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

ABSTRACT: 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.To improve the phytase expression, we use modification of P AOX1 and the ?-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.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 ?-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.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.Using modification of P AOX1 and the ?-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.

SUBMITTER: Li C

PROVIDER: S-EPMC4584009 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Li Cheng C Lin Ying Y Zheng Xueyun X Pang Nuo N Liao Xihao X Liu Xiaoxiao X Huang Yuanyuan Y Liang Shuli S

BMC biotechnology 20150926

<h4>Background</h4>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.<h4>Methods</h4>To improve the phytase expression, we use modification ...[more]

PMID: 26410558

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Project description: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 pPink?-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.

Project description:Alkaline phytase isolated from pollen grains of Lilium longiflorum (LlALP) possesses unique catalytic and thermal stability properties that suggest it has the potential to be used as a feed supplement. However, substantial amounts of active enzymes are needed for animal feed studies and endogenous levels of LlALP in lily pollen are too low to provide the required amounts. Active rLlALP2 (coded by LlAlp2, one of two isoforms of alkaline phytase cDNA identified in lily pollen) has been successfully expressed in intracellular compartments of Pichia pastoris, however enzyme yields have been modest (25-30 mg/L) and purification of the enzyme has been challenging. Expression of foreign proteins to the extracellular medium of P. pastoris greatly simplifies protein purification because low levels of endogenous proteins are secreted by the yeast. In this paper, we first describe the generation of P. pastoris strains that will secrete rLlALP2 to the extracellular medium. Data presented here indicates that deletion of native signal peptides at the N- and C-termini of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion by four-fold; chicken egg white lysozyme signal peptide was ineffective in the extracellular secretion of rLlALP2. Second, we describe our efforts to increase expression levels by employing a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) in place of the strong, tightly controlled promoter of alcohol oxidase 1 gene (PAOX1). PGAP enhanced the extracellular expression levels of rLlALP2 compared to PAOX1. Finally, we report on the optimization of the culture medium to enhance yields of rLlALP2. The strength of PGAP varies depending on the carbon source available for cell growth; secreted expression of rLlALP2 was highest when glycerol was the carbon source. The addition of histidine and Triton X-100 also enhanced extracellular expression. Taken together, the employment of PGAP under optimized culture conditions resulted in approximately eight-fold (75-80 mg/L) increase in extracellular activity compared to PAOXI (8-10 mg/L). The P. pastoris expression system can be employed as a source of active alkaline phytase for animal feed studies.

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

Publications

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

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