Redirecting reductant flux into hydrogen production via metabolic engineering of fermentative carbon metabolism in a cyanobacterium

Abstract

Some aquatic microbial oxygenic photoautotrophs (AMOPs) make hydrogen (H(2)), a carbon-neutral, renewable product derived from water, in low yields during autofermentation (anaerobic metabolism) of intracellular carbohydrates previously stored during aerobic photosynthesis. We have constructed a mutant (the ldhA mutant) of the cyanobacterium Synechococcus sp. strain PCC 7002 lacking the enzyme for the NADH-dependent reduction of pyruvate to D-lactate, the major fermentative reductant sink in this AMOP. Both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) metabolomic methods have shown that autofermentation by the ldhA mutant resulted in no D-lactate production and higher concentrations of excreted acetate, alanine, succinate, and hydrogen (up to 5-fold) compared to that by the wild type. The measured intracellular NAD(P)(H) concentrations demonstrated that the NAD(P)H/NAD(P)(+) ratio increased appreciably during autofermentation in the ldhA strain; we propose this to be the principal source of the observed increase in H(2) production via an NADH-dependent, bidirectional [NiFe] hydrogenase. Despite the elevated NAD(P)H/NAD(P)(+) ratio, no decrease was found in the rate of anaerobic conversion of stored carbohydrates. The measured energy conversion efficiency (ECE) from biomass (as glucose equivalents) converted to hydrogen in the ldhA mutant is 12%. Together with the unimpaired photoautotrophic growth of the ldhA mutant, these attributes reveal that metabolic engineering is an effective strategy to enhance H(2) production in AMOPs without compromising viability.

FIG. 1.

Fermentative metabolism of glucose derived from intracellular, reduced sugars in Synechococcus 7002 based on the sequenced genome and the measured metabolite concentrations for WT and ldhA mutant cultures. Tables indicate excreted metabolite concentrations in the media after 4 days (mol/10¹⁷ cells). 1, enzymes of glycolysis or pentose phosphate pathway; 2, enzymes of the TCA cycle from malate dehydrogenase to succinate dehydrogenase; 3, d-lactate dehydrogenase (insertionally inactivated in ldhA mutant); 4, alanine dehydrogenase; 5, pyruvate:ferredoxin (flavodoxin) oxidoreductase; 6, Fd:NADP⁺ oxidoreductase (FNR); 7, hydrogenase; 8, acetate-CoA ligase.

FIG. 2.

The kinetics of autofermentative metabolite excretion from WT (○) and ldhA mutant (□) cells of Synechococcus 7002. Lactate, alanine, and acetate were determined by ¹H-NMR. Hydrogen in the headspace was measured by gas chromatography. The ordinate gives the concentration per 10¹⁷ cells. Error bars represent plus and minus one standard deviation (SD) from biological triplicates.

FIG. 3.

Comparison of excreted product yields from autofermentation of wild-type versus ldhA mutant cells of Synechococcus 7002. Relative areas of the dashed circles indicate the amount of carbon (a) or reductant (b) originating from endogenous, reduced sugars catabolized over 4 days of autofermentation. Relative areas of the pie graphs represent integrated yields from all excreted metabolites produced after 4 days of autofermentation normalized to amount of carbon (a) or reductant (b) equivalents needed to produce the metabolite. CO₂ is assumed to be equivalent to acetate in concentration, as described in the text. WT cultures (left) excrete the same amount of product as is catabolized, while almost 60% of the balance of catabolized carbon and reductant is not accounted for by excreted products in the ldhA mutant cultures (right).

FIG. 4.

Intracellular NAD⁺ concentration (a) and ratio of NADH/NAD⁺ (b) for WT (○) and ldhA mutant (□) cells of Synechococcus 7002. Time zero was measured at the onset of anoxic conditions. Concentrations of NAD⁺, NADP⁺, NADH, and NADPH were measured with LC-MS. The concentration of the total NAD(H) pool at 96 h was below the detection limit of our instrument. The concentration of NADP⁺ was over an order of magnitude lower than the concentration of NAD⁺ and decreased to below the detection limit by 96 h. The concentration of NADPH remained below the detection limit throughout the experiment. Error bars represent plus and minus one SD from biological triplicates.