Improving Engineered<em> Escherichia coli</em> strains for High-level Biosynthesis of Isobutyrate

• Main Authors: Mingyong Xiong, Ping Yu, Jingyu Wang, Kechun Zhang
• Format: Article
• Language: English
• Published: AIMS Press 2015-05-01
• Series: AIMS Bioengineering
• Subjects: synthetic biology; isobutyrate; isobutanol; fermentor; <i>E.coli</i>
• Online Access: http://www.aimspress.com/Bioengineering/article/294/fulltext.html

Isobutyrate is an important platform chemical with various industrial applications. Previously, a synthetic metabolic pathway was constructed in <em>E. coli</em> 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 <em>E. coli</em> strain. Besides <em>yqhD</em>, the knockouts of three genes <em>eutG</em>,<em> yiaY </em>and<em> ygjB </em>increased isobutyrate production in shake flasks. Furthermore, the introduction of an additional <em>padA</em> on a medium copy number plasmid under the constitutive promoter significantly reduced isobutanol formation. The IBA15-2C strain (BW25113, D<em>yqhD, </em>D<em>ygjB</em>; carrying two copies of <em>padA</em>) produced 39.2% more isobutyrate (0.39 g/glucose yield, 80% of the theoretical maximum yield) than IBA1-1C strain (BW25113, D<em>yqhD</em>; carrying one copy of <em>padA</em>). 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)₂ 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 <em>E. coli</em> to achieve biosynthesis of a nonnative compound with the highest titer and opened up the possibility of the industrial production of isobutyrate.