Redirecting carbon flux towards products in cyanobacteria by engineering the regulatory mechanism of the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase reaction

Introduction

Among prokaryotes oxygenic photosynthesis is a unique feature in cyanobacteria. In the context of establishing a sustainable bioeconomy, they receive growing interest as biocatalysts in photo-biotechnological applications. To rationally engineer cyanobacteria and to direct metabolic fluxes towards chemical products, it is important to consider native molecular processes that control metabolism. Recently, the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM) has been shown to serve as a central control point determining the carbon flux from the Calvin-Benson-Bassham cycle to lower glycolysis. In particular, PGAM activity is controlled by the small protein PirC that also binds to the central regulatory protein P_II. Upon PirC-binding, PGAM is inactivated leading to glycogen accumulation within the cell.¹

Here we aim to target the PirC-PGAM switch in the model strain Synechocystis sp. PCC 6803. In particular, flux through PGAM shall be blocked to increase product yields that derive from gluconeogenic routes.

Material and Methods

Expression of the genes encoding PGAM and PirC was manipulated by using controllable promoters that respond to copper ions. To reduce PGAM activity we aimed for an inducible downregulation of pgam and simultaneous upregulation of pirC. Similar cloning strategies were used to rationally design sucrose production strains, achieved by overexpression of the sps gene and by installing an invertase gene knockout (similar to Du et al.²). Glycogen and sucrose formation were analyzed in the obtained strains.

Results

Recombinant Synechocystis strains showing Cu2+-inducible downregulation of pgam and overexpression of pirC were cloned and glycogen accumulation was analyzed in the corresponding cultures. In both mutant strains an accumulation of glycogen after addition of copper ions was determinable. This glycogen accumulation did not occur in the wild type control or in the mutant strains before changing expression through addition of copper ions. These data confirm that PGAM activity can be controlled through downregulation of pgam expression and overexpression of pirC routing higher amounts of the fixed carbon into glycogen.

¹T. Orthwein, J. Scholl, P. Spät, S. Lucius, M. Koch, B. Macek, M. Hagemann, K. Forchhammer. PNAS, 2021, 118(6): e2019988118

²Du, W.; Liang, F.; Duan, Y.; Tan, X.; Lu, X. Exploring the photosynthetic production capacity of sucrose by cyanobacteria. Metabolic engineering 2013, 19, 17–25