Elucidating carbohydrate preference and engineering glucose transport in Caldimonas thermodepolymerans for enhanced polyhydroxyalkanoate production.

Caldimonas thermodepolymerans DSM 15344, a moderately thermophilic bacterium, has emerged as a promising candidate for next-generation industrial biotechnology (NGIB) due to its ability to utilize lignocellulose-derived sugars for polyhydroxyalkanoate (PHA) production. This study assesses its metabolic potential by evaluating the utilization of various plant-derived sugars and their mixtures, with a focus on xylose, glucose, and cellobiose. The results indicate that C. thermodepolymerans exhibits a strong preference for xylose (3.97 g/L PHB) over glucose (2.28 g/L PHB) but demonstrates even greater efficiency in metabolizing cellobiose (4.96 g/L PHB). However, extracellular hydrolysis of cellobiose leads to glucose accumulation, which constrains overall productivity. Our findings suggest that the primary limitation in glucose metabolism is inefficient glucose transport rather than intracellular catabolism. To address this bottleneck, the glf glucose facilitator gene from the mesophilic bacterium Zymomonas mobilis was introduced into C. thermodepolymerans, enhancing its glucose utilization capacity. The engineered strain (Cald_GLF3) exhibited significantly improved PHA productivity, particularly when cultivated on sugar mixtures containing cellobiose. Despite being grown at suboptimal temperatures due to the thermal instability of Glf from Z. mobilis, Cald_GLF3 outperformed the wild-type strain, achieving notably high PHA yields when cultivated with cellobiose as the sole carbon source (9.26 g/L PHB). These findings highlight the critical role of glucose transport in the metabolism of C. thermodepolymerans and suggest that targeted engineering can further enhance its biotechnological potential. This study establishes C. thermodepolymerans as a promising thermophilic chassis for PHA production from lignocellulosic sugars, contributing to sustainable biopolymer synthesis. KEY POINTS: C. thermodepolymerans DSM 15344 produces PHA from lignocellulose-derived sugars Xylose and cellobiose are preferred substrates, while glucose is poorly utilized Deficient glucose transport in DSM 15344 restored by Zymomonas mobilis glf gene.