Engineering <i>Pseudomonas putida</i> KT2440 for the production of isobutanol

Abstract

We engineered <i>P. putida</i> for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native <i>ilvC</i> and <i>ilvD</i> genes, and implementation of the feedback-resistant acetolactate synthase AlsS from <i>Bacillus subtilis</i>, ketoacid decarboxylase KivD from <i>Lactococcus lactis</i>, and aldehyde dehydrogenase YqhD from <i>Escherichia coli</i>. The resulting strain <i>P. putida</i> Iso2 produced isobutanol with a substrate specific product yield (<i>Y</i> _Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from <i>Carnobacterium maltaromaticum</i> to be a suitable alternative for isobutanol production, since replacement of <i>kivD</i> from <i>L. lactis</i> in <i>P. putida</i> Iso2 by the variant from <i>C. maltaromaticum</i> yielded an identical Y_Iso/S. Although <i>P. putida</i> is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non-growing conditions.