Bacteria produce a wide range of exopolysaccharides (EPS) which are synthesized via different biosynthesis pathways. The genes responsible for synthesis are often clustered within the genome of the respective production organism. A better understanding of the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is critical toward genetic, metabolic and protein-engineering approaches to produce tailor-made polymers. These designer polymers will exhibit superior material properties targeting medical and industrial applications. Exploiting the natural design space for production of a variety of biopolymer will open up a range of new applications. Here, we summarize the key aspects of microbial exopolysaccharide biosynthesis and highlight the latest engineering approaches towards the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications.

The enzyme subclass of glycosyltransferases (EC 2.4) currently comprises 97 families as specified by CAZy classification. One of their important roles is in the biosynthesis of disaccharides, oligosaccharides and polysaccharides by catalyzing the transfer of sugar moieties from activated donor molecules to other sugar molecules. In addition glycosyltransferases also catalyze the transfer of sugar moieties onto aglycons, which is of great relevance for the synthesis of many high value natural products. Bacterial glycosyltransferases show a higher sequence similarity in comparison to mammalian ones. Even when most glycosyltransferases are poorly explored, state of the art technologies, such as protein engineering, domain swapping or computational analysis strongly enhance our understanding and utilization of these very promising classes of proteins. This perspective article will focus on bacterial glycosyltransferases, especially on classification, screening and engineering strategies to alter substrate specificity. The future development in these fields as well as obstacles and challenges will be highlighted and discussed.

Application of state-of-the-art genome editing tools like CRISPR-Cas9 drastically increase the number of undomesticated micro-organisms amenable to highly efficient and rapid genetic engineering. Adaptation of these tools to new bacterial families can open up entirely new possibilities for these organisms to accelerate as biotechnologically relevant microbial factories, also making new products economically competitive. Here, we report the implementation of a CRISPR-Cas9 based vector system in Paenibacillus polymyxa, enabling fast and reliable genome editing in this host. Homology directed repair allows for highly efficient deletions of single genes and large regions as well as insertions. We used the system to investigate the yet undescribed biosynthesis machinery for exopolysaccharide (EPS) production in P. polymyxa DSM 365, enabling assignment of putative roles to several genes involved in EPS biosynthesis. Using this simple gene deletion strategy, we generated EPS variants that differ from the wild-type polymer not only in terms of monomer composition, but also in terms of their rheological behavior. The developed CRISPR-Cas9 mediated engineering approach will significantly contribute to the understanding and utilization of socially and economically relevant Paenibacillus species and extend the polymer portfolio.