.png)
Imagine if your favorite gadgets, your phone, smartwatch, or coffee maker, were crafted as a single, unchangeable piece. If something broke, you’d have to toss it and buy a new one. No upgrades. No recycling. Frustrating, right? That’s what synthetic organelle design often feels like today.
Synthetic organelles, tiny engineered compartments inside cells, are unlocking revolutionary biotech applications (see our previous chapters for more).
From organizing enzymes to streamlining complex processes, these cellular wonders hold immense potential. But here’s the catch: most researchers still rely on “direct fusion” to build them. Think of it as duct-taping parts together. It works…until it doesn’t.
Modular design offers a better way. Flexible, reusable, and robust, modularity is the perfect foundation for the synthetic biology revolution. But despite its promise, modularity remains rare in this field. Why? And how can it unlock the full potential of synthetic organelles? Let’s dive in.
Keep updated and don’t forget to subscribe!
Direct fusion sounds simple: stick two proteins together, one assembly domain for phase separation, another domain for your desired function, and voilà! Your organelle is ready. Except… it rarely works as planned.
Take this case: researchers developed a light-controlled enzyme clustering system, termed OptoClusters, enabling reversible enzymatic pathways. But instead of tunable clustering, some proteins clumped together all the time. As the authors noted in their paper (2019):
“The propensity to cluster can be strongly influenced by fusion to particular metabolic enzymes.”
In another study in 2020, researchers added fused enzymes to agglomerated spider-silk proteins in E. coli, hoping to boost catalytic efficiency. Instead, the fused enzymes underperformed, delivering lower yields than the normal, free-floating ones.
How direct fusions work, and examples where they fail.
And these are just the experiments that made it to publication. Imagine how many failed attempts never saw the light of day.
Phase separation, a powerful mechanism for forming micron-scale compartments, is a game-changer for synthetic organelles. But it’s also a double-edged sword. Many phase-separating proteins are aggregation-prone, difficult to purify, and true troublemakers (J Mol Biol, 2018).
More importantly, direct fusion, despite its wide application for decades, has fundamental flaws by itself.
In a report published in 2016, Clp proteases, key for protein degradation in bacteria E. coli, when fused to different fluorescent proteins, had various distribution patterns, causing artifacts in research. (Nature Method, 2013).
If it doesn’t even work reliably for GFPs, how can we expect it to work for complex enzymes?
Here’s where modularity changes the game:
It’s like building with LEGO bricks instead of pouring concrete. It just makes more sense.
Modular design avoids direct fusion’s pitfalls by separating the problematic regions that form the organelle structure from the functional components. Need a function in organelles? Assemble components modularly, only when required. This approach offers control at your fingertips.
A simple comparison between direct fusion and modular designs.
With this spirit, the design of synthetic organelles can follow these modular principles:
For example, the modular design of our RNA organelle, TEARS, follows this recipe:
Despite its benefits, modularity remains rare in liquid-liquid phase separation (LLPS) studies. Why? Many researchers trained in classic genetic engineering are familiar with designing fusion proteins but lack exposure to modular principles. Without adopting the mindset of synthetic biology, aka "genetic engineering 2.0", the complexity of designing modular assemblies may seem daunting or simply go unnoticed.
On the other hand, synthetic biologists often focus on genetic circuits and modules, rarely delving into biophysics or molecular phase transitions.
But in interdisciplinary work bridging LLPS and synthetic biology, modular designs have shown promise from the start:
In 2017, the iGEM Paris Bettencourt team created RNA organelles in E. coli to organize split GFP systems, reusing existing tools and demonstrating modularity’s power.
In 2018, the iGEM Peking team proposed SPOT, a modular design for recruiting enzymes using nanobodies in yeast. That same year, another team of researchers developed an in vitro droplet to recruit cargos tagged with SYNZIP.
As more synthetic biologists learn LLPS and more biophysicists adopt modularity, a new trend of engineered modular biocondensates is emerging.
When your organelle design has a modular “application programming interface”, everything gets easier.
Take violacein biosynthesis as an example, a violet color pigment with activity for anti-cancer and anti-fungus. When Zhao et al. fused each enzyme with phase-separating domains in the OptoClusters, adding more enzymes required careful fine-tuning of gene expression to avoid system instability. In contrast, when we applied TEARS to control violacein pathways, we modularly recruited enzymes to the organelle via binding domains, avoiding any changes in organelle assembly entirely.
Then, scaling complexity becomes a breeze. We could recruit the entire translational machinery, including over 30 distinct molecules, with a single ribosomal binding site. In another project, a complex procedure was designed in organelles to enable incorporation of unnatural amino acids for codon expansion.
And debugging? Modular systems make it simple as well! In our experiment to synthesize lycopene (the anti-aging color pigment that makes tomato red), initially, I designed the study expecting an increase in production. Clustering enzymes close to each other should improve the processing of metabolic intermediates. In reality, the reaction is repressed, by a lot! Modular RNA organelles helped us pinpoint the issue. It turns out, these enzymes required membrane-binding to function. If we’d used direct fusion, we might still be scratching our heads.
Modular design isn’t just a buzzword. It’s a practical principle that makes bioengineering easier, faster, and more scalable. And thanks to modularity, today’s components may unlock unexpected applications tomorrow.
With better tools (like AI-driven design and high-throughput experiments), the potential compounds exponentially. As synthetic organelle research evolves, modularity will be the difference between organelles that merely work and those that truly excel.
Let’s build organelles as adaptable as the challenges they’re meant to solve!
Ailurus is a pioneering biocomputer company, programming biology as living smart devices, with products like PandaPure® that streamline protein expression and purification directly within cells, eliminating the need for columns or beads. Our mission is to make biology a general-purpose technology - easy to use and as accessible as modern computers.
