Short Feature: Creating Molecules Out of Thin Air

How did life emerge? What does it mean for something to be alive? These questions may be well suited for a philosophy seminar, but they have also been asked in science labs throughout history. In 1859, Charles Darwin published his book On the Origin of Species, explaining how life came to be with the theory of evolution. In 1953, the infamous Miller-Urey experiment studied how life started in the first place under the conditions of Earth’s early atmosphere. Likewise, in 2018, Princeton chemistry professor Michael Hecht’s lab is examining these philosophical questions through the lens of science. According to Hecht, the central question his lab is investigating is: “What are the minimal requirements for life?”

To explore this inquiry, Hecht and his lab are making artificial proteins that can facilitate life-sustaining chemical processes. Former Princeton graduate students Ann Donnelly and Katie Digianantonio, postdoctoral fellow Grant Murphy, and Hecht recently created the first artificial protein that can catalyze the reactions necessary for life both in the lab and in living cells: Syn-F4.

The artificial Syn-F4 protein sustains life by functioning as an enzyme. Life is sustained by myriad chemical reactions, each of which requires an enzyme to catalyze it. Without enzymes, biological reactions would not occur quickly enough for life to exist.

To make proteins from scratch, the researchers synthesized DNA sequences to code for countless random variations of amino acid sequences, which are the building blocks of proteins. From this massive collection of different artificial DNA sequences, they screened for DNA sequences that could potentially replace previously known genes that E. coli need to survive. “Finding that random DNA sequences can do something productive goes against the prevailing thought that DNA sequences have to be optimized over millions of years to do something productive,” said Digianantonio.

In this study, the Hecht team tested a synthesized DNA sequence that encoded the artificial protein Syn-F. First, they created a strain of E. coli that was missing the essential gene that encodes the Fes enzyme, which is involved with iron uptake. While iron, a nutrient E. coli requires to survive, is abundant naturally, it exists in a form that is not easily accessible. Organisms have special molecules that they use to access and collect iron, one of which is called enterobactin, but they need an additional tool like Fes to extract the iron from these molecules. When the scientists offered iron to modified E. coli, all the colonies remained red, indicating that the iron was still held by the enterobactin . Without Fes, this modified E. coli strain could not liberate the iron from the enterobactin on its own. However, when the researchers replaced the missing essential gene that encodes Fes with a synthetic DNA gene that encodes Syn-F4, the E. coli colonies changed color from red to white, indicating thatt the cells successfully accessed the iron, and suggesting that Syn-14 acted as an enyzme in place of Fes to catalyze the release of iron from enterobactin.

While Donnelly was the first to describe Syn-F4’s enzymatic mechanism, her astonishment in the wake of the discovery motivated her to repeat the experiment herself and further ask both Digianantonio and Murphy to repeat it. All of the results confirmed that Syn-F4 did indeed function enzymatically. Since 2011, the Hecht lab has been able to delete four essential E. coli genes and replace them with synthesized DNA sequences that encode artificial proteins. While the previous three artificial proteins did not function as enzymes and instead worked indirectly to sustain E. coli life, Syn-F4 made a huge breakthrough as the first artificial protein to act as an enzyme.

Artificial proteins like Syn-F4 open doors that researchers ddid not previously know existed. “Nature is merely building with what it already has. If we in the lab can give organisms totally new sequences to work with, what could happen?” Digianantonio said. For instance, enzymes help speed up the industrial production of food, fuel, and medicine, but these industries often repackage pre-existing, natural enzymes that have evolved over billions of years. “We can do much more if we do not limit ourselves to proteins that already exist in nature,” said Hecht.

In addition, the Hecht team is taking the first step towards creating an artificial proteome—the complete set of all the proteins expressed by an organism—that can sustain life. Thinking ahead, Hecht said, “Can you replace an entire genome with novel sequences? That would be creating new cells.” While artificially making new cells in a lab may sound like science fiction, Hecht believes that chemistry involves exploring the boundary between the possible and the impossible. d“Molecular biologists are studying life that exists, evolutionary biologists are studying life that was, and chemists are studying that which might be possible” Hecht concluded.