초록
<P>Catabolism of glycogen stored by cyanobacteria occurs during anaerobic auto-fermentation and produces a range of C1–C3 fermentation products and hydrogen <I>via</I> hydrogenase. We investigated both augmenting and rerouting this carbon catabolism by engineering the glycolysis pathway at the NAD<SUP><I>+</I></SUP>-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 (<I>gap1::aphII</I>) and overexpression (<I>gap1</I><SUP><I>+</I></SUP>) strains of <I>Synechococcus</I> sp. strain PCC 7002 were constructed in order to produce more NADPH (<I>via</I> rerouting carbon through OPP) and more NADH (<I>via</I> opening the glycolytic bottleneck), respectively. For <I>gap1::aphII</I> 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 CO<SUB>2</SUB><I>vs.</I> 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 <I>gap1</I><SUP><I>+</I></SUP> 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<SUP>+</SUP> + NADH] pool, foreshadowing an increased catabolic capacity. Indeed, the rate of glycogen catabolism during subsequent dark auto-fermentation increased significantly (58%) <I>vs.</I> WT, resulting in increases in both NADH (4.0-fold) and NADPH (2.9-fold) pools, and terminal fermentation products, hydrogen (3.0-fold) <SMALL>D</SMALL>-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 (<I>gap1::aphII</I>) and 1.1 (<I>gap1</I><SUP><I>+</I></SUP>) 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.</P><BR><BR><P>Graphic Abstract</P><P>Metabolic engineering of GAPDH-1 of glycolysis and OPP pathways for increased NAD(P)H and hydrogen production, under dark-anoxic fermentative conditions.<BR><IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c3ee42206b'><BR></P>