Probing the bacterial cell wall with chemical biology tools

After DNA and proteins, carbohydrates are the third language of life. Chapter 1 introduces the reader to this class of biomolecules, also called sugars or glycans, that can be found on the outer surface of almost all cells and plays a critical role as the social messengers of a cell. Although our knowledge about the role of glycans in eukaryotic cells has increased considerably in recent decades, our understanding of the glycan layer on bacterial cells is still very limited. Besides the carbohydrates that are present in both eukaryotes and prokaryotes an additional wide range of unique (e.g. microbial sialic acid), often very complex (e.g. pseudaminic acid), carbohydrates is present in prokaryotes. This chapter briefly introduces two research fields, carbohydrate chemistry and chemical biology, that when combined provide a powerful way to investigate the biological role of these unique bacterial carbohydrates at the molecular level. This chemistry-based approach, termed chemical microbiology, often starts with the development of a chemical synthesis for a target bacterial carbohydrate. Subsequently, the synthetic route towards this target allows for the introduction of unnatural functional groups, like chemical reporters, that result in the molecular tools needed to study their biological function. The studies described in this thesis, focus on developing such molecular tools to study the role of glycans and glycoconjugates in human gut bacteria and human-associated bacteria. Chapter 2 provides an overview of metabolic oligosaccharide engineering (MOE) a popular chemical biology technique to label glycans in living cells. In MOE, carbohydrates derivatives are synthesised with unnatural chemical reporters and used to study their incorporation in glycans of eukaryote to prokaryote species. The progress in this field over the last 6 years is reviewed in detail with a special emphasis on the synthesis of the unnatural carbohydrates from commercially available sources. The principle behind MOE is that these unnatural carbohydrates with e.g. azide, alkyne, cyclopropene, or isonitrile chemical reporter groups, are still recognised by the endogenous enzymes in the cell that salvage this new carbohydrate. In this way they can enter the associated biochemical pathways and end up in newly biosynthesised cellular glycans. Subsequent labelling techniques, such as strain promoted azide alkyne cycloaddition or tetrazine ligation, enable the visualisation of these incorporated unnatural carbohydrates with for instance fluorescence microscopy. Metabolic labelling is further explored in chapter 3. Key cell envelope glycoconjugates in the mucin-degrading gut microbiota member, Akkermansia muciniphila, were subjected to chemistry-based functional analysis, with Escherichia coli being used as a control species. Two novel non-toxic peptidoglycan (PG) probes were designed and synthesised to investigate the presence of PG in this species. Their design was based on the natural d-alanine dipeptide motif found in PG. Inspired by the fact that d-alanine dipeptide-derivatives were previously reported to be incorporated in newly synthesised PG, we synthesised a cyclopropene and isonitrile d-alanine dipeptide. Our probes proved to be non-toxic, as shown by growth and viable count analysis, and were therefore superior over existing PG probes. Another beneficial property was that the probes also did not influence the specific growth rate of A. muciniphila or E. coli. The PG probes were successfully incorporated into the peptidoglycan layer of A. muciniphila and visualised using a tetrazine click-ligation with a fluorophore. Our analysis proved for the first time that A. muciniphila has a PG layer. Besides PG labelling, we also investigated metabolic labelling of other glycoconjugates on the outer surface of A. muciniphila. This part of the study showed that azido-monosaccharide derivatives of N-acetylglucosamine, N-acetylgalactosamine, and fucose are successfully processed by A. muciniphila salvage pathways and incorporated into its surface glycoconjugates. Especially 6-azido-fucose was readily processed by the recently discovered l-fucose salvage pathway of A. muciniphila. The two compatible labelling techniques were next combined in a dual labelling experiment. Our isonitrile dipeptide peptidoglycan probe and 6-azido-fucose were successfully incorporated into A. muciniphila. Subsequent fluorescent labelling with bio-orthogonal techniques resulted in dual labelling of peptidoglycan and fucose-containing glycans in live A. muciniphila cells. With the positive results of MOE in A. muciniphila in hand, chapter 