Harnessing Microbial Power

On opposite wings of Rice’s George R. Brown Hall, microbiologist George Bennett and biochemical engineer Ka-Yiu San ’78 found themselves sharing not only equipment but a curiosity that would fuel a decades-long collaboration. Their labs, connected by a second-floor breezeway, became the setting for a partnership that would redefine microbial fermentation.

“I was on the west side, and he was on the east side,” Bennett recalled. “There were special doors you could go through to go between wings while staying on the same floor. We used some of the autoclaves and other instruments in common and just started talking to each other sometimes.”

Those hallway conversations led to a bold effort to reengineer E. coli to produce succinate, a compound with immense industrial promise. While microorganisms typically metabolize carbohydrates through fermentation in the absence of oxygen, the team sought to produce succinate — a valuable byproduct — under aerobic and anaerobic conditions and at the maximum theoretical yield. Published in 2005, that successful effort marked a high point in a partnership where microbiology met modeling and ideas became industrially viable technologies.

A Chance Encounter Catalyzes Collaboration

Their decades-long partnership began in the early ’90s with a simple idea. Bennett had been studying pH-regulated promoters in E. coli — genetic switches that turn genes on or off in response to changes in acidity. This is a more precise and controllable method than simply adding a chemical, as the pH can be adjusted throughout the fermentation process.

San immediately saw the engineering potential. Bennett recalled San’s reaction. “He thought, well, yeah, that’s very interesting. You could control that in a fermenter, whereas other ways to induce genes would just be like dumping in some inducer, but once you put it in, you can’t take it out.” San’s reaction? “Very interesting to do.”

With grants and students on board, they began to build pH-regulated vectors, tools used to introduce new genetic material into an organism. By making these vectors pH-regulated, they could precisely control when the new genes were turned on. “That got us interested in other ways to enhance larger-scale fermentation processes,” Bennett said. “That’s what Dr. San was an expert in.”

Their strengths were complementary. San explained that Bennett was “very strong in the biological side in terms of microbiology and genetic regulations,” while San was “more interested in the engineering side.” The difference in perspective was not a limitation but a design-enhancing synergy. As San said, “Either one of us might not have been able to move the project so fast.”

In their early work, Bennett and San’s labs had been focused on manipulating the central metabolic pathways in E.coli to mitigate the problem of acetate accumulation. In many microbial cultures, acetate, a natural byproduct of E.coli metabolism, would build up in the reactor to levels that had a detrimental effect on cell growth and yield. This was an industrial problem because acetate buildup limited yields of pharmaceutical proteins called biologics that were produced in microbes at the time.

“We thought that the succinate network was a reasonable network to study, because you had quite a few knobs or valves that you could control,” said Bennett.

From Acetate to a Building-Block Chemical

The team’s deep understanding of these pathways made them uniquely equipped to pursue a new and promising challenge: producing succinate. This strategic shift was a direct result of a 2004 U.S. Department of Energy (DOE) report that drew attention to the need for bioproduction and identified succinate as one of 12 “building block” chemicals. As a foundational compound that could be derived from renewable biomass instead of petroleum, succinate was a key part of a broader initiative to reduce reliance on fossil fuels. For Bennett and San, who were already experts in engineering these metabolic systems, the report provided a new and important goal for their ongoing research.

Bennett viewed succinate as a crucial compound, noting, “It’s a counterion in a lot of pharmaceuticals. Some of it can be used as a de-icer, and it can be converted to a lot of other compounds.” He said the team was well-positioned to pursue the research because: their previous work had provided a deep understanding of the metabolic pathways that connected in a key junction called the pyruvate node; they had already patented methods for enhancing reductive reactions in vivo by recycling formate; and they had developed techniques for co-factor engineering.

By the time the DOE report came out, Bennett and San were well underway on their projects. “We’d already been in position for several years and had made significant progress,” Bennett said.

The project’s success hinged on two key challenges: first, to genetically modify the microbe to produce succinate efficiently, and second, to then grow it robustly at large scale.

Engineering a Dual-Pathway Solution

To achieve high efficiency succinate production, they needed to overcome the limitations of anaerobic fermentation and the natural tendency of E. coli to produce acetate and other byproducts. Their solution was a dual-pathway approach. Think of a microbe’s metabolic network as a complex highway system where different routes lead to different products. Bennett and San’s goal was to redesign this system, blocking off unproductive routes and opening new ones to direct all the ‘traffic’ toward the desired product — succinate.

