An official website of the United States government
Enzymatic cascades play a crucial role in energy conversion and chemical transformations in living organisms. Due to enzymes’ high selectivity in catalysis and eco-friendly nature, operating enzymatic cascades in cell-free systems has the potential to improve existing chemical transformations. However, applications involving enzymes are often limited by their poor stability in cell-free environments, and the impact of immobilization on the kinetics of enzymatic cascades remains relatively underexplored, largely due to challenges in determining the support structure and controlling the enzyme immobilization process. In this work, we developed NU-1510-Cr, a chromium-based mesoporous metal–organic framework (MOF) with the mtn topology to encapsulate an enzyme cascade that oxidizes ethanol to acetaldehyde and subsequently to acetic acid. The crystalline hierarchical pores provide spatial control of the enzymes within the host, where the impact of the spatial organization on the cascaded reaction kinetics was assessed. The findings offer valuable insights for those aiming to immobilize and compartmentalize biocatalytic or chemocatalytic cascade systems and develop efficient microscale and nanoscale bioreactors.
more »
« less
This content will become publicly available on August 15, 2026
Hydrogel-Immobilized Multienzyme Systems for Cell-Free Chemical Bioproduction
Cell-free gene expression systems derived from bacterial lysates enable the expression of biosynthetic pathways from inexpensive and easily prepared DNA templates. These systems hold great promise for modular and on-demand bioproduction of valuable small molecules in resource-limited settings but are constrained in their long-term stability, reusability, and deployability. In this work, we demonstrate that multiple cell-free expressed enzymes can be co-immobilized in biocompatible hydrogels made from poly(ethylene glycol) diacrylate (PEGDA) with added glycerol for enhanced gel integrity. Using small-angle X-ray scattering (SAXS), we show that the mesh size of PEGDA-glycerol hydrogels is comparable to the globular sizes of many proteins and enzymes, which could be used for protein entrapment. We found that the combination between entrapment and chemical ligation of the enzymes was effective to retain proteins. By employing a method for direct fluorescence measurement from hydrogels, we found that proteins can be retained in PEGDA-glycerol for at least a week. By separating the cell-free enzyme expression from the immobilization step, we successfully fabricated enzyme-laden hydrogels with three heterologous cell-free enzymes for the bioconversion of pyruvic acid to malic acid, an industrially valuable and versatile precursor chemical. Both heterologous and endogenous enzymes from the lysate remain functional in photo-cross-linked hydrogels and can be reused for multiple biocatalytic cycles. Moreover, we also found that the immobilized enzymes exhibit up to 1.6-fold higher activity and 2-fold longer lifetimes than free enzymes in liquid reactions. These results could advance the deployment of cell-free synthetic biology because they show that reusable, stable, and durable multienzyme systems can be created using readily available materials and fabrication techniques.
more »
« less
- Award ID(s):
- 2029249
- PAR ID:
- 10654538
- Publisher / Repository:
- American Chemical Society, ACS Synthetic Biology
- Date Published:
- Journal Name:
- ACS Synthetic Biology
- Volume:
- 14
- Issue:
- 8
- ISSN:
- 2161-5063
- Page Range / eLocation ID:
- 3091 to 3104
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Not AvailableFormate, a biologically accessible form of CO2, has attracted interest as a renewable feedstock for bioproduction. However, approaches are needed to investigate efficient routes for biological formate assimilation due to its toxicity and limited utilization by microorganisms. Cell-free systems hold promise due to their potential for efficient use of carbon and energy sources and compatibility with diverse feedstocks. However, bioproduction using purified cell-free systems is limited by costly enzyme purification, whereas lysate-based systems must overcome loss of flux to background reactions in the cell extract. Here, we engineer an E. coli-based system for an eight-enzyme pathway from DNA and incorporate strategies to regenerate cofactors and minimize loss of flux through background reactions. We produce the industrial di-acid malate from glycine, bicarbonate, and formate by engineering the carbon-conserving reductive TCA and formate assimilation pathways. We show that in situ regeneration of NADH drives metabolic flux towards malate, improving titer by 15-fold. Background reactions can also be reduced 6-fold by diluting the lysate following expression and introducing chemical inhibitors of competing reactions. Together, these results establish a carbon-conserving, lysate-based cell-free platform for malate production, producing 64 μM malate after 8 h. This system conserves 43 % of carbon otherwise lost as CO2 through the TCA cycle and incorporates 0.13 mol CO2 equivalents/mol glycine fed. Finally, techno-economic analysis of cell-free malate production from formate revealed that the high cost of lysate is a key challenge to the economic feasibility of the process, even assuming efficient cofactor recycling. This work demonstrates the capabilities of cell-free expression systems for both the prototyping of carbon-conserving pathways and the sustainable bioproduction of platform chemicals.more » « less
-
RationalePurification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI). MethodsA protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis. ResultsSmall proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected. ConclusionsThe immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.more » « less
-
Polyurethanes (PUs) are a highly adaptable class of biomaterials that are among some of the most researched materials for various biomedical applications. However, engineered tissue scaffolds composed of PU have not found their way into clinical application, mainly due to the difficulty of balancing the control of material properties with the desired cellular response. A simple method for the synthesis of tunable bioactive poly(ethylene glycol) diacrylate (PEGDA) hydrogels containing photocurable PU is described. These hydrogels may be modified with PEGylated peptides or proteins to impart variable biological functions, and the mechanical properties of the hydrogels can be tuned based on the ratios of PU and PEGDA. Studies with human cells revealed that PU–PEG blended hydrogels support cell adhesion and viability when cell adhesion peptides are crosslinked within the hydrogel matrix. These hydrogels represent a unique and highly tailorable system for synthesizing PU-based synthetic extracellular matrices for tissue engineering applications.more » « less
-
The widespread consumption of PET worldwide has necessitated the search for environment‐friendly methods for PET degradation and recycling. Among these methods, biodegradation stands out as a promising approach for recycling PET. The discovery of duo enzyme system PETase and MHETase in 2016, along with their engineered variants, has demonstrated significant potential in breaking down PET. Previous studies have also demonstrated that the activity of the enzyme PETase increases when it is immobilized on nanoparticles. To achieve highly efficient and complete PET depolymerization, we immobilized both FAST‐PETase and MHETase at a specific ratio on magnetic nanoparticles. This immobilization resulted in a 2.5‐fold increase in product release compared with free enzymes. Additionally, we achieved reusability and enhanced stability of the enzyme bioconjugates.more » « less
