Claisen Condensation Reaction Mediated Pimelate Biosynthesis via the Reverse Adipate‐Degradation Pathway and Its Isoenzymes

Abstract

Pimelic acid is an important seven‐carbon dicarboxylic acid, which is broadly applied in various fields. The industrial production of pimelic acid is mainly through a chemical method, which is complicated and environmentally unfriendly. Herein, we found that pimelic acid could be biosynthesized by the reverse adipate‐degradation pathway (RADP), a typical Claisen condensation reaction that could be applied to the arrangement of C−C bond. In order to strengthen the supply of glutaryl‐CoA precursor, PA5530 protein was used to transport glutaric acid. Subsequently, we discovered that the enzymes in the BIOZ pathway are isoenzyme of the RADP pathway enzymes. By combining the isoenzymes of the two pathways, the titer of pimelic acid reached 36.7 mg ⋅ L−1 under the optimal combination, which was increased by 382.9 % compared with the control strain B‐3. It was also the highest titer of pimelic acid biosynthesized by Claisen condensation reaction, laying the foundation for the production of pimelic acid and its derivatives.

Rational Design of the Substrate Tunnel of β-Ketothiolase Reveals a Local Cationic Domain Modulated Rule that Improves the Efficiency of Claisen Condensation

“…Biotechnological applications benefit from highly active and thermostable enzymes to perform catalysis effectively and economically. , In our previous study, Thermobifida fusca β-ketothiolase Tfu_0875 exhibited excellent catalytic properties for the biosynthesis of dicarboxylic acids. − Herein, we demonstrated that Tfu_0875 possesses high thermostability and enzyme activity. In order to rationally design the substrate tunnel of Tfu_0875 to further improve enzyme activity, we applied the deep learning approach (DLKcat) to predict k cat values based on the high-resolution three-dimensional (3D) protein structure .…”

Enhancing the activity and succinyl-CoA specificity of 3-ketoacyl-CoA thiolase Tfu_0875 through rational binding pocket engineering

“…Tfu_0875 belongs to the thiolase I which prefers medium-chain length substrates [ 8 ]. Therefore, we selected acetyl-CoA, malonyl-CoA, succinyl-CoA, and glutaryl-CoA as substrates to condensation with an extension unit acetyl-CoA by Tfu_0875 for evaluating the substrate specificity [ [14] , [15] , [16] ]. As a result, Tfu_0875 demonstrated a relatively broad substrate range, spanning from C2 to C5 carbon chain lengths.…”

“…Recently, we developed a reverse adipate degradation pathway (RADP) for the production of dicarboxylic acids, including glutaric acid [ 14 ], adipic acid [ 15 ], and pimelic acid [ 16 ], which play an important role as biofuels [ 11 ] and nylon monomers 9 . The 3-ketoacyl-CoA thiolase, a member of the thiolase I family, serves as the initial and rate-limiting enzyme in the RADP pathway.…”

“…The 3-ketoacyl-CoA thiolase, a member of the thiolase I family, serves as the initial and rate-limiting enzyme in the RADP pathway. It catalyzes the Claisen condensation reaction between substrates such as malonyl-CoA, succinyl-CoA, or pimeloyl-CoA, and extension unit acetyl-CoA, resulting in the formation of a 3-oxoacyl-CoA intermediates of dicarboxylic acids [ [15] , [16] , [17] ]. However, the wide substrate spectrum of 3-ketoacyl-CoA thiolase often generates mixed products which limited their application in biosynthesis of specific compounds [ 2 ].…”

Mid–Long Chain Dicarboxylic Acid Production via Systems Metabolic Engineering: Progress and Prospects

“…The yield of adipic acid reached 2.50 g/L in a controlled bioreactor with a high initial glycerol concentration. Recently, Bao et al 100 also achieved a high concentration of pimelic acid (36.7 mg/L) using the Claisen condensation reaction. This was accomplished by adopting glutaryl coenzyme A as a precursor and expressing RADP and BioZ pathway isoenzymes in E. coli.…”