The biodiversity of phytate cycling in soils

Phytic acid, myo-inositol hexakisphosphate, InsP6, is the major storage form of phosphate in seeds and grains that constitute a major part of the diets of monogastric animals such as swine and poultry. Monogastrics lack enough enzymes in the right part of the gut to digest dietary phytate. Consequently, phytases, a group of enzymes capable of releasing inorganic phosphate from phytate, are added to commercial poultry and swine diets. These adjunct phytases are a major sector of the global enzyme market with an estimated value of $5 billion in 2015. There is continued commercial interest in the discovery and development of more effective and cost-effective enzymes. The soil environment is microbially diverse and therefore offers significant potential for the isolation of novel phytases. In this thesis, I have developed new methods for the culture-dependent isolation of phytases from different soil environments by first analysing phytase activity of the soil microbiome using HPLC. The isolation of a multiple inositol polyphosphate phosphatase, MINPP, from Acinetobacter sp. represents one of the first phytases of its kind to be isolated from the soil environment. This study provides a robust characterisation of the protein, identifying an outstanding long-term stability at room temperature and activity from 37.6-101.3% over 755 days. The expression of the phytase was examined using β-galactosidase and qPCR assays which showed that while expression was enhanced in the presence of impure phytate by β-galactosidase, it was significantly repressed in the qPCR experiment. Additionally, a long-term phytase isolation experiment was performed using well-characterised Rothamsted soils. Of the sixty-six isolates that were re-streaked from mixed plates, seventeen showed a diverse array of phytase degradation profiles, highlighting the diversity of phytase activity in the soil environment. I have also undertaken metagenomic analysis to examine the diversity of phytases in environmental and enteric environments. This highlighted the dominance of MINPP genes in enteric environments above all other known classes of phytases. In soil and aquatic metagenomes, the relative abundances were significantly less than in enteric environments and here the Multiple Inositol Polyphosphate Phosphatase (MINPP) gene was not the overwhelming majority, instead the Beta-Propeller Phytase (BPPhy), Histidine Acid Phytase (HAPhy) and Protein Tyrosine Phosphatase-like Phytase (PTPhy) were equal to or even higher in abundances. Additionally, in this analysis I also examined the prospect of horizontal gene transfer in the MINPP dataset using the program T-rex. Here I identified multiple HGT events occurring between both enteric and environmental bacteria, with one transfer occurring between environments