The utilisation of microbes for the production of enzymes and pharmaceutical proteins is an important step towards a sustainable future. However, industrial fermentation processes require energy for agitation, aeration and cooling. This energy requirement increases with the increasing microbial cell mass. To address this technological challenge, we previously developed a so-called ‘breathing’ fermentation vessel that allows effective gas exchange. The present study aimed to explore the potential application of the breathing vessel for enzyme production by the Gram-positive bacterial cell factory Bacillus subtilis. In particular, we compared production of the secreted α-amylase AmyQ by the genome-reduced B. subtilis strain IIG-Bs-27-31 during parallel culturing in breathing vessels and shake flasks. Enzyme yields and the cellular and extracellular proteome compositions were compared.