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Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi

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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.

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Acknowledgments

We are indebted to professors Alexander Idnurm and Giuseppe Ianiri for their kind suggestions on AMT, to professors Xiaofeng Dai and Tianhong Wang for providing us Agrobacterium tumefaciens AGL1, to professor Lianghui Ji for providing us pTHR1.

This study was funded by the National Natural Science Foundation of China (3150100585), the China Postdoctoral Science Foundation (2016 M591419), and the Educational Commission of Liaoning Province of China (2016 018).

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Authors and Affiliations

  1. School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood, Dalian, 116034, People’s Republic of China

    Xinping Lin, Sasa Liu, Ruiqi Bao, Ning Gao & Rongqian Zhu

  2. Division of Biotechnology, Dalian Institute of Chemical Physics, Dalian, 116023, People’s Republic of China

    Sufang Zhang & Zongbao Kent Zhao

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  1. Xinping Lin
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  2. Sasa Liu
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  3. Ruiqi Bao
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  4. Ning Gao
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  5. Sufang Zhang
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  7. Zongbao Kent Zhao
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Correspondence to Xinping Lin.

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Lin, X., Liu, S., Bao, R. et al. Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi . Appl Biochem Biotechnol 183, 867–875 (2017). https://doi.org/10.1007/s12010-017-2469-5

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  • DOI: https://doi.org/10.1007/s12010-017-2469-5

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