title: Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.
abstract: 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.
authors.affiliation: Waksman Institute, Rutgers University, New Brunswick, New Jersey.|Department of Microbiology and Biochemistry, Rutgers University, New Brunswick, New Jersey.
authors.forename: Xiao
authors.initials: X
authors.lastname: Qian
authors.affiliation: Waksman Institute, Rutgers University, New Brunswick, New Jersey.
authors.forename: G Kenchappa
authors.initials: GK
authors.lastname: Kumaraswamy
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, Pennsylvania.
authors.forename: Shuyi
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Waksman Institute, Rutgers University, New Brunswick, New Jersey.
authors.forename: Colin
authors.initials: C
authors.lastname: Gates
authors.affiliation: Waksman Institute, Rutgers University, New Brunswick, New Jersey.
authors.forename: Gennady M
authors.initials: GM
authors.lastname: Ananyev
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, Pennsylvania.|Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana.
authors.forename: Donald A
authors.initials: DA
authors.lastname: Bryant
authors.affiliation: Waksman Institute, Rutgers University, New Brunswick, New Jersey. dismukes@chem.rutgers.edu.|Department of Chemistry and Biological Chemistry, Rutgers University, New Brunswick, New Jersey, 08901. dismukes@chem.rutgers.edu.
authors.forename: G Charles
authors.initials: GC
authors.lastname: Dismukes
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D055786:Gene Knockout Techniques; D006859:Hydrogen / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D050901:Nitrate Reductase / Q000235:genetics / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D046940:Synechococcus / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009566:Nitrates; D009573:Nitrites; D009243:NAD; D009249:NADP; D006859:Hydrogen; D050901:Nitrate Reductase
keywords: Synechococcus 7002; fermentation; hydrogen; nitrate reductase; reducing equivalent