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.

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

“…Owing to their ease of genetic modification, many cyanobacteria have been engineered to improve CO 2 capture and light utilization (Ducat et al, 2011; Lan and Liao, 2012; Yu et al, 2013). The capacity to improve these functions depends on how rapidly and completely the downstream steps can utilize the primary metabolic intermediates, thus fundamental studies are needed to understand key metabolic chokepoints and regulatory sites in carbon and energy metabolism (Krishnan et al, 2015b; Qian et al, 2015; Zhang et al, 2015). …”

Proteomic Response to Rising Temperature in the Marine Cyanobacterium Synechococcus Grown in Different Nitrogen Sources

“…Nitrogen is essential for protein synthesis and urea allows cells to redirect saved equivalents to produce more proteins as reported (Qian et al, 2016). However, our results indicated that rising temperature caused negative effects on cellular proteins and ribosomal subunits in both nitrate-and urea-grown cells (Figures 4A,B and Table 1).…”

A Hybrid Flux Balance Analysis and Machine Learning Pipeline Elucidates Metabolic Adaptation in Cyanobacteria

“…Ferredoxins are iron and sulfur-containing proteins that act as electron carriers during many important metabolic processes including oxidation-reduction, photosynthesis, and nitrogen fixation (Lea-Smith et al, 2016). More specifically, nitrate ferredoxin acts as a significant electron sink during the reversible, redox inter-conversion of nitrite to nitrate during photosynthesis (Flores et al, 2005;Qian et al, 2016).…”

“…NADPH dehydrogenases are among the top negative coefficients for all three flux datasets. Cyanobacteria consume a large amount of the cofactors NADPH and NADH whilst reducing nitrate to catabolize glycogen under dark anaerobic conditions (Qian et al, 2016). NADH dehydrogenase (type II) is a protein that catalyzes the electron transfer from NADH to a quinone molecule via a flavin co-factor (Heikal et al, 2014).…”