The globalization that began during the 1990s and flourished into this millennia reshaped aspects of our lives in ways we are only now appreciating. Many of these changes were net positive: cheaper goods increased domestic purchasing power, ultimately improving the quality of life for many Americans. More concerningly, as globalization moved where materials were actually manufactured, Westerners could externalize the immediate impact of the greenhouse emissions and pollution expended for our everyday items that had conveniently been moved to foreign, lower cost locations. Calls for sustainable supply chains were largely drowned out by an intoxication for commodity-level pricing.
Then COVID-19 permeated the world. As governments around the world responded to COVID’s spread, responses ranged from indifference (Brazil) to fierce lockdowns (China). The varying political responses highlighted some of the weaknesses inherent in globalized supply chains. Governments and multinational organizations began looking to find ways of securing supply chains within borders, without compromising on price and local, deleterious environmental impacts.
Biomanufacturing has been tasked with bridging this gap as it offers both 1) cheaper costs and 2) reduced environmental impact. Abundant, sustainable feedstocks provide carbon-negative practices to produce end-products at lower prices. For example, simple sugars power automated chemical synthesis by little biofactories (e.g. microbes) instead of petrochemicals performing the same chemical transformations at high temperatures with significantly more input energy and fossil fuels. The high-tech jobs and reduced emissions to run these fermentation facilities are the politician’s bonus.
Two forms of biomanufacturing essentially dominate conversation among scientists today — the classic bio-fermentation or “whole-cell” approach, versus the more modern, “cell-free” bioproduction system.
In regards to the first approach, whole-cell fermentation is nothing new. Microbes have been used for centuries to make our breads, wines, spirits, and more. However, the introduction of synthetic biology to genetically engineer those microbes, the workhorses of fermentation, to create valuable end products is a much newer concept, dating back ~ 40 years. Genentech’s foundation was based around using microbes to biomanufacture the first recombinant protein drug in growth hormone in 1985. Their approach has expanded into the biologics therapeutic market that we know today. Nowadays, the toolkit to read and write these microbes are being commoditized, allowing for high-throughput design and engineering of programmable bugs. Ginkgo and Zymergen have been the flagship companies of synthetic biology’s approach for whole-cell bioproduction over the last decade.
In contrast, cell-free biological production is a much newer concept. The Economist has done an excellent job capturing its potential here and here. We, at KdT, view cell-free technologies as one of the current edges of biomanufacturing for chemicals and materials. In theory, cell-free chemical synthesis systems offer lots of advantages over traditional fermentation. Rather than engineering the microbe to produce a certain reaction, cell-free biomanufacturing isolates the specific enzymes involved and runs them through the reaction many times over as long as the isolated enzymes remain stable and active (Figure 1).
Cell-free systems offer many advantages in bioproduction; they require significantly less energy and less complexity — it’s both easier and simpler to optimize a bug to create a certain molecule if the microbe need not also keep itself alive (and allocate resources accordingly). Additionally, cell-free systems, if done properly, minimize the downstream processing required for isolation of the end-product. In many microbial systems, the end product or its building blocks are toxic to the microbe itself, which limits production of the end product. Again, not needing to keep the microbe alive, cell-free systems avoid this concern entirely. These advantages make cell-free systems a very attractive format for bio-based chemical and materials production.
This entrance of cell-free systems into mainstream bioproduction with successes like Solugen, Sutro Biopharma, and Debut Biotech has formed the landscape of a bioproduction battle. Some view the future of chemical production as entirely cell-free; whereby chemical synthesis is a series of enzyme catalysis reactions (e.g. Solugen) or a series of cartridges (e.g. Debut Biotech). Others view biomanufacturing as a diversified landscape — where some products can easily be made cell-free and others are preferred to be made through whole cell fermentation, as cell-free has limitations.
Investors and industry professionals now have to ask two key questions:
- Which chemicals and materials are better suited for traditional, whole-cell fermentation versus cell-free bioproduction?
- What tools are limiting cell-free biomanufacturing from becoming even more mainstream?
Which situations do not currently work well for cell-free biomanufacturing & what innovations are required to advance cell-free biomanufacturing?:
High number of synthetic steps: This one might appear obvious — the more steps, the less likely that a cell-free system is a practical alternative. With each additional step, there are a corresponding number of optimizations that need to take place both for that step, but more critically, for the entire system. The practical limitation is clear when you consider you need to grow up microbes to isolate (and potentially purify) the appropriate enzyme for your chemical synthesis. A recent paper in Science showed an 11-step conversion of carbon dioxide to starch was six times faster than what is capable in corn (a whole cell system).
