Multi-gram enzymatic production of complex glycans by flow processes

通过流动工艺多克酶法生产复杂聚糖

• 批准号: 10642651
• 负责人: Joseph Tang
• 金额: $ 113.79万
• 依托单位: ZYMTRONIX CATALYTIC SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-03 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642651
• 关键词: 3D Print; Capital; Carbohydrates; Catalysis; Cell surface; Cells; Complex; Cost Measures; Cost Savings; Development; Engineering; Ensure; Enzyme Stability; Enzymes; Expenditure; Extracellular Matrix; Fermentation; Food Additives; Goals; High Pressure Liquid Chromatography; Human Milk; Immobilization; Immobilized Enzymes; Immune; In Situ; Individual; Infant; Infant formula; Kinetics; Length; Magnetism; Mediator; Methods; Modeling; Natural regeneration; Nucleotides; Oligosaccharides; Phase; Polysaccharides; Preventive treatment; Probiotics; Process; Production; Reaction; Reagent; Recycling; Research; Role; Series; System; Technology; Therapeutic; Transferase; Uridine Diphosphate Galactose; Vertebral column; Work; carbohydrate structure; chemical synthesis; cofactor; cognitive development; commercial application; commercialization; cost; design; disorder prevention; enzyme activity; glycosylation; improved; infant nutrition; interest; manufacturing scale-up; novel; pathogen; pre-clinical; public health relevance; research clinical testing; scale up; screening; sialylation; stem; sugar; tripolyphosphate

Abstract: Zymtronix proposes to significantly improve production efficiency of glycans via new flow processes based on chemoenzymatic catalysis to take significant steps towards broader commercial access. The first glycans of interest are human milk oligosaccharides (HMOs), which have large commercial relevance in infant nutrition, disease prevention and therapeutics. The primary hurdle towards the broader application of complex synthetic glycans in research, preclinical and commercial applications is their affordability. The goal of the proposed research is to lower the production costs of large and complex HMOs (>5 DP) by continuous flow processes where each step of synthesis is conducted in modules. Zymtronix's technology enables (i) high enzyme activity and stability ensuring the reuse of enzyme and (ii) enables the co-immobilization of multiple enzymes to incorporate enzymatic recycling for sub-stoichiometric use of reagents to impart significant cost- reductions. For this fast-track, we will produce large human milk oligosaccharides (HMOs), the third largest component of breast milk that are particularly commercially relevant glycan models. While some simple probiotic HMOs can be effectively produced via fermentation for infant formula (2'FL, 3 DP), complex and branched HMOs are elusive and have been marginally produced at high cost via chemoenzymatic catalysis. Zymtronix's technology solution will significantly improve production of glycans, starting with HMOs, while imparting significant cost savings. Fast-track Phase I specific aims are to immobilize 6 enzymes and to combine them in a defined sequence to generate sialylated lacto-N-neopentaoses. (I) Aim 1: Immobilize 6 individual enzymes on sintered beads using ZymTrap3D technology. 4 transferases and 2 activated-sugar-producing enzymes for in-situ synthesis of GlcNAc-UDP, Gal-UDP and Neu5Ac-CMP, (II) Aim 2: Demonstrate sequential, modular synthesis of 1 mg of α- 2,3- and α-2,6-sialyl lacto-N-pentaoses on beads. (III) Aim 3: Produce 100 mg of α-2,3-sialyl lacto-N- neopentaose using a flow cell. Fast-track Phase II aims are to scale up production of three linear fucosyl- and sialyl-lacto-N-neopentaoses from Aim1. (I) Aim 1: Development of HMO fucosylation and branching modules towards the 100 mg production of 6 biantennary lacto-N-neohexaoses and heptaoses. The modularity of branching followed by three possible `functionalization' modules, and a final elongation step allows the production of 6 biantennary HMOs. (II) Aim 2: Development of flow cells with in situ reagent recycling for multi-gram scale HMO production. Production and cost metrics will be assessed. (III) Aim 3: Scaling up engineering and production of 10 g and then 100 g of fucosyl- and sialyl-lacto-N-neopentaoses. Work will include in-house enzyme production and HPLC purification of HMOs.

摘要：Zymtronix 提出通过新的流程显着提高聚糖的生产效率 基于化学酶催化，为更广泛的商业准入采取重大步骤。第一个 感兴趣的聚糖是母乳寡糖 (HMO)，它在婴儿中具有很大的商业相关性 营养、疾病预防和治疗。复合体更广泛应用的主要障碍 合成聚糖在研究、临床前和商业应用中的优势在于其经济性。的目标 拟议的研究是通过连续流动降低大型复杂 HMO（> 5 DP）的生产成本 每个合成步骤都在模块中进行的过程。 Zymtronix 的技术能够 (i) 高 酶活性和稳定性确保酶的重复使用，并且（ii）能够共同固定多种酶 酶将酶回收用于试剂的亚化学计量使用，从而显着降低成本 减少。为了这个快速通道，我们将生产大母乳低聚糖 (HMO)，这是第三大母乳寡糖 母乳的成分，是特别具有商业相关性的聚糖模型。虽然有些简单 益生菌 HMO 可以通过婴儿配方奶粉（2'FL、3 DP）、复合配方奶粉和复合配方奶粉的发酵来有效生产。 支链 HMO 难以捉摸，并且通过化学酶催化以高成本少量生产。 Zymtronix 的技术解决方案将从 HMO 开始显着提高聚糖的产量，同时 显着节省成本。 快速通道第一阶段的具体目标是固定 6 种酶并将它们按确定的顺序组合起来 产生唾液酸化的乳-N-新五糖。 (I) 目标 1：使用以下方法将 6 种单独的酶固定在烧结珠上 ZymTrap3D 技术。 4 种转移酶和 2 种活性糖生成酶，用于原位合成 GlcNAc-UDP、Gal-UDP 和 Neu5Ac-CMP，(II) 目标 2：演示 1 mg α- 的顺序、模块化合成 珠子上的 2,3- 和 α-2,6-唾液酸乳糖-N-五糖。 (III)目标3：生产100毫克α-2,3-唾液酸乳-N- 使用流动池的新五糖。 快速通道第二阶段的目标是扩大三种线性岩藻糖基-和唾液酸-乳-N-新五糖的生产 来自目标1。 (I) 目标 1：开发 HMO 岩藻糖基化和分支模块，直至 100 mg 产生6种双触角乳-N-新六糖和七糖。分支的模块化 三个可能的“功能化”模块，最后的延伸步骤允许生产 6 个双触角 HMO。 (II) 目标 2：开发具有原位试剂回收功能的流动槽，用于多克规模的 HMO 生产。 将评估生产和成本指标。 (III) 目标 3：扩大 10 克和 10 克的设计和生产 然后100克岩藻糖基-和唾液酸-乳-N-新五糖。工作将包括内部酶生产和 HMO 的 HPLC 纯化。