Double blocking of carbon metabolism causes a large increase of Calvin-Benson cycle compounds in cyanobacteria

Abstract

Carbon-flow-regulator A (CfrA) adapts carbon flux to nitrogen conditions in nondiazotrophic cyanobacteria. Under nitrogen deficiency, CfrA leads to the storage of excess carbon, which cannot combine with nitrogen, mainly as glycogen. cfrA overexpression from the arsenite-inducible, nitrogen-independent ParsB promoter allows analysis of the metabolic effects of CfrA accumulation. Considering that the main consequence of cfrA overexpression is glycogen accumulation, we examined carbon distribution in response to cfrA expression in Synechocystis sp. PCC 6803 strains impaired in synthesizing this polymer. We carried out a comparative phenotypic analysis to evaluate cfrA overexpression in the wild-type strain and in a mutant of ADP-glucose pyrophosphorylase (ΔglgC), which is unable to synthesize glycogen. The accumulation of CfrA in the wild-type background caused a photosynthetic readjustment although growth was not affected. However, in a ΔglgC strain, growth decreased depending on CfrA accumulation and photosynthesis was severely affected. An elemental analysis of the H, C, and N content of cells revealed that cfrA expression in the wild-type caused an increase in the C/N ratio, due to decreased nitrogen assimilation. Metabolomic study indicated that these cells store sucrose and glycosylglycerol, in addition to the previously described glycogen accumulation. However, cells deficient in glycogen synthesis accumulated large amounts of Calvin-Benson cycle intermediates as cfrA was expressed. These cells also showed increased levels of some amino acids, mainly alanine, serine, valine, isoleucine, and leucine. The findings suggest that by controlling cfrA expression, in different conditions and strains, we could change the distribution of fixed carbon, with potential biotechnological benefits.