title: Agrobacterium tumefaciens-mediated transformation of Penicillium expansum PE-12 and its application in molecular breeding.
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.
authors.affiliation: SJTU-Cornell Institute of Sustainable Agriculture and Biotechnology, Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, PR China.
authors.forename: Tian
authors.initials: T
authors.lastname: Zhang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Qi
authors.forename: Yueyue
authors.initials: Y
authors.lastname: Wang
authors.forename: Fangyuan
authors.initials: F
authors.lastname: Zhang
authors.forename: Renyong
authors.initials: R
authors.lastname: Li
authors.forename: Qingsheng
authors.initials: Q
authors.lastname: Yu
authors.forename: Xiangbin
authors.initials: X
authors.lastname: Chen
authors.forename: Huojun
authors.initials: H
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Xiong
authors.forename: Kexuan
authors.initials: K
authors.lastname: Tang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001233:Aspergillus nidulans / Q000235:genetics; D032621:DNA Shuffling / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005827:Genetics, Microbial / Q000379:methods*; D042822:Genomic Instability; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D010407:Penicillium / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; C036483:T-DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D008049:Lipase