6 1.5 16:4(4,7,10,13) 10.1 6 2.3 10.0 6 1.3 2.3 6 0.…”

“…TAG contains both C16 and C18 fatty acids, which are provided from either ER lipids or chloroplast lipids. The 16:0 fatty acids in TAG are likely to be provided by diacylglyceryl-N,N,N-trimethylhomoserine, whereas the 16:4 fatty acid is probably provided by monogalactosyl diacylglycerol (MGDG), because MGDG is the sole lipid class that contains 16:4 as an abundant fatty acid (Sakurai et al, 2014, and many other studies). This does not mean that TAG containing 16:4 fatty acid is present in the chloroplast, as stated by Fan et al (2011).…”

Algal Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase, Triacylglycerol Accumulation Regulator1, Regulates Accumulation of Triacylglycerol in Nitrogen or Sulfur Deficiency

“…The amount of TAG was quantified by gas chromatography-mass spectrometry after derivatization to fatty acid methyl esters using heptadecanoic acid (C17:0) as the internal standard (Chen et al, 2008). Lipid classes were separated by two-dimensional TLC, and their contents were quantified by gas chromatography-mass spectrometry as described previously Sakurai et al, 2014). To estimate the relative amount of neutral lipids, a 200-mL sample of each N-deficient cell culture was mixed with 5 mL of AdipoRed solution (Lonza) and incubated for 5 min in the dark to stain neutral lipids.…”