Characterisation of the butyrate production pathway in probiotic MIYAIRI588 by a combined whole genome-proteome approach

Butyrate is a short chain fatty acid with important industrial applications produced by chemical synthesis. With consumer demand for green products, the fermentative production of butyric acid by microorganisms such as Clostridium is attracting interest. Clostridium butyricum ferments non-digested dietary fibre in the colon to produce butyrate which has multiple health benefits, and certain strains are exploited as probiotics, such as MIYAIRI588 (CBM588). Knowledge of the genes encoding enzymes involved in butyrate production and determining those that are rate-limiting due to low concentrations, could enable strain engineering for higher yields. To this end whole genome sequencing of CBM588 was performed and a circular chromosome, a megaplasmid and the previously reported cryptic plasmid, pCBM588, identified. All genes involved in the butyrate production pathway were found on the chromosome. To identify rate-limiting steps, the relative abundance of the encoded enzymes was assessed by liquid chromatography-mass spectrometry (LC-MS) of total cytosolic proteins. Phosphotransbutyrylase (Ptb) was the least abundant closely followed by butyrate kinase (Buk) and crotonase (Crt). Analysis of upstream regulatory sequences revealed the potential importance of an intact Shine-Dalgarno sequence. Results of this study can now guide bioengineering experiments to improve butyrate yields and enhance the performance of CBM588 as a probiotic. ### Competing Interest Statement The authors have declared no competing interest.