OBJECTIVES: Clostridium butyricum ferments non-digested dietary fibre in the colon to produce butyric acid. Butyrate is a four-carbon, short-chain fatty acid (SCFA) which has multiple health benefits. Many microbial products of pharmaceutical or industrial interest, such as butyrate, are produced in low quantities due to rate-limiting steps in their metabolic pathway, including low abundance or low activity of one or more enzymes in the pathway. By identifying the former, appropriate enzymes can be over-expressed to increase product yields, however, methods to determine these enzymes are laborious. To improve butyrate production in C. butyricum probiotic strain, CBM588, a novel rapid genome-proteome approach was deployed. METHODS: First, whole genome sequencing was performed and the 8 genes involved in butyrate production identified on the chromosome. Second, the relative abundance of these enzymes was investigated by liquid chromatography-mass spectrometry (LC-MS) analysis of total cytosolic proteins from early stationary phase cultures. RESULTS: Phosphotransbutyrylase (Ptb), butyrate kinase (Buk) and crotonase (Crt) were found to be the least abundant. Over-expression episomally of the corresponding genes individually or of the ptb-buk bicistron led to significant increases in butyrate titre per density of culture from 10 to 24 h, compared to the wild type. CONCLUSIONS: Our findings pave the way for over-expressing these genes chromosomally to generate a recombinant probiotic with improved butyrate production and potentially enhanced gut health properties.