Engineering and Evolution in Biotechnology

FROM THE OCTOBER ISSUE: Advancements in yeast optimization are gaining momentum. New companies are entering the space and veterans are improving their processes.
By Lisa Gibson | September 20, 2017

Yeast strains used in the ethanol industry have remained largely static — even genetic engineering for favorable traits has taken hold widely just in the past few years. Now, multiple companies provide upgraded strains and continue research on further improvements, while even more enter the market. Some, meanwhile, are going a step further to isolate and breed new strains.

“Yeast has been stagnant,” says Peter Halling, vice president of commercial biofuels for Novozymes. “But there has been a lot of development in the past few years.” Earlier this year, Novozymes announced it will begin offering yeast strains to the ethanol industry, in addition to its well-known enzyme offerings. It’s a natural progression for a company with extensive experience in microorganisms, Halling says. “We’ve been playing in this space for more than 40 years. We know this by heart and we believe we have something to contribute.”

Pauline Teunissen, a DuPont Industrial Biosciences scientist, says yeast has, indeed, remained the same for quite some time. When genetic modification proved a viable pathway to achieve desirable traits, the industry had to wait for regulatory approval. “It means you also have to deal with DDGs that are derived from genetically modified yeast,” Teunissen points out. Engineering and evolution go hand in hand to modify yeast, she says, and a large team at DuPont is dedicated to exploring all avenues. “There are a lot of different areas that we are focusing on and using a lot of different techniques to do so.”

New and Existing Strains
DuPont is one of many companies that engineers yeast strains, mating different types of yeast to take advantage of certain properties, Teunissen says, adding that DuPont’s team also makes use of natural evolution techniques. “Natural evolution is using a strain you already have or a ‘new’ strain from a culture collection and then expose the strain to the conditions you want the strain to perform in — for instance, if you want a thermotolerant strain, you subject a strain to high temperatures — and then go to multiple rounds of selection to obtain a strain with improved properties,” she says.

Matt Richards, director of application technology for Lallemand Biofuels & Distilled Spirits, says Lallemand uses a mixture of classical biology techniques — cell mating, directed evolution, screening and isolation of strains from environments with desired properties — and advanced metabolic engineering techniques to develop yeast with desired traits for the ethanol industry.

Novozymes is taking a different approach. With a partner Halling declines to name, the company is using a unique breeding platform on a number of new strains the ethanol industry hasn’t seen before, he says. “That gives us the opportunity to not only go in and tweak the individual strain, but also to really look at a much broader portfolio of strains and select the ones that are unique and provide a different performance compared to what you see today.” The technique will involve Novozymes’ enzymes, also, he says, leveraging the synergies to deliver an optimal solution.

Novozymes has customers using its C5 yeast for cellulosic ethanol, but now plans to get into the starch-based ethanol market, offering strains for commercial processes next year. Raízen in Brazil uses the C5 yeast to make ethanol from sugarcane bagasse. Halling says the starch-based ethanol yeast is being tested by potential customers now.

The Main Objectives
Yeast modification can be done for multiple reasons, but one of the main goals is ethanol yield optimization, both Halling and Teunissen say. DuPont has a strain that accomplishes this by reducing the number of byproducts of yeast. “We’ve been able to develop a yeast that can increase ethanol yield up to 3 percent,” Teunissen says. The pathway introduces three enzymes to the yeast that reroute the carbon around alcoholic fermentation, thereby producing less carbon dioxide. The next step in the research is to reduce byproducts even further, she says. Lallemand also aims to reduce byproducts for increased yield in its yeast offerings, focusing on glycerol, Richards says.

Engineered yeast can increase corn oil yield and upgrade coproducts, too, Teunissen says. “Are there certain enzymes you can express in the yeast that help generate certain coproducts?

“There are different ways you can look at yeast engineering for lots of different reasons,” she says, adding improving stability and robustness toward common stressors in the ethanol production process is another main goal. Halling and Richards say it’s a major component of their research, too. 

Dennis Bayrock, global director of fermentation research in the Phibro Ethanol Performance Group and adjunct professor at the University of Minnesota, outlines two main concerns in yeast tolerance and robustness: vitality and viability. “When yeast viability and vitality are optimized, the yeast population as a whole can outcompete bacterial contamination for nutrients and growth,” he says. “The yeast are better able to tolerate the organic acids produced by bacterial contamination, survive process upsets at the plant, and better handle stressful conditions at the plant.

“In general, genetic engineering of fuel ethanol yeast strains have — up to now —focused primarily on yeast production characteristics — some of which include native production of amylases, native production of cellulosic enzymes, reduction in glycerol production and utilization of both C5 and C6 sugars,” Bayrock adds. While these characteristics are important to ethanol production, yeast modifications focused on tolerance to high temperatures, organic acid, fusel stress, osmotic stresses and other stressors represent the next opportunity for improvement, he says. “The next evolution of [genetically modified] yeast will need to better incorporate aspects of yeast viability and vitality to raise the fuel ethanol industry to the next level of production.”

Many ethanol producers also would like to see yeast with an increased capacity to handle solids, Halling says. “We ask our customers what their main pain points are. Our hope is that we’ll be able to provide different types of yeast for the specific visions of the customers. That doesn’t mean we produce 200 strains; it means we look at the market needs, the customers’ needs, and tailor our portfolio to match that.”

For the Future
With this momentum behind yeast optimization, the future looks bright for a facet of ethanol production that hasn’t seen much change. And this could be only the beginning. Bayrock says future engineering could help with yeast nutrition, oil extraction, urea reduction, phytic acid hydrolyzation, digestibility of phosphorous in distillers dried grains, or increased membrane protection. In the far future and with much more research, he adds, perhaps targeting and triggering production and activation of desired enzymes could be an achievable goal.

Richards says he predicts further improvement to byproduct reduction. “As more producers and innovators focus on conversion of corn fiber to higher-valued products, I would also predict that the development of yeast strains will keep pace to allow for utilization of all sugars present in fiber, as well as to allow for more economical breakdown of the corn fiber.”

But the efforts taking place now to improve yeast production characteristics have the capacity to increase profits at existing fuel ethanol plants, Bayrock says. DuPont, Lallemand and Novozymes are on the front lines of that research, and are ready to ramp up to go much further.

“We’ll hopefully take the industry in a new direction and take performance to a new level,” Halling says.

Author: Lisa Gibson
Managing Editor, Ethanol Producer Magazine