Award details

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

Reference	BB/R008744/1
Principal Investigator / Supervisor	Professor Kimberly Watson
Co-Investigators / Co-Supervisors	Professor Dimitris Charalampopoulos, Professor Glenn Gibson
Institution	University of Reading
Department	Sch of Biological Sciences
Funding type	Research
Value (£)	417,082
Status	Completed
Type	Research Grant
Start date	04/04/2018
End date	31/12/2021
Duration	45 months

Abstract

Bifidobacteria are a predominant bacteria in the intestines of breast-fed infants, rapidly colonising within a week after birth, compared to bottle-fed infants. The selective growth of this bacteria is attributed to the oligosaccharides (excluding lactose) contained in human milk - known as human milk oligosaccharides (HMOs). HMOs comprise three basic units: lactose, lacto-N-biose I and N-acetyllactosamine. These core structures are frequently modified by the addition of fucose and sialic acid. This fucosylation step, in the biosynthesis of these oligosaccharides, is typically accomplished by a family of enzymes known as fucosyltransferases. One such product, 2'-Fucosyllactose (2'FL), is one of most abundant functional oligosaccharides in human milk and has been associated with many health benefits, including a lower risk of many types of diarrhoea in breast-fed infants. Current methods for production of fucosylated HMOs have been largely limited to; i) genetic modification of whole bacteria, which is both expensive and non-specific with respect to the types of oligosaccharide products obtained or ii) the use of fucosyltransferases, also limited in specificity of the products formed. In work leading up to this proposal, we have found that an isolated, protein engineered beta-galactosidase enzyme has the capacity to produce fucosylated oligosaccharides (FucOS), when provided with lactose and fucose, or GOS and fucose, as substrates. Our data show that there are approximately 20 different FucOS products that could be isolated and/or further optimised for use as a authentic HMOs. Using computational approaches, at least 4 mutations (Glu447Asp, Glu533Asp, Glu599Asp and Trp591Phe) have been identified, which are predicted to lead to enhanced FucOS production, as HMO mimics. To our knowledge, the sole use of an engineered beta-galactosidase enzyme to produce FucOS has not been shown previously, providing a unique approach for production of authentic HMO supplements.

Summary

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.

Impact Summary

Demand for human milk oligosaccharides (HMOs) as a food ingredient is on the increase, particularly in the infant formula market. In 2014, global demand for HMOs was ~21 kg and it is expected increase to ~82 kg by 2022, representing revenue of ~54M and ~135M USD, respectively; thus, small volume, high value products. Increasing awareness of the benefits of HMOs and a better understanding of their roles in health is expected to fuel its demand over the next seven years. The high mortality rate of infants throughout the world is one of the major factors driving the burgeoning HMO market. HMOs help protect infants from bacterial and viral infections, as well as the appropriate functioning of the neuronal system, and influencing gut flora. Moreover, changing lifestyles and increasing health concerns are expected to augment further growth in the wider HMO market (in functional food & beverage products and food supplements) alongside the knowledge that HMOs can help in the treatment of certain diseases and in high risk patients. There remain significant efforts to produce synthetic HMOs for use in infant formulas (and medical nutrition). This project will contribute significant knowledge to the development of protein engineering and bioprocessing technologies for the production of HMOs and functional food ingredients. The potential and expected impacts from this research are described below. Commercial and economic impact: The food industry stands to benefit from this project through a completely unique and, consequently, more cost effective route for the production of authentic HMOs. Specifically, our industrial partner Clasado Ltd. will benefit from the development of a new product range, which will enable them to enter into a growing market - HMOs and infant formula. Clasado estimates that as little as two years following successful project completion, it will have penetrated 10 per cent of the competitive market generating revenue of 22M Euros. The outputs from this project will also add significant value to Clasado's manufacturing partners in the UK and France, through additional sales of authentic HMOs, and ultimately will contribute to economic growth (through production, sales, employment and expansion) in the UK and Europe. In the longer term, our integrated pipeline approach has the potential to impact the wider food and bioprocessing industries. This project (workflow and outputs) can be translated more generally to the provision of HMOs as functional ingredients in daily consumed food products, such as juices, breads and yogurts with potential beneficial health gains. Previous high costs of production of HMOs have made this prohibitive. Environmental and societal impact: Significantly, our structure-function pipeline (developed previously with Clasado for Bimuno prebiotic production) provides a significant reduction in waste water during the manufacturing process. This is due to a reduction in the filtration processing needed for purification of a crude product. A pure product mixture (through optimisation of the FucOS product) is one of the key objectives of this project. This is a directly favourable and measurable environmental impact and would be of benefit to similar processes where waste water and waste materials are problematic. Importantly, this research has the potential for global societal impact, as it directly addresses human health and disease prevention, through the development of a range of specific FucOS, as authentic HMO supplements with beneficial effects on gut microbiota. Promoting 'good gut bacteria' has been shown to contribute to the prevention of diseases, such as IBS, obesity, diabetes and autism, and to improving immunity. Our pathway to impact includes opportunities to engage with 'conscious consumers' and those with an interest in providing scientific evidence behind authentic HMOs in functional foods, supplements and infant formulas.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Industrial Biotechnology, Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Industrial Partnership Award (IPA)

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