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High-level expression of Thermomyces dupontii thermophilic lipase in Pichia pastoris via combined strategies.

ABSTRACT: In this study, a combined strategy was used to improve the production of Thermomyces dupontii lipase (TDL) in Pichia pastoris. First, the native gene of TDL was optimized based on the codon usage of P. pastoris, ligated to pPICZ?A and transformed in P. pastoris X33. A recombinant strain designated X33-T23 with the highest activity (1020 U/mL in shake flasks) amongst 216 recombinant colonies was selected for further investigations. To further increase the production of TDL, nine different secretion helper factor genes were transformed in the recombinant strain, X33-T23. The recombinant strain co-expression with the gene encoding protein disulfide isomerase, designated X33-T23-PDI, exhibited the highest activity in shake flasks (1760 U/mL) and in 5 L bioreactor (57521 U/mL) which were 1.67- and 1.46-fold higher, respectively, than for strain X33-T23. Additionally, the optimization of the inducers (temperature and pH) for the recombinant strain X33-T23-PDI in 5 L bioreactor produced, as expected, much higher lipase activity (81203 U/mL). The results of this study will provide an effective method to produce TDL and give some clues on how to improve production of heterologous proteins in P. pastoris.

SUBMITTER: Wang J

PROVIDER: S-EPMC6361030 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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High-level expression of Thermomyces dupontii thermophilic lipase in Pichia pastoris via combined strategies.

Wang Jianrong J Wu Zongze Z Zhang Tianyu T Wang Yonghua Y Yang Bo B

3 Biotech 20190201 2

In this study, a combined strategy was used to improve the production of Thermomyces dupontii lipase (TDL) in Pichia pastoris. First, the native gene of TDL was optimized based on the codon usage of P. pastoris, ligated to pPICZαA and transformed in P. pastoris X33. A recombinant strain designated X33-T23 with the highest activity (1020 U/mL in shake flasks) amongst 216 recombinant colonies was selected for further investigations. To further increase the production of ...[more]

PMID: 30729086

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Project description: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 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.ResultsThe 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.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 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.

Dataset Information

High-level expression of Thermomyces dupontii thermophilic lipase in Pichia pastoris via combined strategies.

Publications

High-level expression of <i>Thermomyces dupontii</i> thermophilic lipase in <i>Pichia pastoris</i> via combined strategies.

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