Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Abstract

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell's survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13 C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/ MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System

“…Furthermore, ATP overproduction negatively impacts CFE efficiency . Thus, for any given protein, an optimal range of initial reaction PEP concentrations exists where PEP energizes but is not inhibitory toward TX/TL. − This range is further defined by central metabolic reactions that consume ATP in an unproductive manner (i.e., not contributing to protein expression). ,, In the case of NRPS CFE, the A domain that is characteristic of these enzymes could also deplete the ATP pool, further contributing to expression limitations. , The TC/FlAsH protocol was thus tested as a potential strategy for assessing the sensitivity of BpsA expression to PEP concentrations, and for investigating whether the catalytic A domain also competes for this TX/TL resource.…”

Rewiring cell-free metabolic flux inE. colilysates using a block—push—pull approach

“…Contrary to the previous report ( 29 ), no formate was detected in any of the modified extracts or in the unmodified extract. PflB activity in lysates has been described in the past, so it is likely that formate was only accumulated here at undetectable levels or was actively consumed by formate dehydrogenases ( 29 , 30 , 37 ). Since it has been previously shown that LdhA and PflB can be simultaneously purified from the lysate by this procedure, decreasing lactate buildup was used as a proxy for evaluating the purification of both proteins ( 29 ).…”

“…Conventionally, for 1 mol glucose consumed, glycolysis generates 2 mol of NADH that is primarily recycled to NAD + via the lactate-forming LdhA enzyme ( Figure 1A ). Because this reaction sustains glycolysis by providing NAD + , lactate has been observed as a major fermentation product in the lysate metabolism of glucose ( 30 ). To redirect flux toward ethanol, an alternative ethanol-forming, [NAD + ]/[NADH] balancing pathway can be activated through the pyruvate dehydrogenase complex and aldehyde-alcohol dehydrogenase (PDHC–AdhE) pathway module ( 29 , 31 ).…”

“…depleted lysates) were normalized to the same protein concentration and used to prepare CFME reactions that were fed with a glucose substrate. All metabolite concentrations quantified in incubated reactions were normalized against time-zero controls, which were prepared as described previously ( 30 ). Furthermore, the variability of the depletion approach was assessed by performing this method in triplicate from the same batch of lysate and then using the resulting ‘depletion replicates’ to prepare replicate cell-free reactions ( Figure 2 ).…”

“…Reactions were incubated at 37°C for 20 h unless otherwise stated. For each replicate, a control time-zero reaction was prepared as described previously ( 30 ). Briefly, lysate metabolic activity was terminated with 5% trichloroacetic acid (TCA) prior to the addition of glucose to prepare controls.…”