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PreprintCited by 2Enzyme Catalysis and ImmobilizationMass Spectrometry Techniques and ApplicationsMicrobial Natural Products and Biosynthesis

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A High-Throughput Biocatalytic Platform for Screening Isomeric Kainoid Natural Products

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Shepherd, Robert A., Ramachandra, Manasa, Hopiavuori, Austin R., Jones, Melanie C., Fihn, Conrad A., Tabag, Alex J., Manjunath, Ananya, Gad, Limar Y., Whipple, Chloe R., McKinnie, Shaun M. K., Sanchez, Laura M.·2025·Cell Reports Physical Science
DOI 10.1101/2025.09.07.674781PMC PMC12633004

Preprint — published as 10.1016/j.xcrp.2025.103092 · Paper page

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Abstract

Random mutagenesis generates large, diverse enzyme libraries, however high-throughput screening is challenging when transformations yield isomeric small molecule products. We developed a matrix-assisted laser desorption/ionization–trapped ion mobility spectrometry–mass spectrometry (MALDI-TIMS-MS) platform for rapid, label-free screening of libraries of kainoid synthases, Fe/αKG-dependent cyclases that produce the isomeric neurotransmitters kainic acid (KA) and kainic acid lactone (KAL). This platform achieves near-baseline separation of KA and KAL directly from microbial biomass, with parallel reaction monitoring–PASEF (PRM-PASEF) enhancing signal-to-noise for isomeric product ratio analysis. Using this workflow, we screened a 318-variant DsKabC/GfKabC DNA shuffle library in under 30 minutes, identifying seven variants with improved KAL conversion. Two variants showed a near-complete shift toward KAL production with greater substrate consumption, while retaining favorable expression profiles compared to GfKabC. These results establish MALDI-TIMS-MS as a generalizable platform for high-throughput isomeric product screening and provides new kainoid synthase variants to probe structure–function relationships for further development as biocatalysts. Importantly, this screening approach can be broadly applied to interrogate enzyme variants acting on small molecules, delivering rapid and accurate quantitative insights without reliance on chromatography, enzyme purification, or substrate derivatization.