They built upon an earlier system that used a highly active glyoxylate cycle, a specific metabolic route that allows organisms to convert fats or simple carbon compounds into glucose. While groundbreaking, this earlier system was a ‘leaky’ process, with valuable carbon diverted into undesirable byproducts, which prevented it from achieving maximum theoretical yield.

Bennett and San’s new system was designed with four key genetic mutations. First, they knocked out several genes that would normally divert metabolic flow away from succinate and into other products like acetate. Then, they opened up a second pathway for succinate production through the oxidative arm of the TCA cycle (a core part of a cell’s energy production), creating a two-route system that allowed the microbe to simultaneously use both pathways to balance reductive cofactor metabolism and the use of carbon.

“We devised the route where it would go through both of these paths at the same time,” Bennett explained. “Basically, it would go two-thirds of the metabolism through one path and then about one-third through the other. That was some interesting network partitioning flow to examine.”

The result was a system that demonstrated higher productivity and could achieve the maximum theoretical succinate yield.

To guide other potential genetic changes, they brought in mathematical modeling. Steve Cox, then a professor in the computational and applied math department, used these models to predict the optimal split of metabolic flow to generate the best yields of succinate. The team then used metabolic flux analysis — a technique that measures the rate at which different molecules are being used and produced within a cell’s metabolic network. This allowed the team to precisely see where the carbon was flowing and confirm the models’ predictions.

“They would do these very sophisticated culture studies and then do these analyses to show exactly where the flow was going,” Bennett said. “Sometimes that would point to a new potential mutation we could introduce.”

From a systems biology standpoint, their approach was holistic. San explained, “We were not only looking at just one reaction, but we looked at the whole central pathway, starting from the feedstock and going to different products. For the succinate project, we wanted to redesign it to produce only the succinic acid without any other side products.”

The collaboration wasn’t just a merging of ideas but a merging of teams. “We had group meetings almost every single week,” San said. “Not only the people involved in this project but the whole two groups together. It was a rewarding and fruitful collaboration.” San described their process as a continuous cycle of improvement. “It was the design and then the experimental work, followed by analysis, which then fed back to the model. Then we would ask the questions: ‘What is the next step?’ and ‘Can we improve it further to make the whole process more efficient?’”

From Design to Industrial Viability

Once the team had successfully designed and engineered the microbes, the next crucial challenge was ensuring the modified bacteria could grow robustly and produce the desired product at scale.

Their iterative approach to metabolic engineering extended to practical culturing, where they carefully managed the organism’s growth to optimize performance. This balancing act was important. “Sometimes you would find a certain construct or condition that was very efficient at producing succinate, but it would grow so slowly that it was not something you could practically use,” Bennett said. “Rather soon, we reached optimal configurations that made the strain very robust and grow well.”

From the start, they were thinking not only about yield, but about industrial potential. They worked closely with Rice’s Office of Technology Transfer and initially with industry partners like AgRenew and Roquette and later had licenses of certain patents to other companies. “Seeing it from some initial idea all the way to licensing the technology and seeing that it had that kind of potential, that was a very nice era to be involved in,” Bennett said.

The potential for commercialization was real, but it also presented a new set of challenges, particularly with scale-up. “We were worried about the scale-up, because when you try to go from a one-liter culture up to a hundred or a thousand liters, it turns out that that’s very hard to replicate exactly what’s going on there,” Bennett said. “But if they followed these directions and were monitoring the culture, it would work very well. The team scaled it up without any problem, and we were very pleased.”

A Legacy of Collaboration and Innovation

Today, Bennett looks back on the project as a complete scientific arc. “It was a very good cooperation between Natural Sciences and Engineering,” he said. “The project showcased the unique strengths and perspectives in going about what, early on, was more of a fundamental scientific project of how you could manipulate different knobs in this network to give a desired flow, to then actually making one that’s really highly productive and had some industrial potential. So that was pretty neat.”

For all its rigor, the story began with something simple: Two researchers meeting in a breezeway, asking each other, “What if?” As San put it, “Sometimes we would sit together and then just daydream and pick up different crazy ideas to see whether they work or not.”

In this case, their crazy idea worked, paving the way for a more sustainable industry.