Engineered aldolase for the fermentative production of L-fucose and other rare sugars.

用于发酵生产 L-岩藻糖和其他稀有糖的工程醛缩酶。

• 批准号: 8979634
• 负责人: Yuka Bannai
• 金额: $ 17.58万
• 依托单位: ZUCHEM, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8979634
• 关键词: 3-hydroxybutanal; Aldehyde-Lyases; Aldehydes; Algae; Amino Acids; Anti-Inflammatory Agents; Anti-inflammatory; Anticoagulants; Antioxidants; Antiviral Agents; Bacteria; Biological Assay; Biological Process; Blood Group Antigens; Bypass; Candidate Disease Gene; Cell Survival; Communities; Complement; Complex; Cosmetics; Deoxy Sugars; Development; Dihydroxyacetone; Dihydroxyacetone Phosphate; Engineering; Enzymes; Escherichia coli; Exhibits; Fermentation; Frequencies; Fructose; Fucose; Galactose; Genes; Genetic Engineering; Glucose; Glucosidase Inhibitor; Glycolipids; Goals; Growth; Guanosine Diphosphate; Guanosine Diphosphate Sugars; Hexoses; Human; Human Milk; Industry; Infant Health; Isomerase; Kilogram; L Forms; Libraries; Link; Liver diseases; Marines; Methods; Mutagenesis; Mutation; Nature; Oligosaccharides; Operative Surgical Procedures; Organism; P-Selectin; Pathogenesis; Pathway interactions; Pharmacologic Substance; Phase; Phosphoric Monoester Hydrolases; Physical condensation; Polymers; Polysaccharides; Procedures; Process; Production; Property; Proteins; Reaction; Rhamnose; Screening Result; Skin; Stomach; Sugar Phosphates; System; Therapeutic; Virulence Factors; Work; anti aging; blood lipid; chemotherapy; cost; expression vector; high throughput screening; human disease; in vivo; infant nutrition; inorganic phosphate; large scale production; macromolecule; meetings; metabolic engineering; monomer; mutant; novel; programs; protective effect; public health relevance; ribulose; sugar; tagatose

 DESCRIPTION (provided by applicant): The goal of this proposal is to develop a fermentative strategy for the large-scale production of L-fucose and other rare sugars. L-fucose (6-deoxy-L-galactose) is an important hexose deoxysugar found in a variety of organisms attached to an array of macromolecules. These L-fucose containing glycans exhibit a wide range of medicinal properties including supporting infant health (L-fucose containing human milk oligosaccharides); anticoagulant and antithrombotic, antivirus, antitumor, anticancer and immunomodulatory, anti-inflammatory, blood lipids reducing, antioxidant, activitiy against hepatopathy, uropathy and renalpathy, gastric protective effects and therapeutic potential in surgery (L-fucose containing polymers). The L-fucose monomer has therapeutic properties such as inhibiting virulence factors and is also an invaluable synthetic starting material for a wide range of molecules including human milk oligosaccharides, blood group antigens, E and P-selectin antagonists, and functionalized L-fucose derivatives. In addition to pharmaceutical relevance, L-fucose also possesses topical properties attractive to the cosmetic industry including anti-aging, wrinkle reducing and is safe for sensitive skin. Despite the impressive range of bioactivity discovered thus far, L-fucose remains prohibitively expensive and unavailable in the scale needed to support these applications. We feel a fermentative approach is needed to meet these large-scale requirements and to provide the glycoresearch community with this important building block needed to prepare scarce or unavailable glycans. A key aspect of our strategy is the production of an engineered L-fuculose-1-phosphate aldolase that no longer requires the phosphorylated donor substrate, dihydroxyacetone phosphate. Instead, the novel L-fuculose-1-phosphate aldolase (FucA) will catalyze the condensation between dihydroxyacetone and L-lactaldehyde to form L-fuculose, thereby bypassing the typical sugar-phosphate intermediate. Through metabolic engineering, we envision an E. coli system capable of integrating this engineered L-fuculose-1-phosphate for the specific synthesis of L-fucose. In Phase I we will demonstrate the feasibility of a fermentative L-fucose system by identifying and engineering a mutant FucA enzyme capable of condensing dihydroxyacetone and L-lactaldehyde to form L-fuculose for the ultimate production of L-fucose. In Phase II we will focus on the genetic and metabolic engineering of E. coli for the production and accumulation of the substrates needed for the engineered L-fuculose-1-phospahte aldolase. We propose a yield of 50-100 g/L production of L-fucose after integration of the engineered L-fuculose-1-phosphate aldolase and subsequent optimizations. In Phase III we will commercialize L-fucose as well as other rare sugars produced by the engineered L-fuculose-1-phosphate aldolase using various acceptor aldehyde substrates.

 描述（由适用提供）：该提案的目的是制定一种发酵策略，以大规模生产L-观糖和其他稀有糖。 L-凝血糖（6-脱氧-L-半乳糖）是在附着在一系列大分子上的各种生物体中发现的重要己糖脱氧。这些L-柠檬糖含有聚糖具有广泛的医疗特性，包括支持婴儿健康（含有人牛奶的L-凝血糖寡糖）；抗凝剂和抗血栓形成，抗病毒，抗肿瘤，抗癌和免疫调节，抗炎，血脂降低，抗氧化剂，抗氧化剂，抗肝病，尿道疗法和肾脏对，胃保护作用和胃保护作用和治疗效果（含有l-coldemers）（含有L-COLEREDER cONEREDE）。 L-糖粉单体具有诸如抑制病毒因子之类的治疗特性，并且也是多种分子的宝贵合成起始材料，包括人乳寡糖，血液组抗原，E和P-链纤维蛋白拮抗剂和功能化的L-凝血酶衍生物。除了药物相关性外，L-浓度还具有对化妆品行业有吸引力的局部特性，包括抗衰老，减少皱纹，对敏感皮肤安全。尽管到目前为止发现了令人印象深刻的生物活性范围，但在支持这些应用所需的规模上，L-upose仍然在昂贵且无法使用。我们认为需要一种发酵方法来满足这些大规模要求，并为Glycinesearch社区提供准备稀缺或无法获得的聚糖所需的重要组成部分。我们策略的一个关键方面是生产工程的L-核糖1-磷酸醛糖酶，不再需要磷酸化的供体底物二羟基丙酮磷酸盐。取而代之的是，新型的L-戊糖1-磷酸醛酶（FUCA）将催化二羟基乙酮与L-甲醛之间形成L-纤维素之间的凝结，从而绕过典型的糖磷酸盐中间体。通过代谢工程，我们设想了一个大肠杆菌系统，能够整合该工程的L-五核酸1-磷酸，以进行L-浓度的特定合成。在第一阶段，我们将通过鉴定和工程来证明发酵L凝血液系统的可行性，并在II期中凝结能够凝结的突变体Fuca酶，我们将重点介绍大肠杆菌的遗传和代谢工程，用于生产和积累，用于生产和积累工程的L-凝血素-1-磷酸化体质量质量。我们提出在整合工程的L-凝血酶1-磷酸醛糖酶和随后的优化后，提出了50-100 g/L的产生L-谷物糖的产量。在第三阶段，我们将使用各种受体醛醛底物商业化L-羟基糖和其他稀有糖。