title: Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi.
abstract: Oleaginous yeast Lipomyces starkeyi, a promising strain of great biotechnical importance, is able to accumulate over 60% of its cell biomass as triacylglycerols (TAGs). It is promising to directly produce the derivatives of TAGs, such as long-chain fatty acid methyl esters and alkanes, in L. starkeyi. However, techniques for genetic modification of this oleaginous yeast are lacking, thus, further research is needed to develop genetic tools and functional elements. Here, we used two exogenous promoters (pGPD and pPGK) from oleaginous yeast Rhodosporidium toruloides to establish a simpler Agrobacterium-mediated transformation (AMT) method for L. starkeyi. Hygromycin-resistant transformants were obtained on antibiotic-contained plate. Mitotic stability test, genotype verification by PCR, and protein expression confirmation all demonstrated the success of this method. Furthermore, the strength of these two promoters was evaluated at the phenotypic level on a hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. The PGK promoter strength was 2.2-fold as that of GPD promoter to initiate the expression of the hygromycin-resistance gene. This study provided an easy and efficient genetic manipulation method and elements of the oleaginous yeast L. starkeyi for constructing superior strains to produce advanced biofuels.
authors.affiliation: School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian, 116034, People's Republic of China. yingchaer@163.com.
authors.forename: Xinping
authors.initials: X
authors.lastname: Lin
authors.affiliation: School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian, 116034, People's Republic of China.
authors.forename: Sasa
authors.initials: S
authors.lastname: Liu
authors.affiliation: School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian, 116034, People's Republic of China.
authors.forename: Ruiqi
authors.initials: R
authors.lastname: Bao
authors.affiliation: School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian, 116034, People's Republic of China.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, Dalian, 116023, People's Republic of China.
authors.forename: Sufang
authors.initials: S
authors.lastname: Zhang
authors.affiliation: School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian, 116034, People's Republic of China.
authors.forename: Rongqian
authors.initials: R
authors.lastname: Zhu
authors.affiliation: Division of Biotechnology, Dalian Institute of Chemical Physics, Dalian, 116023, People's Republic of China.
authors.forename: Zongbao Kent
authors.initials: ZK
authors.lastname: Zhao
mesh_terms: D060054:Agrobacterium / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D055160:Lipomyces / Q000201:enzymology / Q000235:genetics*; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: Agrobacterium-mediated transformation; Biofuels; Genetic engineering; Lipomyces starkeyi; Promoters