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Constitutive Expression of Yarrowia lipolytica Lipase LIP2 in Pichia pastoris Using GAP as Promoter

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Abstract

A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for 48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99% after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.

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Acknowledgments

This work was funded by the National Natural Science Foundation of P.R. China (NSFC) (nos. 31070089 and 31170078), the National High Technology Research and Development Program of China (863 Program) (nos. 2010AA101501 and 2011AA02A204), and the Natural Science Foundation of Hubei Province (2009CDA046).

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  1. Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, 430074, China

    Xiaofeng Wang, Yongchuan Sun, Feng Ke, Heyun Zhao, Tao Liu, Li Xu, Yun Liu & Yunjun Yan

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  1. Xiaofeng Wang
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  2. Yongchuan Sun
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  3. Feng Ke
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Correspondence to Yunjun Yan.

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Wang, X., Sun, Y., Ke, F. et al. Constitutive Expression of Yarrowia lipolytica Lipase LIP2 in Pichia pastoris Using GAP as Promoter. Appl Biochem Biotechnol 166, 1355–1367 (2012). https://doi.org/10.1007/s12010-011-9524-4

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