Researchers at the Indian Institute of Science (IISc) have developed a highly efficient cell-free enzyme system that converts fatty acids into 1-alkenes – versatile hydrocarbons that can serve as "drop-in" biofuels, polymer feedstocks, or pharmaceutical precursors.
The team leveraged a membrane-bound bacterial enzyme called UndB and improved its performance using enzyme engineering, co-substrate recycling, and molecular simulation-guided redesign.
"Our goal is the bioproduction of hydrocarbons using these really interesting metalloenzymes," says Debasis Das, corresponding author and Associate Professor at the Department of Inorganic and Physical Chemistry (IPC), IISc. "We want to understand the fascinating chemistry that these enzymes have in terms of catalysis, and also harness their powerful features for hydrocarbon production."
The team previously demonstrated a whole-cell biosynthetic system that used E. coli to express UndB as a fusion enzyme with another enzyme called catalase. But this approach had a few drawbacks: maintaining optimal reaction conditions was difficult, large quantities of expensive cofactors were necessary, and the enzyme was potentially toxic to the bacterial cells at high concentrations.
To solve this, the team integrated UndB into a self-sustaining cell-free system that mimics the biological reaction environment without relying on living cells. They broke apart E. coli cells and extracted the UndB-containing membrane fraction, then combined this in a solution with catalase, cofactors, and a pair of cofactor-recycling enzymes. Fatty acid feedstock could then be added directly to this cell-free reaction mixture.
"A cell-free system has a lot of advantages," explains Das. "You can control the reaction better, optimise the system more easily, and avoid the regulatory challenges that accompany whole-cell biocatalysts."
By decoupling from cellular constraints, the team achieved a remarkable 262-fold increase in the enzyme's turnover number (a measure of catalytic performance), and a ~13-fold increase over the previous whole-cell system. They also achieved a drastic reduction in the consumption of expensive cofactors, and near-complete substrate conversion (up to 98% yield) even with minimal concentrations of the enzyme. The reaction conditions required were also mild – ambient temperature and neutral pH – and no toxic byproducts were generated, making the process environment-friendly.
But the system still struggled with processing long-chain fatty acids, which are more abundant compared to medium-length fatty acids. When the team examined UndB variants across bacterial species, they discovered that there were two distinct classes that preferred fatty acid substrates of different lengths. They turned to molecular dynamics simulations to understand why.
"We found that subtle structural changes modulate the tunnel cavity inside the enzyme so that it can accommodate longer-chain fatty acids," says Abhishek Sirohiwal, co-corresponding author and Assistant Professor at IPC, who led the computational analysis.
By introducing targeted structural changes – replacing a helical region far from the catalytic site – the team engineered a version of UndB that could efficiently process fatty acids of greater lengths. "Enzyme engineering along with computational studies gave us a hold on the residues dictating the substrate specificity," says Subashini Murugan, joint first author and PhD student at IPC. This led to higher yields of longer 1-alkenes like 1-pentadecene, which are important pharmaceutical and biomembrane precursors.
Fatty acids are inexpensive and abundant, especially in organic waste; their value multiplies several-fold when converted to alkenes. The team's biocatalytic platform provides an alternative to synthesis from petrochemicals. "If we can convert waste oils to alkenes, which can be used as fuels or building blocks for polymers or lubricants, then we achieve both environmental and economic benefits," says Das.
The team has been granted a patent for this technology and is exploring industrial collaborations for large-scale production.
