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.

• Keywords
• Caldimonas thermodepolymerans, gene deletion, genetic engineering, polyhydroxyalkanoates, thermophiles,
• MeSH
• Gene Editing * methods   MeSH
• Electroporation MeSH
• Genome, Bacterial MeSH
• Homologous Recombination MeSH
• Metabolic Engineering methods   MeSH
• Polyhydroxyalkanoates * biosynthesis   metabolism   MeSH
• DNA Transposable Elements MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Polyhydroxyalkanoates * MeSH
• DNA Transposable Elements MeSH

To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering remain scarce. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous xylose isomerase pathway remained unresolved. Here, we elucidate the xylose metabolism and establish a foundation for further engineering followed by ALE. First, native glycolysis is derepressed by deleting the local transcriptional regulator gene hexR. We then enhance the pentose phosphate pathway by implanting exogenous transketolase and transaldolase into two lag-shortened strains and allow ALE to finetune the rewired metabolism. Subsequent multilevel analysis and reverse engineering provide detailed insights into the parallel paths of bacterial adaptation to the non-native carbon source, highlighting the enhanced expression of transaldolase and xylose isomerase along with derepressed glycolysis as key events during the process.

• MeSH
• Metabolic Engineering MeSH
• Pentose Phosphate Pathway MeSH
• Pseudomonas putida * genetics   MeSH
• Transaldolase genetics   MeSH
• Xylose * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Transaldolase MeSH
• Xylose * MeSH