Researchers have created a novel synthetic three-stranded molecule that functions just like a natural enzymes

Enzymes are nature’s powerhouses.

Found in the cells of all animals, plants, and every other living organism, they accelerate the chemical reactions that trigger thousands of biological functions – from forming neurons to digesting food.

They perform their jobs so selectively and so quickly – millions of times faster than a blink of the eye – that the field of biomimetic chemistry has emerged over the past few decades with the goal of designing artificial enzymes that can mimic the powers of natural enzymes in industrial settings.

Artificial enzymes could, for example, convert corn into ethanol or help create new drugs more quickly, cheaply, and effectively.

Moving one step closer to achieving that goal, Rajeev Prabhakar, a computational chemist at the University of Miami, and his collaborators at the University of Michigan have created a novel, synthetic, three-stranded molecule that functions just like a natural metalloenzyme, or an enzyme that contains metal ions.

“It wasn’t clear that they could be made, but we made them. And, then we used them to successfully catalyze reactions,” said Prabhakar, a professor of chemistry who studies enzyme reactions in hopes of designing their artificial analogues.

“This is an incremental but important step in the development of artificial enzymes, which has long been considered chemistry’s holy grail.

Unfortunately, as good as natural enzymes work in our bodies and other life forms, they don’t tolerate other settings very well.

They’re also very expensive and not easy to prepare and purify.”

For their groundbreaking study published in Nature Chemistry this week, Prabhakar and graduate student Vindi M. Jayasinghe-Arachchige joined forces with Vincent L. Pecoraro, a University of Michigan chemistry professor, to improve the performance of the artificial enzymes Pecoraro’s lab pioneered over the years.

The Michigan researchers had previously created simpler synthetic metalloenzymes that successfully catalyzed a number of chemical reactions.

But those artificial macromolecules were designed with three identical, or symmetrical “homotrimeric” strands, which, Prabhakar said, limited their catalytic abilities.

In the new molecule, which Jayasinghe-Arachchige designed on the University of Miami’s supercomputer with Prabhakar’s guidance, the third strand differs in structure from the other two strands.

Her quantum mechanical calculations showed that the more complex, non-symmetrical, three-stranded structure, known as a “heterotrimeric” coil, expanded the catalytic performance of homotrimeric artificial metalloenzymes – a finding that Pecoraro and his team confirmed with experiments in his Michigan lab.

“Our techniques are different, but complimentary,” Prabhakar said. “What we do the Pecoraro group cannot do, and what they do, we cannot do.

We model molecules on the computer so we can predict their structural properties and the mechanism of their formations.

They use our models to build the real thing, and in this case that is the first example of a natural heterotrimeric molecule.”

Most lay people would probably find the study as incomprehensible as its title: “Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys)3 template mediated strategy.”

But the bottom line, Prabhakar said, is that the collaborative research conducted in Miami and Michigan opens the door to a new strategy for achieving the creation of artificial enzymes that work as well as natural enzymes.

In addition to Pecocaro, Prabhakar, and Jayasinghe-Arachchige, other co-authors of the study include Prabhakar’s former graduate student, Thomas J. Paul, now at the University of Michigan; Audrey E. Tolbert, Catherine S. Ervin, and Kosh P. Neupane, also from the University of Michigan; and Leela Ruckthong, from King Mongkut’s University of Technology, in Thailand.

Now in her last year of study for her doctorate in chemistry, Jayasinghe-Arachchige said she remains fascinated by the advances in computational chemistry techniques that allowed her to model the chemical structures and reactions of the new molecule.

“I’m excited that our findings will create new avenues toward the development of efficient artificial enzymes that can be used to enhance the quality of life,” said Jayasinghe-Arachchige, “and as a woman in a field where women are underrepresented, I hope this study will motivate women to join the fascinating world of STEM fields.”

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Synthetic Biology offers innovative approaches for engineering new biological systems or re-designing existing ones for useful purposes (see Figure 1). It has been described as a disruptive technology at the heart of the so-called Bioeconomy, capable of delivering new solutions to global healthcare, agriculture, manufacturing, and environmental challenges (Cameron et al., 2014; Bueso and Tangney, 2017; French, 2019).

However, despite successes in the production of some high value chemicals and drugs, there is a perception that synthetic biology is still not yet delivering on its promise.

