Citrobacter amalonaticus Phytase on the Cell Surface of Pichia pastoris Exhibits High pH Stability as a Promising Potential Feed Supplement

Cheng, Li; Lin, Ying; Huang, Yuanyuan; Liu, Huan; Liang, Shuli

doi:10.1371/journal.pone.0114728

Cited by 11 publications

(6 citation statements)

References 44 publications

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“…Plasmid construction in this work is illustrated in Additional file 1 . The vector pPICZαA-phy [ 7 ], which carries the phytase gene of C. amalonaticus CGMCC 1696, had the zeocin resistance gene replaced by the HIS4 and kanamycin resistance genes from plasmid pPICHKA [ 22 ] to create plasmid pPICHKA-phy (Phy). The combination of cis-acting elements with P AOX1 , a deletion of P AOX1 nucleotides −777 and −712 and the addition of −203 and −190 [ 10 ], was performed to create plasmid pAOX1 d1+201 -α-phy-HKA (AOXm).…”

Section: Resultsmentioning

confidence: 99%

“…The phytase gene of C. amalonaticus CGMCC 1696, PHY [GenBank: ABI98040.1], was from the vector pPICZαA-phy [ 7 ]. The vectors pPICHKA and pPICZA-HAC1 were gifts from Dr. Han (South China University of Technology) [ 22 ].…”

Section: Methodsmentioning

confidence: 99%

“…Among these, the phytase from C. amalonaticus CGMCC 1696 has the highest specific activity, 3548 U/mg [ 6 ]. The C. amalonaticus phytase expressed by P. pastoris also has an optimal temperature of 60 °C and pH of 3.0 [ 7 ], which meets the requirements of industrial applications. However, the high cost of phytase remains a barrier to its use in industrial applications.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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“…To test the Cre/ loxP Zeo R marker recycling vectors, the genes PHY that encodes the phytase from Citrobacter amalonaticus CGMCC 1696 (Phy) 33 , 34 , XYN that encodes the xylanase A from Bacillus halodurans C-125 (Xyn) 10 and ARL that encodes lipase from Acinetobacter radioresistens CMC-1 (Arl) 35 , which are three important kinds of enzymes applied in industry 10 , 34 – 37 , were used as reporter genes. Considering the advantages of the most commonly used promoter P AOX1 for expression of heterologous proteins in P. pastoris , e.g., strong transcription level and tightly regulated (recently reviewed by Ahmad 38 ), and advantages of the secreted expression system, e.g., simple purification, reduced potential degradation of heterologous proteins and toxicity to hosts by accumulation of secreted heterologous proteins 39 , 40 , the vector pZACH was chosen for secreted heterologous proteins expression.…”

Section: Resultsmentioning

confidence: 99%

Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris

Cheng

Lin

Zheng

et al. 2017

Sci Rep

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Pichia pastoris is a widely used heterologous protein production workhorse. However, with its multiple genetic modifications to solve bottlenecks for heterologous protein productivity, P. pastoris lacks selectable markers. Existing selectable marker recycling plasmids have drawbacks (e.g., slow growth and conditional lethality). Here, zeocin-resistance marker recycling vectors were constructed using the Cre/loxP recombination system. The vectors were used to (i) knock in heterologous phytase, xylanase and lipase expression cassettes, (ii) increase the phytase, xylanase and lipase gene copy number to 13, 5, and 5, respectively, with vector introduction and (iii) engineer the secretion pathway by co-overexpressing secretion helper factors (Sly1p and Sec1p) without introducing selectable markers, giving a phytase field of 0.833 g/L. The vectors allow selectable marker recycling and would be a useful tool to engineer P. pastoris for high heterologous protein productivity.

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“…Display of lipase on P. pastoris has been extensively reported for biodiesel production (refer to Yan et al for a detailed review) or synthesis of various kind of esters . Other enzymes displayed on P. pastoris include cellulases, phytase, glycosidases, etc. (Figure D).…”

Section: P Pastoris As a Whole‐cell Biocatalystmentioning

confidence: 99%

Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives

Zhu

Sun

2019

Biotechnology Journal

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With nearly three decades of development, Pichia pastoris (P. pastoris) has become a powerful eukaryotic protein expression system for the expression of thousands of proteins both on a laboratory and industrial scale. In addition, it has also been extensively used as a cell factory for the production of a variety of chemicals. This review summarizes the bottlenecks and solutions in achieving high-level secretory protein expression with P. pastoris and then outlines its applications on chemical production with an emphasis on its role as whole-cell biocatalyst. Furthermore, current challenges and future directions of this important system are also discussed.

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Citrobacter amalonaticus Phytase on the Cell Surface of Pichia pastoris Exhibits High pH Stability as a Promising Potential Feed Supplement

Cited by 11 publications

References 44 publications

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

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

Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris

Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives

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