Comprehensive analysis of vanillin toxicity in Escherichia coli identifies targets for engineering improved biovanillin production

One of the key issues affecting yields of biovanillin in cell factories such as Escherichia coli is product toxicity, the mechanisms of which are poorly understood. To identify targets for engineering improved strains, we have studied mechanisms of vanillin toxicity in E. coli using adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome sequencing. Vanillin-tolerant strains isolated by ALE had distinct non-synonymous SNPs in the citrate synthase gene gltA, in addition to strain specific mutations in cpdA, rob and marC. One strain had a large ~10 kb deletion including the marRAB region. Our data provide new understanding and novel gene targets for engineering vanillin-tolerant strains of E. coli, including increasing citrate synthase activity using variant enzymes.