Of the over 450,000 land plant species, only a few thousand are studied, and fewer have been sequenced. “There’s a huge trough of plants in completely different families and phyla that are not as close in terms of relationship to Arabidopsis,” explained Sebastian Cocioba, an independent plant bioengineer.

And that’s just the untapped plant diversity. Synthetic biology researchers and companies are now looking beyond model organisms to find novel molecules and insights in previously understudied species across the tree of life.

Research on non-model organisms doesn’t have to be limited to discovering new metabolites. “Let's say there are metabolic pathways that are unique to this one phylum of plants. Instead of trying to get that gene circuit in Arabidopsis or tobacco, why don't we domesticate the plant that produces it to better harness that native ability,” said Cocioba.

Tinkering with Non-Model Organisms

“Non-model microbes tend to have a lot of phenotypes that you just can't build easily in the model systems,” said Carrie Eckert, a synthetic biologist at Oak Ridge National Laboratory. However, the model organisms have genetic tools that must be built for non-model organisms.

Over the last decade, the fall in sequencing costs has greatly boosted research on non-model organisms. It has made sequencing entire genomes and transcriptomes feasible. “Now we can look at base pair scale instead of these kilobases of regions that look important,” said Katharine Grabek, CSO of Fauna Bio, a Berkeley-based biotech working on non-model mammalian systems. 

However, sequencing plants can still be challenging, especially for cash-strapped labs or early-stage startups. Many plant genomes are rich in repeats and transposons and are polyploid. As a result, “most plants are several times the size of a human genome. Doing the assembly of that requires more computing,” said Cocioba.

However, if the plant is being developed as a specialist model, Cocioba added, being able to culture it and study its gene expression could be sufficient. Compared to a general model, a specialist model is when an organism is developed to study a particular trait, such as drought tolerance. Regardless, recalcitrance, or the ability to resist tissue culture, can be a major challenge when working with new plant species.

To overcome this barrier, researchers have developed tools based on insights into the role of transcription factors in plant regeneration. “There are technologies like Baby Boom, which is a chimera of two different transcription factors, where when you put those factors into the plant, they start producing shoots,” said Coicioba. Breaking recalcitrance makes non-model plants more receptive to genetic engineering, regardless of how little is known about culturing them. 

Similarly, with transforming non-model bacteria, “a main limiting factor is that they recognize any foreign DNA you're putting into them and are active against it,” said Eckert. The bacteria look at methylation patterns to discriminate between self and foreign DNA. “What we can do is find the enzymes that make those methylation patterns and express those in E. coli that we're using for our cloning DNA,” Eckert added. This fools the target bacteria to take in foreign DNA, making it easier to engineer them.

Precise and efficient gene editing tools, such as CRISPR, are also enabling research into various non-model organisms. For culturing plants, it can be used to overcome REcalcitrance. “If there’s a repressor involved in the recalcitrance mechanism, CRISPR can edit it out,” said Cocioba.

In non-model organisms, CRISPR interference can be used to reveal the functions of novel genes. Moreover, genome-wide CRISPR screens reveal which genes are important for fitness, the metabolic pathways involved in metabolite production, and the links between them.

When faced with an abundance of choice, high-throughput functional and comparative genomics could also inform which non-model organism to work with. Berkeley-based biotech Arcadia does this by tracking patterns across the tree of life at different biological scales.

“Since not all genes have the same evolutionary history as the organism as a whole, you can find interesting patterns where they depart from what you might expect,” said Arcadia CEO Seemay Chou. “And maybe examples of novelty, like a sudden expansion of a gene family or an emergence of a gene family.” Based on the desired capabilities, the company’s platform makes predictions on which organisms could be ideal.

Applications of Non-Model Organisms

Often, a non-model organism may exhibit lower fitness when grown in a lab. Altering the genome to keep only what is necessary could overcome this challenge. By mapping genotype-phenotype relations with genome-wide CRISPR screens, scientists can identify which genes are essential to a non-model system. This allows the creation of a genome-reduced chassis with improved genetic stability, resource utilization, transformation efficiency, and, in turn, greater yield of the desired product. 

Non-model organisms, particularly microbes, are particularly suited to environmental protection. This is because they have high-stress tolerance and unique metabolic pathways adapted to living in polluted sites. For example, scientists have discovered multiple enzymes that can break down plastics in microbes. While isolating these enzymes is an option, using these microbes to convert plastic and other waste could improve efficiency.

Eckert’s research looks at the genetic factors that lead to the ability of an organism to be tolerant to something or to be able to use something. One of the microbes Eckert works with is Pseudomonas putida, a non-model soil bacterium. “We have developed genetic tools for it, and it's becoming one of the model systems that can break down lignin,” said Eckert.

Research on non-model organisms could also benefit human health. Speaking on Arcadia’s work on ticks, Chou said, “We have identified a set of molecules that are really effective against blocking itch.” Ticks have evolved to bite animals without being pesky, and the key is molecules in their saliva.

Fauna Bio performs single-cell transcriptomics on tissues from the thirteen-lined ground squirrel, an animal that can hibernate for over six months a year. During hibernation, these squirrels can withstand body temperatures as low as 4-8°C, resulting in cellular damage. Insights into how they do this could offer hints into how to reverse cellular damage in human diseases. The company looks at what genes are differentially expressed during hibernating and compares it to the genomes of humans and other mammals.

“We're adding in other animals with extreme physiology, such as the spiny mouse,” said Grabek. The mouse has impressive regenerative potential and can heal multiple tissues without scars. In another project, Fauna Bio is exposing ground squirrels to long-term low-dose radiation to ground squirrels, mimicking what astronauts would experience in space to investigate if hibernation has a protective effect. 

Scaling Up

Cocioba experimented with different white flowers in search of a template for designing flowers. The species that finally clicked was an unlabeled petunia that his mother bought at a farmer's market. “I struggled with petunias in the past, as many commercial cultivars were very recalcitrant. I tried this one, and it's the fastest tissue culture plant I've ever worked with,” said Cocioba.

This anecdote illustrates how non-model organisms can offer surprises that could benefit how synthetic biology is done. Additionally, they could help overcome the challenge of scaling up. “Maybe the way to increase scaling ability by an order of magnitude is not to make E. coli slightly different. It could be to start from a totally different chassis that might have better ingredients for success,” said Chou.

As synthetic biology advances make it easier to engineer organisms, researchers will explore more diverse and resilient life forms. These will have applications in sustainability, human health, and industrial applications. In a positive feedback loop, these non-model organisms could also reveal genetic components that will expand the scope and scale of synthetic biology.