Research ArticleSYNTHETIC BIOLOGY

High-throughput mapping of CoA metabolites by SAMDI-MS to optimize the cell-free biosynthesis of HMG-CoA

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Science Advances  05 Jun 2019:
Vol. 5, no. 6, eaaw9180
DOI: 10.1126/sciadv.aaw9180
  • Fig. 1 A generalized approach for capturing CoA-bound metabolites.

    (A) A peptide with an N-terminal cysteine readily reacts with the thioester of CoA metabolites, creating a stable amide bond with the acyl group. After capture, the thiol of the peptide can then be used to immobilize the analyte and peptide onto a maleimide-presenting monolayer. (B) Five-hundred micromolar CoA conjugates of acetyl, acetoacetyl, HMG, and succinyl was reacted with the peptide CAK(Me3)SA. The resulting SAMDI spectra show that all analytes can be efficiently detected. m/z, mass/charge ratio.

  • Fig. 2 A cell-free metabolic pathway from glucose to isoprenoid intermediate HMG-CoA.

    (A) Cellular overexpression of Ac-CoA acetyltransferase and HMG-CoA synthase and subsequent lysis produces enzyme-enriched lysates, which can convert glucose to HMG-CoA, as well as acetate and glutamate. The pathway includes Ac-CoA and AA-CoA intermediates. SAMDI can capture metabolites from crude lysates. NADH, reduced form of NAD+. (B) Cell-free reactions containing lysates, cofactors, salts, and substrate were performed in standard 384-well microtiter plates. Reactions were then quenched, and any CoA-bound products were captured by incubation with the sensor peptide. Complex reaction mixtures were printed onto monolayer arrays using liquid-handling robotics for isolation and detection. HMG-CoA accumulates in the reaction over time for both (C) acetate salts and (D) glutamate salts. The dominant carbon source used for HMG production was determined by feeding cell-free reactions 13C-labeled glucose and 13C-labeled acetate and monitoring isotopic incorporation into the HMG product. The concentration of cofactors ATP, NAD+, and CoA was set to 1 mM each.

  • Fig. 3 G3P can be detected.

    The initial steps of glycolysis were reconstituted in situ using purified enzymes and fed glucose. When sufficient enzymes are present, G3P is captured and detected.

  • Fig. 4 Derivatives of non–CoA bound G3P were captured by the sensor peptide.

    (A) A possible mechanism for detection of G3P as N-lactyl-peptide is via pyruvaldehyde, which is known to be generated from G3P and can undergo rearrangement with thiols to form lactyl-thioesters. (B) Pyruvaldehyde can also undergo pH-dependent aldol condensation to yield a six-carbon species that overlaps in mass with the desired HMG product.

  • Fig. 5 Parallel measurement of metabolites.

    Ac-CoA, HMG-CoA, and G3P production was profiled in 768 unique cofactor conditions. Each product was quantified by calculating percent conversion relative to the unreacted peptide sensor using the area under the curve for each species. All reactions proceeded for 2 hours at 37°C.

  • Fig. 6 HMG-CoA concentration and carbon source shifts in response to cofactor conditions.

    (A) To analyze HMG-CoA yield across all conditions, the dataset was normalized to an internal standard, a peptide of similar sequence to the sensor without an N-terminal Cys, present at a constant concentration across all reactions. 13C-labeled glucose was used to concurrently monitor the fraction of HMG-CoA product coming from glucose. HMG production was also visualized as four-dimensional plots for both (B) acetate and (C) glutamate systems. For the three highest yielding conditions in each system, average [HMG] was determined. In these plots, each point represents a specific concentration condition of cofactors with [ATP], [NAD+], [CoA] on the x, y, and z axes, respectively. Color of each point represents yield with the highest yield represented by red. From acetate to glutamate, the red region shifts from high CoA and high ATP to moderate CoA and low ATP.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/6/eaaw9180/DC1

    Fig. S1. Second-order kinetics for the reaction of CoA metabolites with the peptide.

    Fig. S2. Description of spectral analysis.

    Fig. S3. The pathway intermediate, AA-CoA, is not observed.

    Fig. S4. Limit of detection for HMG-CoA.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Second-order kinetics for the reaction of CoA metabolites with the peptide.
    • Fig. S2. Description of spectral analysis.
    • Fig. S3. The pathway intermediate, AA-CoA, is not observed.
    • Fig. S4. Limit of detection for HMG-CoA.

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