Moreover, there are some concerns from governments that synthetic biology expands the pool of agents of concern, which increases the need to develop detection, identification and monitoring systems, and proactively build countermeasures against chemical and biological threats (Wang and Zhang, 2019).

The participation of representatives from various government organizations at this meeting is testament to their commitment to maintaining an active dialogue with the synthetic biology community. In this way, they aim to keep abreast of the changing nature of threats and provide the best advice to government about investment in science and technology and the introduction or amendment of regulatory processes.

The cost of DNA sequencing and synthesis have decreased dramatically (Carlson, 2014; Kosuri and Church, 2014) and we have access to more genetic information and more powerful genetic engineering capabilities than ever before.

Critical investments in infrastructure are bearing fruit and, as is described below, synthetic biology is increasingly becoming, at least part of, the solution to many of our present and future needs in medicine, food and energy production, remediation, manufacturing, and national security. So what is the potential of synthetic biology and what challenges does it still face to realize this?

Advances in Synthetic Biology: The State of Play

Small Molecules: Production on Demand a Reality

Despite the lack of predictability in biology, and current technical constraints that limit data collection and analyses, we can now produce small molecules on demand using synthetic biology approaches.

Probably the most impressive examples come from the Foundry at the Broad Institute of MIT and Harvard. When the Defense Advanced Research Projects Agency (DARPA) put the MIT-Broad Institute Foundry’s design capabilities to the test, its researchers were able to deliver 6 out of 10 molecules of interest to the US Department of Defense in 90 days. This “pressure test” confirms the potential of synthetic biology to address shortages of key compounds quickly (Casini et al., 2018).

Indeed, many labs can now design and construct relatively complex gene networks capable of generating a wide variety of “designer” molecules in a range of host cells; however, this is often a slow iterative process of trial and error.

As yet, very few small molecules in medicine are manufactured using a synthetic biology process; it remains very difficult to engineer microbes to carry out processes that Nature did not intend.

This is to be expected: the performance of microbes is “good enough” from an evolutionary perspective. Microbes evolved to address the specific needs and challenges of their natural environments not those of industrial fermenters and bioreactors.

Gene Transfer from one system to another may sound easy but in practice is hard work and rarely generates sufficient reward (i.e., increased yield) to justify the investment made. The application of automation and artificial intelligence (e.g., in designing and building plasmids) may help to reduce the time and cost—and improve return on investment—in the future (Zhang et al., 2018).

“Scale up is product specific – we need more synthetic biology in the production process”

Plants make alternative production platforms. Improvements in mining plant genomes and the development of effective transient expression systems have enabled large-scale production of, for example, vaccines in tobacco plants in just a few weeks (Dirisala et al., 2017; Emmanuel et al., 2018). Directing the production of synthetic biological materials to plant chloroplasts also shows promise (Boehm and Bock, 2019).

The photosynthetic reducing power generated in plant chloroplasts can be harnessed for the light-driven synthesis of bioactive molecules such as dhurrin, which protects plants against insects (Gnanasekaran et al., 2016).

However, underlying all these platforms is a knowledge gap in our understanding in how nature works. This makes it very hard to apply the design/build/test/learn cycles used in conventional engineering to the production of synthetic biological materials whatever the production platform (yeast, bacteria, plants, or human cells) if the platform itself is not well-understood (Sauro et al., 2006).

What we need now are instruments able to measure and characterize outputs, assisted by progress in robotics and automation, and the application of machine learning approaches to analyse the data generated. This will help us to generate more robust models of biological systems, so we can improve experimental design for future engineering strategies.

“We can do ‘build’. ‘Test’ is the challenge when we want to learn from the iterative design process”

Healthcare: Reimagining Medicine

Synthetic biology is driving significant advances in biomedicine, which will lead to transformational improvements in healthcare. Already, patients are benefiting from so-called CAR (for chimeric antigen receptor) technology, which engineers the immune cells (T-cells) of the patient to recognize and attack cancer cells (June et al., 2018).

Genetically engineered viruses are being used to correct defective genes in patients with inherited diseases such as Severe Combined Immune Deficiency (SCID) or epidermolysis bullosa (Dunbar et al., 2018).

