MALDI-MS Screening of Microbial Colonies With Isomer Resolution to Select Fatty Acid Desaturase Variants

Themes: Conversion

Keywords: Lipidomics, Mass Spectrometry

Citation

Choe, K.Jindra, M.A.Hubbard, S.C.Pfleger, B.F.Sweedler, J.V. May 3, 2023. “MALDI-MS Screening of Microbial Colonies with Isomer Resolution to Select Fatty Acid Desaturase Variants.” Analytical Biochemistry. DOI: 10.1016/j.ab.2023.115169.

Overview

Overall workflow of MALDI-MS screening of the Δ6 desaturase random library and validation of Δ8-active variants by GC-MS.

Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput assays, limiting the scale of redesign efforts to <200 variants. Here, we report a rapid mass spectrometry (MS) assay for profiling the positions of double bonds on membrane lipids produced by Escherichia coli colonies after treatment with ozone gas. By MS measurement of the ozonolysis products of Δ6 and Δ8 isomers of membrane lipids from colonies expressing recombinant Thunbergia alata desaturase, we screened a randomly mutagenized library of the desaturase gene at 5 s per sample. Two variants with altered regiospecificity were isolated, indicated by an increase in 16:1 Δ8 proportion. We also demonstrated the ability of these desaturase variants to influence the membrane composition and fatty acid distribution of E. coli strains deficient in the native acyl-ACP desaturase gene, fabA. Finally, we used the fabA deficient chassis to concomitantly express a non-native acyl-ACP desaturase and a medium-chain thioesterase from Umbellularia californica, demonstrating production of only saturated free fatty acids.

Data

Download (1.1 GB) includes:

  • MALDI data
  • Desat dry LB plate data
  • Membrane composition
  • FFA production strains