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

Abstract

Background: Phytase 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.

Methods: To improve the phytase expression, we use modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) 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.

Results: The phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (P AOX1 ) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of P AOX1 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 HAC1 (i) (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.

Discussion: The 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.

Conclusions: Using modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) 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.

Fig. 1

Quantitative PCR assay of the PHY copy number in recombinant yeast strain genomic DNA. The threshold value (horizontal dashed line) was set at 0.2. The values indicate the average ± standard deviations from three independent qPCR experiments

Fig. 2

Expression of the phytase from C. amalonaticus CGMCC 1696 in P. pastoris. a: Time dependence of the activity of phytase, cell growth and phytase protein content of Gs115/Phy after induction with methanol, using GS115/HKA as the background (control) sample. b: SDS-PAGE assay of the culture supernatant containing C. amalonaticus CGMCC 1696 phytase (stained with Coomassie Blue) after methanol induction for 96 h. Lane 1: culture supernatants from recombinant P. pastoris GS115/HKA; lane 2: culture supernatants from recombinant P. pastoris Gs115/Phy; lane 3: culture supernatants from recombinant P. pastoris Gs115/Phy after PNGase F treatment; lane 4: culture supernatants from recombinant P. pastoris GS115/HKA after PNGase F treatment

Fig. 3

Different tactics for enhancing the expression of Phy in P. pastoris. a: Effect of modification of P_AOX1 and the signal peptide, increasing gene copy number, and overexpression of Kar2p, Ero1p, Pdi1p, or Hac1p on phytase production in recombinant strains carrying six Phy gene copies after 96-h induction with methanol. All activities used GS115/HKA as the background measurement. b: SDS-PAGE assay of the collected culture supernatant from the most effective strains after 96-h induction with methanol. Lane M: protein marker; lane 1: GS115/HKA; lane 2: GS115/Phy; lane 3: GS115/AOXm; lane 4: GS115/αE10; lane 5: GS115/6c; lane 6: 6c/HAC1; lane 7: 0.05 mg/mL BSA; lane 8: 0.2 mg/mL BSA; lane 9: 0.3 mg/mL BSA. c: The phytase protein content of the most effective strains after 96-h induction with methanol

Fig. 4

Growth and phytase production time course of P. pastoris 6c/HAC1 in a 10-L fermenter. a: Time dependence of phytase activity, cell density and phytase protein content after induction with methanol. The phytase activity was detected using collected culture supernatant. The sample was boiled for 10 min was used as the background sample. b: SDS-PAGE assay of the collected culture supernatant. All the collected supernatants were diluted 1:40 with 100 mM sodium acetate buffer (pH5.5) (v/v). Lane M: protein marker; lane 1: induction for 24 h; lane 2: induction for 48 h; lane 3: induction for 72 h; lane 4: induction for 96 h; lane 5: induction for 120 h; lane 6: induction for 144 h; lane 7: 0.05 mg/mL BSA; lane 8: 0.2 mg/mL BSA; lane 9: 0.3 mg/mL BSA

Fig. 5

Changes in phytase activity of strain 6c/HAC1 after ten cultivations. The phytase activity of strain 6c/HAC1 after ten sub-cultivations with 96-h methanol induction. All activities were compared with the activity of the original clone, with GS115/HKA as the background sample