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Cell-Free Biomanufacturing for Cheaper, Cleaner Chemicals

Biotech startup Greenlight Biosciences has a cell-free approach to microbial chemical production.

Biotechnologists have genetically engineered bacteria and other microbes to produce biofuels and chemicals from renewable resources. But complex metabolic pathways in these living organisms can be difficult to control, and the desired products can be poisonous to the microbes. What if you could eliminate the living cell altogether?

Greenlight Biosciences researchers
Brewing biochemistry: Greenlight Biosciences researchers Kamil Gedeon (seated) and Stephanie DeMarino (standing) monitor a culture of bacteria engineered to produce enzymes for cell-free chemical synthesis.

Greenlight Biosciences, a Boston-area startup, engineers microbes to make various enzymes that can produce chemicals and then breaks open the bugs to harvest those enzymes. The scientists don’t have to go to the trouble of isolating the enzymes from the other cellular material; instead, they add chemicals to inhibit unwanted biochemical reactions. By mixing slurries based on different microbes with sugars and other carbon-based feedstocks, the company can generate complex reactions to produce a variety of chemicals. Greenlight say its technology enables the company to make cheaper versions of existing chemicals and has already produced a food additive, drug products, and pesticides and herbicides.

The biggest motivation in starting the company was to figure out how to produce such compounds in a more environmentally friendly way, says CEO Andrey Zarur. But Greenlight’s products also have to be cheaper than those produced by chemical- or cell-based manufacturing, he says, or industries will be reluctant to use them.

Greenlight’s strategy is a departure from classic fermentation processes that depend on vats of living microbes. It is also unlike a different approach to genetic engineering, often called synthetic biology, that tweaks the pathways in microbes so that they are optimized to fabricate desirable compounds. Several companies are engineering bacteria and yeast to produce specialty chemicals, but for the most part, these groups keep the bugs alive. Amyris, for example, can make biofuels, medicines, and chemicals used in cosmetics and lubricants by engineering microbes with new sets of enzymes that can modify sugars and other starting materials (see “Amyris Announces Commercial Production of Biochemicals” and “Microbes Can Mass-Produce Malaria Drug”). Metabolix has engineered bacteria to produce biodegradable plastic (see “A Bioplastic Goes Commercial”).

A problem with that strategy is that when bacteria and other microbes are turned into living chemical factories, they still have to put some resources into growing instead of chemical production, says Mark Styczynski, a metabolic engineer and systems biologist at the Georgia Institute of Technology. Furthermore, even in a seemingly simply bacterium, metabolism is complicated. “Metabolic pathways have complex regulation within them and across them,” he says. Changing one metabolic pathway to improve chemical production can have broad and sometimes negative consequences for the rest of the cell.

Thus, separating the production pathway from the needs of the cell could be a huge advantage, he says. Greenlight doesn’t completely avoid microbes. In the company’s sunny lab space north of Boston, researchers use bubbling bioreactors to grow bacteria in liquid culture, maintaining different species and strains that can produce a variety of enzymes. Once the bugs have reached a certain density, the researchers send them through a high-pressure extruder to break them into pieces. Then they add drugs to the resulting gray slurry to turn off most of the cells’ metabolic enzymes; the useful enzymes are unaffected because they have been engineered to resist the drugs.

The technology that keeps the exposed metabolic pathways working was developed by James Swartz, a biochemical engineer at Stanford University who left his position as a protein engineer at the biotechnology company Genentech to develop cell-free methods for producing pharmacological proteins (insulin is an example of a druglike protein that can be produced by biotechnology). Seeking more control over the biological machinery that produces proteins, Swartz figured out how to give that machinery the biochemical environment it needed even outside its normal home in a cell. Not only did his methods enable him to make more complex proteins, but it turned out they could also be used to control biological machinery to make small molecules and chemicals. “We’ve found that by reproducing the chemical conditions that occur inside the cell, we activate a lot of metabolic processes, even ones people thought were too complicated,” he says.

Greenlight can troubleshoot and tweak the metabolic production of chemicals in ways more akin to chemical engineering than anything found in typical microbial engineering. The cell-free slurries are active for 96 hours before the enzymes begin to break down. At that point, a new batch of microbes must be grown.

“One of the beauties of the cell is it is self-replicating,” says David Berry, a principle at Flagship Ventures, who cofounded the biofuels companies LS9 and Joule Unlimited. (Berry is a 2007 MIT Technology Review Innovator Under 35.) But even though a cell-free system misses out on that advantage, there are other benefits, such as superior flexibility. “There is the potential to work with more inputs and to work around situations where certain pathways currently don’t work because of the needs of the cell,” Berry says.

Zarur says Greenlight could have its first product on the market at the beginning of next year. It will be a food supplement with health benefits, he says.

The company has also received a $4.5 million grant from ARPA-E to develop a system for converting methane, the main ingredient in natural gas, to liquid fuel. The agency says such technology could “enable mobile fermenters to access remote sources of natural gas for low-cost conversion of natural gas to liquid fuel.”

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