A multistrategy approach for improving the expression of E. coli phytase in Pichia pastoris
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GENETICS AND MOLECULAR BIOLOGY OF INDUSTRIAL ORGANISMS - ORIGINAL PAPER
A multistrategy approach for improving the expression of E. coli phytase in Pichia pastoris Yuankun Helian1,2,3 · Yuanming Gai2,3 · Huan Fang2,3 · Yumei Sun1 · Dawei Zhang2,3,4 Received: 30 March 2020 / Accepted: 7 September 2020 © Society for Industrial Microbiology and Biotechnology 2020
Abstract Phytase is an additive in animal feed that degrades phytic acid in plant material, reducing feeding costs, and pollution from fecal phosphorus excretion. A multistrategy approach was adopted to improve the expression of E. coli phytase in Pichia pastoris. We determined that the most suitable signal peptide for phytase secretion was an α-factor secretion signal with an initial enzyme activity of 153.51 U/mL. Increasing the copy number of this gene to four increased phytase enzyme activity by 234.35%. PDI overexpression and Pep4 gene knockout increased extracellular phytase production by 35.33% and 26.64%, respectively. By combining favorable factors affecting phytase expression and secretion, the enzyme activity of the phytaseengineered strain was amplified 384.60% compared with that of the original strain. We also evaluated the potential for the industrial production of the engineered strain using a 50-L fed-batch fermenter and achieved a total activity of 30,246 U/ mL after 180 h of fermentation. Keywords Phytase · Heterologous expression · Secretion · Pichia pastoris
Introduction Phytic acid, or phytate (myo-inositol hexakisphosphate), is the primary stored form of phosphorus in most plants and is abundant in seeds and legumes [1, 2]. Monogastric animals cannot utilize phosphorous from ingested phytate due Yuankun Helian and Yuanming Gai contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10295-020-02311-6) contains supplementary material, which is available to authorized users. * Yumei Sun [email protected] * Dawei Zhang [email protected]
to the lack of the necessary enzymes in their digestive tracts [3]. The supplementation of inorganic phosphates in animal feed is one solution used to compensate for the deficiency of absorbable phosphorus; however, it results in increased feed costs as well as environmental costs (due to excessive phosphorus in animal excrement) [4]. Phytic acid also acts as an antinutritional factor by reducing the bioavailability of some mineral ions, proteins, and enzymes via the chelation of mineral ions and precipitation of proteins and enzymes. Consequently, phytic acid limits the absorption of these important ions and inhibits the activities and digestion of 2
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, People’s Republic of China
3
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, People’s Republic of China
4
University of Chin
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