For industrial bacteria like Escherichia coli, the tricarboxylic acid cycle (TCA cycle) plays a crucial role in their aerobic growth process, converting carbon sources into cellular biomass. Any attempt to redirect carbon flux from cell growth to the production of desired products can disrupt natural metabolism and potentially impact carbon efficiency.
In theory, blocking the TCA cycle and its bypasses could reduce carbon dissipation, facilitating chemical bio-synthesis in aerobic fermentation. However, the inhibition of the TCA cycle often interferes with the bacterium's natural growth.
Biological Transformation Vessel. Photo by Tao Yong.
Recently, the team led by Tao Yong made significant progress in the study of aerobic fermentation-based decarbonization chassis, redesigning a novel Escherichia coli chassis cell with an incomplete tricarboxylic acid (TCA) cycle to minimize carbon loss during aerobic fermentation, thus achieving the goal of microbial fermentation decarbonization. The related paper was published in Nature Communications.
"With this chassis cell, all α-ketoglutarate-dependent enzyme catalytic reactions can be optimized. It's like hitting multiple targets with one stone," said Tao Yong, co-corresponding author of the paper and researcher at the Institute of Microbiology, Chinese Academy of Sciences.
A challenging decision
The tricarboxylic acid cycle is a common metabolic pathway present in aerobic organisms, breaking down organic substances into carbon dioxide and water while generating a large amount of high-energy molecules. It serves as the final metabolic pathway for sugars, lipids, and amino acids.
Lin Baixue, co-corresponding author of the paper and project researcher at the Institute of Microbiology, Chinese Academy of Sciences, told Chinese Science Bulletin: "The tricarboxylic acid cycle is essential for aerobic biological life. Interrupting the tricarboxylic acid cycle in this study was a challenging task."
This originated from a challenge encountered in production practices a decade ago. In 2013, penicillin gradually fell out of favor among patients due to its tendency to cause allergies. However, China's penicillin production capacity remained intact.
Tao Yong's team, which has always focused on industry, noticed that cephalosporin, a less allergenic alternative to penicillin, is structurally similar to penicillin. If penicillin could be converted into cephalosporin, it could solve the problem of penicillin's sluggish sales and replace chemical reactions in the original production process with biosynthesis, reducing heavy metal pollution.
"There is an enzyme that can convert penicillin into the nucleus of cephalosporin. This enzyme naturally acts on penicillin N, while what we produce is penicillin G," explained Tao Yong. "At that time, Yang Keqian, a researcher at the Institute of Microbiology, Chinese Academy of Sciences, improved the enzyme's affinity for penicillin G, but its catalytic activity was still low."
The team's research found that to enhance the catalytic activity of this enzyme, α-ketoglutarate, a common substrate, is essential. However, α-ketoglutarate is an intermediate metabolite of the tricarboxylic acid cycle. The cost of adding it externally was too high, posing a major challenge to the green and efficient supply of α-ketoglutarate to the catalytic reaction from penicillin to cephalosporin.
Biological fermentation tank. Photo by Tao Yong
"Since exogenous sources are not feasible, let's try endogenous," said Tao Yong. In the catalytic reaction from penicillin to cephalosporin, alpha-ketoglutarate produces succinate, while in the tricarboxylic acid cycle, alpha-ketoglutarate also produces succinate. The team couldn't help but imagine: if alpha-ketoglutarate in the tricarboxylic acid cycle could be converted to succinate in the catalytic reaction, wouldn't that solve the endogenous supply problem?
Based on this, researchers disrupted the pathway from alpha-ketoglutarate to succinate in the tricarboxylic acid cycle, coupling the enzyme-catalyzed reaction from penicillin to cephalosporin into the cycle. This not only provided the necessary substrate for the enzyme while the cell grew normally but also addressed the catalytic deficiency of a series of enzymes dependent on alpha-ketoglutarate.
"We only modified this step, and we increased the enzyme catalytic efficiency by 11 times," said Lin Baixue. In 2015, this research was published in the Proceedings of the National Academy of Sciences (PNAS).
Survival of the Fittest: "Smart" Cells
New challenges emerged. "After the tricarboxylic acid cycle was disrupted, cells could only grow in nutrient-rich beef extract peptone broth medium provided in the laboratory. Once transferred to the inorganic salt medium used in industrial production, the cells stopped growing," recalled Tao Yong.
The team immediately began exploring further industrialization pathways.
