(311c) Leveraging Genome-Scale Metabolic Models and Synthetic Biology Tools to Engineer UDP-Sugar Metabolism and Exopolysaccharide Synthesis in Komagataeibacter Xylinus

Domestication of alternative host organisms with unique biosynthetic capability is an attractive alternative to extensive rewiring of model organisms. Domestication promises to preserve complex genetic systems associated with tolerance and biosynthesis while enabling genomics-driven metabolic engineering and synthetic biology approaches to optimize and diversify the native products. This is particularly true of natural biomaterials like bacterial nanocellulose (BNC), an attractive starting point for constructing engineered living biomaterials. BNC is found in kombucha tea and nata de coco - and may be used in wound dressings, field electronic transistors, and tissue engineering. Yet, metabolic engineering and synthetic biology tools for tuning BNC biosynthesis are limited. Therefore, we have domesticated a BNC-producing strain, DS12, from active kombucha culture.

Based on 16S genotyping, we identified DS12 as a strain of Komagataeibacter xylinus, which is a gram-negative acetic acid bacterium. We then conducted growth and yield experiments, demonstrating that K. xylinus DS12 can produce pure, highly-crystalline BNC from a variety of sugars and sugar alcohols. Yet, the current understanding of BNC-producing bacteria does not satisfactorily explain the mechanism behind the varying cellulose yields and properties from different carbon sources. To this end, we have sequenced the genome of K. xylinus DS12. Drawing from the genome-scale metabolic model iMR640 based upon K. xylinus strain E25, we seek to expand and query a metabolic model with our growth and cellulose production data to elucidate a mechanistic explanation for the substrate preferences.

To facilitate metabolic engineering of BNC biosynthesis, we have successfully transformed K. xylinus DS12 and developed a synthetic biology parts toolkit. In order to leverage the power of a modern DNA assembly standard, we have adapted the CIDAR MoClo standard to K. xylinus DS12. To do this, we designed a MoClo compatible broad-based BBR1 origin and replaced the ampicillin selectable marker with spectinomycin to avoid observed ampicillin resistance. With these modifications, we were able to characterize MoClo compatible promoters in K. xylinus DS12 with flow cytometry. We believe this toolkit has several advantages over existing BioBrick compatible toolkits for BNC producing bacteria. In sum, domestication of K. xylinus DS12 is a key step towards rationally designed living cellulosic biomaterials.