Solution: While there is currently not an ideal solution for this, groups are taking a more systematic approach to designing biosynthetic pathways. By utilizing extensive data and biosynthetic pathway design tools to map out the most efficient routes, they are able to prioritize pathways and foresee issues in regards to purification and downstream processing (see below about optimizing to scale up for industrial production).
Presence of endogenous, harmful intermediates: Cells protect their component parts from each other through compartmentalization — shielding certain components from the toxic effects of other cellular processes. When complex reactions take place in cell-free reactions, the absence of compartmentalization of reactions can kill or disturb other enzymes and co-factors in the system. Without a repair pathway or compartmentalizing these intermediate products that are created, enzymes and cofactors can unintentionally be inactivated or inhibited, leading to disastrous effects. Thus, reactions need to be carefully charted out to see if intermediate products inhibit other enzymes and cofactors that are vital to the cell-free process.
Solution: Strategies employed by companies like Debut Biotech where single cell-free steps are confined to individual “cartridges’’ help create compartments to protect activity levels of enzymes in reaction series versus “one pot” reactions (where all enzymes are confined in a single step).
Systems requiring an extensive number of co-factors: Cofactors are expensive biomolecules that help enzymes initiate reactions. The cost of these cofactors can range from immaterial to economic crippling — depending on the scarcity and manufacturability of the cofactor. They vary widely. Thus, those cofactors that have short half-lives and quickly biodegrade have the potential to be the Achilles’ heel of cell-free. When a large number of cofactors are needed, it can also complicate reaction pathways, leading to an impractical biomanufacturing process. Nonetheless, given the importance of cofactors in these pathways, groups are optimizing pathways to lessen the burden of an increased reliance on cofactors.
Solution: To tackle this, companies like HydRegen are developing a novel hydrogen-powered NADH recycling technique that could be translated to recycle cofactors in biosynthetic reactions in the future. Synthetic biology and humanity more broadly would benefit from increased attention in solving this cofactor barrier.
High protein stability & turnover: Proteins are very sensitive to their surrounding environment and will only fold (and function) under a narrow set of conditions. In cell-free reactions, you have to harvest enzymes to drive these reactions. If the enzyme degrades quickly as the reaction proceeds, more must be continuously reharvested to ensure the reaction runs to completion.
This continuous reharvesting may eventually lead to larger underlying costs on the cell-free system, ultimately creating an insurmountable barrier to broader adoption of cell free systems.
Solution: However there is promise. Companies like Aperiam Bio are working on developing an AI platform to engineer for protein stability to overcome these challenges and lower costs. Ultimately, for cell-free chemistry, enzymes with high stability that react quickly will be instrumental to avoid enzyme turnover and keep costs manageable as it scales.
Scaling up for industrial production: Like all manufacturing efforts, cell-free systems need to demonstrate an ability to scale to commercial quantities to reach their potential. Much like whole cell systems, the methodology used to create a cell-free model can differ significantly between a small prototype model and an industrial scale. The ability to scale is the difference between a novel idea and a transformational company.
Solution: To alleviate these issues, groups like Doulix are developing a rapid prototyping platform that can design, test, and optimize biosynthetic platforms for cell-free systems. This extensive in silico prototyping can discover competing endogenous metabolism reactions, different co-factors can be modeled to compare costs and product yields, and pathways can quickly be tested in living cells and can be further optimized to scale up to industrial levels.
Despite the promise of cell-free synthetic biology, there are still challenges to overcome. Below (Table 1) we have highlighted a few companies that are working to enable cell-free biomanufacturing to become mainstream.
As the world of biotech goes through its Cambrian explosion, we at KdT are incredibly excited for the future of biomanufacturing. We believe the next generation of green chemicals and end-consumer goods will be produced with the combination of centuries-old fermentation processes and cell-free biomanufacturing. We’re thrilled to support the most ambitious founders and companies as they reinvent manufacturing from a computational biology perspective. If you are interested in creating for improving the future of biomanufacturing, feel free to reach out!
By Anirudh Sudarshan and Phil Grayeski, KdT Ventures