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。奶粉含有蛋白质、碳水化合物、脂肪、矿物质和维生素等营养物质，可以促进骨骼、牙齿、皮肤的健康，有助于肌肉的生长，还有助于预防一些疾病，如骨质疏松症、心血管疾病甚至癌症。直到本世纪初，奶妈一直是母乳喂养的唯一安全替代品。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。但由于情绪和/或身体状况，如产后手术、精疲力竭或营养不良，并不是所有的女性都能母乳喂养。然而，母乳仍然是婴儿营养的黄金标准，制造商的目标是让婴儿配方奶粉尽可能接近人类母乳。牛奶中的一组碳水化合物被称为低聚半乳糖(GOS)，是婴儿配方奶粉中帮助弥合部分差异的主要添加剂。GOS由糖分子(称为半乳糖)的短链组成，由另一种乳糖(乳糖)由称为乳糖酶或β-半乳糖苷酶的酶合成。这些可溶性的、不可消化的碳水化合物被添加为益生菌，试图刺激肠道中的“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，并可能参与过敏和感染预防，以及增加钙、铁、镁和锌等重要矿物质的吸收。然而，益生菌GOS的组成与人类乳寡糖(HMOS)中的糖相似，但并不完全相同；半乳糖并不自然地存在于母乳中。相比之下，岩藻糖化的糖，即2‘-岩藻糖基乳糖，在分泌型女性(她们将血型抗原分泌到体液和组织中，有助于免疫)的乳汁中大量存在。在婴儿配方奶粉中使用GOS和低聚果糖(果糖分子的短链)，两者都不能模仿HMO。与这一建议相关的是，一些β-半乳糖苷酶可以利用岩藻糖来产生岩藻糖化低聚糖(FucOS；岩藻糖基糖分子的短链)，这是母乳的重要成分。然而，β-半乳糖苷酶产生岩藻糖苷酶的机制以及这一功能可能如何与其结构相关尚不清楚。我们最近使用β-半乳糖苷酶(称为BbgIII)优化GOS产品的工作，为直接影响酶功能和后续产品形成的特定修饰提供了重要的见解。我们开发了新型的BbgIII酶，可以产生理想的GOS产品，以提高益生菌补充剂Bimuno(一种GOS)的生产。在这项工作中，我们还发现，通过进行特定的修改，两种酶的变体能够合成20种不同的FucOS产品，这些产品可以更接近地模仿HMO。这个项目的总体目标是优化我们的BbgIII酶变体，以提供特定和理想的FucOS；可以说是更真实的HMOS，这对婴儿配方奶粉和医疗营养中使用的肠道微生物群具有有益的影响。我们的方法是独一无二的，因为目前生产HMO的方法成本很高，而且所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化的正宗HMO提供了强大的行业和社会驱动因素。

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.