Combinatorial metabolic engineering enabled the development of efficient microbial cell factories for modulating gene expression to produce desired products. Here, we report the combinatorial metabolic engineering of Corynebacterium glutamicum to produce butyrate by introducing a synthetic butyrate pathway including phosphotransferase and butyrate kinase reactions and repressing the essential acn gene-encoding aconitase, which has been targeted for downregulation in a genome-scale model. An all-in-one clustered regularly interspaced short palindromic repeats interference system for C. glutamicum was used for tunable downregulation of acn in an engineered strain, where by-product-forming reactions were deleted and the synthetic butyrate pathway was inserted, resulting in butyrate production (0.52 +/- 0.02g/L). Subsequently, biotin limitation enabled the engineered strain to produce butyrate (0.58 +/- 0.01g/L) without acetate formation for the entire duration of the culture. These results demonstrate the potential homo-production of butyrate using engineered C. glutamicum. This method can also be applied to other industrial microorganisms.