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Acetate is a biological anion with many applications in the chemical and food industries. In addition to being a common microbial fermentative end-product, acetate can be produced by photosynthetic cyanobacteria from CO2 using solar energy. Using wild-type cells of the unicellular model cyanobacterium Synechocystis PCC 6803 only low levels of acetate are observed outside the cells. By inserting a heterologous phosphoketolase (PKPa) in the acs locus, encoding acetyl-CoA synthetase responsible for the irreversible conversion of acetate to acetyl-CoA, an increased level of 40 times was observed. Metabolite analyses indicate an enhanced Calvin-Benson-Bassham cycle, based on increased levels of glyceraldehyde 3-phosphate and fructose-1,6-biphosphate, while the decreased levels of 3-phosphoglycerate and pyruvate suggest a quick consumption of the fixed carbon. Acetyl-P and erythrose-4-phosphate showed significantly increased levels, as products of phosphoketolase, while acetylCoA remained stable through the experiment. The results of intra- and extra-cellular acetate levels clearly demonstrate an efficient excretion of produced acetate from the cells in the engineered strain. Knock-out of ach and pta showed a reduction in acetate production however, it was not as low as in cells with a single knock-out of ach. Overexpressing acetyl-CoA hydrolase (Ach) and acetate kinase (AckA) did not significantly increase production. In contrast, overexpressing phosphotransacetylase (Pta) in cells containing an inserted PKPa resulted in 80 times more acetate reaching 2.3 g/L after 14 days of cultivation.

Background: Photosynthetic microorganisms, such as cyanobacteria, are promising candidates for sustainable production of chemicals. Photosynthesis is a unique process where light energy is used to convert CO₂ into carbon metabolites that sustain the cell`s metabolism. One of these products is acetate, a chemical with various applications in industry. Metabolic engineering can be used to increase the titer of extracellular acetate in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis).

Results: Simultaneous expression of phosphoketolase (PK) and phosphotransacetylase (Pta) resulted in an enhanced acetate titer in Synechocystis cells (Roussou et al. Metab Eng 88:250-260) [1]. In the present study these two enzymes were expressed in different locus in the genome as well as expressed in the same locus organized as a single operon. The latter design reached higher acetate production. Attempts to further optimize the production through the creation of fused protein did not result in significant higher values than 2.3 g/L previously reported. However, the production was further increased when acetate kinase (AckA) was additionally overexpressed. Cultivation of this strain in high density cultivation (CellDEG system) led to high levels of acetate with a maximum of 7.1 g/L cumulative acetate production after 12 days of experiment when the cultures were sampled every day.

Conclusions: Synechocystis sp. PCC 6803 is a candidate for sustainable acetate production driven by sunlight and CO₂. The high level of acetate production is result of combining genomic integration of heterogenous genes in the cell and overexpression of native genes through self-replication vector. The production level achieved through the high-density cultivation reveal the strain capabilities when the growth conditions are optimal.

Cyanobacteria are promising microorganisms as cell factories due to their ability to perform oxygenic photosynthesis. During this process the cell produces metabolites through CO₂ fixation powered by light energy. The main carbon fixation pathway in cyanobacteria is the Calvin-Benson-Bassham (CBB) cycle. Cyanobacteria, for example Synechocystis PCC 6803 (thereafter Synechocystis) have the ability to produce carbon compounds, for example acetate, as side products of their metabolism. Acetate production can be increased through the insertion of a phosphoketolase and overexpression of a phosphotransacetylase. In the present study the production of acetate was further tuned by overexpression of selected enzyme(s) of CBB-cycle. The enzymes selected was aldolase (FBA) and its combination with either fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) or transketolase (TK). The higher increase was noticed in the strain overexpressing FBA, 1.5 times fold increase, followed by the strain overexpressing both FBA and FBP/SBPase, while the overexpression of both FBA and TK did not influence the acetate production. However, when CP12, a small regulatory protein of the CBB-cycle, was knocked out, the strain overexpressing FBA and TK showed increased acetate titers while the other two combinations showed moderate increase. These results indicate the capability to optimize the acetate production through overexpression of FBA and emphasize the dynamic regulation of the CBB-cycle enzyme(s).

Cyanobacteria are one of the most promising microorganisms to produce biofuels and renewable chemicals due to their oxygenic autotrophic growth properties. However, to rely on photosynthesis, which is one of the main reasons for slow growth, low carbon assimlation rate and low production, is a bottleneck. To address this challenge, optimizing the Calvin-Benson-Bassham (CBB) cycle is one of the strategies since it is the main carbon fixation pathway. In a previous study, we showed that overexpression of either aldolase (FBA), transketolase (TK), or fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), enzymes responsible for RuBP regeneration and vital for controlling the CBB carbon flux, led to higher production rates and titers in ethanol producing strains of Synechocystis PCC 6803. In the present study, we investigated the combined effects of the above enzymes on ethanol production in Synechocystis PCC 6803.

The ethanol production of the strains overexpressing two CBB enzymes (FBA + TK, FBP/SBPase + FBA or FBP/SBPase + TK) was higher than the respective control strains, overexpressing either FBA or TK. The co-overexpression of FBA and TK led to more than 9 times higher ethanol production compared to the overexpression of FBA. Compared to TK the respective increase is 4 times more ethanol production. Overexpression of FBP/SBPase in combination with FBA showed 2.5 times higher ethanol production compared to FBA. Finally, co-overexpression of FBP/SBPase and TK reached about twice the production of ethanol compared to overexpression of only TK. This study clearly demonstrates that overexpression of two selected CBB enzymes leads to significantly increased ethanol production compared to overexpression of a single CBB enzyme.

The application of synthetic phototrophic microbial consortia holds promise for sustainable bioenergy production. Nevertheless, strategies for the efficient construction and regulation of such consortia remain challenging. Applying tools of genetic engineering, this study successfully constructed a synthetic community of phototrophs using Rhodopseudomonas palustris (R. palustris) and an engineered strain of Synechocystis sp PCC6803 for acetate production (Synechocystis_acs), enabling the production of biohydrogen and fatty acids during nitrogen and carbon dioxide fixation. Elemental balance confirmed carbon capture and nitrogen fixation into the consortium. The strategy of circadian illumination effectively limited oxygen levels in the system, ensuring the activity of the nitrogenase in R. palustris, despite oxygenic photosynthesis happening in Synechocystis. When infrared light was introduced into the circadian illumination, the production of H₂ (9.70 μmol mg^–1) and fatty acids (especially C16 and C18) was significantly enhanced. Proteomic analysis indicated acetate exchange and light-dependent regulation of metabolic activities. Infrared illumination significantly stimulated the expression of proteins coding for nitrogen fixation, carbohydrate metabolism, and transporters in R. palustris, while constant white light led to the most upregulation of photosynthesis-related proteins in Synechocystis_acs. This study demonstrated the successful construction and light regulation of a phototrophic community, enabling H₂ and fatty acid production through carbon and nitrogen fixation.