Scientists have dreamt of rationally engineering microorganisms since the 1960s. Back then, while studying the lac operon in Escherichia coli, Francois Jacob and Jacques Monod proposed that cells possessed regulatory circuits that underpinned their environmental responses. In the 1990s, the genomics revolution and the rise of systems biology led to the development of a rigorous engineering discipline for creating, controlling, and programming cellular behavior. Then, in 2012, Emmanuelle Charpentier and Jennifer Doudna brought CRISPR to the world, enabling scientists to edit the genome by removing and inserting any piece of DNA, resulting in cells with novel biological properties.
These developments in molecular and cellular biology have contributed to the rise of synthetic biology, a field in which engineering principles—such as the construction of complete systems from standardized parts—are applied to living systems. The field has expanded dramatically over the last two decades, transforming biotechnology for applications in human health and beyond.
Refactoring the genetic code with noncanonical amino acids
Daniel de la Torre, PhD, is creating new classes of molecules that have never been seen before in nature. As a graduate student in the laboratory of Jason W. Chin, PhD, at the Medical Research Council’s Laboratory of Molecular Biology, de la Torre used a synthetic E. coli called Syn61 to generate nonnatural molecules.
Syn61 is special because it translates DNA into proteins using only 61 codons, instead of the usual 64, to code for the natural, proteinogenic amino acids. The three “spare” codons can be used to code for noncanonical amino acids—nonproteinogenic amino acids that are either found naturally in organisms or are synthetically made in a laboratory. In his work with Syn61, de la Torre used genome writing and reengineered protein translation machinery to make molecules that incorporate noncanonical amino acids.
This technology has vast applications, from drug development to materials science. For example, de la Torre’s refactored Syn61 can be used to modify antibody-drug conjugates (by incorporating site-specific nonnatural amino acids that can act as molecular glue for coupling payloads, such as toxins) or to improve therapeutic efficacy and stability.
“By taking a self-propagating biological system and expanding its chemical toolbox, we can access these applications in a way that we would otherwise be capable of doing only in the chemistry laboratory—and not in a biologically sustainable manner,” says de la Torre, who is now the head of research and development at Constructive Bio.
At the biotechnology startup, de la Torre and his team are employing synthetic genomics and noncanonical amino acids to craft “advanced biopolymers.” But what excites de la Torre most is the idea of letting nature figure out how to best use the new genetic code instead of directly rewriting genes.
“You could generate genotypic diversity by encoding the cellular polymerization of weird combinations of weird monomers to make weird polymers, and then apply Darwinian selection to fish out materials or polymers that have the right properties, and then go back and trace the genotype that gave rise to that material,” de la Torre suggests. “This is currently science fiction. We need to be really good at recoding cells to create genetic space, but we also need to build a very robust engine for engineering the translational machinery of cells to really make the components really good at accepting substrates beyond what nature has given them.”
Reprogramming cells for medical and industrial applications
While training as a neurosurgeon at the University of Cambridge in 2006, Mark Kotter, MD, PhD, dreamt of being able to create therapies for spinal cord injuries. But that vision stalled when Kotter learned that the protocols available to generate oligodendrocytes took about half a year.
“Imagine starting a small laboratory to explore this interest and then, six months in, telling your graduate students that you might have a cell that could be used in some experiments—or that you might not because [the protocol] wasn’t working out,” Kotter says. “If you want to create a differentiation protocol, you’re basically running down Waddington’s landscape with a torch, trying to see where the branch points are and how you can bias them.”
Then, auspiciously, Kyoto University’s Shinya Yamanaka published his work on the reprogramming of fibroblasts to generate induced pluripotent stem cells (iPSCs). Kotter became interested in this research on cell reprogramming and started looking through the literature when he came across the work of Robert L. Davis, Harold Weintraub, and Andrew B. Lassar, researchers at Fred Hutchinson Cancer Research Center. In 1987, they reported the cloning of MyoD, a gene capable of turning fibroblasts into muscle cells.
“[Cellular programming] inherently is a synthetic biology process,” Kotter observes. “You’re engineering cells and switching on genes, and the identity of the cell changes.”
Kotter is currently an academic neurosurgeon at the University of Cambridge. His research forms the scientific basis of two spinout companies: bit.bio, a synthetic biology company focused on cellular reprogramming, and Meatable, a cultured-meat startup. According to Kotter, synthetic biology has three main components: reading, writing, and executing genetic information. Kotter believes that the field has already done a lot of heavy lifting in the reading and writing domains and that the problem is really in the execution piece.
“When you just throw viral vectors onto stem cells to make them change from one cell type to another cell type, you see two things: random integration and gene silencing,” Kotter points out. “If you use genomic safe harbors, you can make this deterministic—suddenly, you have a single-step protocol for any cell type that you want.”
