定向进化策略提高豌豆苗来源尿苷二磷酸糖焦磷酸化酶底物耐受的研究

The functional groups with biological activity or leading compounds are glycosylated named the glycosylation in vitro is a common and effective way in drug molecule modification. Chemical synthesis would provide a feasible but inefficient route to glycosylation, however, the protection and the deprotection of hydroxy groups are unavailable and requires lots of organic solvents. So a more economic and cleaner route is enzymatic synthesis. The glycosylation mediated by glucosyltransferase is a specific, simple, high efficiency and green reaction, avoiding the protection and the deprotection of hydroxy groups. But UDP-sugars as activated form of monosaccharide and exclusive sugar donor of glucosyltransferase are extremely expensive, limiting its industrial application. A UDPsugar pyrophosphorylase gene had benn cloned from pea (Pisum sativum L.) sprouts genome (PsUSP) and the efficient synthesis of some UDP-sugar could be catalyzed by PsUSP with sugar-1-phosphate and UTP as substrates. Although the PsUSP owns a great many merits, such as, broad substrate spectrum, high specific activity, excellent thermal stability and high-efficiency expression in prokaryote, its substrate-tolerant is very poor. The industrialization application of PsUSP is limited. So the aim of this project is to improve substrate-tolerant of PsUSP considerably by directed evolution, research mechanisms between enzyme molecular structure and biocatalytic function based on protein characterization with multiple ways, and optimize fermentation and expression conditions of the ideal host in 100 L fermentor. All our work will be the theoretical bases of UDP-sugar industrialization.

体外糖基化是将具有生物活性的基团或先导化合物合成为糖基化衍生物,是药物结构改造和修饰的常用手段之一。化学法可以实现糖基化，但效率低，无法避免繁琐的糖基保护和去保护过程，大量有机溶剂的使用对环境压力很大；糖基转移酶催化的转糖基反应具有反应条件绿色温和，糖基化位点专一，避免糖基的保护和去保护等优势。但该酶的糖基供体必须使用极其昂贵的核苷酸糖，这使其无法真正应用于产业化。源于豌豆苗尿苷二磷酸糖焦磷酸化酶以UTP和一磷酸单糖为底物，高效合成UDP-单糖，该酶具有底物谱广，活力高，热稳定好，能在原核表达系统中高效表达等诸多优点。但我们研究发现该酶底物耐受度很低，这成为其产业化应用最大的障碍。因此本项目拟大幅提高该酶的底物耐受度为目标，采取定向进化技术改造该酶的基因，表征并探讨酶分子结构与催化功能的科学机制，选择最佳表达宿主在100L发酵罐中确定其最优发酵表达条件，为UDP糖产业化提供可靠的理论依据。

体外糖基化是将具有生物活性的基团或先导化合物合成为糖基化衍生物,是药物结构改造和修饰的常用手段之一。化学法可以实现糖基化，但效率低，无法避免繁琐的糖基保护和去保护过程，大量有机溶剂的使用对环境压力很大；糖基转移酶催化的转糖基反应具有反应条件绿色温和，糖基化位点专一，避免糖基的保护和去保护等优势。但该酶的糖基供体必须使用极其昂贵的核苷酸糖，这使其无法真正应用于产业化。源于豌豆苗尿苷二磷酸糖焦磷酸化酶以UTP和一磷酸单糖为底物，高效合成UDP-单糖，该酶具有底物谱广，活力高，热稳定好，能在真核表达系统中高效表达等诸多优点。但我们研究发现该酶底物耐受度很低，这成为其产业化应用最大的障碍。因此本项目拟大幅提高该酶的底物耐受度为目标，首先将PsUSP基因构建到pYES2质粒上，在酿酒酵母中表达。采取利用计算机对该酶半理性分子设计，预测酶活性相关氨基酸位点，采取代饱和突变，定向改造PsUSP基因；同时利用易错PCR技术，对PsUSP基因开展随机突变。兼顾筛选的效率和准确性，兼顾筛选的效率和准确性，建立双酶耦合紫外显色快速（一分钟）筛选模型，对突变文库进行高通量筛选，累计筛选1万个突变重组子。同时建立离子色谱复筛的方法，定量筛选有价值的正负突变重组子。通过对比正负突变重组子的氨基酸序列，我们确定H185和V284是PsUSP蛋白催化的关键氨基酸位点，对改位点的突变可以实现酶的底物耐受度。我们优化了酿酒酵母的发酵培养条件，利用连续补料策略在10L发酵罐中实现了高密度水平发酵。我们的工作结果为UDP-糖产业化提供可靠的理论依据。