Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002
- Waksman Institute Rutgers University New Brunswick New Jersey, Department of Microbiology and Biochemistry Rutgers University New Brunswick New Jersey
- Waksman Institute Rutgers University New Brunswick New Jersey
- Department of Biochemistry and Molecular Biology The Pennsylvania State University Pennsylvania
- Department of Biochemistry and Molecular Biology The Pennsylvania State University Pennsylvania, Department of Chemistry and Biochemistry Montana State University Bozeman Montana
- Waksman Institute Rutgers University New Brunswick New Jersey, Department of Chemistry and Biological Chemistry Rutgers University New Brunswick New Jersey 08901
ABSTRACT To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H 2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H 2 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, Δ narB 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, Δ narB 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 Δ narB , in contrast to the wild type (WT) control. The Δ narB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H 2 and d ‐lactate, by 6‐fold (2.8% yield) and 2‐fold (82.3% yield), respectively, than WT. Continuous removal of H 2 from the fermentation medium (milking) further boosted net H 2 production by 7‐fold in Δ narB , at the expense of less excreted lactate, resulting in a 49‐fold combined increase in the net H 2 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. Biotechnol. Bioeng. 2016;113: 979–988. © 2015 Wiley Periodicals, Inc.
- Sponsoring Organization:
- USDOE
- Grant/Contract Number:
- DOE‐EERE; DE‐EE0003373
- OSTI ID:
- 1400451
- Journal Information:
- Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Vol. 113 Journal Issue: 5; ISSN 0006-3592
- Publisher:
- Wiley Blackwell (John Wiley & Sons)Copyright Statement
- Country of Publication:
- United States
- Language:
- English
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