基于DHAP依赖型醛缩酶的酿酒酵母稀有糖合成平台的构建及优化调控

Most rare sugars are currently still expensive, and their synthetic routes are limited and the yields are low, which restrict their functional study and utilization. In addition, bacteria presently applied in rare sugar synthesis are non-generally regarded as safe (GRAS) microorganisms, which may cause security problems. Saccharmoyces cerevisiae could be potentially one of the most suitable microorganism for rare sugar synthesis due to its clear genetic backgrounds, good productive characters, secure fermentation products and incapability of metabolizing rare sugars. Among aldolase family, dihydroxyacetone phosphate (DHAP)-dependent aldolases are particularly attractive for the synthesis of many kinds of carbohydrates. However, for this group of aldolases, the main drawback is that the substrate DHAP is a rather expensive and unstable compound, which limits its use in large-scale preparation. This proposal is based on our previous study on the synthesis of rare sugars with DHAP-dependent aldolase in vitro. Firstly, DHAP is generated in S.cerevisiae via glycolytic pathway with glucose as "green" carbon source and accumulated in the cell through metabolic engineering. Meanwhile, the co-expression of DHAP-dependent aldolase and phosphatase is optimized and regulated in this engineered S.cerevisiae strain, and the corresponding aldelyde is supplemented into the medium. As a result, the desired products could come out from the cell membrane making the purification process easier and the phosphate group could be regenerated in the cell simultaneously. This research will provide theory and method for the production of rare sugars in metabolized engineered S.cerevisiae.

目前大多数稀有糖由于价格高、合成路线有限、产率低等问题限制了其功能研究和开发利用。此外当前应用于稀有糖合成中的细菌为非安全性微生物, 这可能存在隐患。酿酒酵母具有遗传背景清楚、生产性状良好、发酵产品安全、不能代谢稀有糖等优点，成为潜在的合成稀有糖的最适微生物之一。在醛缩酶家族中，磷酸二羟基丙酮（DHAP）依赖型醛缩酶广泛应用于各种糖类化合物的合成，但DHAP因价格昂贵且不稳定，限制了该酶类大规模的合成应用。本项目拟在前期利用DHAP依赖型醛缩酶在体外进行稀有糖合成的基础上，以葡萄糖为"绿色"碳源，利用酿酒酵母的糖酵解途径并通过代谢工程的方法在胞内积累DHAP，同时在胞内优化调控DHAP依赖性醛缩酶和磷酸酶的共表达并在培养基中添加相应的醛，生成的产物可以分泌到胞外便于分离和纯化，同时磷酸基团在胞内再生。该研究将为代谢工程改造酿酒酵母生产稀有糖提供理论和方法依据。

目前大多数稀有糖由于价格高、合成路线有限、产率低等问题限制了其功能研究和开发利用。在醛缩酶家族中，磷酸二羟基丙酮（DHAP）依赖型醛缩酶广泛应用于各种糖类化合物的合成，但DHAP因价格昂贵且不稳定，限制了该酶类大规模的合成应用。为了优化前期建立的稀有糖“一釜四酶法”合成，肺炎链球菌来源的磷酸甘油氧化酶(GPOS.pne)进行了纯化和表征。利用(GPOS.pne)和DHAP依赖型醛缩酶采用“一釜四酶法”高效合成了包括稀有糖在内的多种酮糖。近些年来，嗜热菌来源的酶类在生物合成方面显示了巨大的潜力，利用了Thermotoga maritima MSB8 来源的 RhaDT.mari 醛缩酶进行了多种稀有糖的高效合成，这是首次利用嗜热的RhaD醛缩酶进行稀有糖的合成。在利用DHAP依赖型醛缩酶在体外合成稀有糖的基础上，将体外催化反应成功在大肠杆菌工程菌中实现。和体外催化相比，解决了如下问题：1) 通过利用微生物的代谢途径解决了DHAP的来源问题；2) 避免了大量纯化醛缩酶和磷酸酶；3) 在胞内实现了羟醛缩合产物的脱磷酸化，同时磷酸基团在胞内再生；4）生成的产物可以分泌到胞外便于分离和纯化。重要的是，该合成平台能够以多种醛分子作为受体，能够以克级合成多种稀有糖及其衍生物。此外，基于酿酒酵母孢子所特有的孢子壁结构，利用孢子固定化酶建立了从葡萄糖高效合成稀有糖D-阿洛酮糖的两步法。如上研究将为利用微生物及相关酶类生产稀有糖提供理论和方法依据。