Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene

Abstract

Citric acid secretion by fluorescent pseudomonads has a distinct significance in microbial phosphate solubilization. The role of citrate synthase in citric acid biosynthesis and glucose catabolism in pseudomonads was investigated by overexpressing the Escherichia coli citrate synthase (gltA) gene in Pseudomonas fluorescens ATCC 13525. The resultant~2-fold increase in citrate synthase activity in the gltA-overexpressing strain Pf(pAB7) enhanced the intracellular and extracellular citric acid yields during the stationary phase, by about 2-and 26-fold, respectively, as compared to the control, without affecting the growth rate, glucose depletion rate or biomass yield. Decreased glucose consumption was paralleled by increased gluconic acid production due to an increase in glucose dehydrogenase activity. While the extracellular acetic acid yield increased in Pf(pAB7), pyruvic acid secretion decreased, correlating with an increase in pyruvate carboxylase activity and suggesting an increased demand for the anabolic precursor oxaloacetate. Activities of two other key enzymes, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase, remained unaltered, and the contribution of phosphoenolpyruvate carboxylase and isocitrate lyase to glucose catabolism was negligible. Strain Pf(pAB7) demonstrated an enhanced phosphate-solubilizing ability compared to the control. Co-expression of the Synechococcus elongatus PCC 6301 phosphoenolpyruvate carboxylase and E. coli gltA genes in P. fluorescens ATCC 13525, so as to supplement oxaloacetate for citrate biosynthesis, neither significantly affected citrate biosynthesis nor caused any change in the other physiological and biochemical parameters measured, despite approximately 1.3-and 5-fold increases in citrate synthase and phosphoenolpyruvate carboxylase activities, respectively. Thus, our results demonstrate that citrate synthase is rate-limiting in enhancing citrate biosynthesis in P. fluorescens ATCC 13525. Significantly low extracellular citrate levels as compared to the intracellular levels in Pf(pAB7) suggested a probable limitation of efficient citrate transport.

Artificial Citrate Operon Confers Mineral Phosphate Solubilization Ability to Diverse Fluorescent Pseudomonads

“…P. fluorescens possesses H + -dependent citrate transporter [34], [35], which normally supports their growth on citrate as a sole carbon source. Our results suggest that this transporter seems to be inefficient in the efflux of citrate as also observed in our previous studies with the wild type citrate synthase gene [12]. The active pH homeostasis favors this in mesophilic bacteria [36].…”

“…In an earlier investigation, P. fluorescens ATCC13525 overexpressing E. coli wild type cs gene (allosterically inhibited by NADH) reported to increase CS activity by 2 fold with accumulation of 18.3 mM intracellular citrate [12]. Since native CS enzyme is subject to allosteric inhibition by NADH, it was hypothesized that over-expression of NADH insensitive variants of the CS would lead to further improvement of the citrate production in the genetically modified strains.…”

“…Preparation of cell free extracts and enzyme activities for G-6-PDH, PYC, ICDH and ICL assays as well as whole cell suspension for GDH assay were carried out as described earlier [12], [28]. All enzymes were assayed at room temperature (30±2°C) against appropriate controls without the substrate and enzyme source in the reaction mixture.…”

“…Previously, our studies demonstrated accumulation of high levels of citric acid but low secretion upon heterologous overexpression of phosphoenolpyruvate carboxylase ( ppc ) and citrate synthase ( cs ) genes in Pseudomonas fluorescens ATCC13525 [12], [13]. Overexpression of these genes independently and together showed that CS activity chiefly regulates citric acid biosynthesis.…”

Evaluation of cytotoxic, membrane, and DNA damaging effects of Thymus revolutus Célak essential oil on different cancer cells

“…One unit of enzyme activity represents the reduction of 1 µmol GSSG/min. G6PD activities were determined by following the reduction of NADP at 340 nm (28).…”