Engineering Methylotrophic Yeast for in vivo Production of Deoxysugars from DHAP

该提案的目标是开发一种发酵系统，以生产各种稀有和 使用混杂的DHAP依赖性醛缩酶的脱氧糖。形成碳-碳键的醛缩酶反应 对精细化学品、中间体和生物活性化学品的制备具有重要意义。的 负责这种C-C键形成的醛缩酶催化不对称羟醛缩合 其由醛和酮的偶联组成。磷酸二羟丙酮（DHAP）依赖性 醛缩酶是有吸引力的，因为存在四种具有不同立体特异性的不同类型。而 受体的底物特异性是灵活的，它们需要DHAP作为供体。不幸的是，DHAP是 非常不稳定和昂贵，使其不适用于工业用途。 我们建议开发一种新的方法来生产DHAP使用代谢修饰的菌株， 多形汉逊酵母，一种甲基营养型酵母。H. polymorpha具有独特的新陈代谢能力，能够创造 C6和DHAP仅来自包括甲醇的混合碳源。突变体积累了 DHAP的前体，高产率的二羟基丙酮已被鉴定，并将用于增强DHAP 生产 在第一阶段，我们将证明使用细胞内DHAP体内转化为 产生模型化合物L-岩藻糖。该反应由岩藻糖-1-磷酸醛缩酶（FucA）催化。 具体而言，我们将证明使用H.多形藻无细胞提取物， 工程师H。为提高DHAP的生产，克隆和表达岩藻糖-1-磷酸醛缩酶 进入H。多形藻，并最终证明L-岩藻糖在体内的生产。 在第二阶段，我们将进一步优化系统，敲除将DHAP转化为GAP的基因， 引入岩藻糖异构酶生产L-岩藻糖。然后，我们将克隆其他主要的依赖于DHAP的 醛缩酶对醛类具有更广泛的特异性，并能产生数十种脱氧糖， 糖和碳水化合物使用不同的醛受体。在第三阶段，该技术将 通过授权生产DHAP和醛缩酶的菌株并使用 用于药物发现候选物的生成和药物中间体的制备的系统。

The goal of this proposal is to develop a fermentation system to produce a variety of rare and deoxysugars using promiscuous DHAP-dependent aldolases. Carbon-carbon bond forming aldolase reactions are of great relevance to the preparation of fine chemicals, intermediates and bioactive chemicals. The aldolase enzymes responsible for this kind of C-C bond formation catalyze an asymmetric aldol condensation which consists of coupling an aldehyde and a ketone. Dihydroxyacetone phosphate (DHAP) dependent aldolases are attractive because there are four different types available with different stereo-specificities. While substrate specificity for the acceptor is flexible, they require DHAP to be the donor. Unfortunately, DHAP is very unstable and expensive, making it impractical for industrial use. We propose to develop a novel approach to produce DHAP using a metabolically modified strain of Hansenula polymorpha, a methylotrophic yeast. H. polymorpha has a unique metabolism with the ability create C6 and DHAP solely from mixed carbon sources including methanol. Mutants that that accumulate the precursor to DHAP, dihydroxyacetone at high yields have been identified and will be used to enhance DHAP production. In Phase I we will demonstrate the feasibility of using an in vivo conversion of intracellular DHAP to produce the model compound L-fuculose. This reaction is catalyzed by Fuculose-1-phosphate aldolase (FucA). Specifically, we will demonstrate the ability to produce L-fuculose using H. polymorpha cell-free extracts, engineer H. polymorpha for improved DHAP production, clone and express the fuculose-1-phosphate aldolase into H. polymorpha, and finally demonstrate the production of L-fuculose in vivo. In Phase II we will optimize the system further, knock-out genes that convert DHAP to GAP, and introduce fuculose isomerase for production of L-fucose. We will then clone the other main DHAP-dependent aldolases with broader specificity to aldehydes and demonstrate production of dozens of deoxysugars, rare sugars and carbohydrates using different aldehyde acceptors. In Phase III the technology will be commercialized by licensing DHAP and aldolase producing strains and carrying out contract R&D using the system for the generation of drug discovery candidates and the preparative of pharmaceutical intermediates.