(253f) Multi-Plasmid Cotransformation to Combinatorial Engineer Multiple Histidine Kinases in Clostridium Acetobutylicum

Clostridium acetobutylicum harbors bulks of functional-like genes which belong to paralogous gene family and are involved in the same metabolic or regulatory pathway. Multigene disruption is key and urgent demand for investigating these genes function to engineer excellent strain, while conventionally performed by successive and time-consuming rounds. Here, we firstly demonstrate a multi-plasmid co-transformation strategy for simultaneously combinatorial disruption of histidine kinases (HKs) in C. acetobutylicum, which HKs function in controlling phosphorylation state of transcriptional regulator Spo0A. This strategy reliably produces stable mutants without the limitation of plasmids incompatibility. After optimization, a mutant library (71.0% of theoretical capacity) was generated with an efficiency of 47.5% and 4 weeks, boosting the screening of superior butanol-producing strain. Phenotypic and morphological analysis further elucidated the systematic regulation of HKs on cell growth, solvent metabolism and developmental attributes. Collectively, our strategy will facilitate the genetic dissection of complex regulatory network and the strain development.