Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress.

Diatoms are unicellular algae that accumulate significant amounts of triacylglycerols as storage lipids when their growth is limited by nutrients. Using biochemical, physiological, bioinformatics, and reverse genetic approaches, we analyzed how the flux of carbon into lipids is influenced by nitrogen stress in a model diatom, Phaeodactylum tricornutum. Our results reveal that the accumulation of lipids is a consequence of remodeling of intermediate metabolism, especially reactions in the tricarboxylic acid and the urea cycles. Specifically, approximately one-half of the cellular proteins are cannibalized; whereas the nitrogen is scavenged by the urea and glutamine synthetase/glutamine 2-oxoglutarate aminotransferase pathways and redirected to the de novo synthesis of nitrogen assimilation machinery, simultaneously, the photobiological flux of carbon and reductants is used to synthesize lipids. To further examine how nitrogen stress triggers the remodeling process, we knocked down the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The strain exhibits 40-50% of the mRNA copy numbers, protein content, and enzymatic activity of the wild type, concomitant with a 43% increase in cellular lipid content. We suggest a negative feedback sensor that couples photosynthetic carbon fixation to lipid biosynthesis and is regulated by the nitrogen assimilation pathway. This metabolic feedback enables diatoms to rapidly respond to fluctuations in environmental nitrogen availability.

Natural osmolytes are much less effective substrates than glycogen for catabolic energy production in the marine cyanobacterium Synechococcus sp. strain PCC 7002.

ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.

Altered carbohydrate metabolism in glycogen synthase mutants of Synechococcus sp. strain PCC 7002: Cell factories for soluble sugars.

Glycogen and compatible solutes are the major polymeric and soluble carbohydrates in cyanobacteria and function as energy reserves and osmoprotectants, respectively. Glycogen synthase null mutants (glgA-I glgA-II) were constructed in the cyanobacterium Synechococcus sp. strain PCC 7002. Under standard conditions the double mutant produced no glycogen and more soluble sugars. When grown under hypersaline conditions, the glgA-I glgA-II mutant accumulated 1.8-fold more soluble sugars (sucrose and glucosylglycer-(ol/ate)) than WT, and these cells spontaneously excreted soluble sugars into the medium at high levels without the need for additional transporters. An average of 27% more soluble sugars was released from the glgA-I glgA-II mutant than WT by hypo-osmotic shock. Extracellular vesicles budding from the outer membrane were observed by transmission electron microscopy in glgA-I glgA-II cells grown under hypersaline conditions. The glgA-I glgA-II mutant serves as a starting point for developing cell factories for photosynthetic production and excretion of sugars.