酿酒酵母细胞工厂半乳糖调控的重构优化研究

Construction of highly productive microbial cell factories needs to avoid the conflict between cell growth and product accumulation. However, the leakage expression of inducible system would aggravate the conflict and reduce product yield. It is reported that the application of promoter engineering enables promoters to obtain desired regulatory characteristics, which allows precise control of heterologous gene expression. Using the galactose regulation system in Saccharomyces cerevisiae cell factory as an example, the applicant proposes to fine-tune the key transcription factors of galactose regulation by using artificial promoter library, which is constructed by screening in vivo promoters via genome-wide transcriptional profiling analysis and applying hybrid promoter engineering, aiming to achieve precise control of gene expression and ultimately increase productivity. This project intends to mainly focus: 1) using GAL4 as the target, screening for GAL4 regulatory elements that intensify glucose repression to precisely control the timing expression of GAL4; 2) using GAL80 as the target, screening for GAL80 regulatory elements that reduce galactose dependency to precisely control the timing expression of GAL80; 3) reconstructing the galactose regulation in yeast cell factory by using the above regulatory elements to improve the yield of target product. This study provides a new insight and solution for reducing the leakage of inducible expression system and realizing the efficient production of natural products using yeast cell factories with precise galactose regulation.

构建高生产力的微生物细胞工厂需要避免细胞生长和产物积累的冲突，然而诱导系统的泄漏表达会加重冲突、降低产物得率。研究表明利用启动子工程能使启动子获得期望调控特征从而精确控制异源基因表达。申请者以酿酒酵母细胞工厂的半乳糖调控系统为例，设想通过全基因组转录谱分析筛选体内启动子并结合杂合启动子工程构建人工启动子文库，对半乳糖调控系统中的关键转录因子进行精细改造，从而实现基因表达的精确控制，最终提高生产力。主要研究：1）以GAL4为靶点，筛选强化葡萄糖抑制的GAL4调控元件，精确控制GAL4时序表达量；2）以GAL80为靶点，筛选弱化半乳糖依赖的GAL80调控元件，精确控制GAL80时序表达量；3）应用上述调控元件库重构酵母细胞工厂的半乳糖调控体系，提高产物得率。该研究为降低诱导表达系统泄漏，实现利用精准半乳糖调控的酵母细胞工厂高效生产天然产物提供了新的思路和解决办法。

构建高生产力的微生物细胞工厂需要避免细胞生长和产物积累的冲突，然而诱导系统的泄漏表达会加重冲突、降低产物得率。本研究针对“如何优化重构半乳糖调控系统实现对基因表达的精确控制”的关键科学问题，以酿酒酵母细胞工厂的半乳糖调控系统为研究对象，按照“全基因组转录谱分析筛选时序启动子到目标启动子文库构建和差异表型筛选”的模式对半乳糖调控系统中的关键转录因子进行精细改造，系统完成了：1）以GAL4为靶点，筛选获得三个强化葡萄糖抑制的GAL4调控元件（PADY2、PYAT1、PYOR019W），实现精确控制GAL4时序表达量；2）以GAL80为靶点，筛选了弱化半乳糖依赖的GAL80调控元件尝试精确控制GAL80时序表达量；3）应用上述调控元件重构酵母细胞工厂的半乳糖调控体系，首次发现YAT1启动子是强化葡萄糖抑制的高效GAL4调控元件，它能够有效抑制诱导系统的表达泄漏，应用该元件将番茄红素和虾青素细胞工厂的产量分别提高了14.9%和29.5%，实现了通过精确控制基因表达显著提升细胞工厂生产力。本研究为降低诱导表达系统泄漏，实现利用精准半乳糖调控的酵母细胞工厂高效生产天然产物提供了新的思路和解决办法。