Construction of a Pseudozyma antarctica strain without foreign DNA sequences (self-cloning strain) for high yield production of a biodegradable plastic-degrading enzyme

• Published in: Bioscience, Biotechnology, and Biochemistry
• Main Authors: Sameshima-Yamashita, Yuka, Watanabe, Takashi, Tanaka, Takumi, Tsuboi, Shun, Yarimizu, Tohru, Morita, Tomotake, Koike, Hideaki, Suzuki, Ken, Kitamoto, Hiroko
• Other Authors: Project of the NARO Bio-oriented Technology Research Advancement Institution
• Format: Article in Journal/Newspaper
• Language: English
• Published: Informa UK Limited 2019
• Subjects: Antarc*; Antarctica
• Online Access: http://dx.doi.org/10.1080/09168451.2019.1571898; http://academic.oup.com/bbb/article-pdf/83/8/1547/36827638/bbb1547.pdf

ABSTRACT The basidiomycetous yeast Pseudozyma antarctica GB-4(0) esterase (PaE) is a promising candidate for accelerating degradation of used biodegradable plastics (BPs). To increase safety and reduce costs associated with the use of PaE, we constructed a self-cloning strain with high-PaE productivity. A Lys12 gene (PaLYS12)-deleted lysine auxotroph strain GB4-(0)-L1 was obtained from GB-4(0) by ultraviolet mutagenesis and nystatin enrichment. Subsequently, the PaE gene (PaCLE1) expression cassette consisting of GB-4(0)-derived PaCLE1, under the control of a xylose-inducible xylanase promoter with PaLYS12, was randomly introduced into the GB4-(0)-L1 genome. A PaE high-producing strain, PGB474, was selected from among the transformants by high throughput double-screening based on its ability to degrade emulsified polybutylene succinate-co-adipate. Quantitative PCR revealed that four copies of the PaE gene expression cassette were introduced into the PGB474 genome. PGB474 produced 2.0 g/L of PaE by xylose-fed-batch cultivation using a 3-L jar fermentor for 72 h.