Evaluation of Strategies to Narrow the Product Chain-Length Distribution of Microbially Synthesized Free Fatty Acids

Themes: Conversion

Keywords: Genomics


Jindra, M.A.Choe, K., Chowdhury, R., Kong, R., Ghaffari, S., Sweedler, J.V.Pfleger, B.F. March 1, 2023. “Evaluation of Strategies to Narrow the Product Chain-Length Distribution of Microbially Synthesized Free Fatty Acids.” Metabolic Engineering. DOI: 10.1016/j.ymben.2023.02.012.


(A) Pipeline for preparation, screening, and validation of BTE variants with enhanced C12 FFA production capability using MALDI-ToF MS screen. (B) Heatmaps showing in vivo results of BTE variants selected after MALDI-ToF analysis with total FFA titer on the left and the C12 to C14 ratio within the product distribution on the right. Titers represent methyl ester derivatives of bulk cell cultures, including FFA and membrane lipids. One asterisk indicates a p-value of less than 0.05 when subjecting C12 to C14 ratio of the respective variant and the WT to a one-sided t-test. Two asterisks indicate a p-value of less than 0.01.

The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by heterologous hosts is expression of a selective acyl-acyl carrier protein (ACP) thioesterase. However, few of these enzymes can generate a precise (greater than 90% of a desired chain-length) product distribution when expressed in a microbial or plant host. The presence of alternative chain-lengths can complicate purification in situations where blends of fatty acids are not desired. We report the assessment of several strategies for improving the dodecanoyl-ACP thioesterase from the California bay laurel to exhibit more selective production of medium-chain free fatty acids to near exclusivity. We demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was an effective library screening technique for identification of thioesterase variants with favorable shifts in chain-length specificity. This strategy proved to be a more effective screening technique than several rational approaches discussed herein. With this data, we isolated four thioesterase variants which exhibited a more selective FFA distribution over wildtype when expressed in the fatty acid accumulating E. coli strain, RL08. We then combined mutations from the MALDI isolates to generate BTE-MMD19, a thioesterase variant capable of producing free fatty acids consisting of 90% of C12 products. Of the four mutations which conferred a specificity shift, we noted that three affected the shape of the binding pocket, while one occurred on the positively charged acyl carrier protein landing pad. Finally, we fused the maltose binding protein (MBP) from E. coli to the N – terminus of BTE-MMD19 to improve enzyme solubility and achieve a titer of 1.9 g per L of twelve-carbon fatty acids in a shake flask.


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