Bioengineering a synthetic enzyme for the production of authentic human milk oligosaccharides.

人们普遍认为牛奶对我们有好处。母乳中含有蛋白质、碳水化合物、脂肪、矿物质和维生素等营养素，有助于骨骼、牙齿、皮肤的健康，有助于肌肉的生长，有助于预防骨质疏松症、心血管疾病甚至癌症。直到世纪，奶妈一直是母乳喂养唯一安全的替代品。这是因为母乳的组成与牛奶不同;蛋白质的组成不同，碳水化合物的结构多样性也明显不同。但并非所有女性都能够母乳喂养，由于情绪和/或身体状况，如产后手术，疲惫或营养不良。然而，母乳仍然是婴儿营养的黄金标准，制造商的目标是使婴儿配方奶粉尽可能接近母乳。牛奶中的一组碳水化合物称为低聚半乳糖（GOS），是婴儿配方奶粉中的主要添加剂，有助于弥合一些差异。GOS由糖分子（称为半乳糖）的短链组成，并通过称为乳糖酶或β-半乳糖苷酶的酶从另一种乳糖（乳糖）合成。这些可溶的、不易消化的碳水化合物作为益生元添加，试图刺激肠道中“健康细菌”的生长，并复制人类母乳的有益特性。在婴儿营养方面，GOS已被证明可降低特应性皮炎的风险，并可能参与预防过敏和感染，并增加钙、铁、镁和锌等重要矿物质的吸收。然而，益生元GOS的组成与人乳低聚糖（HMO）中发现的糖相似，但不完全相同;半乳糖糖不会天然存在于母乳中。相比之下，岩藻糖基化糖，即2 '-岩藻糖基乳糖，在分泌型女性的乳汁中大量存在（分泌型女性将其血型抗原分泌到体液和组织中，有助于免疫）。在婴儿配方奶粉中使用GOS和低聚果糖（基于果糖的糖分子的短链），两者都不模拟HMO。与此相关的是，一些β-半乳糖苷酶可以利用岩藻糖产生岩藻糖基化低聚糖（FucOS;基于岩藻糖的糖分子的短链）;母乳的重要成分。然而，β-半乳糖苷酶能够产生FucOS的机制以及这种功能如何与其结构相关尚不清楚。我们最近的工作是使用β-半乳糖苷酶（称为BbgIII）优化GOS产品，为直接影响酶功能和后续产物形成的特定修饰提供了重要见解。我们开发了新的BbgIII酶，其产生期望的GOS产物，用于改善益生元补充剂Bimuno（一种GOS）的生产。在这项工作的过程中，我们还发现，通过进行特定的修饰，该酶的两种变体能够合成20种不同的FucOS产物，这些产物可以更接近地模拟HMOs。可以说是更真实的HMO，其对用于婴儿配方食品和医疗营养的肠道微生物组表现出有益的影响。我们的方法是独特的，因为目前生产HMO的方法成本高昂，而且生产的碳水化合物缺乏结构多样性，这为开发更具成本效益和结构多样性的正宗HMO提供了强大的行业和社会驱动力。

It is generally accepted that milk is good for us. It contains nutrients, such as protein, carbohydrates, fat, minerals and vitamins that are known to promote healthy bones, teeth, skin and help build muscle and can help in the prevention of some diseases, such as osteoporosis, cardiovascular disease and even cancer.Until the turn of the century, a wet nurse was the only safe alternative to breastfeeding. This is because breast milk composition is different from cow's milk; the composition of proteins is different and the structural diversity of the carbohydrates is distinctly different. But not all women are able to breastfeed, due to emotional and/or physical conditions such as postpartum surgery, exhaustion or poor nutrition. And yet, breast milk remains the gold standard for infant nutrition and manufacturers aim to make infant formulas as close to human milk as possible. One group of carbohydrates in cow's milk called galactooligosaccharides (GOS) is the primary additive in infant milk formulae to help bridge some of the difference. GOS consist of short chains of sugar molecules (called galactose), and are synthesised from another milk sugar (lactose) by enzymes called lactase or beta-galactosidase. These soluble, non-digestible carbohydrates are added as a prebiotic in an attempt to stimulate growth of 'healthy bacteria' in the gut and replicate the beneficial properties of human breast milk. In infant nutrition, GOS have been shown to decrease the risks of atopic dermatitis and may be involved in allergy and infection prevention and to increase the absorption of important minerals such as calcium, iron, magnesium and zinc. However, prebiotic GOS composition is similar, but not identical, to the sugars found in human milk oligosaccharides (HMOs); galactose sugars do not naturally occur in breast milk. In contrast, fucosylated sugars, namely 2'-fucosyllactose, are richly found in the milk of Secretor women (who secrete their blood type antigens into body fluids and tissues, contributing to immunity). The use of GOS and fructooligosaccharides (short chains of fructose-based sugar molecules) in infant formulas, neither of which mimic HMOs.Relevant to this proposal, some beta-galactosidases can utilise fucose sugars to yield fucosylated-oligosaccharides (FucOS; short chains of fucose-based sugar molecules); an important component of breast milk. However, the mechanism by which beta-galactosidases are able to produce FucOS and how this function might be related to their structure are unknown. Our recent work, toward optimisation of GOS products, using a beta-galactosidase enzyme (known as BbgIII), has provided significant insight into specific modifications that directly affect enzyme function and subsequent product formation. We developed novel BbgIII enzymes that give rise to desirable GOS products for improved production of the prebiotic supplement Bimuno (a type of GOS). During the course of this work, we also discovered that by making specific modifications, two variants of the enzyme were able to synthesise 20 different FucOS products that could more closely mimic HMOs.The overall aim of this project is to optimise our BbgIII enzyme variants to provide specific and desirable FucOS; arguably more authentic HMOs, that exhibit a beneficial effect on the gut microbiome for use in infant formulae and medical nutrition. Our approach is unique, as current methods to produce HMOs are costly and lack structural diversity in the carbohydrates produced, which provides strong industry and societal drivers to develop more cost effective and structurally diverse authentic HMOs.