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提议通过新的流动过程显着提高聚糖的生产效率 基于化学酶催化，采取了重大步骤，以朝着更广泛的商业通道迈进。第一个 感兴趣的聚糖是人乳寡糖（HMO），它们在婴儿中具有很大的商业意义 营养，预防疾病和治疗。更广泛应用复杂的主要障碍 在研究中，临床前和商业应用中的合成聚糖是它们的负担能力。目标的目标 拟议的研究是通过连续流量降低大而复杂的HMO（> 5 dp）的生产成本 在模块中进行每个合成步骤的过程。 Zymtronix的技术使（i）高 酶的活性和稳定性确保酶的再利用，（ii）可以使多种 酶纳入酶促回收，以用于对试剂的亚化学计量使用，以赋予大量成本 - 减少。对于此快速轨道，我们将生产大型的人乳寡糖（HMOS），第三大 母乳的成分特别是商业相关的聚糖模型。虽然很简单 益生菌HMO可以通过针对婴儿配方（2'fl，3 dp），复合物和复合物和 分支的HMO难以捉摸，并且通过化学酶催化以高成本生产。 Zymtronix的技术解决方案将从HMOS开始显着改善聚糖的产量，而 节省大量成本。 快速轨道I期的特定目的是固定6个酶，并将其组合为定义的序列 产生溶解的乳酸 - n-神经。 （i）目标1：使用6种单独的酶固定在烧结的珠子上 Zymtrap3D技术。 4种转移和2种激活的产生酶，用于原位合成 glcnac-udp，gal-udp和neu5ac-cmp，（ii）目标2：证明1 mgα-的顺序，模块化合成 珠子上的2,3-和α-2,6-盐乳酸-N-戊二醇。 （iii）目标3：产生100毫克的α-2,3-盐lacto-n- 使用流动池的Neopentaose。 Fast-Track II期的目标是扩大生产三个线性烟素和siAllyl-lacto-n-neopentaiss 来自AIM1。 （i）目标1：开发HMO岩藻糖基化和向100 mg的分支模块 产生6个Biantennary乳酸乳糖果和七肠。分支的模块化，然后 三个可能的“功能化”模块，最后一个伸长步骤允许生产6个Biantennary HMOS。 （ii）目标2：具有原位试剂回收的流动细胞的开发，用于多克量表HMO生产。 将评估生产和成本指标。 （iii）目标3：扩展工程和生产10 g，并且 然后100 g的岩藻糖基和siAllyl-lacto-n-神经毒素。工作将包括内部酶生产和 HMO的HPLC纯化。