GABA Production as a Potential Wound Healing Agent in Synechocystis sp. PCC6803 With Inactivated Glutamate and Ketoglutarate Decarboxylase Pathways

GABA (γ-aminobutyric acid) is a four carbon non-protein amino acid produced by the decarboxylation of glutamate which is also known as GAD pathway. GABA production is ubiquities in all life forms ranging from prokaryotes to eukaryotes. It functions as an inhibitory neurotransmitter in mammalian brain, whereas it is known to have various environmental stress relieving roles in plants and bacteria. One of the important roles of GABA in mammals is the healing process of cutaneous wounds especially during early wound healing stage. Because of the beneficial functions of GABA and increasing commercial demand, various attempts have been made for chemical and biological synthesis of GABA. However biological synthesis of GABA is considered as a more promising method due to the simple catalytic reaction, cost effectiveness and environmental compatibility. In blue-green algae Synechocystis sp. PCC6803, the GAD (glutamate decarboxylase) and KGD (alpha-ketoglutarate decarboxylase) pathways are known to complete the tricarboxylic acid cycle (TCAC). However there are evidences that in bacteria and plants, polyamine catabolism in part also contributes to the metabolic pool of TCAC via GABA formation. A novel polyamines degradation pathway was discovered/studied in Synechocystis by disrupting both the GAD and KGD pathways by genetic engineering to generate a double mutant strain (∆gad:∆kgd). The novel pathway might leads to GABA production and subsequently connects to TCAC in Synechocystis. The ∆gad:∆kgd was found not only viable but also having higher intracellular alpha-ketoglutarate and polyamines content as compared to wild type. Gene expression analysis using RT-PCR indicated that in ∆gad:∆kgd strain the γ-aminobutanal dehydrogenase (gabdh) pathway (the catalytic pathway for polyamine catabolism to GABA) was highly up-regulated. To further validate the association of polyamine catabolism with GABA synthesis, another mutant strain called as ∆gabdh was created by disrupting gabdh pathway. Metabolite analysis in ∆gabdh strain indicated very high intracellular GABA, glutamate, succinate and spermidine accumulation. For the first time, the study has demonstrated the link between polyamine degradation and GABA synthesis via gabdh pathway in blue-green algae. The metabolites and gene expression analyses indicate that the gabdh pathway contributes to upregulate the flux from alpha-ketoglutarate to spermidine and subsequently to GABA in the absence of GAD and KGD pathways in Synechocystis sp. PCC6803. The gabdh pathway might also serve as the sole contributor in providing precursor substrate to keep the TCAC intact in Synechocystis. This work forms the basis for further development of GABA production by employing cyanobacteria that are safe and eco-friendly microorganisms.

کلیدواژه ها:

γ-Aminobutyric acid ، Metabolism ، Polyamines ، γ-Aminobutanal dehydrogenase ، Synechocystis PCC6803