Biotechnological approaches to the phosphorus cycle: Recombinant phytase production in bacillus subtilis

Phosphorus (P) is classified as one of the most essential macronutrients for all living organisms. It plays a key role in many vital processes such as growth, protein synthesis, repair and energy utilization. Phosphate (P2O5), the common inorganic form of P, is mostly found as phytic acid or phytate form (myo-inositol 1,2,3,4,5,6-hexakisphosphate), which cannot be consumed by plants and most of the animals. In order to prevent P deficiency, inorganic phosphate fertilizers are excessively used in agriculture. This strategy is unsustainable since these fertilizers are mostly dependent on phosphate rocks. Besides, the use of fertilizers causes eutrophication in aquatic environments, which results in impairment of the ecosystem. Phytase (3.1.3.8) is an enzyme from the phosphatase family, which catalyzes the hydrolysis of phytic acid into myo-inositol and lower phosphate esters. Especially microbial phytase enzyme is used as a fertilizer or sometimes as a food/feed additive to overcome P deficiency. Therefore, studies have been focused on high yield enzyme production, which also has contribution to both phosphorus cycle and relevant industries. In this study, Bacillus subtilis was chosen as the host for recombinant phytase production due to its ability of extracellular protein secretion. Phytase gene was cloned from B. subtilis 168 strain and transferred to B. subtilis RIK1285, a protease deficient strain. Considering the importance of the signal peptide for the secretion, as an integral part of the cloning strategy, signal peptides have been screened from the transformants with varying phytase activity. In total, twenty-nine B. subtilis transformants were obtained with a phytase activity range of 0.03-0.3 U/mL. Sequencing of the selected recombinant plasmids revealed that the colonies contained the signal peptides, lipB or dltD. Also, comparison of different growth media showed that the presence of rice bran increased the efficiency of enzyme production. This study underlines the importance of not only the modifications on a molecular level but also the choice of growth medium for increasing the efficiency of recombinant enzyme production.