An initial genome editing toolset forCaldimonasthermodepolymerans, the first model of thermophilic polyhydroxyalkanoates producer

The limited number of well-characterized model bacteria cannot address all the challenges in a circular bioeconomy. Therefore, there is a growing demand for new production strains with enhanced resistance to extreme conditions, versatile metabolic capabilities, and the ability to utilize cost-effective renewable resources while efficiently generating attractive biobased products. Particular thermophilic microorganisms fulfill these requirements. Non-virulent Gram-negative Caldimonas thermodepolymerans DSM15344 is one such attractive thermophile that efficiently converts a spectrum of plant biomass sugars into high quantities of polyhydroxyalkanoates (PHA) - a fully biodegradable substitutes for synthetic plastics. However, to enhance its biotechnological potential, the bacterium needs to be “domesticated”. In this study we established effective homologous recombination and transposon-based genome editing systems for C. thermodepolymerans . By optimizing the electroporation protocol and refining counterselection methods, we achieved significant improvements in genetic manipulation and constructed the AI01 chassis strain with improved transformation efficiency and a Δ phaC mutant that will be used to study the importance of PHA synthesis in Caldimonas . The advances described herein highlight the need for tailored approaches when working with thermophilic bacteria and provide a springboard for further genetic and metabolic engineering of C. thermodepolymerans , which can be considered the first model of thermophilic PHA producer.