“All this tremendous beauty and complexity of the biological world all comes about through this one simple, beautiful design algorithm, and what I do is use that algorithm to build new biological things.”
– Frances Arnold
The Nobel Prize in Chemistry 2018 Interview
By now nobody in the chemical industry will have missed that Dr. Frances Arnold has been awarded the Nobel Prize for Chemistry. Here at EnginZyme we are absolutely delighted by this news — not only couldn’t the prize go to a more well-deserving recipient, but it will also be a boost for the field of biocatalysis as a whole!
Many articles have been written about Dr. Arnold in the past weeks, but few of them go into much detail about her work and career, and fewer still are written with an audience of chemists in mind — a deficiency we thought needed to be addressed!
Through the power of evolution driven by natural selection, life has developed from a collection of molecules encased in a simple lipid membrane into a rich array of complex multicellular organisms.
This process has acted on populations for millions of years, continuously changing organisms to better suit their environment; imagine increasing the rate of that process and harnessing it to create new molecules fit for purpose.
It took the fresh, if unconventional, approach of Dr. Frances Arnold’s brilliant mind to consider transforming Darwinian principles into practice, directing rapid protein evolution to make better biocatalysts.
Dr. Arnold, who has spent the last thirty years conducting research and teaching new generations of students as the Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry and the Director of the Donna and Benjamin M. Rosen Bioengineering Center at the California Institute of Technology (Caltech), began her journey in the academic world at Princeton studying mechanical and aerospace engineering.
During her undergraduate work, Frances worked on solar energy at the Center for Energy and Environmental Studies at Princeton. After graduating, she did engineering work in South Korea and Brazil.
Returning to the U.S., Frances continued to work as an engineer at the Solar Energy Research Institute in Chicago before starting her Ph.D. in chemical engineering at the University of California, Berkeley.
She initially intended to focus on biofuels. However, when the U.S. administration changed from President Carter to President Reagan, there was a shift in attitude towards alternative fuels which precipitated Frances’s desire to change directions.
She started moving into biotechnology. In her graduate thesis work, Frances worked with proteins for the first time, studying protein separation using affinity chromatography. Following the completion of her Ph.D., Dr. Arnold accepted an invitation to Caltech.
At Caltech Dr. Arnold turned from chemical engineering to biotechnology. Fascinated by enzymes, she built a research program focused on enzyme engineering. When she started her work in biotechnology, the genetic engineering revolution had just begun (with important contributions from her fellow 2018 Nobel laureates).
The methods of the time formed the basis of how she approached enzyme engineering. Rational design – considering a wide variety of information to generate hypotheses about how to improve an enzyme – was the standard approach.
The field was full of biochemists and molecular biologists tinkering with genetic sequences. As an outsider, Dr. Arnold said “I basically knew nothing about the field. Otherwise I probably wouldn’t have done it, because I would have known how hard it was.”
Dr. Arnold chose her first target: a protease. Subtilisin E is a serine protease that is commonly found in detergents and food processing applications. Dr. Arnold wanted to engineer subtilisin E so that it could function in dimethylformamide (DMF), a powerful organic solvent in which the catalytic mechanism of subtilisin E could be harnessed to synthesize peptides.
She began with the classical approach. Charged amino acids on the surface of subtilisin E molecules were substituted using site-directed mutagenesis. The work was laborious, as the number of variants that had to be generated containing different combinations of mutations was astronomical.
This incredible challenge led Dr. Arnold to consider an interesting new approach: harnessing the fundamental force of evolution. What if we simply mutate the gene randomly and then work backwards, testing the mutants for activity and figuring out what mutations had resulted in better enzyme performance?
She began to use random mutagenesis to modify subtilisin E before screening for catalytic activity in DMF. She identified new versions of the enzyme which performed better than the original enzyme. She then sequenced these mutant genes to identify two beneficial mutations: Q103R and D60N.
The effect was additive. Combining both mutations into a new version of subtilisin E resulted in even better performance. Seeing the effectiveness of random mutagenesis and the additive effect of these mutations, Dr. Arnold decided to try running the process again on the improved mutant to find more of these additive effects, an idea criticized “because nature had never gone there before.”
This led, in 1993, to the publication of a paper titled “Tuning the Activity of an Enzyme for Unusual Environments: Sequential Random Mutagenesis of Subtilisin E for Catalysis in Dimethylformamide”.
This paper, written by Dr. Arnold and her student Keqin Chen, is the first publication describing the use of directed evolution for protein engineering for which Dr. Arnold won the 2018 Nobel Prize in Chemistry. The paper describes their use of sequential cycles of random mutagenesis and performance screening to optimize the function of subtilisin E in DMF.
