A two-step screening platform based on trans -aconitic acid assimilation unlocks novel bacterial resources for trans -aconitic acid production

ABSTRACT trans -Aconitic acid (TAA) possesses significant advantages in bioactivity, biosafety, and chemical modifiability, leading to its wide application in biopesticides, pharmaceuticals, and biobased synthesis. However, the scarcity of microbial resources for TAA production limits its large-scale commercial production, making it difficult to replace currently costly and low-yield chemical synthesis and plant extraction methods. In bacteria, aconitate isomerases (AIs) catalyze the biosynthesis of TAA. AIs are bifunctional enzymes that can also facilitate the reverse reaction, using TAA as the sole carbon source for bacterial growth. This provides a readily observable marker for the selection of TAA-producing bacteria. Using this dual functionality, we developed a novel two-step screening strategy comprising: (i) high-throughput screening of TAA-assimilating strains using a minimal medium with TAA as the sole carbon source and (ii) secondary screening of these assimilating strains to detect their ability to produce TAA in citrate medium. A total of 219 TAA-assimilating bacteria were isolated from a variety of environmental samples ( n = 135). Subsequent LC-Q-TOF-MS analysis identified 19 strains (8.68%) capable of producing TAA at concentrations ranging from 34.5 to 621.9 μM. Taxonomic analysis revealed two new orders, five new families, and seven new genera of TAA-producing bacteria. This study established an efficient AI-directed strategy for discovering novel TAA-producing bacteria and significantly expanded taxonomic diversity, providing a sustainable resource for microbial technology development and strain innovation, thereby promoting the industrial biomanufacturing of TAA and its applications in multiple fields. IMPORTANCE This study proposes a novel method for constructing a high-throughput screening platform using aconitate isomerase, which catalyzes both the biosynthesis and assimilation of trans -aconitic acid (TAA), to target the discovery of microbial TAA-producing strains. Using TAA assimilation as a phenotypic marker for rapid screening, this strategy overcomes the key bottleneck of scarce microbial resources. The results show that this method effectively identified seven previously unknown genera of TAA-producing bacteria, greatly expanding the phylogenetic diversity and revealing the widespread presence of TAA metabolism across different taxa. Significant yield variation among different isolates underscores their potential as chassis for strain improvement. These resources provide a sustainable driving force for constructing efficient microbial cell factories, facilitating industrial bioproduction of TAA to meet the growing demands of agricultural biocontrol and advanced biomanufacturing.