Multi-gram enzymatic production of complex glycans by flow processes

翻译后摘要：Zymtronix提出，通过新的流程，显着提高生产效率的聚糖 基于化学酶催化，朝着更广泛的商业准入迈出重要步伐。第一 感兴趣的聚糖是人乳低聚糖（HMO），其在婴儿中具有很大的商业相关性。 营养、疾病预防和治疗。更广泛地应用复杂 合成聚糖在研究、临床前和商业应用中的最大优势是它们的可负担性。的目标 建议的研究是通过连续流降低大型和复杂的HMO（>5 DP）的生产成本 其中合成的每个步骤在模块中进行的工艺。Zymtronix的技术使（i）高 酶活性和稳定性，确保酶的重复使用，以及（ii）能够共固定多个 酶结合酶循环用于试剂的亚化学计量使用，从而带来显著的成本- 减少。在这个快速通道中，我们将生产第三大人乳低聚糖（HMO）， 母乳中的主要成分是商业上特别相关的聚糖模型。虽然一些简单的 益生菌HMO可以通过发酵有效地生产，用于婴儿配方（2 'FL，3DP），复合物和 支链HMO是难以获得的，并且通过化学酶催化以高成本少量生产。 Zymtronix的技术解决方案将显著改善聚糖的生产，从HMO开始， 从而显著节省成本。 快速通道I期的具体目标是筛选6种酶，并将它们以确定的顺序联合收割机组合， 产生唾液酸化的乳糖-N-新戊糖。(I)目的1：使用固定化酶将6种单独的酶固定在烧结珠上。 ZymTrap 3D技术。4种转移酶和2种活性糖产生酶，用于原位合成 GlcNAc-UDP、Gal-UDP和Neu 5Ac-CMP，（II）目标2：证明1 mg α-GlcNAc-UDP、Gal-UDP和Neu 5Ac-CMP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)目的3：生产100 mg α-2，3-唾液酸乳糖-N- 使用流动池测定新戊糖。 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-乳糖-N-新戊糖的生产 目标1 (I)目的1：开发HMO岩藻糖基化和分支模块，以达到100 mg 生产6种双触角乳糖-N-新己糖和七糖。分支的模块化， 三个可能的“官能化”模块，最后的延伸步骤允许产生6个双触角 健康维护组织(II)目的2：开发用于多克规模HMO生产的具有原位试剂再循环的流动池。 将评估生产和成本指标。(III)目标3：按比例扩大10克和100克的工程和生产 然后加入100 g岩藻糖基-和唾液酸基-乳糖-N-新戊糖。工作将包括内部酶生产和 HMO的HPLC纯化。

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.