Optimization of Recombinant Expression of Synthetic Bacterial Phytase in Pichia pastoris Using Response Surface Methodology

Abstract

Background:Escherichia coli phytase is an acidic histidine phytase with great specific activity. Pichia pastoris is a powerful system for the heterologous expression of active and soluble proteins which can express recombinant proteins in high cell density fermenter without loss of product yield and efficiently secrete heterologous proteins into the media. Recombinant protein expression is influenced by expression conditions such as temperature, concentration of inducer, and pH. By optimization, the yield of expressed proteins can be increase. Response surface methodology (RSM) has been widely used for the optimization and studying of different parameters in biotechnological processes.Objectives:In this study, the expression of synthetic appA gene in P. pastoris was greatly improved by adjusting the expression condition.Materials and Methods:The appA gene with 410 amino acids was synthesized by P. pastoris codon preference and cloned in expression vector pPinkα-HC, under the control of AOX1 promoter, and it was transformed into P. pastoris GS115 by electroporation. Recombinant phytase was expressed in buffered methanol-complex medium (BMMY) and the expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and enzymatic assay. To achieve the highest level of expression, methanol concentration, pH and temperature were optimized via RSM. Finally, the optimum pH and temperature for recombinant phytase activity was determined.Results:Escherichia coli phytase was expressed in P. pastoris under different cultivation conditions (post-induction temperature, methanol concentration, and post-induction pH). The optimized conditions by RSM using face centered central composite design were 1% (v/v) methanol, pH = 5.8, and 24.5°C. Under the optimized conditions, appA was successfully expressed in P. pastoris and the maximum phytase activity was 237.2 U/mL after 72 hours of expression.Conclusions:By optimization of recombinant phytase expression in shake flask culture, we concluded that P. pastoris was a suitable host for high-level expression of phytase and it can possess high potential for industrial applications.

Statistical Design of Experiments for Synthetic Biology

“…For example, product yield from an engineered metabolic pathway might be sensitive to culture incubation temperature, the pH of the growth media and the concentration of a chemical inducer. 28 An experimental treatment would therefore consist of a measurement of pathway yield from a culture grown in the presence of a set concentration of inducer, at a defined temperature and pH. Alternatively, yield might be tuned by changing the strength of the promoters that regulate each of the constituent genes in a pathway; 17 a treatment in that instance would consist of a measurement of pathway yield from an engineered strain in which the relevant promoter strengths were defined.…”

“…28,99−104 For example, Akbarzadeh et al used a Central Composite Design (CCD) to explore the effect of culture incubation temperature, inducer concentration and postinduction media pH on recombinant protein yield in Pichia pastoris. 28 Similarly, RSM was used by Kusuma et al to optimize media composition, inducer concentration and induction timing for heterologous expression of the Mycobacterium tuberculosis MPT64 antigen in E. coli, 99 and by Lee et al to optimize media composition, culture temperature, aeration and agitation for heterologous expression of a carboxylase, also in E. coli. 100 Song et al also employed RSM in the form of a Box−Behnken design to optimize geranylgeraniol (GGOH) overproduction in an engineered strain of S. cerevisiae.…”

A novel fungal beta-propeller phytase from nematophagous Arthrobotrys oligospora: characterization and potential application in phosphorus and mineral release for feed processing

“…Briefly, 100 μL of the diluted enzyme solution at 8.8 µg/mL and 900 μL of substrate solution (2 mM sodium phytate in 100 mM Tris-HCl buffer, pH 7.0) were mixed, and then incubated at 37 °C for 30 min. The reaction was immediately stopped by adding 1 mL of 10% trichloroacetic acid, followed by the addition of 2 mL AMES solution, and measurement of the absorbance at λ 700 nm [20]. The experiment was performed in triplicate.…”

Biosynthesis, purification, kinetic and thermodynamic analysis of phytase from Aspergillus oryzae

“…For this reason, low residual phytate content in the feed is desired (Mendoza-Avandano et al, 2019). Similarly, Phytates, six ringed structures, have 50-80% phosphorous in a bound form, which cannot be broken by the endogenous enzyme of the poultry (Akbarzadeh et al, 2015, Dersjant-Li et al, 2015. Phytase use as a feed additive is approved as GRAS (generally regarded as safe) in 22 countries to improve the nutritional value of feed (Rosenfelder et al, 2020).…”