Engineering Methylotrophic Yeast for in vivo Production of Deoxysugars from DHAP

工程化甲基营养酵母用于体内从 DHAP 生产脱氧糖

• 批准号: 10082147
• 负责人: Leila Aminova
• 金额: $ 19.31万
• 依托单位: MIDWEST BIOPROCESSING CENTER, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082147
• 关键词: 3-hydroxybutanal; Aldehyde-Lyases; Aldehydes; Carbohydrates; Carbon; Cell Communication; Cell-Free System; Cells; Chemicals; Contracts; Coupling; Deoxy Sugars; Development; Dihydroxyacetone; Dihydroxyacetone Phosphate; Disease; Engineering; Enzymes; Fermentation; Formaldehyde; Fucose; Generations; Genes; Glycerol; Glycerone kinase; Goals; Industrialization; Inflammation; Isomerase; Ketones; Knock-out; Licensing; Metabolic; Metabolism; Methanol; Modeling; Modification; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physical condensation; Pichia; Plasmids; Play; Preparation; Process; Production; Reaction; Role; Source; Specificity; Substrate Specificity; System; Technology; Testing; Therapeutic; Xylose; Yeasts; drug discovery; enzyme activity; flexibility; improved; in vivo; inhibitor/antagonist; inorganic phosphate; mutant; novel strategies; overexpression; promoter; research and development; sugar

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.

这项提案的目标是开发一种发酵系统，以生产各种稀有和 脱氧糖使用DHAP依赖的杂交醛缩酶。形成缩醛酶反应的碳-碳键 与精细化学品、中间体和生物活性化学品的制备密切相关。这个 负责这种C-C键形成的醛缩酶催化不对称的羟醛缩合 它由醛和酮偶联而成。磷酸二羟丙酮(DHAP)依赖 醛缩酶之所以有吸引力，是因为有四种不同的类型可供选择，它们具有不同的立体声特性。而当 底物专一性对于受体来说是灵活的，它们需要DHAP作为供体。不幸的是，达普 非常不稳定和昂贵，使其不切实际的工业用途。 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 多态汉逊酵母，一种富含甲醇的酵母。多形藻具有独特的新陈代谢能力，具有创造 C6和DHAP仅来自包括甲醇在内的混合碳源。这些突变体积累了 DHAP的前体二羟基丙酮已被鉴定为高产率，并将用于增强DHAP 制作。 在第一阶段，我们将演示使用细胞内DHAP体内转化为 生产模型化合物L-岩藻糖。该反应是由1-磷酸果糖缩醛酶(FucA)催化的。 具体地说，我们将展示使用多形杆菌无细胞提取物生产L岩藻糖的能力， 工程菌改良DHAP生产，克隆和表达岩藻糖-1-磷酸缩醛酶 转化为多形藻，最后演示了L-岩藻糖的体内生产。 在第二阶段，我们将进一步优化系统，将DHAP转化为GAP的敲除基因，以及 引进岩藻糖异构酶生产L岩藻糖。然后我们将克隆另一个主要依赖于DHAP的 对醛具有更广泛专一性的醛缩酶，并证明能产生数十种脱氧糖，稀有 糖和碳水化合物使用不同的醛受体。在第三阶段，这项技术将 通过许可DHAP和醛缩酶生产菌株并使用 用于生成候选药物和制备药物中间体的系统。