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

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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.