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

## TL;DR

Researchers improved a thermophilic bacterium's ability to produce bioplastics by engineering it to better use glucose, a key step in using plant-based sugars for sustainable production.

## Contribution

Engineering glucose transport in Caldimonas thermodepolymerans using a gene from Zymomonas mobilis to enhance PHA production from lignocellulosic sugars.

## Key findings

- Caldimonas thermodepolymerans prefers xylose and cellobiose over glucose for PHA production.
- Glucose transport limitation was overcome by introducing the glf gene from Zymomonas mobilis.
- The engineered strain achieved high PHA yields even when cellobiose was the sole carbon source.

## Abstract

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.

C. thermodepolymerans DSM 15344 produces PHA from lignocellulose-derived sugarsXylose and cellobiose are preferred substrates, while glucose is poorly utilized Deficient glucose transport in DSM 15344 restored by Zymomonas mobilis glf gene

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