熊蜂生假丝酵母启动子的筛选及强度分析

Screening and intensity analysis of promoters in Starmerella bombicola

摘要:

[背景] 熊蜂生假丝酵母（Starmerella bombicola）作为一种非常规假丝酵母菌株，因其具备高产槐糖脂生物表面活性剂的能力而受到广泛关注。然而，由于自身的表达系统并不完善，限制了该菌株的代谢工程改造。[目的] 克隆、筛选及鉴定新的系列内源启动子表达元件。[方法] 通过对比分析熊蜂生假丝酵母全基因组及9种功能已知目的基因信息，并结合启动子预测网站，筛选获得系列启动子候选序列，以SbGFP（密码子优化后的酵母增强型绿色荧光蛋白）为报告基因进行整合表达，通过绿色荧光蛋白强度及转录水平分析鉴定启动子强度。[结果] 在分别以葡萄糖和油酸作为唯一碳源的条件下，启动子P_TEF1和P_GPD在不同碳源培养条件下均显示出较高的转录水平。启动子P_CYP52M1、P_UGTA1、P_UGTB1及P_MOB在以油酸为唯一碳源时具有弱转录活性，而在以葡萄糖为唯一碳源时则未检测到它们具有转录活性，推测它们是油酸诱导型启动子。进一步利用实时荧光定量PCR （RT-qPCR）对SbGFP进行转录水平分析，检测结果与绿色荧光表达水平一致。[结论] 获得了系列熊蜂生假丝酵母内源性启动子，进一步丰富了该菌株的表达元件，为菌株的代谢工程改造及基因的表达与调控奠定了理论基础。

关键词:熊蜂生假丝酵母;启动子;密码子优化;绿色荧光蛋白;基因表达与调控

Abstract:

[Background] Starmerella bombicola, as an unconventional yeast strain, has attracted wide attention owing to its ability to produce sophorolipids biosurfactant. However, its expression system is not well defined, which limits the development of metabolic engineering. [Objective] A panel of endogenous promoters were cloned and characterized from S. bombicola. [Methods] In this study, through the comparative analysis of the whole genome of S. bombicola and 9 target genes, combined with the promoter prediction website, a series of promoter candidate sequences were screened and obtained, and SbGFP (codon-optimized yeast enhanced green fluorescent protein for S. bombicola), as the reporter gene, was integrated and expressed in S. bombicola. The promoter strength was identified by analyzing the intensity of green fluorescent protein and its transcriptional levels. [Results] When glucose and colleseed oil were respectively used as the sole carbon source, the promoters P_TEF1 and P_GPD showed higher transcription levels under both conditions. The promoters P_CYP52M1, P_UGTA1, P_UGTB1, and P_MOB had weak transcriptional activity when colleseed oil was used as the sole carbon source, but no transcriptional activity was detected when cultured with glucose. It was speculated that they were colleseed oil-inducible promoters. Transcriptional level of SbGFP was further analyzed by real-time fluorescence quantitative PCR (RT-qPCR), the result was consistent with the expression level of SbGFP.[Conclusion] A panel of different promoters were screened and characterized, which would further enrich its expression elements and lay a theoretical foundation for the metabolic engineering for S. bombicola.

Key words:Starmerella bombicola;promoter;codon optimization;SbGFP;gene expression and regulation