Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi
Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi
复制标题
农杆菌介导的转化方法的开发和产油酵母斯氏油脂酵母中两种外源组成型启动子的评估
DOI：
10.1007/s12010-017-2469-5
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发表时间：
2017-11-01
影响因子：
3
通讯作者：
Zhao, Zongbao Kent
中科院分区：
文献类型：
--
作者：
Lin, Xinping;Liu, Sasa;Zhao, Zongbao Kent
斯氏油脂酵母是一种具有重要生物工程意义的油脂酵母，其细胞生物量的60%以上可转化为三酰甘油。利用乳酸菌直接生产长链脂肪酸甲酯、烷烃等TAG衍生物具有广阔的应用前景。starkeyi。然而，这种产油酵母的遗传修饰技术缺乏，因此，需要进一步的研究来开发遗传工具和功能元件。本研究利用酵母Rhodosporidium toruloides的两个外源启动子（pGPD和pPGK），建立了一种简单的农杆菌介导的L. starkeyi。在含抗生素的平板上获得潮霉素抗性转化子。有丝分裂稳定性试验、PCR基因型验证和蛋白表达确认均证明了该方法的成功。此外，还在潮霉素梯度平板上在表型水平上以及通过实时定量PCR在转录水平上评估了这两个启动子的强度。PGK启动子启动潮霉素抗性基因表达的能力是GPD启动子的2.2倍。本研究为产油酵母L.用于构建生产先进生物燃料的上级菌株。
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.