4 describes the further investigation of MOE. After successful validation of our Ac4FucAz probe for MOE in Bacteroides fragilis we continued their application in other human gut microbiota members, including the butyrate-producing Anaerostipes rhamnosivorans, Intestimonas butyriciproducens, and Eubacterium hallii. Labelling of these human gut microbes proved to be rather challenging with a-specific cellular labelling with the fluorophore being the major problem. Initial results, however, did show that a 6-azido-l-rhamnose probe resulted in fluorescent labelling of A. rhamnosivorans, which provides initial evidence for the existence of an as of yet undocumented salvage pathway. In this species the 6-azido-fucose probe was not salvaged. Via confocal microscopy and flow cytometry analysis we observed that the 6-azido-rhamnose probe was selective for A. rhamnosivorans in the presence of A. muciniphila. Such a co-culture experiment is a first step in mimicking the complex human gut microbiome. For E. hallii Ac4GalNAz gave clear metabolic labelling and the majority of the cell population could be labelled with the fluorescent dye after a strain-promoted azide alkyne cycloaddition. Other glycan probes (Ac4GlcNAz, Ac4FucAz, and Neu5Az) also resulted in labelling, but not as prominent as Ac4GalNAz. Surprisingly, MOE has never been reported for the common lab strain Escherichia coli MG1655. Curious to investigate this in more detail we started MOE in E. coli. However, no labelling was obtained when Ac4GlcNAz probe was added to E. coli, most likely due to the fast growth, metabolism and turnover. Only, when fresh Ac4GlcNAz probe was added every 30 minutes, metabolic labelling in E. coli was observed. To further investigate the influence of GlcNAc metabolism in E. coli on MOE, single-gene knock-outs of E. coli GlcNAc metabolism from the Keio collection were investigated. Labelling was observed for NagA (N-acetyl glucosamine 6 P deacetylase) and NagK (N-acetyl-d-glucosamine kinase) E. coli mutants. Both enzymes are involved in the last step of the biosynthesis towards UDP-N-acetylglucosamine. When the overall E. coli metabolism was inhibited, after addition of the respiration inhibitor sodium azide, no metabolic labelling was observed. These results indicate that MOE in E. coli is possible, but challenging and can only be performed under specific circumstances. An investigation into the total synthesis of pseudaminic acid, a sialic acid produced by specific human-associated prokaryotes, is described in chapter 5. Sialic acids are typically found at the terminal positions of surface glycoconjugates in both eukaryotes and prokaryotes. Other related microbial sialic acids are legionaminic and acinetaminic acid. The total synthesis of these microbial sialic acids is notoriously difficult, as exemplified by the fact that only a few chemical synthesis routes towards them are currently known. Our total synthesis of pseudaminic acid started from the readily available amino acid l-threonine that was transformed into a key versatile Garner aldehyde derivative intermediate. With this aldehyde in hand, the Henry nitro-aldol condensation reaction was investigated. After studying numerous conditions, such as asymmetric catalysis or elongated reaction times, and extensive optimisation efforts we were never able to obtain the Henry reaction product to continue with this route. As an alternative, a tethered aminohydroxylation was investigated for its ability to introduce the key functional group and stereochemistry onto an intermediate obtained from the Garner aldehyde derivative. This reaction indeed gave the desired amino-alcohol motif in the correct stereochemistry, but another diastereomer proved very difficult to separate from the desired product. After some additional transformations and protection steps we obtained a derivative in which the primary alcohol could be oxidised to provide a hexose intermediate that resembles the hexose intermediate present in pseudaminic acid biosynthesis. This key hexose intermediate will likely enable a subsequent Barbier reaction, a chain elongation step, in future studies. With most of the key transformations accomplished, the completion of a pseudaminic total synthesis based on l-threonine should soon be possible. Besides finishing the total synthesis, future work should also focus on adapting this synthesis route to allow installation of chemical reporter groups on pseudaminic acid for its application in MOE. Chapter 6 is the general discussion about all the work mentioned in the other chapters. It also contains additional information and suggestions for further research in the field of chemical microbiology.</p

