Dataset Information

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris.

ABSTRACT:

Background

Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains.

Results

Potential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter.

Discussion

The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

SUBMITTER: Navone L

PROVIDER: S-EPMC7836175 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris.

Navone Laura L Vogl Thomas T Luangthongkam Pawarisa P Blinco Jo-Anne JA Luna-Flores Carlos C Chen Xiaojing X von Hellens Juhani J Speight Robert R

Microbial cell factories 20210107 1

<h4>Background</h4>Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critic ...[more]

PMID: 33494776

Similar Datasets

Project description: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.

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.

Dataset Information

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris.

Background

Results

Discussion

Publications

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris.

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