Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803

Abstract

Background: Cyanobacteria can be metabolically engineered to convert CO 2 to fuels and chemicals such as ethylene. A major challenge in such efforts is to optimize carbon fixation and partition towards target molecules. Results: The efe gene encoding an ethylene-forming enzyme was introduced into a strain of the cyanobacterium Synechocystis PCC 6803 with increased phosphoenolpyruvate carboxylase (PEPc) levels. The resulting engineered strain (CD-P) showed significantly increased ethylene production (10.5 ± 3.1 µg mL −1 OD −1 day −1) compared to the control strain (6.4 ± 1.4 µg mL −1 OD −1 day −1). Interestingly, extra copies of the native pepc or the heterologous expression of PEPc from the cyanobacterium Synechococcus PCC 7002 (Synechococcus) in the CD-P, increased ethylene production (19.2 ± 1.3 and 18.3 ± 3.3 µg mL −1 OD −1 day −1 , respectively) when the cells were treated with the acetyl-CoA carboxylase inhibitor, cycloxydim. A heterologous expression of phosphoenolpyruvate synthase (PPSA) from Synechococcus in the CD-P also increased ethylene production (16.77 ± 4.48 µg mL −1 OD −1 day −1) showing differences in the regulation of the native and the PPSA from Synechococcus in Synechocystis. Conclusions: This work demonstrates that genetic rewiring of cyanobacterial central carbon metabolism can enhance carbon supply to the TCA cycle and thereby further increase ethylene production.

Recent Advances in Developing Artificial Autotrophic Microorganism for Reinforcing CO2 Fixation

“…With the goal of enhancing CO 2 assimilation efficiency, much interest has been devoted to creating stoichiometrically and thermodynamically feasible routes. Starting with CO 2 capturing enzymes, oxygen-insensitive and kinetically superior carboxylases or reductases have been identified, for instance, PEP carboxylase (EC 4.1.1.31) ( Durall et al, 2020 ), pyruvate decarboxylase (EC 6.4.1.1) ( Bar-Even et al, 2010 ), CoA-dependent carboxylase and metal-dependent FDH ( Schwander et al, 2016 ; Cotton et al, 2018 ). Next, a series of CO 2 fixation pathways were predicted in silico by evaluating the stoichiometric and thermodynamic feasibility of theoretical pathways, from which to recruit a route characterized by high energy-efficiency.…”

Engineered cyanobacteria with additional overexpression of selected Calvin-Benson-Bassham enzymes show further increased ethanol production

“…Every second day the pH was adjusted (7.5) with the addition of 37% HCl (Sigma-Aldrich) and NaHCO 3 was added (50 ​mM final concentration). These two steps were vital for the successful cultivation of the cells as when HCO 3 − is diffused in the carboxysome, it leads to the increase of CO 2 close to RubisCO ( Durall et al., 2020 ) and increased concentration of OH − and eventually alkalization of the pH, that leads to the starvation of the cells. To induce the P nrsB promoter, and thereby the ethanol production, 2.5 ​μM of Ni 2+ (NiCl 2 ) was used and it was added in the cultures every second day.…”

Genetic, Genomics, and Responses to Stresses in Cyanobacteria: Biotechnological Implications

“…Analysis of glutathione synthesis [333] Analysis of heme oxygenase encoding genes [334] Photoproduction of poly-hydroxybutyrate (PHB) biodegradable bioplastics [335] Analysis of Flv3 flavodiiron protein [336] Photoproduction of pinene [337] Analysis of promoters and ribosome binding sites [338] Development of the CRISPR technologies for gene deletion or silencing [339] Photoproduction of ethylene [340] slr0846 and slr2030-slr2031 intergenic region Photoproduction of glutamate, linalool, and valencene [341]…”