Open innovation in high value manufacturing

The aim of this paper is to examine the concept of open innovation and understand if it occurs and how it occurs within the High Value Manufacturing (HVM) context. There is a key theoretical relevance since open innovation has not been explored from a network based perspective. Similarly, there is a strong practical relevance for this research since policy makers in the EU (especially in the UK) are focusing on strengthening HVM in their economies but the role innovation, and especially open innovation, is not fully understood. The methodology adopts an exploratory case approach within four manufacturing firms that we consider to be operating within a HVM context. Interviews with ten technical managers across the four cases were collected. NVivo analysis and data structuring based on Gioia et al. (2012) form the basis of the data analysis. The findings suggest that many different ‘modes’ and types of innovation take place within the HVM context. Open innovation is witnessed more commonly from an ‘outside in’ perspective i.e. firms draw knowledge or technology from external sources into their internal innovation process. Our findings also suggest that open innovation occurs mainly in closed networks, with other firms within their supply chain. However, our findings also highlight that the maturity of technology and sector ‘norms’ may also have an influence on degree of openness

Linking manufacturing ecosystems theory with servitization : a 'nested' solutions-based approach

Vanderwerme et al. (1988) identified servitization as manufacturing companies making the shift from selling purely goods to selling bundles of goods, services, support, and knowledge. This shift represents a customer-centric approach where a focus on delivering features and functionality within a tangible product is replaced with a focus on delivering value in use. This shift usually represents a strategic change in a company’s opera3ons (Baines and W. Lightfoot, 2014) and is often driven by new technology (Xu et al., 2018). More recently digitalization has introduced a new technology pathway for manufacturers to extend their servitization strategy (Coreynen et al., 2017). As digital servitization is a relatively new area of study, business models for the deployment of digital product service bundles have still to be characterised (Paschou et al., 2020). This paper seeks to address this gap by using manufacturing ecosystems theory developed by Sminia et al. (2019), Sminia et al. (2022), Paton et al. (2023), and Ates et al. (2023) within a case-based approach to analyse the strategic development and operational implementation of a digitally enabled servitization business model by a manufacturing company

Firm strategy and the continuity and change in ecosystems

The notion of 'industry', defined as all firms that produce close substitutes (Porter, 1980), has served us well. What it did is to focus our attention on competition and more specifically on the importance of value appropriation as well as capability development (Barney, 1991; Teece, Pisano, & Shuen, 1997) for firms as these compete in an industry. Recent developments made us recognize the importance of complementarity, that is that the value of a combination of products and services exceeds the value of products and services when taken separately (Brandenburger & Nalebuff, 1996, 2021). This has various consequences for our conceptualization of the relevant environment for firms. Taking complementarity into account, firm performance is not only a function of how the firm competes but also of how the firm cooperates. The term of co-opetition expresses this new reality (Bengtsson & Kock, 2000; Brandenburger & Nalebuff, 1996). There are also more actors that have become relevant bar the ones that have been captured by Porter’s (1980) 'five forces'. Consequently, the notion of 'ecosystem' has gained traction as an alternative to 'industry', with various definitions being proposed (Adner, 2006; Gawer, 2014; Jacobides, Cennamo, & Gawer, 2018). To us an ecosystem is a set of activities that generates complex functionality in the form of a product-service bundle for a system-of-use (Sminia, Ates, Paton, & Smith, 2019). Complex functionality as a coherent solution (Hannah & Eisenhardt, 2018) is what represents value for end-users. Generating complex functionality normally involves many different actors including buyers, suppliers, competitors, possible entrants, subsitutors, but also complementors, orchestrators, platform leaders, financiers, insurers, regulators, and government agencies. Every ecosystem is arranged in its own idiosyncratic way as it accommodates the three dynamics of capability, governance, and appropriation (Paton, Ates, Sminia, & Smith, 2021; Sminia et al., 2019). The arrangement that characterizes an ecosystem therefore contains a capability configuration, a governance structure, and an appropriation regime. The capability configuration tells which firms are contributing what capability to create the complex functionality. The governance structure governs the relationships between the actors in the ecosystem as well as provides the rules, regulations, and standards that must be complied with. The appropriation regime determines how firms capture the value that the complex functionality represents. The three dynamics have a bearing on each other. Consequently, individual firm performance is a consequence of the firm's position in, the capability configuration, the governance structure, and the appropriation regime. Capability, governance, and appropriation are 'dynamics' because these are not stable entities. The ecosystem arrangement is inherently fluid because firms are constantly trying to improve their position and in doing so perpetuate or change the arrangement. Furthermore, ecosystem activity is stratified in that there is a basic process by which firms and other actors perform within the existing arrangement while there simultaneously is a underlying process going on by which the existing arrangement is maintained or changed (Lawrence, Leca, & Suddaby, 2009; Sminia & de Rond, 2012). Firm activity therefore has dual consequences. On the surface, every move serves a purpose for utilizing a firm's position in the existing arrangement. Simultaneously this activity has the additional effect of either conforming to and maintaining the existing arrangement, or of deviating from, undermining, and possibly changing the existing arrangement, all to improve the firm's position in the arrangement. Ecosystem activity can include overt initiatives that are aimed at transforming an ecosystem, potentially changing it beyond recognition when change becomes so fundamental that it transforms the complex functionality and how this is valued by the system-of-use. All of this means that questions regarding how an ecosystem emerges, develops, and changes must be posed and answered in terms of how this volatility plays out, recognizing the stratified nature of ecosystem activity. In turn, this allows us to appreciate firm strategy not only as a firm utilizing its position in an existing ecosystem arrangement, but maybe and more importantly to also see strategy as actively engaging with and changing the ecosystem arrangement to its advantage. For this reason, we posit that ecosystems do not emerge from nothing but that ecosystems morph and transform on occasion giving the impression something completely new has developed because of the inherent volatility present in an ecosystem, especially if a string of initiatives succeeds that in effect alter the complex functionality that is being generated. This paper develops this dynamic understanding of the notion of an ecosystem and puts forward four propositions about how firms can deal with ecosystem volatility. It does so by first explaining about ecosystem dynamics in more detail. The layered nature of ecosystem strategy will be explicated second, which allows us to develop our four propositions. Our elaboration of ecosystems and what it means for firm strategy will be illustrated using a firm that we refer to by the name of SpaceCo. SpaceCo is the fictitious name of a company we currently work with in a knowledge exchange project. The paper will finish by discussing implications and suggesting further research

