Metal Oxide Mediated Extracellular NADPH Regeneration Improves Ethanol Production by Engineered Synechocystis sp. PCC 6803

Abstract

The ethanol synthesis pathway engineered Synechocystis sp. PCC 6803 (hereafter Synechocystis ) was used to investigate the influence of metal oxide mediated extracellular NADPH regeneration on ethanol synthesis. The in-vitro studies proved that the metal oxides have the potential to generate the NADPH in the presence of electron donor, the usual components of photoautotrophic growth conditions. When the NADPH regeneration was applied in Synechocystsis , the strain showed improved growth and ethanol production. This improved ethanol synthesis is attributed to the increased availability of NADPH to the ethanol synthesis pathway and redirection of closely related carbon metabolism into the ethanol synthesis. Under optimized light intensity and NADP addition, the maximum ethanol production of 5,100 mg/L was observed in MgO mediated extracellular NADPH regeneration after 25 days of cultivation, which is 2-fold higher than the control. This study indicates the feasibility of metal oxide mediated extracellular NADPH regeneration of Synechocystis to increase the production of ethanol.

Evaluation of New Genetic Toolkits and Their Role for Ethanol Production in Cyanobacteria

“…Another approach could include replicating plasmids with native origins of replication for ethanol production, which demonstrated higher transgene expression compared to genomic integration. To address the imbalanced enzymatic activity of adh SYN and pdc ZM , either their expression may be uncoupled from each other or different gene combinations could be tested in order to avoid the accumulation of the metabolic intermediate acetaldehyde, which is toxic to the cells [31,32,71,72,[74][75][76].…”

“…In another study, the co-overexpression of a heterologous pyruvate kinase gene to increase the pyruvate pool in a lactic acid producing Synechocystis strain lead to an almost doubled lactic acid titre [82]. Those measures combined with the supplementation of metal oxides, to enhance the NADP(H) regeneration, could improve the metabolic flux towards the ethanol synthesizing pathway [74].…”

“…The endogenous NADP(H) dependent aldehyde reductase slr0941 (alr SYN ) replaced adh SYN and the pyruvate decarboxylase PDC1 (pdc SC ) of Saccharomyces cerevisiae the pdc ZM , both under the control of the P psbA1 promoter. Overall, 4.85 g L −1 and 5.10 g L −1 were reached after 25 days of cultivation, which correspond to 0.194 g L −1 d −1 and 0.204 g L −1 d −1 STY respectively, with either MgO or Fe 2 O 3 [74]. In order to increase the available pyruvate pool for the ethanol synthesizing pathway, a knock-out of the PHB (polyhydroxy butyrate) pathway with the ethanol synthesis expression cassette was tested, but unexpectedly this did not increase the ethanol yield [31].…”