Following three rounds in which the best performing subtilisin E mutant was subjected to further random mutagenesis and screening, Dr. Arnold and Keqin Chen found an enzyme that was 256 times more efficient than the original enzyme. This new variant had 8 more amino acid substitutions.
Nobody could have hypothesized the additive effects of Q103R and D60N, let alone the benefits of 8 additional substitutions. Having used cycles of random mutation and screening to cause the accumulation of beneficial mutations, Dr. Arnold had “directed” evolution for the first time.
K Chen & F Arnold (1991) Enzyme Engineering for Nonaqueous Solvents: Random Mutagenesis to Enhance Activity of Subtilisin E in Polar Organic Media. Bio/Technology 9, 1073–1077.
As Dr. Arnold began running with this conceptual leap, the broad potential of this technology started to become clearer. Dr. Arnold set out to direct the evolution of enzymes towards improved thermostability for industrial use. She used subtilisin E as a model.
Previous attempts made to engineer more thermostable enzymes typically didn’t give the desired result. Amino acid substitutions that helped an enzyme function at a higher temperature typically compromised the performance of the enzyme at lower temperatures, making it less useful.
Dr. Arnold figured that with the appropriate selection pressures and screening assays, she could make an enzyme with high performance at both low and high temperatures.
To accomplish this, Dr. Arnold built on the work of Willem Stemmer, who demonstrated the use of DNA shuffling (a method of in vitro recombination) to evolve a beta-lactamase in 1994. Dr. Arnold and her student Huimin Zhao demonstrated a new process for in vitro DNA recombination inspired by the polymerase chain reaction (PCR).
The staggered extension process (StEP) was designed to be fast and user-friendly. The StEP process began with randomly mutated template DNA added to a PCR reaction mixture.
The reaction was then rapidly cycled between high (denaturing) and low (annealing) temperatures. Short cycling (~5 seconds) only allows the polymerase to add small stretches of DNA from whichever mutant a gene copy has annealed to in a given cycle.
The resulting mutated gene copies contain information from different DNA templates, allowing even more sequence space to be explored. In 1998, Dr. Arnold and Huimin showed that this process, in combination with random mutagenesis and screening, generated a new subtilisin E gene containing 8 amino acid substitutions which improved the stability of the enzyme at 65°C by more than 200-fold. Graduating that year, Dr. Zhao then went on to lead a team at Dow until starting his independent career applying directed evolution in the emerging field of synthetic biology, carrying on the legacy of Dr. Arnold’s work.
K Chen & F Arnold (1993) Tuning the Activity of an Enzyme for Unusual Environments: Sequential Random Mutagenesis of Subtilisin E for Catalysis in Dimethylformamide. Proc Natl Acad Sci U S A. 90(12), 5618-22.
H Zhao & F Arnold (1999) Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Eng. 12(1) 47-53.
H Zhao, L Giver, Z Shae, JA Affholter, F Arnold (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16(3) 268-61.
Throughout her research career, Dr. Arnold has been a teacher and a mentor for many students. One former student, Dr. Jeffrey Moore, was among the first people to use directed evolution in her lab and, in 2002, went on to found Codexis, a company that uses directed evolution to engineer enzymes.
Today, Codexis has over 800 patents or pending patents on its technology, including its enzyme engineering platform called CodeEvolver. Serving as an advisor to the company, Dr. Arnold helped to guide the company and support her former student for years.
Over the 16 years since its founding, the company has applied directed evolution technology to the development of enzymes for industries including food and beverages, biotherapeutics, and pharmaceuticals.
Showing its impact in the field, Codexis has won several awards for the expert use of directed evolution technology to engineer biocatalysts.
In 2006, the company won the President’s Green Chemistry Challenge for engineering an enzyme that catalyzes a reaction to produce a key intermediate in the synthesis of the cholesterol-lowering drug Lipitor, famously the first drug to generate over $10 billion in annual sales.
By evolving three enzymes to work together, Codexis created a green and economical process. Codexis again won the President’s Green Chemistry Challenge in 2010 for work performed with Merck on the anti-diabetes type 2 drug, sitagliptin.
Codexis engineered a transaminase enzyme to recognize the chiral amine of the prositagliptin ketone substrate, eliminating the need for a rhodium catalyst. In a triumph of green chemistry using biocatalysis, Merck was able to use this newly engineered enzyme to double the production of sitagliptin cost-effectively.
Following her success as an advisor for Codexis, Dr. Arnold took a more active role in supporting the green chemistry and biocatalysis aspirations of her students.
