期刊
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
卷 183, 期 3, 页码 867-875出版社
HUMANA PRESS INC
DOI: 10.1007/s12010-017-2469-5
关键词
Lipomyces starkeyi; Agrobacterium-mediated transformation; Promoters; Genetic engineering; Biofuels
资金
- National Natural Science Foundation of China [3150100585]
- China Postdoctoral Science Foundation [2016 M591419]
- Educational Commission of Liaoning Province of China [2016 018]
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.
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