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Figure 1. Overview of KabC structure and activity. A) Abbreviated mechanism for the conversion of prekainic acid to both kainic acid (KA) and kainic acid lactone (KAL) by KabC. The mechanistic branch point putatively occurs after pyrrolidine ring formation, with deprotonation resulting in KA formation, and nucleophilic addition of the side chain carboxylate resulting in the formation of KAL. B) The predicted substrate binding pockets of DsKabC and GfKabC are highly similar in composition, reflecting their overall high sequence similarity and identity (84 and 73%, respectively). C) LC-MS extracted ion chromatograms of m/z 212.09 ± 0.2, demonstrating the differing production of KA and KAL between DsKabC and GfKabC. D) Seven sites chosen for rational SDM based on multiple sequence alignment of KabC orthologs. Boxed in red is the Y347F mutant carried forward as an additional positive control for screening development. E) In vitro isomeric distribution and percent substrate conversion (represented by the gray diamonds) for DsKabC and the four prioritized single-point KabC variants via LC-MS (n = 1). The displayed LC-MS data were collected on a Bruker AmaZon SL.
Figure 1. Overview of KabC structure and activity. A) Abbreviated mechanism for the conversion of prekainic acid to both kainic acid (KA) and kainic acid lactone (KAL) by KabC. The mechanistic branch point putatively occurs after pyrrolidine ring formation, with deprotonation resulting in KA formation, and nucleophilic addition of the side chain carboxylate resulting in the formation of KAL. B) The predicted substrate binding pockets of DsKabC and GfKabC are highly similar in composition, reflecting their overall high sequence similarity and identity (84 and 73%, respectively). C) LC-MS extracted ion chromatograms of m/z 212.09 ± 0.2, demonstrating the differing production of KA and KAL between DsKabC and GfKabC. D) Seven sites chosen for rational SDM based on multiple sequence alignment of KabC orthologs. Boxed in red is the Y347F mutant carried forward as an additional positive control for screening development. E) In vitro isomeric distribution and percent substrate conversion (represented by the gray diamonds) for DsKabC and the four prioritized single-point KabC variants via LC-MS (n = 1). The displayed LC-MS data were collected on a Bruker AmaZon SL.CC BY-NC-ND · PubMed Central
Figure 2. Optimization of TIMS-MS parameters for monitoring KA and KAL. A) Optimization of TIMS resolution for KA and KAL. Increasing the ramp time and decreasing the 1/K0 range of acquisition maximized resolution for our analysis. B) Comparison between MALDI- and ESI-TIMS-MS for measuring the relative abundance of KA and KAL. When measuring the same mixture of standards, it was observed that ESI-TIMS-MS displayed a 65% decrease in the relative intensity of KAL, suggesting that MALDI-TIMS-MS is more reliable for relative kainoid quantitation. C) Employment of PRM-PASEF-MS significantly reduced chemical noise for our MALDI-based analysis, simplifying and increasing confidence in downstream data visualization.
Figure 2. Optimization of TIMS-MS parameters for monitoring KA and KAL. A) Optimization of TIMS resolution for KA and KAL. Increasing the ramp time and decreasing the 1/K0 range of acquisition maximized resolution for our analysis. B) Comparison between MALDI- and ESI-TIMS-MS for measuring the relative abundance of KA and KAL. When measuring the same mixture of standards, it was observed that ESI-TIMS-MS displayed a 65% decrease in the relative intensity of KAL, suggesting that MALDI-TIMS-MS is more reliable for relative kainoid quantitation. C) Employment of PRM-PASEF-MS significantly reduced chemical noise for our MALDI-based analysis, simplifying and increasing confidence in downstream data visualization.CC BY-NC-ND · PubMed Central
Figure 3. Optimization of on-agar DsKabC expression via MALDI-TIMS-MSI. A) Ion images of BL21 (DE3) E. coli expressing WT DsKabC, grown on LB/KAN agar containing 1 mM PKA substrate. The amount of IPTG and the temperature of incubation is varied between all four biological replicates and all images were collected during the same MALDI-MSI acquisition. B) Box and whisker plots highlighting the distribution of intensity values for m/z 212.0923 normalized by root mean square (RMS). The box and whisker plots are overlaid with violin plots highlighting the density and spread of normalized intensity values within each ion image. Significance was determined using Tukey’s post-hoc test, following a two-way ANOVA with temperature and IPTG presence serving as the independent variables. C) Ion images of BL21 DE3 E. coli expressing either WT DsKabC or DsKabC Y347F, grown on LB/KAN agar containing 1mM PKA substrate (m/z 212.0891). D) The average relative abundance of KA and KAL across all individual spectra for both WT DsKabC (n = 3052) and DsKabC Y347F (n = 3744). Y347F continues to display increased production of KAL relative to WT DsKabC, consistent with the initial in vitro assessment of Y347F activity via LC-MS (Figure 1E).
Figure 3. Optimization of on-agar DsKabC expression via MALDI-TIMS-MSI. A) Ion images of BL21 (DE3) E. coli expressing WT DsKabC, grown on LB/KAN agar containing 1 mM PKA substrate. The amount of IPTG and the temperature of incubation is varied between all four biological replicates and all images were collected during the same MALDI-MSI acquisition. B) Box and whisker plots highlighting the distribution of intensity values for m/z 212.0923 normalized by root mean square (RMS). The box and whisker plots are overlaid with violin plots highlighting the density and spread of normalized intensity values within each ion image. Significance was determined using Tukey’s post-hoc test, following a two-way ANOVA with temperature and IPTG presence serving as the independent variables. C) Ion images of BL21 DE3 E. coli expressing either WT DsKabC or DsKabC Y347F, grown on LB/KAN agar containing 1mM PKA substrate (m/z 212.0891). D) The average relative abundance of KA and KAL across all individual spectra for both WT DsKabC (n = 3052) and DsKabC Y347F (n = 3744). Y347F continues to display increased production of KAL relative to WT DsKabC, consistent with the initial in vitro assessment of Y347F activity via LC-MS (Figure 1E).CC BY-NC-ND · PubMed Central
