High-level Secretive Expression of a Novel Achieved Talaromyces Cellulolyticus Endo-polygalacturonase in Pichia Pastoris by Improving Gene Dosage for Hydrolysis of Natural Pectin

Overview

Journal World J Microbiol Biotechnol

Publisher Springer

Specialties Biotechnology
Microbiology

Date 2019 May 29

PMID 31134444

Citations 3

Authors

Xiao-Bo Peng

Guang-Jun Chen

Zhen-Gang Han

Jiang-Ke Yang

Affiliations

Soon will be listed here.

Abstract

Pectin is a type of complex hydrophilic polysaccharide widely distributed in plant resources. Thermal stable pectinase has its advantage in bioapplication in the fields of food processing, brewing, and papermaking, etc. In this study, we enzymatically characterized a putative endo-polygalacturonase TcPG from a Talaromyces cellulolyticus, realized its high-level expression in Pichia pastoris by in vitro constructing of a series of multi-copy expression cassettes and real time quantitative PCR screening. The secretive expression level of TcPG was nonlinear correlated to the gene dosage. Recombinants with five-copy TcPG gene in the host genome showed the highest expression. After cultivation in a bioreactor for about 96 h, the enzyme activity reached 7124.8 U/mL culture. TcPG has its optimal temperature of 70 °C. Under the optimized parameters, the pectin could be efficiently hydrolyzed into oligosaccharides.

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References

Vassileva A, Chugh D, Swaminathan S, Khanna N . Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Expr Purif. 2001; 21(1):71-80. DOI: 10.1006/prep.2000.1335. View

Kashyap D, Vohra P, Chopra S, Tewari R . Applications of pectinases in the commercial sector: a review. Bioresour Technol. 2001; 77(3):215-27. DOI: 10.1016/s0960-8524(00)00118-8. View

Federici L, Caprari C, Mattei B, Savino C, di Matteo A, De Lorenzo G . Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein). Proc Natl Acad Sci U S A. 2001; 98(23):13425-30. PMC: 60887. DOI: 10.1073/pnas.231473698. View

van Pouderoyen G, Snijder H, Benen J, Dijkstra B . Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger. FEBS Lett. 2003; 554(3):462-6. DOI: 10.1016/s0014-5793(03)01221-3. View

Maharajh D, Roth R, Lalloo R, Simpson C, Mitra R, Gorgens J . Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica. Appl Microbiol Biotechnol. 2008; 79(2):235-44. DOI: 10.1007/s00253-008-1420-7. View

Damasio A, da Silva T, Maller A, Jorge J, Terenzi H, Polizeli M . Purification and partial characterization of an exo-polygalacturonase from Paecilomyces variotii liquid cultures. Appl Biochem Biotechnol. 2009; 160(5):1496-507. DOI: 10.1007/s12010-009-8682-0. View

Cregg J, Tolstorukov I, Kusari A, Sunga J, Madden K, Chappell T . Expression in the yeast Pichia pastoris. Methods Enzymol. 2009; 463:169-89. DOI: 10.1016/S0076-6879(09)63013-5. View

Abad S, Kitz K, Hormann A, Schreiner U, Hartner F, Glieder A . Real-time PCR-based determination of gene copy numbers in Pichia pastoris. Biotechnol J. 2010; 5(4):413-20. DOI: 10.1002/biot.200900233. View

Nakkeeran E, Umesh-Kumar S, Subramanian R . Aspergillus carbonarius polygalacturonases purified by integrated membrane process and affinity precipitation for apple juice production. Bioresour Technol. 2010; 102(3):3293-7. DOI: 10.1016/j.biortech.2010.10.048. View

10.

Yuan P, Meng K, Wang Y, Luo H, Shi P, Huang H . A protease-resistant exo-polygalacturonase from Klebsiella sp. Y1 with good activity and stability over a wide pH range in the digestive tract. Bioresour Technol. 2012; 123:171-6. DOI: 10.1016/j.biortech.2012.07.037. View

11.

Liu M, Dai X, Bai L, Xu X . Cloning, expression of Aspergillus niger JL-15 endo-polygalacturonase A gene in Pichia pastoris and oligo-galacturonates production. Protein Expr Purif. 2013; 94:53-9. DOI: 10.1016/j.pep.2013.10.025. View

12.

Liu H, Ma Y, Chen N, Guo S, Liu H, Guo X . Overexpression of stress-inducible OsBURP16, the β subunit of polygalacturonase 1, decreases pectin content and cell adhesion and increases abiotic stress sensitivity in rice. Plant Cell Environ. 2013; 37(5):1144-58. PMC: 4286026. DOI: 10.1111/pce.12223. View

13.

Fujii T, Hoshino T, Inoue H, Yano S . Taxonomic revision of the cellulose-degrading fungus Acremonium cellulolyticus nomen nudum to Talaromyces based on phylogenetic analysis. FEMS Microbiol Lett. 2013; 351(1):32-41. DOI: 10.1111/1574-6968.12352. View

14.

Kataoka M, Akita F, Maeno Y, Inoue B, Inoue H, Ishikawa K . Crystal structure of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) GH family 11 xylanase. Appl Biochem Biotechnol. 2014; 174(4):1599-1612. PMC: 4177571. DOI: 10.1007/s12010-014-1130-9. View

15.

Tu T, Luo H, Meng K, Cheng Y, Ma R, Shi P . Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions. Appl Environ Microbiol. 2015; 81(19):6938-44. PMC: 4561696. DOI: 10.1128/AEM.01363-15. View

16.

Zeni J, Pili J, Cence K, Toniazzo G, Treichel H, Valduga E . Characterization of novel thermostable polygalacturonases from Penicillium brasilianum and Aspergillus niger. Bioprocess Biosyst Eng. 2015; 38(12):2497-502. DOI: 10.1007/s00449-015-1468-6. View

17.

Okuda N, Fujii T, Inoue H, Ishikawa K, Hoshino T . Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain. Biosci Biotechnol Biochem. 2016; 80(10):2065-8. DOI: 10.1080/09168451.2016.1189315. View

18.

Sreekrishna K, Brankamp R, Kropp K, Blankenship D, Tsay J, Smith P . Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene. 1997; 190(1):55-62. DOI: 10.1016/s0378-1119(96)00672-5. View

19.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View