Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris

Abstract

BackgroundPhytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have been expressed in Pichia pastoris, among which the phytase from Citrobacter amalonaticus CGMCC 1696 had high specific activity (3548 U/mg). Improvement of the phytase expression level will contribute to facilitate its industrial applications.MethodsTo improve the phytase expression, we use modification of PAOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1i to enhance folding and secretion of the protein in the endoplasmic reticulum. The genetic stability and fermentation in 10-L scaled-up fed-batch fermenter was performed to prepare for the industrial production.ResultsThe phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (PAOX1) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of PAOX1 and the α-factor signal peptide increased the phytase yield by 35 and 12 %, respectively. Next, on increasing the copy number of the Phy gene to six, the phytase yield was 141 % higher than in the strain containing only a single gene copy. Furthermore, on overexpression of HAC1i (i indicating induced), a gene encoding Hac1p that regulates the unfolded protein response, the phytase yield achieved was 0.75 g/L with an activity of 2119 U/mL, 412 % higher than for the original strain. The plasmids in this high-phytase expression strain were stable during incubation at 30 °C in Yeast Extract Peptone Dextrose (YPD) Medium. In a 10-L scaled-up fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL.DiscussionThe production of a secreted protein will reach its limit at a specific gene copy number where further increases in transcription and translation due to the higher abundance of gene copies will not enhance the secretion process any further. Enhancement of protein folding in the ER can alleviate bottlenecks in the folding and secretion pathways during the overexpression of heterologous proteins in P. pastoris.ConclusionsUsing modification of PAOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1i to enhance folding and secretion of the protein in the endoplasmic reticulum, we have successfully increased the phytase yield 412 % relative to the original strain. In a 10-L fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. Large-scale production of phytase can be applied towards different biocatalytic and feed additive applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0204-2) contains supplementary material, which is available to authorized users.

High-Level Expression and Biochemical Properties of A Thermo-Alkaline Pectate Lyase From Bacillus sp. RN1 in Pichia pastoris With Potential in Ramie Degumming

“…There are also some restrictions that hinder further production. For example, a plateau effect occurred when the gene dosage exceeded a certain range due to metabolic burden (Gasser et al, 2008;Li et al, 2015). Consequently, engineering of protein folding, ERAD (endoplasmic reticulum-associated protein degradation) systems and transport systems should be performed to further improve protein production (Zahrl et al, 2017).…”

Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris

“…The optical density of Phy band was calculated and the RNC aliquots from the same samples with proteinase K digestion were normalized to those with no proteinase K digestion. b Quantitative analysis of protein aggregates extracted from wild-type and RP deletion strains transformed with Phy which is glycosylated in P. pastoris [57]. The samples were subject to SDS-PAGE separation followed by Coomassie blue staining, and Phy was visualized by western blotting.…”

Characterization of the Catalytic Structure of Plant Phytase, Protein Tyrosine Phosphatase-Like Phytase, and Histidine Acid Phytases and Their Biotechnological Applications

“…It belongs to the class of hydrolases and thereby hydrolyzes phytate (phytic acid) to release inositol phosphates, phosphorus, inositol, and other essential nutrients required for absorption ( Figure 1 ). Moreover, it is an important component of a variety of metabolic processes as released phosphorus favors development, formation and mineralization of animal bone, cellular metabolism, and protein synthesis [ 31 – 35 ].…”