Figure 4. Schematic of MALDI-TIMS-MS screening platform. A) Following mutagenic PCR, the genes are ligated into a pET28a(+) expression plasmid and transformed into E. coli. The transformation plates contain 1 mM of PKA substrate to allow for KabC expression and substrate conversion in the same incubation period. The resulting colonies are directly transferred to MALDI plates, and matrix applied for downstream analysis. B) MALDI-TIMS-MS acquisition is performed, generating mobilograms that can subsequently be compiled into *.csv files using the MPP software. C) The generated *.csv files are fed into an in-house python script, generating heatmaps of variant fold change. D) The relative abundance of KAL for each variant and template controls is calculated first. The fold change is calculated by dividing the relative KAL abundance for each variant by the average of 4 DsKabC template control spots (red circle) for a 96-spot MALDI target. Blue indicates high fold-change, white represents zero-fold change, and black represents spots in which the total TIMS peak area for KA + KAL was less than a defined threshold (1000 was used for our screening). Variants within 3 standard deviations of the average relative KAL abundance of the template controls are marked with a red X.
Figure 4. Schematic of MALDI-TIMS-MS screening platform. A) Following mutagenic PCR, the genes are ligated into a pET28a(+) expression plasmid and transformed into E. coli. The transformation plates contain 1 mM of PKA substrate to allow for KabC expression and substrate conversion in the same incubation period. The resulting colonies are directly transferred to MALDI plates, and matrix applied for downstream analysis. B) MALDI-TIMS-MS acquisition is performed, generating mobilograms that can subsequently be compiled into *.csv files using the MPP software. C) The generated *.csv files are fed into an in-house python script, generating heatmaps of variant fold change. D) The relative abundance of KAL for each variant and template controls is calculated first. The fold change is calculated by dividing the relative KAL abundance for each variant by the average of 4 DsKabC template control spots (red circle) for a 96-spot MALDI target. Blue indicates high fold-change, white represents zero-fold change, and black represents spots in which the total TIMS peak area for KA + KAL was less than a defined threshold (1000 was used for our screening). Variants within 3 standard deviations of the average relative KAL abundance of the template controls are marked with a red X.CC BY-NC-ND · PubMed Central
Figure 5. Overview of selected DNA-shuffled KabC variants. A) Isomeric distribution of KA and KAL represented as relative percent abundance, and percent substrate conversion for the selected DNA-shuffled variants determined via LC-MS in vitro assays. Grey diamonds represent data points collected using the timsTOF fleX mass spectrometer; pink diamonds represent datapoints collected using a Bruker AmaZon SL mass spectrometer. B) Correlation of measured relative KAL abundance between MALDI-TIMS-MS and LC-MS for the DNA shuffle variants studied in vitro. A high correlation was observed for KAL/[KA+KAL] between LC-MS and MALDI-TIMS-MS analysis on the seven purified shuffle library hits and WT DsKabC (R2 = 0.95). C) Simplified multiple sequence alignment schematic of the 7 shuffled variants. D) Comparison between the predicted structures of DsKabC (blue) and GfKabC (orange) with that of the consensus between all seven shuffled variants tested in vitro.
Figure 5. Overview of selected DNA-shuffled KabC variants. A) Isomeric distribution of KA and KAL represented as relative percent abundance, and percent substrate conversion for the selected DNA-shuffled variants determined via LC-MS in vitro assays. Grey diamonds represent data points collected using the timsTOF fleX mass spectrometer; pink diamonds represent datapoints collected using a Bruker AmaZon SL mass spectrometer. B) Correlation of measured relative KAL abundance between MALDI-TIMS-MS and LC-MS for the DNA shuffle variants studied in vitro. A high correlation was observed for KAL/[KA+KAL] between LC-MS and MALDI-TIMS-MS analysis on the seven purified shuffle library hits and WT DsKabC (R2 = 0.95). C) Simplified multiple sequence alignment schematic of the 7 shuffled variants. D) Comparison between the predicted structures of DsKabC (blue) and GfKabC (orange) with that of the consensus between all seven shuffled variants tested in vitro.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Shepherd, R. A., Ramachandra, M., Hopiavuori, A. R., Jones, M. C., Fihn, C. A., Tabag, A. J., Manjunath, A., Gad, L. Y., Whipple, C. R., McKinnie, S. M. K., & Sanchez, L. M.
Affiliations
University of California, Santa Cruz (US)
Corresponding author
  • McKinnie, Shaun M. K.
  • Sanchez, Laura M.
ORCID
Venue
Cell Reports Physical Science
Publisher
openRxiv
Published
2025-09-12
Volume
7
Issue
2
Pages
103092-103092
ISSN
2666-3864 (ISSN-L)
DOI
10.1101/2025.09.07.674781
PMC
PMC12633004
OpenAlex
W7127981111
Cited by
2
References
1
Citations by year
2026: 2
FWCI
4.10
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports Physical Science (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by-nc-nd)
Funders
National Institutes of Health (R21GM148870, K12GM139185)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
Keywords
kainic acid, high-throughput enzyme screening, structure-function relationship, enzyme stability

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