Strategic Thinking for Manufacturing Ecosystems

If people say your manufacturing firm is operating in an ecosystem, what does that mean? Actually, it means quite a lot. It has far-reaching consequences for how you need to think about strategy

Bridging differing perspectives on ecosystems research to understand co-opetition

Management practitioners and scholars alike use a range of terms such as industry, sector, and market to describe and characterise an organisation's environment. More recently, the word ecosystem, a term borrowed from the field of biology, has entered the lexicon. Grand challenges of today are increasingly tackled by new forms of multi-organisational arrangements, representing the ecosystems. Consequently, the opportunities for countries and organisations to cooperate are even larger today—from tackling Covid-19 to climate change. For example, ecosystems explain the rapid progress in monitoring the spread of Coronavirus (e.g., the track and trace apps for Covid-19). Apple and Google’s decision to cooperate in creating contact-tracing technology for Covid19 enabled a rapid response to the pandemic. By sharing user location data across platforms, the two companies cooperated with governments, health organisations (e.g., NHS in the UK) and users to create effective notification apps. Therefore, a better understanding of ecosystems will help today’s businesses, managers, and countries find a better way to work and succeed together (Beaudry et al., 2021; Brandenburger and Nalebuff, 2021). We identified four main approaches to study ecosystems – entrepreneurial ecosystems (Marshall, 1920), business ecosystems (Moore, 1993), innovation ecosystems (Adner, 2006), and platform ecosystems (Kretschmer et al., 2020). Although the ecosystem literature is exponentially growing but also increasingly fragmented. Despite conceptual similarities, different strands with interesting contributions made by scholars from innovation, strategy, and entrepreneurship disciplines develop in silos. Accordingly, the main objective of this research is to bring conceptual clarity to ecosystem notion taking a multidisciplinary perspective

Crafting strategic responses to ecosystem dynamics in manufacturing

This research goes beyond the dyadic view of co-opetition in supply chains and seeks to explore how firms that act as suppliers in a dynamic manufacturing ecosystem establish and sustain their strategic position. We interviewed 31 senior managers in seven firms that were identified by a committee representing government and academia as occupying various advanced manufacturing ecosystems. We argue that as actors within a manufacturing ecosystem interact overt time to co-create the overall product-service offerings, new relationships may be formed, and existing connections may be dissolved, giving rise to three co-opetition dynamics at the ecosystem level - capability configuration, value appropriation, and network governance. Our analysis unveiled eighteen operational tactics that suppliers deploy which combine to produce nine strategic responses that allow them to sustain their position within manufacturing ecosystems. Specifically, we discuss the role of suppliers in manufacturing ecosystems and capture the relationship between ecosystem dynamics and the strategic responses as they accommodate co-opetition. This research indicates that ecosystem performance is essentially a dynamic effort, which is simultaneously collective and distributed. Thus, policymakers should avoid carrying out analysis based on overly linear and single industry conceptualisations of manufacturing value networks