Exploring and increased acetate biosynthesis in Synechocystis PCC 6803 through insertion of a heterologous phosphoketolase and overexpressing phosphotransacetylase

Abstract

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 acetyl-CoA 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.

Keywords: Acetate; Acetyl phosphate; Acetyl-CoA; Calvin-Benson-Bassham; Cyanobacteria; FBP; G3P; Phosphoketolase; Pyruvate.