Unravelling fundamental mechanisms of bacterial exopolysaccharide catabolism for use in food and health applications

Bacteria produce various chemical compounds, many of which are carbohydrates playing key roles in biological processes. One important group, exopolysaccharides (EPS), makes some bacteria smooth and slimy. EPS are crucial for both bacteria and humans. Pathogenic bacteria use EPS in biofilms to evade the immune system and antibiotics. In food production, bacteria secrete EPS to enhance texture and stabilize products. In complex communities like the gut, EPS act as signaling molecules for communication between bacteria and the host. EPS produced by gut bacteria can also serve as nutrients for other bacteria, influencing gut flora composition. In this project, we aim to learn more about EPS, how these complex carbohydrates are broken down by bacteria, and how we can use EPS in new food and health applications. We will produce large quantities of pure EPS of different types (both known and unknown) in our local biorefinery. These EPS will be used as nutrients for bacteria so that we can identify which bacteria can utilize them. By identifying, analyzing, and acquiring the enzyme systems bacteria use to break down EPS, we can use these, along with chemical methods, to create new EPS molecules with new and useful functions. These new molecules will be developed for use in several new applications, for example, to change the composition of the gut flora (from poor to good composition), to alter the texture and taste of food, and as new antimicrobial agents. So far, the project has been a great success. During the first half of the project, we have managed to scale up the production of several lactic acid bacteria that produce new types of EPS. Such scaling involves preliminary experiments on a milliliter scale, pilot experiments up to one liter, and finally large fermentations in an advanced 100-liter fermenter. This has required the development of methods for downstream processing of large volumes, combined with analytical methods such as size-exclusion chromatography, monosaccharide analysis, and NMR analysis to determine purity and structure. Already, we have identified a completely new EPS from the lactic acid bacterium Lactoplantobacillus pentosus, which will be a significant scientific discovery and may also have potential as a food additive (for example, as a prebiotic) or as a basis for a new type of antibiotic. Through the process development for producing and purifying this EPS, we have also discovered new gentle methods for isolating cell wall polysaccharides from Gram-positive bacteria. This is something the field has struggled with for many years, where most have used destructive extraction methods that partially destroy the polysaccharides. Although the study is not yet published, there is already great interest in this finding among our partners, and we see the potential for a breakthrough when this is published scientifically (first, we will consider whether it is possible to protect the finding). Regarding the studies of how EPS are broken down in nature, we are pursuing this in two directions – enzymes for the degradation of the commercially available thickening agent gellan gum and enzymes for the degradation of our new EPS from L. pentosus. Using our extensive expertise in genomics and proteomics, we have now identified many enzymes that we are in the process of characterizing. These are important to us as they will be used to create oligosaccharides, which will then be used in glyco-conjugation chemistry to create fluorescent substrates and potentially antimicrobial variants. The synthesis work is well underway, and we have already produced a number of fluorescent variants of gellan gum oligosaccharides. These are now being used to identify bacteria that can utilize gellan gum as a nutrient. Upon uptake of the fluorescent saccharides, the bacteria will light up, allowing us to isolate them and sequence their genomes to determine their identity. This will be very useful and interesting for understanding how gellan gum affects the gut flora, as this substance is used in many different food products. The project benefits from the interdisciplinary approach that spans from medical microbiology to organic chemistry. This gives us a competitive advantage in the field and allows us to conduct experiments and create products that few others can. This makes us an interesting collaboration partner, and we have already expanded our network to France and Scotland with active collaborative activities. The project has also been actively involved in the education of master’s students. The project is now in its most productive phase, and we look forward to the next phase where scientific production will increase and the first PhD candidate will complete their work.

Bacterial exopolysaccharides (EPS) represent a highly diverse group of biopolymers that are secreted by bacteria. Whereas hundreds of EPS structures have been characterized, few studies exist that have investigated EPS degradation and the enzymes involved. Due to the enormous diversity of EPS molecules, we thus hypothesize that there is a large gap in knowledge of EPS degrading and modifying enzymes. In the present project we will tap into this large reservoir of unexplored enzymes to increase our general understanding of EPS utilization in biological systems and, through an interdisciplinary effort, harness the acquired knowledge to generate novel biotechnological tools and applications. By utilizing an on-site biorefinery, multi-gram amounts of pure EPS from several bacteria will be produced and subsequently used as nutrient sources in bacterial cultivation screens. Here, an expected important outcome is knowledge on how EPS can alter the composition of the gut microbiota. By analysis of EPS degrading isolates or enrichment cultures by a combination of genomics and proteomics, enzymes involved in EPS degradation will be identified and subsequently cloned, expressed and characterized to reveal complete degradation pathways. These pathways are likely to contain multiple new enzyme families and activities. The characterized enzymes represent an EPS toolbox that will allow tailoring of EPS-oligosaccharides, that, by means of chemical synthesis, will be used to generate fluorescent glycoconjugates (e.g. fluorescent glyco-carbon dots) that in turn can be used to identify EPS-binding or degrading bacteria in complex microbial communities. Using the same chemical approach, novel antimicrobial compounds will be synthesized to specifically target EPS degrading pathogens identified earlier in the project timeline. Finally, EPSs and EPS-oligosaccharides will be screened for their immunomodulatory properties to unravel potential EPS-host interactions.