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Applied Biochemistry and Biotechnology

, Volume 169, Issue 4, pp 1160–1172 | Cite as

Impact of Gene Dosage on the Production of Lipase from Rhizopus chinensis CCTCC M201021 in Pichia pastoris

  • Chong Sha
  • Xiao-Wei YuEmail author
  • Fei Li
  • Yan XuEmail author
Article

Abstract

In this work, the high-level expression of the lipase r27RCL was achieved by optimization of the lipase gene copy number in the host strain Pichia pastoris. The copy number of the lipase gene proRCL from Rhizopus chinensis CCTCC M201021 was quantified by real-time quantitative polymerase chain reaction and a range of Mut+ P. pastoris strains carrying one, three, five, and six copies of proRCL were obtained. The maximum lipase activity was achieved at 12,500 U/mL by the five-copy recombinant strain after 96 h of methanol induction in the 7-L fermenter. However, the enzyme activity of the six-copy recombinant strain decreased remarkably. By transcription analysis of proRCL, ERO1, and PDI, it suggested that unfolded protein response seemed to be triggered in the highest copy recombinant strain after 24 h. Thus, elaborate optimization of foreign gene dosage was very important for the high-level expression of foreign proteins in P. pastoris.

Keywords

Lipase Pichia pastoris Real-time quantitative PCR (RT-QPCR) Transcription analysis Gene dosage 

Notes

Acknowledgments

Financial support from the National Key Basic Research and Development Program of China (973 Program; no. 2011CB710800), the National High Technology Research and Development Program of China (863 Program; no. 2012AA022207, 2011AA02A209, and 2011AA02A210), the Fundamental Research Funds for the Central Universities (JUSRP11014), the Programme of Introducing Talents of Discipline to Universities (111 Project; 111-2-06 ), and the Ministry of Education, China, and from NSFC (20802027) are greatly appreciated.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.State Key Laboratory of Food Science and Technology, the Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina

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