Increased tolerance to biomass-derived inhibitors by deletion of the transcriptional regulator mraZ in Cupriavidus necator H16.

Lignocellulosic biomass is an attractive renewable feedstock for sustainable biomanufacturing, but inhibitors generated during pretreatment and saccharification severely limit microbial growth and productivity. Among various strategies to overcome this, we proposed a novel strategy to control a morphology-related transcriptional regulator and compared the wild-type Cupriavidus necator H16 with its mraZ deletion mutant H16 ΔmraZ, in which mraZ functions as a transcriptional regulator influencing cell size, nutrient utilization, and polyhydroxybutyrate (PHB) synthesis under lignocellulose-derived inhibitors such as furfural, vanillin, acetate, and formate. The H16 ΔmraZ strain mostly exhibited higher growth and PHB production than the wild type across all tested inhibitors. Scanning electron microscopy (SEM) revealed that ΔmraZ maintained cell morphology and length after furfural treatment, whereas the wild type displayed significantly decreased cell size. Consistent with these observations, viability and IC50 analyses demonstrated a 3.5-fold increase in viability and a 1.7-fold increase in IC50 in H16 ΔmraZ. When the xylA and xylB genes from Bacillus subtilis 168 were introduced into both H16 and H16 ΔmraZ for cultivation with barley straw- and pine-derived hydrolysates, H16 ΔmraZ showed 1.18-fold higher biomass accumulation and 1.41-fold higher PHB synthesis than the wild type. H16 ΔmraZ showed a higher cyclopropane index in phospholipid fatty acid analysis and increased cfa and H16_A0706 (groEL) expression under furfural stress, suggesting that membrane fatty acid remodeling and chaperone-associated stress responses contributed to improved tolerance. These findings indicate that deletion of the transcriptional regulator mraZ is an effective strategy to enhance stress tolerance and improve bioproduction.