Rerouting of carbon flux in a glycogen mutant of cyanobacteria assessed via isotopically non-stationary 13 C metabolic flux analysis.

Cyanobacteria, which constitute a quantitatively dominant phylum, have attracted attention in biofuel applications due to favorable physiological characteristics, high photosynthetic efficiency and amenability to genetic manipulations. However, quantitative aspects of cyanobacterial metabolism have received limited attention. In the present study, we have performed isotopically non-stationary 13 C metabolic flux analysis (INST-13 C-MFA) to analyze rerouting of carbon in a glycogen synthase deficient mutant strain (glgA-I glgA-II) of the model cyanobacterium Synechococcus sp. PCC 7002. During balanced photoautotrophic growth, 10-20% of the fixed carbon is stored in the form of glycogen via a pathway that is conserved across the cyanobacterial phylum. Our results show that deletion of glycogen synthase gene orchestrates cascading effects on carbon distribution in various parts of the metabolic network. Carbon that was originally destined to be incorporated into glycogen gets partially diverted toward alternate storage molecules such as glucosylglycerol and sucrose. The rest is partitioned within the metabolic network, primarily via glycolysis and tricarboxylic acid cycle. A lowered flux toward carbohydrate synthesis and an altered distribution at the glucose-1-phosphate node indicate flexibility in the network. Further, reversibility of glycogen biosynthesis reactions points toward the presence of futile cycles. Similar redistribution of carbon was also predicted by Flux Balance Analysis. The results are significant to metabolic engineering efforts with cyanobacteria where fixed carbon needs to be re-routed to products of interest. Biotechnol. Bioeng. 2017;114: 2298-2308. © 2017 Wiley Periodicals, Inc.

Cyanobacterial biomass as carbohydrate and nutrient feedstock for bioethanol production by yeast fermentation.

BACKGROUND: Microbial bioconversion of photosynthetic biomass is a promising approach to the generation of biofuels and other bioproducts. However, rapid, high-yield, and simple processes are essential for successful applications. Here, biomass from the rapidly growing photosynthetic marine cyanobacterium Synechococcus sp. PCC 7002 was fermented using yeast into bioethanol. RESULTS: The cyanobacterium accumulated a total carbohydrate content of about 60% of cell dry weight when cultivated under nitrate limitation. The cyanobacterial cells were harvested by centrifugation and subjected to enzymatic hydrolysis using lysozyme and two alpha-glucanases. This enzymatic hydrolysate was fermented into ethanol by Saccharomyces cerevisiae without further treatment. All enzyme treatments and fermentations were carried out in the residual growth medium of the cyanobacteria with the only modification being that pH was adjusted to the optimal value. The highest ethanol yield and concentration obtained was 0.27 g ethanol per g cell dry weight and 30 g ethanol L(-1), respectively. About 90% of the glucose in the biomass was converted to ethanol. The cyanobacterial hydrolysate was rapidly fermented (up to 20 g ethanol L(-1) day(-1)) even in the absence of any other nutrient additions to the fermentation medium. CONCLUSIONS: Cyanobacterial biomass was hydrolyzed using a simple enzymatic treatment and fermented into ethanol more rapidly and to higher concentrations than previously reported for similar approaches using cyanobacteria or microalgae. Importantly, as well as fermentable carbohydrates, the cyanobacterial hydrolysate contained additional nutrients that promoted fermentation. This hydrolysate is therefore a promising substitute for the relatively expensive nutrient additives (such as yeast extract) commonly used for Saccharomyces fermentations.