| The current drive for sustainable routes to industrial chemicals and energy due to depleting fossil reserves has motivated research in the field of biotechnology. Biomass is abundant on earth and photosynthesis, if utilized properly, can lead the way to a sustainable carbon cycle. The recent advances in metabolic engineering, DNA sequencing, and protein engineering are driving research to achieve this goal. In this work, the native leucine and isoleucine biosynthetic pathways in Escherichia coli were expanded for the synthesis of pentanoic acid (PA) and 2-methylbutyric acid (2MB) respectively. Several aldehyde dehydrogenases and 2-ketoacid decarboxylases were studied for the conversion of ketoacids into respective carboxylic acids. The optimal combinations of these enzymes enabled production of 2.6 g/L 2MB with IPDC-AldH and 2.6 g/L of PA with IPDC-KDHba in shake-flask fermentations. The extension of these pathways to synthesize value-added chemicals like hydroxyacids was attempted. The cytochrome P450 BM-3 enzyme was engineered to enhance its ability to oxidize unactivated C-H bonds in the short-chain carboxylic acids. Nine mutants were created by rational. The mutant L437K showed 20 to 30-fold higher activity compared to the wild-type. A screening strategy was developed to evolve isobutyrate-hydroxylating activity in P450s based on the valine degradation pathway in Pseudomonas aeruginosa. As a growth-based selection strategy, it will allow screening of a large library. This work expands the library of chemicals that can be produced biologically as a step forward to the sustainable future. |
