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Krishnan, A, Kumaraswamy GK, Vinyard DJ, Gu H, Ananyev GM, Posewitz MZ, Dismukes GC.  2015.  Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant.. Plant J. 81(6):947-960. Abstract
Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.
Krishnan, A, Zhang S, Liu Y, Tadmori KA, Bryant DA, Dismukes GC.  2016.  Consequences of ccmR deletion on respiration, fermentation and H2 metabolism in cyanobacterium Synechococcus sp. PCC 7002. Biotechnol Bioeng. Abstract
CcmR, a LysR-type transcriptional regulator, represses the genes encoding components of the high-affinity carbon concentration mechanism in cyanobacteria. Unexpectedly, deletion of the ccmR gene was found to alter the expression of the terminal oxidase and fermentative genes, especially the hydrogenase operon in the cyanobacterium Synechococcus sp. PCC 7002. Consistent with the transcriptomic data, the deletion strain exhibits flux increases (30-50%) in both aerobic O2 respiration and anaerobic H2 evolution. To understand how CcmR influences anaerobic metabolism, the kinetics of autofermentation were investigated following photoautotrophic growth. The autofermentative H2 yield increased by 50% in the CcmR deletion strain compared to the wild-type strain, and increased to 160% (within 20 h) upon continuous removal of H2 from the medium ("milking") to suppress uptake. Consistent with this greater reductant flux to H2 , the mutant excreted less lactate during autofermentation (NAD(P)H consuming pathway). To enhance the rate of NADH production during anaerobic metabolism, the ccmR mutant was engineered to introduce GAPDH overexpression (more NADH production) and LDH deletion (less NADH consumption). The triple mutant (ccmR deletion + GAPDH overexpression + LDH deletion) showed 6-8-fold greater H2 yield than the WT strain, achieving conversion rates of 17 nmol 108 cells-1 h-1 and yield of 0.87 H2 per glucose equivalent (8.9% theoretical maximum). Simultaneous monitoring of the intracellular NAD(P)H concentration and H2 production rate by these mutants reveals an inverse correspondence between these variables indicating hydrogenase-dependent H2 production as a major sink for consuming NAD(P)H in preference to excretion of reduced carbon as lactate during fermentation.
Kumaraswamy, GK, Guerra T, Qian X, Zhang S, Bryant DA, Dismukes GC.  2013.  Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: metabolic engineering of NAD+-dependent GAPDH. Energy Environ. Sci.. 6:3722-3731. AbstractWebsite
Catabolism of glycogen stored by cyanobacteria occurs during anaerobic auto-fermentation and produces a range of C1–C3 fermentation products and hydrogen via hydrogenase. We investigated both augmenting and rerouting this carbon catabolism by engineering the glycolysis pathway at the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH-1), its major regulation site at the nexus of two pathways (Oxidative Pentose Phosphate pathway, OPP, and glycolysis/gluconeogenesis). Null (gap1::aphII) and overexpression (gap1+) strains of Synechococcus sp. strain PCC 7002 were constructed in order to produce more NADPH (via rerouting carbon through OPP) and more NADH (via opening the glycolytic bottleneck), respectively. For gap1::aphII quantitative analyses after four days of dark auto-fermentation showed undiminished glycogen catabolism rate, significant increases of intracellular metabolites in both OPP and upper-glycolysis, decrease in lower-glycolysis intermediates, 5.7-fold increase in NADPH, 2.3-fold increase in hydrogen and 1.25-fold increase in CO2vs. wild type (WT). These changes demonstrate the expected outcome of redirection of carbon catabolism through the OPP pathway with significant stimulation of OPP product yields. The gap1+ strain exhibits a large 17% increase in accumulation of glycogen during the prior photoautotrophic growth stage (gluconeogenesis), in parallel with a 2-fold increase in the total [NAD+ + NADH] pool, foreshadowing an increased catabolic capacity. Indeed, the rate of glycogen catabolism during subsequent dark auto-fermentation increased significantly (58%) vs. WT, resulting in increases in both NADH (4.0-fold) and NADPH (2.9-fold) pools, and terminal fermentation products, hydrogen (3.0-fold) D-lactate (2.3-fold) and acetate (1.4-fold). The overall energy conversion yield over four days from catabolized glycogen to hydrogen increased from 0.6 mole of hydrogen per mole of glucose (WT) to 1.4 (gap1::aphII) and 1.1 (gap1+) under headspace accumulation conditions (without hydrogen milking). These findings demonstrate the significant potential of metabolic engineering for redirecting carbon pathways for carbohydrate catabolism and hydrogen production in cyanobacteria.