Arnold Lab Uses Directed Evolution to Create Siloxane-Busting Enzyme

An artist's depiction of an artificially evolved enzyme breaking a silicon-carbon bond.
An artist’s depiction of an artificially evolved enzyme breaking a silicon-carbon bond. [Caltech/Dow]

For the first time, scientists have engineered an enzyme that can break the stubborn man-made bonds between silicon and carbon that exist in widely used chemicals known as siloxanes, or silicones. The California Institute of Technology (Caltech) team, and collaborators, used directed evolution to engineer a bacterial cytochrome P450 variant that could break silicon-carbon bonds in both linear and cyclic volatile methylsiloxanes (VMS), a common subgroup of the siloxane family.

“Nature is an amazing chemist, and her repertoire now includes breaking bonds in siloxanes previously thought to evade attack by living organisms,” said research lead Frances Arnold, PhD, the Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry at Caltech. Arnold won the 2018 Nobel Prize in Chemistry for her pioneering work in directed evolution, a method for engineering enzymes and other proteins using the principles of artificial selection.

The researchers say that while practical uses for their engineered enzyme could still be a decade away or more, its development opens the possibility that manmade siloxanes—which can persist in the environment—could one day be degraded biologically. “For example, natural organisms could evolve in siloxane-rich environments to catalyze a similar reaction, or further improved versions of laboratory-evolved enzymes such as this one could possibly be used to treat siloxane contaminants in wastewater,” Arnold said.

Arnold and colleagues, including Dimitris Katsoulis, PhD, of Dow Inc. reported on their work in Science, in a paper titled “Directed evolution of enzymatic silicon-carbon bond cleavage in siloxanes,” in which they concluded, “Discovery of this so-called siloxane oxidase opens possibilities for the eventual biodegradation of VMS.”

Siloxane chemicals can be found in countless products, including those used in household cleaning, personal care, and the automotive, construction, electronics, and aerospace industries. These non-biodegradable chemicals are produced at megaton-per-year scale, the authors explained…“Linear and cyclic volatile methylsiloxanes (VMS) are anthropogenic compounds with material properties—such as high backbone flexibility and low surface tension—that make them useful in many consumer applications, from detergents and antifoaming agents to lotions, shampoos, and hair conditioners.” Cyclic VMS are also important feedstocks for the synthesis of silicone polymers.

The compounds’ chemical backbone is made of silicon–oxygen bonds, while carbon-containing groups, often methyl, are attached to the silicon atoms. “The silicon–oxygen backbone gives the polymer an inorganic-like character while the silicon–methyl groups give the polymer organic-like characteristics,” Katsoulis noted. “Thus, these polymers have unique material properties, such as high thermal and oxidative stability, low surface tension, and high backbone flexibility among others.” And while silicon-carbon bonds are not used in nature, Katsoulis noted, “… we do and have been for about 80 years. The volatile nature of some of these compounds warrants health and environmental research to properly understand the degradation mechanisms of these materials in the environment.”

Siloxanes are believed to persist in the environment for days to months, and ongoing research aims to provide greater scientific understanding of the health and environmental safety of silicone materials. The authors further added, “… the societal benefits of VMS must be balanced with their potential for environmental contamination, bioaccumulation and toxicity.” The chemicals naturally start to fragment into smaller pieces, especially in soil or aquatic environments, and those fragments become volatile or escape into the air, where they undergo degradation by reacting with free radicals in the atmosphere. Of all the bonds in siloxanes, the silicon–carbon bonds are the slowest to break down. “Degradation of VMS is nontrivial owing to their high thermal stability and lack of functional group handles,” the team added. “Hydrolysis of the Si–O bonds merely leads to speciation, producing silanols and siloxanediols, whereas complete degradation requires cleavage of the relatively inert Si–C bonds.”

Katsoulis approached Arnold to collaborate on efforts to speed up siloxane degradation after he read about her lab’s work in coaxing nature to produce silicon–carbon bonds. In 2016, Arnold and colleagues had used directed evolution to engineer a bacterial protein called cytochrome c to form silicon–carbon bonds, a process that does not occur in nature. That research demonstrated that biology could make these bonds in ways that are more environmentally friendly than those traditionally used by chemists. Directed evolution can be thought of as similar to breeding dogs or horses, in that the process is designed to bring out desired traits.

For their newly reported study the researchers wanted to find ways to break the silicon-carbon bonds, rather than create them. They used directed evolution to evolve a bacterial cytochrome P450 enzyme. They first identified in their enzyme collection a variant of cytochrome P450 that had a very weak ability to break silicon–carbon bonds in linear and cyclic volatile methylsiloxanes. “… a previously unpublished P450BM3 variant evolved for silane and siloxane Si–H hydroxylation designated LSilOx1 (linear siloxane oxidase, generation 1) was chosen as a starting point for evolution of the Si–C bond cleavage activity,” the team wrote.

They mutated the DNA of the cytochrome P450 and tested the new variant enzymes. The best performers were then mutated again, and the testing was repeated until the process generated an enzyme that had enough activity to enable the researchers to identify the products of the reaction and study the mechanism by which the enzyme works.

“Evolving enzymes to break these bonds in siloxanes presented unique hurdles,” explained Tyler Fulton, PhD, co-lead author of the study and a postdoctoral scholar at Caltech in Arnold’s lab. “With directed evolution, we must evaluate hundreds of new enzymes in parallel to identify a few enzyme variants with improved activity.”

One challenge the team had to address involved the siloxane molecules leaching plastic components from the 96-well plates used to screen the variants. To solve this problem, the team created new plates made from common lab supplies. “Another challenge was finding the starting enzyme for the directed evolution process, one with even just a tiny amount of the desired activity,” Arnold said. “We found it in our unique collection of cytochrome P450s evolved in the laboratory for other types of new-to-nature silicon chemistry.”

The final improved enzyme does not directly cleave the silicon–carbon bond, but rather oxidizes a methyl group in the siloxanes in two sequential steps. This means that two carbon–hydrogen bonds are replaced with carbon–oxygen bonds, a change that allows the silicon–carbon bond to break more readily. “… the tandem oxidation of the siloxane methyl group unlocks a mechanism for the enzymatic cleavage of Si–C bonds in VMS and serves as an entryway into future biodegradation efforts,” the scientists commented. “The cleavage of a siloxane Si–C bond catalyzed by the enzymes reported here is a proof of principle and represents a first step toward biodegradation of siloxanes that are currently not considered biodegradable … By engineering enzymes that can cleave Si–C bonds, we take a key step forward toward the eventual biodegradation of VMS.”.

The research draws parallels to studies involving a plastic-eating enzyme, explains Fulton, referring to a polyethylene terephthalate (PET)-degrading enzyme discovered in the bacteria Ideonella sakaiensis in 2016 by a different group of researchers. The authors further explained, “This situation is reminiscent of the discovery of Ideonella sakaiensis—a microorganism collected from sediment near a recycling plant rich in polyethylene terephthalate (PET)—and its evolved PET-degrading enzymes that enable it to grow on PET as the sole carbon source. This discovery inspired a slew of innovations, both enzymatic and microbial, that presented viable routes to PET biodegradation, which are just now coming to fruition.”

Fulton added, “While the PET-degrading enzyme was discovered by nature rather than by engineers, that enzyme inspired other innovations that are finally coming to fruition for plastic degradation. We hope this demonstration will similarly inspire further work to help break down siloxane compounds.”