Metabolic Engineering of Bacillus licheniformis for Sustainable Production of Isobutanol

Isobutanol is regarded as a renewable alternative to fossil fuels. In this study, Bacillus licheniformis DWc9n was able to grow in the presence of 16 g/L isobutanol, showing high resistance to isobutanol. To achieve high-level production of isobutanol, a push–pull–restrain strategy was employed. First, native alcohol dehydrogenase YugJ with the highest synthesis efficiency of isobutanol was identified by comparing different heterologous and endogenous alcohol dehydrogenases. Eliminating competing pathways resulted in a remarkable increase in isobutanol production. Moreover, gene bcd encoding leucine dehydrogenase was identified as the rate-limiting enzyme for isobutanol production, and overexpression of bcd led to a 3.29-fold increase in isobutanol titer, increasing to 5.54 g/L from 1.29 g/L. Subsequently, we conducted chromosome engineering to increase the supply of precursor α-ketoisovalerate and combined cofactor engineering to increase the availability of NADPH. The resultant strain IB13 produced 7.81 g/L isobutanol, increasing by 40.11-fold compared to its parental strain IB1. Finally, 10.80 g/L isobutanol with a yield of 44% (mol/mol glucose) in shake flasks was achieved under the optimal fermentation conditions, which was the highest obtained for Bacillus species. Our results demonstrated the potential to engineer B. licheniformis strain to produce 2-keto acid-derived chemicals such as isobutanol efficiently from renewable feedstocks.