The ability to reprogramme somatic cells from patients into induced pluripotent stem cells is furthering our understanding of their disease, reducing the use of animals in research, and paving the way for the development of personalized medicines and cell therapies. In principle at least, we could engineer a patient’s own cells to multiply, differentiate into different cell types and even self-assemble into new tissues, or even organs, to repair those damaged through disease or injury (Davies and Cachat, 2016; Satoshi et al., 2018).

Work on new vectors that are able to deliver large genetic loads to target tissues is helping to produce more efficient therapeutics and vaccines that will have fewer side effects and a smaller risk of resistance. Furthermore, optimizing antibody or vaccine production, or example, so that they are in an edible format (e.g., plant based), could greatly reduce the cost and increase the speed of vaccine production in an epidemic.

“We have the tools but need the creativity to make stuff that can’t be made without synthetic biology”

In the next few years, genetically engineering pigs to be virus resistant and have human-like immune profiles could make xenotransplantation a clinical reality (Burkard et al., 2018). Engineering the microbiome is expected to lead to the development of synthetic probiotics (Dou and Bennett, 2018).

The synthetic biology initiative known as Human Genome Project-write (HGP-write) has set its sights even higher, rallying scientists to build entire human chromosomes (Boeke et al., 2016). Concerns have been raised about the ethics of creating “synthetic humans” and indeed the scientific and commercial value of such a project. More recently, HGP-write champions have proposed a more focused project to build a virus-resistant chromosome, making at least 400,000 changes to the human genome to remove DNA sequences that viruses use to hijack cells and replicate (Dolgin, 2018).

One of the many exciting opportunities that synthetic biology offers medicine is in the production of theranostic cell lines that can sense a disease state and produce an appropriate therapeutic response (Teixeira and Fussenegger, 2019). Several obstacles need to be overcome to achieve this goal: first, to expand the range of molecules that can be recognized by cellular “sensors” as inputs; and second, to better understand the genetic control factors that regulate gene expression in space and time so we can engineer better activator systems.

“At present we need pills because we can’t swallow a chemistry kit”

Metabolomics is shedding light on many disease biomarkers. Because some biomarkers are shared between seemingly unrelated diseases, an accurate diagnosis will require the detection of multiple markers to provide a more unique “disease fingerprint.” Work in whole cell and cell free systems to develop sensors of multiple disease-specific biomarkers could assist in earlier detection of disease and prognostic monitoring.

To expand the range of biologically detectable molecules, it is possible to design metabolic pathways that transform currently undetectable molecules of interest (e.g., hippuric acid, the prostate cancer biomarker) into molecules for which sensors already exist (in this case benzoic acid) (Libis et al., 2016).

Cybergenetics is an emerging field that is developing experimental tools for the computer control of cellular processes at the gene level in real time. Cybergenetic control can be achieved by interfacing living cells with a digital computer that switches on or off the embedded “genetic switch” using light (optogenetics) or chemicals (Gabriele et al., 2018; Maysam et al., 2019).

Such systems could help to maintain cellular homeostasis by monitoring the state of the body and triggering an appropriate response upon the detection of dysregulation; for example, they could trigger the release of insulin when blood glucose levels rise as detected by a wireless diagnostic tool (Ye et al., 2011).

Advanced Materials: Inspired by Nature, Improved by Synthetic Biology

Synthetic biology offers the opportunity to create responsive and multifunctional materials (Le Feuvre and Scrutton, 2018). The integration of biochemical components from living systems with inorganic components can lead to new materials that are able to sense the environment (or internal signals) and change their properties. These features could be particularly useful for improving protective clothing or building materials.

An issue when using microbes to produce composite materials is regulating the assembly of these materials to achieve specific desired properties. By understanding how microbes communicate with each other, it is possible to make them work better together and combine them with other production systems so that the properties of materials can be tailored for particular functions.

Interestingly, rather than modifying, or improving existing protein-based materials, an alternative approach involves using computational techniques to design completely novel proteins that self-assemble into predicted shapes (Ljubetič et al., 2017). Such “programmable” proteins open up even further opportunities for synthetic biology not only for materials science but also for medicine and chemistry.

“The tools are there, we just don’t know what we want to make”

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More information: Audrey E. Tolbert et al, Heteromeric three-stranded coiled coils designed using a Pb(ii)(Cys)3 template mediated strategy, Nature Chemistry (2020). DOI: 10.1038/s41557-020-0423-6