Directed Evolution of an Artificial Enzyme, a Step toward Nonnatural Metabolism

Representation of the new-to-nature olefin metathesis reaction in E. coli using a ruthenium-based artificial metalloenzyme to produce novel high-added-value chemicals. [NCCR Molecular Systems Engineering]

Industrial chemistry and biological chemistry don’t always mix well within living cells. For example, olefin metathesis, a reaction type that inspired research honored by the 2005 Nobel Prize in Chemistry, relies on organometallic catalysts that tend to fare poorly in aqueous solutions or cellular-like environments. Nonetheless, such catalysts have been brought closer to nature. They have been reconceived as artificial metalloenzymes and generated by means of directed evolution. One artificial enzyme in particular, biot-Ru-SAV, has been produced inside living cells, where it has shown the capacity to catalyze abiotic and industrially significant reactions.

A research team led by scientists at the University of Basel and ETH Zurich created biot-Ru-SAV within Escherichia coli bacteria by using the biotin–streptavidin technology. This method relies on the high affinity of the protein streptavidin for the vitamin biotin, where compounds bound to biotin can be introduced into the protein to generate artificial enzymes.

The scientists described their work in an article (“Directed Evolution of Artificial Metalloenzymes for In Vivo Metathesis”) that appeared August 29 in the journal Nature. The article describes how an abiotic co-factor that incorporated ruthenium was joined with a protein to create the artificial metalloenzyme.

“We report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction without equivalent in nature,” wrote the article’s authors. “Building on previous work on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor.”

This approach allowed the scientists to overcome a difficulty often encountered with metal co-factors. Such co-factors are inhibited by cellular components and require purification of the scaffold protein. Unfortunately, the need for purification limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism.

“The main breakthrough was the idea to use the periplasm of Escherichia coli as a reaction compartment, whose environment is much better suited for an olefin metathesis catalyst,” said Markus Jeschek, M.Sc., the Nature article’s first author and a researcher at ETH Zurich. The periplasm, the space between the inner cytoplasmic membrane and the bacterial outer membrane in Gram-negative bacteria, contains low concentrations of metalloenzymes inhibitors, such as glutathione.

Having found ideal in vivo conditions, the authors went a step forward and decided to optimize biot-Ru-SAV by applying principles of directed evolution, a method that mimics the process of natural selection to evolve proteins with enhanced properties or activities. “We could then develop a simple and robust screening method that allowed us to test thousands of biot-Ru-SAV mutants and identify the most active variant,” explained Thomas R. Ward, Ph.D., the article’s senior author and a professor of chemistry at the University of Basel.

“This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants,” asserted the authors of the Nature article. “The evolved metathase compares favorably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow.”

Not only could the authors markedly improve the catalytic properties of biot-Ru-SAV, but they could also show that organometallic-based enzymes can be engineered and optimized for different substrates, thus producing a variety of different chemical products. “The exciting thing about this is that artificial metalloenzymes like biot-Ru-SAV can be used to produce novel high-added-value chemicals,” concluded Dr. Ward. “It has a lot of potential to combine both chemical and biological tools to ultimately utilize cells as molecular factories.”