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

Abstract

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 C metabolic flux analysis (INST- 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.

Genome Features and Biochemical Characteristics of a Robust, Fast Growing and Naturally Transformable Cyanobacterium Synechococcus elongatus PCC 11801 Isolated from India

“…PCC 6803 (henceforth referred to as PCC 7942 and PCC 6803, respectively) have been engineered to produce some compounds such as alkanes 5 , hydrogen 6 , ethanol 7 , butanol 8 , 2,3-butanediol 9 , acetone 10 , ethylene 11 , fatty acids 12 , and isoprene 13 . Metabolic flux analysis of the model cyanobacterial strains has provided useful information on strain engineering 14 , 15 .…”

An improved method for extraction of polar and charged metabolites from cyanobacteria

“…To obtain accurate transient labeling data, it is required to quantitate the mass isotopologues which can be present in very minute quantities and therefore the efficiency of extraction is extremely important. To perform isotopic non-stationary 13 C MFA for the strains PCC 11801 and PCC 7942, we started out with a reported extraction method (method 1) which has also worked in our hands for the strain PCC 7002 [ 10 ]. This method primarily involves quenching of filtered cells with cold methanol and extraction in methanol-chloroform-water mixture in 1:2:1 ratio (refer to Fig 1 and the methods section).…”

“…This method primarily involves quenching of filtered cells with cold methanol and extraction in methanol-chloroform-water mixture in 1:2:1 ratio (refer to Fig 1 and the methods section). The cyanobacterial cultures grown under photoautotrophic conditions were filtered rapidly in the presence of light to ensure integrity of the light sensitive metabolites and then the filter paper was transferred to cold methanol to quench the metabolism [ 10 , 39 ]. The cells were removed from the filter paper using chloroform solution and the cell suspension in methanol-chloroform mixture was vortexed to break the cells and extract the metabolites.…”

CO2 to succinic acid – Estimating the potential of biocatalytic routes

“…However, due to the low titers and productivities, competitive production with cyanobacteria might currently only be achieved for fine chemicals ( Savakis and Hellingwerf, 2015 ). Succinate production faces particular challenges, because the cyanobacterial TCA cycle has a low activity and the conversion of 2-oxoglutarate to succinate is low ( Hendry et al, 2017 , Young et al, 2011 , Zhang and Bryant, 2011 ).…”