Some microorganisms in nature naturally lack the tricarboxylic acid cycle but can survive under aerobic conditions. This excited Zhou Hang, the first author of the paper and a doctoral student at the time at the Institute of Microbiology, Chinese Academy of Sciences: could bacteria with a disrupted tricarboxylic acid cycle adapt to the inorganic salt environment through artificial evolution?
Zhou Hang embarked on an 11-week adaptive evolution experiment. "We first let the bacteria without a complete tricarboxylic acid cycle grow in beef extract peptone broth medium. After seeing the bacteria grow, we diluted them tenfold and transferred one-tenth to glucose medium. After 45 rounds of cycles, we found that bacteria without a complete tricarboxylic acid cycle could grow normally in inorganic salt medium." Subsequently, the team members began genome analysis.
In 2017, Tao Yong went to the United States to attend a conference. Maciek R. Antoniewicz, a scholar in bacterial metabolic flux analysis and professor at the University of Delaware, showed great interest in the research Tao Yong's team was conducting and offered to help them with isotopic labeling for localization. Based on the analysis of carbon-13 metabolic flux data, Tao Yong's team finally discovered the mystery of cells' ability to grow normally with an incomplete tricarboxylic acid cycle.
Among the 12 molecules provided by central metabolism in cells, alpha-ketoglutarate, oxaloacetate, and succinyl coenzyme A are essential for the tricarboxylic acid cycle.
"Through metabolic flux analysis, we found that besides where we artificially blocked the cycle, there are two places where there is no flow, and the normal tricarboxylic acid cycle flows clockwise, but in the evolved bacteria, one step flows counterclockwise," Tao Yong told the Chinese Science Bulletin. "Cells are smart. We don't let them go this way, but they have to survive, so they have to mutate another step of the reaction, reverse the direction, to maintain the production of succinyl coenzyme A."
Thus, they succeeded in enabling cells with an incomplete tricarboxylic acid cycle to survive in inorganic salt medium in the laboratory.
Achieving the Biomanufacturing of High-Value Chemicals
From design to evolution to analysis, this was already a mature research discovery. But Zhou Hang was not satisfied.
"Escherichia coli, like many aerobic microorganisms, converts glucose taken in into carbon dioxide in the tricarboxylic acid cycle," Zhou Hang said, "The emission of carbon dioxide reduces the carbon flux directly used for product synthesis, thereby negatively affecting product yield. Therefore, blocking the tricarboxylic acid cycle and its bypasses can reduce carbon dissipation and promote chemical biogenesis in aerobic fermentation. This is also the issue scientists have been trying to improve, the production efficiency in aerobic fermentation."
Is there a way to make cells not require succinyl coenzyme A and increase production efficiency in industrial production? Zhou Hang once again delved into the literature for clues.
Finally, they discovered, "By introducing an exogenous acetyl coenzyme A-dependent pathway into Escherichia coli, Escherichia coli no longer needs to rely on succinyl coenzyme A," said Lin Baixue. The modified Escherichia coli grew very well in inorganic salt without a tricarboxylic acid cycle.
The team constructed a strain of Escherichia coli without a functional tricarboxylic acid cycle, which could serve as a universal chassis for the biogenesis of chemical substances. Using this chassis cell, they began to attempt the biosynthesis of different chemicals, achieving the biosynthesis of four different chemicals and increasing the conversion rate of products.
The research team further broke through with the biosynthesis of L-carnitine. L-carnitine is a naturally occurring amino acid substance in the human body, widely used in food additives, feed additives, pharmaceutical treatments, and other fields, with a growing market demand. It is estimated that the market size of L-carnitine will reach $250 million by 2025.
Tao Yong's team used the above industrial chassis cells to build microbial cell factories, achieving green and efficient biomanufacturing of L-carnitine. The project has achieved key technologies with independent intellectual property rights, reaching an international advanced level, and has completed technology transfer, achieving industrialization, with considerable economic benefits after production. "The realization of the L-carnitine project transformation shows that this chassis cell can achieve multiple goals and close the loop of research and development," Lin Baixue said.
Tao Yong said that this research result not only breaks the key bottleneck from the laboratory to industrialization but also provides new ideas for reducing carbon emissions in microbial fermentation. In the future, this green and efficient "cell factory" will continue to empower the synthesis of various chemicals. Tao Yong said, "While reducing carbon dioxide emissions and increasing the yield of synthetic products, I think this should be the most ideal goal for us to achieve green biomanufacturing, and it has always been the direction we have been striving for." Paper Information
