Bioengineering a synthetic enzyme for the production of authentic human milk oligosaccharides.
Lead Research Organisation:
University of Reading
Department Name: Sch of Biological Sciences
Abstract
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.
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.
Technical Summary
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.
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.
Planned Impact
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.
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.
| Description | We engineered a number of enzyme variants to assess their ability to produce oligosaccharides for use as prebiotics and/or food ingredients that would have a beneficial effect on gut health. During the course of this research, we synthesised 18 novel variants of one such enzyme, identified, isolated and characterised two further enzymes with the ability to produce oligosaccharides as prebiotics. We identified a further 12 possible variants of these enzymes (including two new enzymes), which we are continuing to charactierise and exploit for their structure-function potential to produce novel prebiotic ingredients. In addtion to these prebiotic producing enzymes we identified, isolated and characterised 9 other enzymes (fucosidases) for production of fucosylated oligosaccharides, as potential human milk oligosaccharides. Work is ongoing, via the studentship, for a more detailed characterisation of the resultant oligosaccharide products from these enzymes. We successfully produced sufficent quantities of novel conjugated oligosaccharides for in vitro batch and gut fermentation experiments, where we have been able to show beneficial effects for two specific reaction mixtures. These results are currently awaiting approval for publication by the company, pending any patent filing (yet to be decided). |
| Exploitation Route | Potential outcomes will be at least 3 publications (review article, novel enzyme/product screening and batch/gut fermentations) are pending approval by the company, as they are in internal discussions regarding any potential patent filing required. Our pipeline method, utilising rational structure-guided protein engineering, has been recognised by the food and bioprocessing industries. In the former case, our expertise in this area has led to additional funding by industry (one part funded and one fully funded PhD student) and we have started a new collaboration with another company collaborator exploring the use of bile acids as nutraceuticals. |
| Sectors | Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology |
| Description | Professor Watson has been working with Clasado Ltd, since 2012, to develop more thermally stable enzymes for use in the production of prebiotics. Her research has led to a novel enzyme being adopted by the company in the manufacture of their flagship product Bimuno®, which enabled higher yields, reduced downstream processing and produced less waste. By 2015, the company had switched production to the new thermostable enzyme, leading to more efficient Bimuno production. Operating the production process significantly higher has helped the company to increase yield of the prebiotic product, while simultaneously reducing the need for additional processing, and reducing waste water. This increased purity also enabled Clasado to increase their product range, now selling Bimuno as pastilles in addition to powdered form. Professor Watson and her team, with the support of this BBSRC grant, have been exploring the potential to use their enzyme, related family members of this class of enzyme, and identification of novel enzymes for other uses. In particular, the team are exploring the use of protein engineering to produce novel enzymes for the synthesis of specific carbohydrates that could stimulate specific gut microbes that give rise to a beneficial effect on the gut microbiome and result in a health benefit. Synthesis and functional characterisation of more than 30 enzyme variants has been completed, during the course of this grant, with identification of 2 new enzymes (from one enzyme class) and 9 new enzymes (from an alternative class), whose structure - function properties are being exploited for the production of novel oligosaccharides that support a healthy gut microbiome. We anticipate at least one major publication to be submitted in 2019 reporting our results to date. This novel work has led to the discovery of additional novel enzymes (and variants of these enzymes, through protein engineering methods) that has led to further novel oligosaccharides that will be tested for their prebiotic effect, in future work. This is leading to a new patent, filed by Clasado, in which Professor Watson is named as Co-Inventor, which has delayed any publication, at this time. |
| First Year Of Impact | 2021 |
| Sector | Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology |
| Impact Types | Societal,Economic |
| Description | PhD Studentship |
| Amount | £120,000 (GBP) |
| Organisation | Clasado BioSciences |
| Sector | Private |
| Country | Jersey |
| Start | 10/2020 |
| End | 09/2023 |
| Description | Protein engineering novel enzymes for prebiotic production |
| Amount | £165,000 (GBP) |
| Organisation | Clasado BioSciences |
| Sector | Private |
| Country | Jersey |
| Start | 10/2023 |
| End | 09/2026 |
| Title | Carbohydrate separation |
| Description | We have improved methods for analytical separation and profiling of novel GOS using GC-MS and IC HPAE-PAD PA210 column, which we anticipate will lead to new method papers. In the latter case, the utilisation of a new PA210 column for enhanced GOS separation and profiling has not been reported in the literature to date. Additionally, we have established a new preparative GOS purification protocol, enabling isolation of different degree of polymerisation (DP) fractions following synthesis reaction. This facilitates large scale production of FucOS and novel GOS and enables testing different pools of molecules of different length for their prebiotic effect. We anticipate that this work also will reveal novel findings, since there is very little published data regarding the differences in colonic fermentation of different degree of polymerisation of GOS. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2020 |
| Provided To Others? | No |
| Impact | This work is newly evolving, so the impacts are too early to descibe. However, we do anticipate at least two publications (one methods based and the other resulting from testing the resulting GOS products in gut models) in medium to high impact journals. This work could have significant impact in the food biotechnology sector as well as have huge impact on potential new health supplements. |
| Description | Bile acids as nutraceuticals |
| Organisation | Dextra Laboratories |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | We have established two new PhD studentships, focused on exploring bile acids as potential nutraceuticals for improving gut health. This has involved cross-collaboration and expertise between the schools of Biological Sciences and Food Biosciences, at Reading. The company has provided expertise and materials to enable the study of novel bile acid derivatives for improving gut health. |
| Collaborator Contribution | The wider collaborating team, through the affiliation with the company, also has brought together a breadth of expertise in synthetic chemistry (Southampton University) and neurodegenerative diseases (University of Sheffield). |
| Impact | A patent will be filed this year securing our efforts to date. We also hope to publish this work in due course, following successful patent application with the company. My team will be listed as co-inventors. |
| Start Year | 2020 |
| Description | OptiBiotix |
| Organisation | Optibiotix Health plc |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | As a result of long term collaboration with Clasado, and by reputation, we were approached by OptiBiotix Ltd, to initiate a research collaboration in the area of probiotics. |
| Collaborator Contribution | OptiBiotix have fully funded a 3 year PhD studentship award, including fees, stipend, consumables and travel to enable this new research collaboration. |
| Impact | No formal outcomes as yet, but publications are expected during and following completion of the PhD programme. It is a multidisciplinary collaboration bringing together Food and Nutritional Sciences and Biomedical Sciences (including cell and molecular and structural biology). |
| Start Year | 2018 |