Having nurtured an interest in biofuels since her undergraduate days, Dr. Arnold used ideas for directing the evolution of enzyme pathways to produce ethanol and isobutanol. This was spun out into Gevo with Dr. Peter Meinhold (a former student), and Dr. Matthew Peters (a former colleague) in 2005.
The key technology behind Gevo was developed in Dr. Arnold’s research group. She described the engineering of ketol-acid reductoisomerase and alcohol dehydrogenase enzymes to function in E. coli using the cofactor NADH rather than NADPH.
This enabled the production of higher alcohols in a reconstituted pathway under anaerobic conditions. By eliminating the need for air-circulation, the process is conducted on a larger scale, with lower operational costs, making it more competitive.
Gevo uses its evolved enzymes to generate alcohol from plant carbohydrates. By generating low-carbon jet fuel from biological sources, the company aims to prevent the emission of greenhouse gases from fossil fuels.
Their low-carbon alcohol-to-jet (ATJ) fuel has been approved by the American Society for Testing and Materials (ASTM) for commercial use and the fuel is being tested by GE Aviation for compatibility with engine combustor components. In 2017, Gevo’s ATJ fuel was used in a demonstration at the O’Hare International airport in Chicago for Fly Green Day.
One of Dr. Arnold’s favourite enzyme families is the cytochrome P450 group. In an interview with Fellows of the American Academy of Microbiology, she said, “My little brother, Eddy, who got me interested in biochemistry, told me, ‘This isn’t an enzyme – this is a city.’ It’s so complex, it does so many interesting things.”
Dr. Arnold’s group has created a library of sequentially and functionally diverse cytochrome P450 enzymes using a technique developed by her group called SCHEMA recombination. SCHEMA recombination uses an algorithm to determine portions of genes that can be recombined without disrupting the 3D structure of the resulting protein.
This improves the efficiency of directed evolution efforts by targeting cross-over sites for recombination between distantly related genes. As a service to the community, Dr. Arnold’s group offers this software package freely to enzyme engineers to help them navigate the intractable landscape of recombination fitness.
The capability to direct the evolution of enzymes, Dr. Arnold has remarked, will encourage others to creatively apply the technology to many different problems.
Dr. Arnold’s fascination with P450 enzymes extends to engineering some of the more challenging chemical reactions that they carry out. One common function of cytochrome P450 enzymes is the insertion of an oxygen atom into a hydrocarbon molecule.
By engineering cytochrome P450 enzymes to catalyze the transfer of carbon atoms instead of oxygen atoms, her group developed variants that could perform remarkable carbene transfer reactions such as cyclopropanation. Dr. Arnold supported the vision of her student, Pedro Coelho, who recognized that many insect pheromones contain, or are built from, cyclopropane groups.
Armed with the new reactivities of these engineered P450 enzymes, Dr. Arnold, Dr. Coelho, and Dr. Meinhold, co-founded Provivi to manufacture insecticides based on insect pheromones. The pheromones produced by Provivi selectively target pest insects, disrupting reproductive behaviours.
The famed primatologist Dr. Jane Goodall has endorsed Provivi and its nature-based approach to pest control. Some of the funds for the start-up of Provivi came from Dr. Arnold having been the first woman to win the prestigious Millennium Technology Prize in 2016.
Her successes in business aside, Dr. Arnold has always been more interested in the research side of her work. She has dabbled in many different aspects of enzyme engineering and directed evolution.
One of her goals has been to “evolve innovation” in the biological field by evolving new species of enzymes and by engineering new chemical reactivities into enzymes. This has been the focus of her recent work.
Her group has evolved enzymes capable of performing “non-natural” chemical reactions including carbon-silicon and carbon-boron bond formation in living cells. With these impressive feats of enzyme engineering, Dr. Arnold has expanded the chemical space that can be explored within biological systems.
CA Voigt, C Martinez, Z-G Wang, SL Mayo, F Arnold (2002) Protein building blocks preserved by recombination. Nat. Struct. Biol. 9, 553-558.
PS Coelho, EM Brustad, A Kannan, F Arnold (2013) Olefin Cyclopropanation via Carbene Transfer Catalyzed by Engineered Cytochrome P450 Enzymes. Science 339(6117) 307-10.
Dr. Arnold has been an innovator in many roles: she is a revolutionary scientist, an award-winning protein engineer, a businesswoman, a mentor, and an outstanding role-model. The far-reaching impact of her pioneering work in directed evolution has made biocatalysis a cornerstone of industrial chemistry. Her dedication to green chemistry and the development of sustainable solutions to real-world problems in biotechnology continues to inspire the next generation of scientists and engineers.