Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: metabolic engineering of NAD+-dependent GAPDH

Kumaraswamy, G. Kenchappa; Guerra, Thacia Rodrigues; Qian, Xiao; Zhang, Shuyi; Bryant, Donald A.; Dismukes, G. Charles

doi:10.1039/c3ee42206b

Cited by 47 publications

(25 citation statements)

References 33 publications

(42 reference statements)

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“…The LC–MS analysis system and methods were described previously (Bennette et al, 2011). Extracellular metabolite profiles were analyzed by HPLC according to a previously described protocol (Kumaraswamy et al, 2013). …”

Section: Methodsmentioning

confidence: 99%

Natural and Synthetic Variants of the Tricarboxylic Acid Cycle in Cyanobacteria: Introduction of the GABA Shunt into Synechococcus sp. PCC 7002

et al. 2016

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For nearly half a century, it was believed that cyanobacteria had an incomplete tricarboxylic acid (TCA) cycle, because 2-oxoglutarate dehydrogenase (2-OGDH) was missing. Recently, a bypass route via succinic semialdehyde (SSA), which utilizes 2-oxoglutarate decarboxylase (OgdA) and succinic semialdehyde dehydrogenase (SsaD) to convert 2-oxoglutarate (2-OG) into succinate, was identified, thus completing the TCA cycle in most cyanobacteria. In addition to the recently characterized glyoxylate shunt that occurs in a few of cyanobacteria, the existence of a third variant of the TCA cycle connecting these metabolites, the γ-aminobutyric acid (GABA) shunt, was considered to be ambiguous because the GABA aminotransferase is missing in many cyanobacteria. In this study we isolated and biochemically characterized the enzymes of the GABA shunt. We show that N-acetylornithine aminotransferase (ArgD) can function as a GABA aminotransferase and that, together with glutamate decarboxylase (GadA), it can complete a functional GABA shunt. To prove the connectivity between the OgdA/SsaD bypass and the GABA shunt, the gadA gene from Synechocystis sp. PCC 6803 was heterologously expressed in Synechococcus sp. PCC 7002, which naturally lacks this enzyme. Metabolite profiling of seven Synechococcus sp. PCC 7002 mutant strains related to these two routes to succinate were investigated and proved the functional connectivity. Metabolite profiling also indicated that, compared to the OgdA/SsaD shunt, the GABA shunt was less efficient in converting 2-OG to SSA in Synechococcus sp. PCC 7002. The metabolic profiling study of these two TCA cycle variants provides new insights into carbon metabolism as well as evolution of the TCA cycle in cyanobacteria.

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Section: Methodsmentioning

confidence: 99%

Natural and Synthetic Variants of the Tricarboxylic Acid Cycle in Cyanobacteria: Introduction of the GABA Shunt into Synechococcus sp. PCC 7002

et al. 2016

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“…One of the recent studies showed that rerouting the carbon pathways for carbohydrate catabolism and hydrogen production in cyanobacteria using metabolic engineering has also significant potential to enhance the yield of biohydrogen. Kumaraswamy et al (2013) engineered the glycolysis pathway at the NAD + -dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH-1) in Synechococcus sp. strain PCC 7002, resulting 2.3-to 3-fold increase in hydrogen than wild type.…”

Section: Strategies To Enhance the Biohydrogen Productionmentioning

confidence: 99%

Photosynthetic Microorganisms

Singh

Sundaram

Kishor

2014

SpringerBriefs in Materials

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“…The cell pellet was saved for intracellular quantification of total carbohydrates. The supernatant was collected for HPLC (Perkin Elmer) analysis of lactate, acetate and succinate as was described in Kumaraswamy et al (2013). Frozen cell pellets were used to quantify total reducing carbohydrates by an adapted anthrone method (Trevelyan and Harrison, 1952).…”

Section: Analytical Assaysmentioning

confidence: 99%

Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002

Qian

Kumaraswamy

Zhang

et al. 2015

Biotech & Bioengineering

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To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations--both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))--were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.

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Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: metabolic engineering of NAD+-dependent GAPDH

Cited by 47 publications

References 33 publications

Natural and Synthetic Variants of the Tricarboxylic Acid Cycle in Cyanobacteria: Introduction of the GABA Shunt into Synechococcus sp. PCC 7002

Natural and Synthetic Variants of the Tricarboxylic Acid Cycle in Cyanobacteria: Introduction of the GABA Shunt into Synechococcus sp. PCC 7002

Photosynthetic Microorganisms

Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002

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