Primary alcohols as substrates or products in whole-cell biocatalysis: Toxicity for Escherichia coli expression strains

Whole-cell biotransformations, either with resting or growing cells, are a method of choice for reactions that involve complex settings, such as multi-enzyme systems, or require complex and costly cofactors. Alcohols may play a central role as reagents, reaction intermediates or products in such a setting but are, as many other organic solvents, toxic to microorganisms. Albeit much is known on the effect of short-and long-chain alcohols on e.g. membrane fluidity, a systematic study on the influence of alcohols with different chain lengths on viability under biotechnological conditions is missing. We close this gap and report on the effects that alcohols exert in typical microbial expression strains. Towards this aim, we tested four different expression strains, Pseudomonas putida KT2440, Escherichia coli (E. coli) BL21(DE3) and Top 10 as well as Bacillus subtilis str. 168, for their tolerance towards short to middle-length aliphatic alcohols. For growing cells in complex media, the toxicity of primary alcohols rose with the chain length with tolerated concentrations of less than 1 vol% for compounds with 5 or more carbon atoms, in good accordance with reported logPO/W-correlated toxicity development. Interestingly, higher alcohol concentrations could be tolerated by growing cells at higher cell density and in resting-cell setups. In an exemplary whole-cell biotransformation to produce 1-hexanol from n-hexane by a recombinant cytochrome P450 monooxygenase system in E. coli Top10, this knowledge is then applied. Our study thus emphasizes the need of careful substrate and product dosing in whole-cell setups to avoid lethal and inactivating effects caused by the substances.

Automated summary

Whole-cell biotransformations are a preferred method for complex reactions involving multi-enzyme systems or costly cofactors. However, the toxicity of alcohols, which can serve as reagents, intermediates, or products, poses a challenge for microbial cells. This study investigates the tolerance of different expression strains to alcohols with varying chain lengths, providing important insights for whole-cell biotransformations.