초록
Isobutyrate is an important platform chemical with various industrial applications. Previously, a synthetic metabolic pathway was constructed in E. coli to produce isobutyrate from glucose. However, isobutanol was found to be a major byproduct. Herein, gene knockouts and enzyme overexpressions were performed to optimize further the engineered E. coli strain. Besides yqhD, the knockouts of three genes eutG, yiaY and ygjB increased isobutyrate production in shake flasks. Furthermore, the introduction of an additional padA on a medium copy number plasmid under the constitutive promoter significantly reduced isobutanol formation. The IBA15-2C strain (BW25113, DyqhD, DygjB; carrying two copies of padA) produced 39.2% more isobutyrate (0.39 g/glucose yield, 80% of the theoretical maximum yield) than IBA1-1C strain (BW25113, DyqhD; carrying one copy of padA). A scale-up process was also investigated for IBA15-2C strain to optimize the conditions for the production of isobutyrate in the fermentor. With Ca(OH)<sub>2</sub> as the base for pH control and 10% dissolved oxygen level, IBA15-2C strain produced 90 g/L isobutyrate after 144 h. This study has engineered E. coli to achieve biosynthesis of a nonnative compound with the highest titer and opened up the possibility of the industrial production of isobutyrate.