Construction of Bacillus velezensis engineering strains for producing 5‐aminolevulinic acid to elicit plant resistance against abiotic stresses and tobacco bacterial wilt disease

BACKGROUND: Crops suffer from numerous biotic and abiotic stresses, leading to substantial economic losses globally. 5-Aminolevulinic acid (ALA) has been shown to stimulate plant resistance, thereby alleviating stress effects. RESULTS: This study aimed to develop stable and multifaceted Bacillus velezensis strains with enhanced ALA production capabilities as potential biocontrol agents. To achieve this, the hemA gene sourced from Bradyrhizobium japonicum was integrated into the genomic DNA of Bacillus velezensis by sequentially replacing existing genes bdh, pgsB, and sinI with hemA, resulting in a recombinant strain, designated R9-3, harboring three copies of the hemA gene. R9-3 achieved an ALA production titer of approximately 22 mg/L. In an effort to further enhance ALA accumulation, we disrupted downstream metabolic pathways by deleting hemB, which is responsible for converting ALA into heme, and sucCD, which participates in diverting succinyl-coenzyme A, a key intermediate in ALA biosynthesis, away from succinic acid production. Unexpectedly, these genetic modifications did not result in additional increases in ALA production. R9-3 proved efficacious in mitigating the detrimental effects of salinity and drought on tobacco plants. By stimulating the activity of vital antioxidative enzymes such as catalase, peroxidase, and superoxide dismutase, and maintaining chlorophyll integrity, this strain reinforced the plants' defense against oxidative stress under harsh environmental conditions. Moreover, the resistance elicited by R9-3 effectively protected tobacco from bacterial wilt disease. CONCLUSIONS: These findings suggest that the engineered strain has significant potential to be developed into a multi-functional biocontrol agent for future agricultural applications, offering both abiotic and biotic stress protection. © 2025 Society of Chemical Industry.