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Sustainable biofuel: Design principles for bioengineered microbe catalysts

The US has been stuck on corn kernels for producing ethanol, rather than woody “cellulosic” material. Efficient microbes for converting cellulose to biofuel could change the game.

Biofuels that are more eco-friendly would be made from cellulose, the tough parts of plants like woody stems and corn stalks—rather than starchy kernels that yield today’s ethanol. But researchers don’t yet have an efficient way to pull the sugars in cellulose apart and then ferment them into fuel. Fei Wen, an assistant professor of chemical engineering at the University of Michigan, has shown how computer modeling could enable the design of genetically-engineered microbes that can do both jobs—break down the cellulose and ferment it.

She and her collaborators recently published a paper on the work in Nature Catalysis. In “Elucidating structure–performance relationships in whole-cell cooperative enzyme catalysis,” Wen and her colleagues establish design principles that could help researchers get beyond the current trial-and-error approach of engineering microbes for producing biofuel. She answered questions about the advance from Michigan Engineering.

What makes it so challenging to engineer microbes to break down cellulose?

In the past, the engineering strategies relied on trial-and-error methods because it was difficult to quantitatively evaluate how enzymes assemble on the surface of microbes. Researchers could only treat these microbes as blackboxes.

We developed experimental tools that enabled us to study how the enzymes were arranged on the membranes of baker’s yeast at the level of individual cells. These quantitative data in turn enabled us to build the first computational model of how these surface enzymes operate, and we uncovered design principles for future efforts.

Why did you choose baker’s yeast?

Baker’s yeast is easy to genetically modify, good at producing ethanol, and industrially very robust.

How did you augment the yeast?

With our computer model, we explored how to control the enzymes on the surface. We screened dozens of microbes—each decorated with suites of enzymes organized in different structures—to identify good candidates. The engineered microbes can break the cellulose all the way down to the smallest sugar unit—glucose—and keep the glucose on the surface of the microbes. This way, all of the glucose is available to be fermented into fuel molecules using the complex metabolic network of the microbes. This is how we achieved “one pot” conversion with the highest titer reported to date, 7.17 grams of ethanol per liter of fermentation broth.

What’s the lure of one-pot biofuel conversion? 

One-pot conversion can greatly simplify the industrial production of biofuels by reducing the number of manufacturing steps. As a result, it can significantly cut down the cost of cellulosic biofuels to make them economically competitive with other forms of transportation fuels, which is critical to achieving energy sustainability. 

One of the reasons why producing cellulosic biofuel is so expensive is because the cellulosic plant material has to be softened up before the microbes can get to work. Could microbes engineered in this way work without preprocessing?

Expanding the suites of enzymes used to decorate the surface of microbes, such as those that can break down hemicellulose and lignin (another two major components of plant biomass), could significantly reduce and ultimately eliminate the need for pre-processing biomass. 

What are some of the takeaways from your study in terms of designing baker’s yeast that is effective at converting cellulose into ethanol? 

The most unexpected finding is that more is not necessarily better! We found that molecular crowding is actually taking place when decorating the baker’s yeast cell surface with enzymes—although baker’s yeast is about 100,000,000 times bigger than enzymes. Therefore, future designs should focus on optimizing how to best organize these enzymes for a maximum packing on the surface to achieve the best performance. 

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Renewable resource switchgrass for heating and production biofuel. Getty Images.

What kind of plant material did you use, and is this generalizable to other types?

We chose one of the most commonly used materials, an acid-treated form of cellulose, in this study to compare the performance of our baker’s yeast with others’. We are currently testing our baker’s yeast on untreated crystalline cellulose, which is tougher to break down, and the results are encouraging. We expect similar performance enhancement for other types of plant materials, such as straw and switchgrass.

Can other researchers use what you learned?

Yes, they can use our model and experimental tools directly if they’d like to explore very different designs of enzymes. And if they’re using the particular design in this work, we provide guiding principles. 

This study was done in close collaboration with Jung-Kul Lee, a professor of chemical engineering at Konkuk University in Seoul, South Korea. Robert Ziff, a professor of chemical engineering at U-M, also contributed to this work.