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Cell-free biofilm materials as catalysts for biomanufacturing

The biofilms allow the assembly of complex biocatalytic manufacturing processes and enzyme-specific control on-the-fly

By Benjamin Boettner

Biomanufacturing is a rapidly growing sector because of its ability to harness biological systems to produce chemicals and materials that our society needs, ranging from pharmaceuticals to polymers to biofuels. In contrast to conventional manufacturing that relies heavily on petroleum-derived starting materials, biomanufacturing can make use of renewable energy sources and raw materials, like cellulosic biomass (i.e. plant waste). Established biomanufacturing methods often hijack the metabolism of microbial cells in order to transform low value feedstocks into higher value chemicals.

However, this approach has some inherent inefficiencies. One problem is that the energy used by microbial fermentation, the process by which bacteria convert carbon-containing sugars from their environment into other chemicals, can not be exclusively dedicated to the biomanufacturing process of interest but must be split three ways between growing the cells themselves, producing the desired chemical, and waste generation. In order to overcome this bottleneck, researchers utilize enzyme immobilization strategies in which all the enzymes needed for the transformation of a starting substance into a desired product are immobilized on a solid substrate in the absence of the rest of the cellular components. This ensures that all of the carbon flux in the system is channeled toward synthesizing a specific product, which also greatly simplifies its purification as no unspecific products related to cell growth and waste production are present in the mix. The challenge with this approach is that the labor and costs of manufacturing, purifying, and immobilizing the relevant enzymes are often quite high.

Wyss Institute researchers are producing de-cellularized biofilms on different surfaces that can perform complex biocatalytical manufacturing processes using multiple, individually replenishable enzymes. Credit: Wyss Institute at Harvard University

To address these issues, Neel Joshi’s team at the Wyss Institute has developed a way to leverage bacterial biofilm-derived proteins as biocatalysis scaffolds. They call their approach Biofilm Integrated Nanofiber Display (BIND), and it essentially converts the workhorse bacterium E. coli into a general platform for the fabrication of nanomaterials. In order to do this, the team exploits the ‘curli amyloid’ system, which secretes a self-assembling protein called CsgA and orchestrates the formation of a robust nanofiber mesh with it outside the cells. Joshi and his group have previously reported BIND as a way to make adhesive biofilm materials that can stick to different surfaces, such as steel, and immobilize various biotechnologically interesting proteins.  In 2015, the researchers managed to permanently immobilize a model enzyme, the type of protein that, among other things, helps transform a substrate into a useful product, to the nanofibers. In a follow-up study, they have now expanded the BIND functionality to reversibly immobilize multiple enzymes in biofilms that, in addition, have been depleted of the cell bodies that initially produced the biofilm materials.

“One advantage of BIND is that it sidesteps the need for bacterial cell growth during the chemical production phase and greatly simplifies the separation of the actual reaction product downstream. Moreover, in advancing BIND, we wanted to engineer more complex systems with more than one enzyme that we could manipulate on-the-fly in case, specific enzymes would lose their activity,” said first-author Martin Nussbaumer, Ph.D., who worked as a Postdoctoral Fellow with Joshi and now is a Researcher at F. Hoffmann-La Roche Ltd. “Many biotechnologically relevant conversions require multi-step reactions to convert a starting substrate to the final product. This means that lots of enzymes are needed, and some of them need expensive cofactors to work efficiently. We can make the whole cell-free system using the cells themselves and program the material to regenerate the cofactors that are needed.”

The team achieved these goals by appending various “connector” domains to CsgA, which assemble together with CsgA into the secreted extracellular nanofibers. Since enzymes are fused with complementary “connector” domains, the researchers can specifically immobilize them in the biofilm’s nanofiber matrix according to the key-lock principle without having to purify them. Furthermore, they demonstrated that different “connector” domains can be used in combination to allow for multiple enzymes to be immobilized simultaneously. This streamlines the process of creating multi-enzyme-based catalytic systems, and makes them potentially cost effective to use on industrial scales. The biofilms’ effectiveness is further enhanced by removal of their producing cells that otherwise would continue to grow and multiply, using up precious energy resources and producing unwanted products.

Another feature of the new system is that the “connector” domains can be disconnected under certain conditions. “This means that we can selectively remove a single enzyme from a multi-enzyme system and refresh it on the surface of the nanofibers. Not all enzymes are equally stable, so our system lets you regenerate a biocatalytic process by replacing a single component rather than the entire system,” said Wyss Core Faculty member Joshi, Ph.D., who also is Associate Professor of Chemical and Biological Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

In a biotechnologically relevant proof-of-principle experiment, the researchers engineered a bi-enzymatic biofilm system by combining two enzymatic reactions using two different connectors. “The first reaction is performed by an enzyme called RADH — an alcohol dehydrogenase enzyme from the bacterium Ralstonia sp., that only works in the presence of a small co-factor molecule known as NADPH and generates a desired chemical product. By incorporating a second enzyme that constantly replenishes the supply of NADPH using an NADPH-byproduct of RADH’s reaction, we managed to design a biofilm capable of simultaneously performing an industrial reduction reaction that regenerates itself in a coupled process,” said Wyss Technology Fellow and co-author Peter Nguyen, Ph.D.

The Wyss researchers think that, in the future, this technology will enable even more complex biosynthetic pathways to be reconstituted using this enzyme immobilization strategy. “This, combined with the fact that the enzyme-modified biofilm materials can be produced cheaply by fermentation and molded into complex shapes, will lead to new bioreactor designs that can address our urgent need for sustainable manufacturing methods,” said Joshi.

The study was published in ChemCatChem.

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