The limited number of well-characterised 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 utilise cost-effective renewable resources while efficiently generating attractive biobased products. Particular thermophilic microorganisms fulfil 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 optimising 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 ΔphaC 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.

• Klíčová slova
• Caldimonas thermodepolymerans, gene deletion, genetic engineering, polyhydroxyalkanoates, thermophiles,
• MeSH
• editace genu * metody   MeSH
• elektroporace MeSH
• genom bakteriální MeSH
• homologní rekombinace MeSH
• metabolické inženýrství metody   MeSH
• polyhydroxyalkanoáty * biosyntéza   metabolismus   MeSH
• transpozibilní elementy DNA MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• polyhydroxyalkanoáty * MeSH
• transpozibilní elementy DNA MeSH

Polyhydroxyalkanoates (PHAs) have emerged as an environmentally friendly alternative to conventional polyesters. In this study, we present a comprehensive analysis of the genomic and phenotypic characteristics of three non-model thermophilic bacteria known for their ability to produce PHAs: Schlegelella aquatica LMG 23380T, Caldimonas thermodepolymerans DSM 15264, and C. thermodepolymerans LMG 21645 and the results were compared with the type strain C. thermodepolymerans DSM 15344T. We have assembled the first complete genomes of these three bacteria and performed the structural and functional annotation. This analysis has provided valuable insights into the biosynthesis of PHAs and has allowed us to propose a comprehensive scheme of carbohydrate metabolism in the studied bacteria. Through phylogenomic analysis, we have confirmed the synonymity between Caldimonas and Schlegelella genera, and further demonstrated that S. aquatica and S. koreensis, currently classified as orphan species, belong to the Caldimonas genus.

• Klíčová slova
• Caldimonas, DSM 15264, DSM 15344T, LMG 21645, LMG 23380T, Next-Generation Industrial Biotechnology, PHAs, Schlegelella, de novo assembly,
• Publikační typ
• časopisecké články MeSH