In vitro transcription-based biosensing of glycolate for prototyping of a complex enzyme cascade

Abstract

In vitro metabolic systems allow the reconstitution of natural and new-to-nature pathways outside of their cellular context and are of increasing interest in bottom-up synthetic biology, cell-free manufacturing, and metabolic engineering. Yet, the analysis of the activity of such in vitro networks is very often restricted by time- and cost-intensive methods. To overcome these limitations, we sought to develop an in vitro transcription (IVT)-based biosensing workflow that is compatible with the complex conditions of in vitro metabolism, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a 27-component in vitro metabolic system that converts CO₂ into glycolate. As proof of concept, we constructed a novel glycolate sensor module that is based on the transcriptional repressor GlcR from Paracoccus denitrificans and established an IVT biosensing workflow that allows us to quantify glycolate from CETCH samples in the micromolar to millimolar range. We investigate the influence of 13 (shared) cofactors between the two in vitro systems to show that Mg²⁺, adenosine triphosphate , and other phosphorylated metabolites are critical for robust signal output. Our optimized IVT biosensor correlates well with liquid chromatography-mass spectrometry-based glycolate quantification of CETCH samples, with one or multiple components varying (linear correlation 0.94-0.98), but notably at ∼10-fold lowered cost and ∼10 times faster turnover time. Our results demonstrate the potential and challenges of IVT-based systems to quantify and prototype the activity of complex reaction cascades and in vitro metabolic networks.

Keywords: CETCH cycle; GlcR; allosteric transcription factors; biosensing; in vitro metabolic system; in vitro transcription; pathway prototyping; screening method.

Graphical abstract

Figure 1.

Characterization of a glycolate-responsive IVT-based biosensor module. (a) The ROSALIND system is based on the controlled expression of the 3WJdB RNA aptamer and the correlating fluorescence signal of the 3WJdB:DFHBI-1T complex. Expression is regulated by a transcriptional repressor that binds to an operator sequence downstream of a T7 promoter. The repression is lifted in a dose-responsive manner by binding an effector molecule to the aTF. This system allows faster and cheaper sample measurement in microtiter plates than analysis by LC–MS, but its precision is yet unknown. Time estimates refer to glycolate quantification. (b, c) Dose–response of the GlcR sensor module to glycolate in the concentration range of 100 nM to 100 mM glycolate (8 h time point, further time points shown in Supplementary Fig. S5e). The sensor shows an operational range from 10 µM to 20 mM glycolate with an excellent correlation between 16 µM and 8 mM glycolate and a 10-fold dynamic range. Note that IVT biosensing responses are highly time sensitive with better correlation at early time points and better sensitivity at late time points (Supplementary Fig. S5; Table S8). (d) Promiscuity assay of GlcR shows a low-level dose response to dl-lactate and dl-glycerate (4 h time point, further data shown in Supplementary Fig. S6). Raw fluorescence data are standardized to MEF (µM fluorescein). Data are the mean of 3 technical replicates ± SD. IVT output without the effector molecule and without MGlcR is shown as horizontal dotted lines.

Figure 2.

Individual influence of CETCH cycle components on IVT in the absence of GlcR. (a) Reaction sequence of the CETCH cycle to convert CO₂ into glycolate [2, 19]. Ten nonenzymatic components are actively involved in the cycle, and three additional components are required to maintain enzyme activity during storage. (b) Titration of ATP, coenzyme B₁₂, MgCl₂, and NADPH concentrations in IVT reactions shows a dose-dependent influence of each component on IVT output after 4 h. Detailed data for nine additional components are shown in Supplementary Fig. S7. Raw fluorescence data are standardized to MEF (µM fluorescein). Data are the mean of n = 3 technical replicates ± SD. (c) Heat map describing the influence of all 13 nonenzyme components of the CETCH cycle on IVT output [as shown in (b) and Supplementary Fig. S7]. Data are normalized to IVT output in the absence of the respective component to indicate inhibition, enhancement, and no effect of the screened component at the indicated concentration.

Figure 3.

Glycolate sensing from CETCH cycle samples with a single component, the concentration of enzyme Mco, varied. (a) Schematic of the experimental setup. (b) LC–MS quantification of glycolate from six CETCH cycle samples with the titrated Mco concentration measured in technical triplicates. (c) Time course of glycolate measurement using the GlcR sensor module. Time points shown in (f) are indicated as vertical dotted lines. (d) Time course of IVT measurement in the absence of GlcR showing no differences in inhibition by CETCH cycle samples. (e, f) Correlation between GlcR sensor module output and LC–MS quantification [as shown in (b)] over time. The quality of the correlation is time sensitive and worsens as soon as the first IVT reactions plateau. Data of 4-h and 8-h time points are exemplarily shown in (f). Raw fluorescence data are standardized to MEF (µM fluorescein). Data are the mean of n = 3 technical replicates ± SD (c, d, f).

Figure 4.

Glycolate sensing from CETCH cycle samples with varied concentrations of nonenzyme components and enzymes. (a) Schematic of the experimental setup. (b) LC–MS quantification of glycolate from six CETCH cycle samples of different compositions (detailed compositions listed in Supplementary Table S6), measured in technical triplicates. (c, i) Time course of glycolate measurement with the GlcR module without and with the addition of 20 mM MgCl₂, respectively. (d, h) Correlation between GlcR module output [4-h time point, indicated as dashed lines in (c, i)] and LC–MS quantification [as shown in (b)]. See Supplementary Fig. S10b for correlation coefficients of hourly time points. (e) Correlation between free Mg²⁺ and IVT output in the absence of GlcR. Free Mg²⁺ is approximated as the concentration difference of added MgCl₂ and Mg²⁺ binding to estimate the change in free Mg²⁺ upon addition of the CETCH sample to the GlcR sensor module. (f) Titration from 0 to 30 mM MgCl₂ showed a dose-dependent IVT output in the presence and absence of GlcR. In the presence of 750 nM GlcR, the leakiness of the repressed module increased linearly, whereas in the absence of GlcR, the IVT output was bell-shaped. (g) Effect of additional 20 mM MgCl₂ on the constitutive repressed and derepressed state of the GlcR sensor module. Elevated MgCl₂ concentrations increased the leakiness of the module but did not affect the response to 1 mM glycolate. Data shown are 4-h time points, normalized to data with only MGlcR added. (c–i) Raw fluorescence data are standardized to MEF (µM fluorescein). Data are the mean of n = 3 technical replicates ± SD.