Surface Engineering of _Escherichia coli_ to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities  
[PERSON]  
1 Biotechnology and Biosciences Research Group, School of Chemical Sciences and Engineering, Autonomous University of Baja California, Tijluna 22390, RCN, Mexico;  
lipoproteins, and autotransporters [5; 6; 7; 8; 9], whereas the most usual carriers for yeast display are glycosylphosphatidylidylinositol (GPI)-dependent cell wall proteins and the Aga1-Aga2 anchor system [6; 10; 11; 12]. This biotechnology has various potential applications, including vaccine development, peptide library screening, metal bioadsorption, and bioconversion using whole-cell biocatalysts [1; 6; 13; 14; 15]. Phytases (EC 3.1.3.8, 3.1.3.26, and 3.1.3.72) belong to the histidine acid phosphatase (HAP) superfamily and catalyze the removal of orthophosphate from phytic acid or its salt (phyrate), which is the primary phosphorus storage compound in seeds. Given that phytase activity is absent in the digestive tract of monogastric animals (e.g., poultry and pigs), phytate is not metabolized and is therefore excreted undegraded, contributing to phosphorus pollution in regions with intensive livestock production [16; 17; 18; 19]. In addition, phytic acid is recognized as an anti-nutrient that can reduce the bioavailability of essential minerals (by chelating calcium, magnesium, zinc, and iron) [20; 21; 22]. Microbial phytases have been widely used as feed additives, resulting in increased phosphorus utilization and mineral availability, thus reducing the negative environmental impacts [23; 24; 25]. _E. coli_ AppA is a periplasmic enzyme that exhibits both acid phosphatase and phytase activity [26]. Optimal phosphatase activity was observed at pH 2.5, using p-nitrophenyl phosphate (pNPP) as a synthetic substrate (K\({}_{\text{M}}\) = 2.8 mM). Moreover, it can hydrolyze the 5'-\(\beta\)-phosphorly residue of ppGpp (K\({}_{\text{M}}\) = 1.8 mM) and the \(\gamma\)-phosphorylyl residue of GTP (K\({}_{\text{M}}\) = 0.35 mM). However, it cannot cleave most phosphomonoester substrates except fructose 1,6-bisphosphate and 2,3-bisphosphoglycerate (K\({}_{\text{M}}\) = 5 mM) [27]. In contrast, the optimal phytase activity was observed at pH 4.5 (K\({}_{\text{M}}\) = 0.63 mM on phytic acid). Moreover, it performs hydrolysis in a stereospecific manner through sequential phosphate removal [26; 28; 29]. Among the bacterial phytases, AppA exhibits the most consistent features and displays significant resistance to proteolysis under physiological conditions, supporting its putative application in monogastric animal nutrition [24; 30; 31]. To date, only a few phytases have been successfully displayed on cell surfaces, using yeast as the preferred host for membrane engineering and whole-cell biocatalysis. PhyA from _Aspergillus niger_ (a fungal phytase) was displayed on both _S. cerevisiae_ and _P. pastoris_, utilizing the C-terminus of yeast \(\alpha\)-agglutin as a carrier [32; 33]. In contrast, Phy from _Citrobacter amalonaticus_ (a bacterial phytase) was displayed on _P. pastoris_ using yeast GCW61, a GPI-anchored cell-wall glycoprotein homolog, as the fusion partner [34]. These specific approaches resulted in engineered yeast cells displaying active phytases, which exhibited promising prospects as whole-cell biocatalysts, but with a chance to improve enzyme stability and activity. More recently, _E. coli_ AppA was displayed on the surface of _Bacillus subtilis_ spores using the outer coat protein CotG as a carrier [18]. Although this approach favored valuable features of displayed AppA (e.g., protease resistance, protein stability, and enzyme efficiency), using appropriate peptide linkers may improve its full operativity. Diverse systems have been developed to display proteins on bacterial surfaces, such as those using either outer membrane protein A (OmpA) or adhesin involved in diffusible adhesion-I (AIDA-I) from _E.