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Microbiol Res. 2013 Mar 30;168(3):130-7. doi: 10.1016/j.micres.2012.11.001. Epub 2012 Dec 21.

Agrobacterium tumefaciens-mediated transformation of Penicillium expansum PE-12 and its application in molecular breeding.

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  • 1SJTU-Cornell Institute of Sustainable Agriculture and Biotechnology, Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, PR China.

Abstract

Lipase produced by Penicillium expansum is widely used in laundry detergent and leather industry; however, the absence of an efficient transformation technology sets a major obstacle for further enhancement of its lipase productivity through advanced gene engineering. In this work, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for P. expansum PE-12 transformation, using hygromycin phosphotransferase (hph) as a selectable marker gene. As a result, we revealed that the frequency of transformation surpassed 100 transformants/10(5)condida, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and all the transformants showed mitotic stability. Facilitated by this newly established method, for the first time, P. expansum PE-12 was genetically engineered to improve the lipase yield, through a homologous expression vector carrying the endogenous lipase gene (PEL) driven by the strong constitutive promoter of the glyceraldehydes-3-phosphate dehydrogenase gene (gpdA) from Aspergillus nidulans. The highest expression level of the engineered strain reached up to 1700 U/mL, nearly 2-fold of the original industrial strain (900 U/mL). Our reproducible ATMT system has not only revealed the great potential of homologous expression-directed genetic engineering, which is more efficient and specific compared to traditional mutagenesis, but also provided new possibilities and perspectives for any other practical applications of P. expansum-related genetic engineering in the future.

Copyright © 2012 Elsevier GmbH. All rights reserved.

PMID:
23265791
[PubMed - indexed for MEDLINE]
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