Regulation of organic acid and hydrogen production by NADH/NAD+ ratio in Synechocystis sp. PCC 6803

Abstract

Cyanobacteria serve as useful hosts in the production of substances to support a low-carbon society. Specifically, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) can produce organic acids, such as acetate, lactate, and succinate, as well as hydrogen, under dark, anaerobic conditions. The efficient production of these compounds appears to be closely linked to the regulation of intracellular redox balance. Notably, alterations in intracellular redox balance have been believed to influence the production of organic acids and hydrogen. To achieve these alterations, genetic manipulations involved overexpressing malate dehydrogenase (MDH), knocking out d-lactate dehydrogenase (DDH), or knocking out acetate kinase (AK), which subsequently modified the quantities and ratios of organic acids and hydrogen under dark, anaerobic conditions. Furthermore, the mutants generated displayed changes in the oxidation of reducing powers and the nicotinamide adenine dinucleotide hydrogen (NADH)/NAD⁺ ratio when compared to the parental wild-type strain. These findings strongly suggest that intracellular redox balance, especially the NADH/NAD⁺ ratio, plays a pivotal role in the production of organic acids and hydrogen in Synechocystis 6803.

Keywords: Cyanobacteria; acetate; fermentation; hydrogen; lactate; organic acids; succinate.

Figure 1

Production of fermentation metabolites in Synechocystis 6803 under dark, anaerobic conditions, with a focus on organic acid and hydrogen, nicotinamide adenine dinucleotide hydrogen (NADH), ferredoxin, and adenosine triphosphate (ATP) production. P designates phosphate. OPP; oxidative pentose phosphate, EMP; Embden–Meyerhof–Parnas, ED; the Entner–Doudoroff, PEP; phosphoenolpyruvate, PEPC; phosphoenolpyruvate carboxylase, PPS; phosphoenolpyruvate synthase, PYK; pyruvate kinase, MDH; malate dehydrogenase, FUM; fumarase, SDH; succinate dehydrogenase, DDH; d-lactate dehydrogenase, PDH; pyruvate dehydrogenase, PFOR; pyruvate: ferredoxin oxidoreductase, Fdx_red; reduced ferredoxin, Fdx_ox; oxidized ferredoxin, ACS; acetyl-CoA synthetase, PTA; phosphotransacetylase, and AK; acetate kinase.

Figure 2

Changes in glycogen levels before and after dark, anaerobic conditions. Synechocystis 6803 GT, Δddh, Δacs, ΔackA, and CitHox were subjected to aerobic conditions with continuous illumination (50 μmol photons m⁻² s⁻¹) for five days. Following this, cells were incubated under dark, anaerobic conditions for three days. Glycogen levels were quantified using the LabAssay Glucose kit. The term “Anaerobic” indicates glycogen levels of the cells after three days of incubation under dark, anaerobic conditions. Data represents the means ± SD from four biologically independent experiments. Asterisks indicate statistically significant differences between glycogen levels before and after dark, anaerobic conditions for each strain (Student’s t-test; *p < 0.05).

Figure 3

Quantification of four-carbon dicarboxylic acids, lactate, and acetate in Synechocystis 6803 GT, Δddh, Δacs, ΔackA, and CitHox under dark, anaerobic conditions. The levels of organic acids excreted from cells after three days of incubation under dark, anaerobic conditions were determined using high-performance liquid chromatography. Total organic acids represent the combined levels of four-carbon dicarboxylic acids, lactate, and acetate. Data is presented as means ± SD from three to four biologically independent experiments. Four-carbon dicarboxylic acids refer to the sum of succinate, fumarate, and malate. ND indicates undetectable levels. Asterisks indicate statistically significant differences between GT and the mutant strains (Student’s t-test; *p < 0.05, **p < 0.005).

Figure 4

Quantification of hydrogen levels in Synechocystis 6803 GT, Δddh, Δacs, ΔackA, and CitHox cells under dark, anaerobic conditions. The concentration of hydrogen was determined using gas chromatography with a thermal conductivity detector. Data is presented as means ± SD from three to four biologically independent experiments. Asterisks indicate statistically significant differences between GT and the mutant strains (Student’s t-test; *p < 0.05, **p < 0.005).

Figure 5

Intracellular NADH/NAD⁺ ratios of Synechocystis 6803 GT, Δddh, ΔackA, and CitHox cells under dark, anaerobic conditions. NADH and the total sum of NAD⁺ and NADH levels were quantified using the NAD/NADH Assay Kit-WST. The intracellular NADH/NAD⁺ ratios were calculated from the NADH levels and the total sum of NAD⁺ and NADH within the cells. The data are presented as relative values, with the values of GT set as 1 after three days of dark, anaerobic incubation. Data is presented as means from four biologically independent experiments. These results represent means from four biologically independent experiments. Asterisks indicate statistically significant differences in the ratios between GT and the mutant strain (Student’s t-test; *p < 0.05, **p < 0.005).

Figure 6

Schematic model of competition for carbon intermediates and reducing power by organic acids and hydrogen production in Synechocystis 6803 under dark, anaerobic conditions. P designates phosphate. OPP; oxidative pentose phosphate, EMP; Embden–Meyerhof–Parnas, ED; the Entner–Doudoroff, PEP; phosphoenolpyruvate, MDH; malate dehydrogenase, DDH; d-lactate dehydrogenase, PDH; pyruvate dehydrogenase, PFOR; pyruvate: ferredoxin oxidoreductase, Fdx_red; reduced ferredoxin, and AK; acetate kinase.