Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains

Abstract

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O 2 evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O 2 evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.Microalgae are able to efficiently convert sunlight, water, and CO 2 into a variety of products suitable for renewable energy applications, including H 2 , carbohydrates, and lipids (11,12,16,21,38,41,44). The unicellular green alga Chlamydomonas reinhardtii has emerged as a model organism for studying algal physiology, photosynthesis, metabolism, nutrient stress, and the synthesis of bioenergy carriers (12,15,19,24,32). During acclimation to nitrogen deprivation, C. reinhardtii cells accumulate significant quantities of starch and form lipid bodies (4,5,8,26,28,30,34,43,46,48). Despite the significance of these products in algal physiology and in biofuels applications, the metabolic, enzymatic, and regulatory mechanisms controlling the partitioning of metabolites into these distinct carbon stores in algae are poorly understood. Several C. reinhardtii starch mutants with various phenotypic changes in starch content and structure have been isolated (2-4). Two of these, the sta6 and sta7 mutants, contain single-gene disruptions that result in "starchless" phenotypes with severely attenuated levels of starch granule accumulation (2,4,34,39,40,48).The disrupted loci in the two isolated starchless mutants are distinct and each mutant has a unique phenotype (7,40). In the sta6 mutant, the small, catalytic subunit of ADP-glucose pyrophosphorylase (AGPase-SS) is disrupted (2,4,48), and this mutant accumulates less than 1% of the starch observed in wild-type (WT) cells under conditions of...

Choreography of Transcriptomes and Lipidomes ofNannochloropsisReveals the Mechanisms of Oil Synthesis in Microalgae

“…The remarkable increase in glycerolipids can result from not only the accelerated partitioning of newly photosynthetically fixed carbon through glycolysis but also from the recycling of carbon fluxes derived from the degradation of existing macromolecules (e.g., carbohydrates and proteins) into the precursors for lipid biosynthesis. Previous studies of the unicellular green alga C. reinhardtii suggested that conversion of storage polysaccharides (e.g., starch) into the precursors for lipid biosynthesis contributed to TAG accumulation under N-conditions (Li et al, 2010b(Li et al, , 2011Work et al, 2010). Microscope observations indicated Nannochloropsis cells lack pyrenoid starch (Vieler et al, 2012;Dong et al, 2013), consistent with the absence of genes responsible for starch biosynthesis in the IMET1 genome.…”

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas

“…Previous studies have assessed the responses of metabolic genes and proteins under non-steady state conditions when TAG accumulates (Miller et al, 2010;Boyle et al, 2012;Msanne et al, 2012;Blaby et al, 2013;Wase et al, 2014) or identified mutants that are defective for TAG accumulation or catabolism (Li et al, 2012;Boyle et al, 2012;Tsai et al, 2014;Xie et al, 2014;Ngan et al, 2015;Kajikawa et al, 2015). The relationship between starch synthesis and TAGs has also been investigated, and it appears that fixed carbon can be shunted toward TAGs when starch synthesis is blocked (Li et al, 2010a(Li et al, , 2010bWork et al, 2010;Siaut et al, 2011;Goodenough et al, 2014). One recently identified regulatory mutant, tar1, is defective in accumulation of lipid bodies in response to nutrient starvation, and the TAR1 locus was shown to encode a YAK subtype DYRK family protein kinase (Kajikawa et al, 2015).…”

The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii

“…Total Glc contained in starch was measured after amyloglucosidase and amylase digestion with the Megazyme total starch analysis kit, similar to Work et al (2010). Briefly, starch pellets remaining after methanol:chloroform (2:1) lipid extraction was autoclaved for 1 h in 0.1 M acetate buffer (pH 4.8) and then was treated with a-amylase and amyloglucosidase for 1 h at 55°C.…”