Enhanced Monensin Biosynthesis in Streptomyces cinnamonensis with Multigene Integration and Overexpression of Newly Identified Genes

Monensin is a polyether antibiotic produced by Streptomyces cinnamonensis . Due to its highly complex biosynthetic pathway, incomplete characterization of key biosynthetic genes has long hindered improvements in industrial-scale monensin production. Based on our previous transcriptomic analysis that key genes involved in monensin biosynthesis were initially identified in a high-yield S. cinnamonensis strain, this study focused on metabolic engineering of this strain through multi-gene integration strategy, targeting primary metabolism, precursor synthesis, ATP supply, stress resistance, and the monensin biosynthetic gene cluster. Results demonstrated that the co-expression of phosphofructokinase ( pfk ), citrate synthase ( cs ), methylmalonyl-CoA mutase subunit β ( mutB ), and methylmalonyl-CoA mutase ( mcm ) rendered the monensin titer from 12.6   g/L to 20.2   g/L, an approximately 60% increase compared with the original strain. Co-expression of the ATP synthase β subunit ( assβ ), the fungal thiol-coupled enzyme ( mac ), and the monensin biosynthetic enzyme ( monE ) allowed the monensin production from 12.6   g/L to 19.4   g/L, corresponding to an increase of above 50%. Furthermore, simultaneous co-expression of the seven genes using the novel pKHBT1 system increased the monensin titer to 26.2   g/L in a 5-L fermenter, more than double that of the original strain. These newly identified key metabolic genes were indeed associated closely with the monensin biosynthesis in S. cinnamonensis , which supplied a clue to metabolic engineering of the monensin production strain for efficient biosynthesis.