At bit.bio, a patented safe harbor gene targeting approach called opti-ox (optimized inducible overexpression) is used to inducibly express transcription factor combinations that reprogram human iPSCs into highly defined and mature human cell types. Kotter and bit.bio have built a discovery platform that combines genetic screening, big data, machine learning, and large-scale experimentation, and they are using it to study how transcription factor codes are combined in human cells of different types.
This work could support medical applications. Moreover, deterministic reprogramming could be extended to cells of other organisms to support various industrial applications. For example, reprogrammed cow cells could be used to produce cultured meat (as Meatable demonstrates) or laboratory-grown leather.
Membraneless organelles for biocomputing
As a graduate student at the University of Paris, Haotian Guo, PhD, studied the spatial regulation of complex biochemical reactions. This kind of regulation is accomplished by living systems that have the ability to organize intracellular components and maintain isolated microenvironments. It is a kind of regulation that bioengineers are keen to emulate.
Guo’s research led him to focus on liquid-liquid phase separation, the creation of membraneless compartments in eukaryotic cells, including P granules, nucleoli, heterochromatin, and stress granules. Liquid-liquid phase separation is the same process that makes emulsion droplets that are integral to many of today’s single-cell sequencing technologies. These “membraneless organelles” concentrate associated biomolecules and increase biochemical reaction efficiency. They can also protect mRNA or proteins to promote cell survival under stress.
“In biosynthetic manufacturing, you need chromatography to purify all the proteins or all the products away from different impurities—but nature doesn’t do this,” Guo says. “Natural plants and organisms have organized to capture their product and push it through a secretion pathway.”
Guo established an approach to generating synthetic membraneless organelles in E. coli by engineering RNAs that can autonomously phase separate. These man-made membraneless organelles are named transcriptionally engineered addressable RNA solvent droplets (TEARS). They make it possible to study the basic biology of organelles and find new drugs that target condensates. They also facilitate the engineering of metabolic pathways that can improve the yield and purity of desired products.
To commercialize TEARS technology, Guo co-founded and became the CEO of Ailurus Biotechnology. Ailurus provides the TEAR-2 development kit to help customers engineer phase separations and spatially control intracellular reactions.
Although Guo sees synthetic biology as a major disruptor to the manufacturing industry, he’s interested in the use of biological systems as information processing tools. “Biocomputation is still a science fiction word for most people,” Guo admits. “But the last wave in the biotechnology industry has been largely driven by genetic information–related technologies, like next-generation sequencing and the synthesis of mRNA vaccines.”
Along those lines, Guo and Ailurus are using biocomputing to develop systems that use biological components to perceive, decide, and act. These biocomputing “programs” can be both miniaturized and parallelized into cells or similar compartments, turning them into microscale laboratories that operate R&D and production tasks automatically. By maintaining spatially distinct compartments, such as TEARS, these microscale laboratories can perform on a massive scale to generate biologically relevant data much more efficiently than humans, robots, and electronic computers.
“Waste is a failure of the imagination”
As a chemical engineer with several decades of experience, Nicole Richards had been thinking about the impact of industries on the environment for quite some time. Then a unique opportunity to help the environment came along. In 2020, she became CEO of Allonnia, a Ginkgo Bioworks spinoff that uses synthetic
biology to advance bioremediation.
“Contaminants from manufacturing processes seep into our soil and our water, but over time, nature evolves to degrade anything in its environment—it works in a circular economy,” Richards says. “What’s so interesting to me about synthetic biology is that it allows you to amplify existing biological functions. It just makes so much sense to amplify the metabolic degradation of contaminants. Our tagline is: ‘Waste is a failure of the imagination.’”
Allonnia starts by visiting a contaminated site and collecting samples. Within these samples there are microorganisms that survive despite the presence of contaminants. There may even be microorganisms that survive because they have developed a way to metabolize the contaminants in their niche and outcompete microorganisms that cannot.
“You can’t take just a nonnative organism and think it’s going to survive,” Richard remarks. “If it doesn’t come from there, all the other organisms will outcompete it.”
After Allonnia finds an organism that has a contaminant-metabolizing advantage in the organism’s own niche, the company begins to identify and engineer strains that would excel in the desired bioremediation application. For example, in an application to support sustainable mining, Allonnia would look for organisms that possess proteins that bind to specific rare earth elements. Such organisms would be used to remove the element from environments such as waste streams.
From her time at Allonnia so far, Richards is convinced that synthetic biology offers tools to restore the health of the world around us, including the water we drink. “I don’t think we should tolerate drinking water that has even trace levels of 1,4-dioxane and 50% polyfluoroalkyl substances,” Richards maintains. “That hurts human health.”
