Metabolic Engineering Strategies to Produce Medium-Chain Oleochemicals via Acyl-ACP:CoA Transacylase Activity
CABBI Theme: Conversion
Keywords: Genome Engineering, Genomics
Citation
Yan, Q., Cordell, W.T., Jindra, M.A., Courtney, D.K., Kuckuk, M.K., Chen, X., Pfleger, B.F. Feb. 24, 2022. “Metabolic-Modeling—Yield-Analysis-of-PhaG.” GitHub Repository, https://github.com/Pfleger-Lab/Metabolic-Modeling—Yield-Analysis-of-PhaG.
(A) Comparison of PhaG homolog activities in vivo by evaluating methyl ketone titers. A two-dimensional cluster map displays the sequence similarity of PhaG variants. Colored boxes and dots indicate the sequences tested. (B) FAME analysis of cultures harboring PhaG variants containing combinatrial point mutations.
Overview
Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation – an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.
Data
Codes for Calculating Theoretical Yields
- FAME Results
- Plasmid Maps
- Titers
- Reactions
Related Publication
Yan, Q., Cordell, W.T., Jindra, M.A., Courtney, D.K., Kuckuk, M.K., Chen, X., Pfleger, B.F. March 25, 2022. “Metabolic Engineering Strategies to Produce Medium-Chain Oleochemicals via Acyl-ACP:CoA Transacylase Activity.” Nature Communications 13:1619. DOI: 10.1038/s41467-022-29218-3.