Work

Glucose-xylose co-utilization and lactate production with engineered thermophilic Parageobacillus thermoglucosidasius

Year: 2026

Type: preprint

Abstract: Background Lignocellulose is a renewable feedstock rich in glucose and xylose, yet most microbes preferentially prioritize the consumption of glucose due to carbon catabolite repression (CCR), which can limit titers and productivity in bioprocesses. In this study, we employed a reverse engineering approach based on a previous adaptive laboratory evolution (ALE) investigation to construct and study Parageobacillus thermoglucosidasius DSM 2542 mutants capable of glucose-xylose co-utilization. Additionally, we rationally engineered xylose uptake and utilization by introducing derepressed operons related to the xylose assimilation pathway. Results To tackle CCR in P. thermoglucosidasius , we introduced a single nucleotide mutation resulting in an amino-acid substitution in PtsG, a component of the glucose phosphotransferase uptake system, and knocked-out the ribose operon repressor ( rbsR ). Mutant strains ptsG R452C, Δ rbsR and ptsG R452C Δ rbsR exhibited improved glucose-xylose co-utilization under microaerobic conditions, which was accompanied by the derepression of multiple operons typically governed by carbon catabolite repression (CCR), as suggested by proteome analysis. Next to the reverse engineering approach, we rationally engineered xylose metabolism, too. Promoter activity studies of the native xylose utilization operon ( xylAB ) and a xylose transporter ( xylFGH ) indicated regulation by both CCR, and xylose induction. Constitutive expression of xylAB and a CCR-unresponsive xylFGH mutant under the P ldhL promoter resulted in faster growth on xylose-containing media. Last, the strains developed in this study were cultivated in bioreactors and evaluated for their glucose-xylose utilization and lactate production. Mutants ptsG R452C, Δ rbsR and ptsG R452C Δ rbsR showed a clear phenotype of reduced CCR, whereas xylAB overexpression improved xylose consumption in the presence of glucose notably leading to almost complete consumption of xylose in mixed-sugar fermentations. WT achieved a lactate yield of 56.76 mol-% on the glucose-xylose mixed substrate, whereas mutants Δ rbsR and P ldhL - xylAB exhibited the highest values, reaching 73 mol-% and 63 mol-%, respectively. Conclusions In summary, carbon catabolite repression in Parageobacillus thermoglucosidasius was addressed through rational and reverse engineering strategies. Constitutive expression of derepressed xylAB and xylFGH improved xylose utilization, yet did not fully remove CCR. Reverse-engineered ALE-derived mutations in ptsG and rbsR facilitated glucose-xylose co-utilization. Bioreactor studies demonstrated that multiple strains could achieve higher lactate yield compared to the WT.

Source: Research Square

DOI: 10.21203/rs.3.rs-10242303/v1