Already used to produce some biofuels and pharmaceuticals, bioproduction through microbial fermentation has the potential to revolutionize the high-value chemical industry by shifting chemical manufacturing to more sustainable pathways independent of fossil fuels.

That’s the idea driving the work of Ahmed Mannan, PhD, a research fellow at the School of Engineering at the University of Warwick, and his colleague Declan Bates, PhD, a professor of bioengineering at the school. But there remain challenges to the widespread adoption of bioproduction of chemicals, particularly in terms of the costs of scaling processes up to industrial scale. Mannan and his colleagues have developed a theoretical biological circuit that reprograms natural systems in microbes to reduce costs and optimize growth and production.

In any microbial fermentation, there’s a tension between cellular growth and the production of the chemical humans want to produce, with both processes dependent on the same pool of carbon. “If you want to re-divert chemical traffic so it’s producing these chemicals, you are taking away from the cell being able to build itself,” Mannan says, “so you get this inherent trade-off.”

One strategy for striking a balance is to adopt a two-phase approach, where microbes are first allowed to grow unmolested to sufficient size and number and are then induced to switch from focusing on growth to focusing on synthesizing chemicals. Inducing that shift from a growth phenotype to a production phenotype requires the use of chemical inducers that trigger genetic control systems in the microbes, and such inducers can be very expensive. The synthetic inducer IPTG can cost up to nearly $1,500 for just 25 g, for example.

“You don’t need a huge amount to induce the switch, but the fact is that as you scale up, the costs go up as well,” Mannan says. And it’s not a one-time application, he adds, since the cells consume the inducer but “when it’s not there anymore the switch is not pressed on anymore. It’s let go and switches back” to focusing on cellular growth.”

Modified microbial control circuit

But in a paper published in Nature Communications, Mannan and Bates describe a modified microbial control circuit that could, in theory, allow for the cheap and irreversible switching of microbes from a growth phenotype to a production phenotype. Taking E. coli as a model organism, they focused on the bacteria’s fatty acid nutrient uptake system and how to re-engineer it to shift the organism from a growth phenotype to a production phenotype in the presence of fatty oleic acid, rather than an expensive synthetic inducer.

E. coli can take advantage of fatty acids when they become available through a nutrient sensor-actuator encoded in their genome, the transcription factor known as FadR. When there are no fatty acids in the environment, FadR suppresses the expressions of enzymes that break down and consume fatty acids, saving the cell energy by not making enzymes it does not need. But when fatty acids become available, a small amount trickles in and is converted into fatty acyl-coenzyme A (acyl-CoA). Acyl-CoA then binds and sequesters FadR, so the now inert transcription factor cannot suppress the expression of the enzymes needed to consume fatty acids.

In their paper, Mannan and Bates show that genetically engineering FadR also represses the enzymes that catalyze chemical production, while simultaneously activating the expression of an enzyme that controls cell growth, creating a switch that flips an E. coli bacterium from cell growth to chemical production in the presence of fatty acid.

But this is activation is only temporary—the “switch” still springs back in the absence of fatty acid. The paper goes on to show how it should be possible to irreversibly suppress FadR and therefore keep the bacterium in the production phenotype with only a temporary addition of fatty acids, through two important factors.

First, Mannan and Bates show that by engineering FadR to activate rather than suppress its own expression, no more FadR will be made once it is sequestered by the intake of fatty acids. As the cells grow and divide over time, the inert FadR dilutes away.

Second, by engineering FadR to suppress another transcription factor known as TetR, and by engineering TetR to suppress the expression of FadR, they create a mechanism where the sequestration of FadR leads to the accumulation of TetR. TetR then suppresses any further expression of FadR. If sufficient time is given for the inert FadR to dilute away, then even once the fatty acids are gone, “there won’t be enough FadR to repress its repressor TetR again, allowing us to retain suppression of FadR expression and so prevent reversion, ”Mannan and Bates note in the paper. The switch locks and the bacterium shifts permanently to the production phenotype.

Such an irreversible switch would not only reduce the cost of bioproduction, according to Mannan, it would also help with the downstream purification process for the chemical produced, “since you completely consume the inducer as well,” he says.  And because the irreversible switch itself is agnostic when it comes to the chemical engineered microbes it will ultimately produce, “it’s widely applicable to the synthesis of almost any chemical.”

Chemical laboratory technicians fitting hose
Researchers have shown that genetically engineering transcription factor FadR represses the enzymes that catalyze chemical production, while simultaneously activating the expression of an enzyme that controls cell growth. This creates a switch that flips an E. coli bacterium from cell growth to chemical production in the presence of fatty acid. [Westend61/Getty Images]
This reprogramming of a natural system to exert dynamic control over microbes could be the key to wider and larger industrial applications for microbial bioproduction according to Di Liu, PhD, a postdoctoral appointee at Sandia National Laboratories who has collaborated with Mannan in the past but was not directly involved in this new research.

“I think this work is an elegantly designed system and really addresses a key bottleneck in the field,” she says, adding that the same approach could work in a wide variety of organisms since fatty acid metabolism is universal among them.

For that matter, Liu adds, Mannan and Bates’ system is not limited to fatty acids. “What’s also important to the field is that the system designed in this study also has the potential to be extended to various nutrients,” she says. “Researchers in the field can follow the design principles in this work and come up with their own designs.”

That’s actually what Mannan considers the crux of this work, “showing that you don’t need to add an expensive chemical, you can exploit all of these natural systems that are in bacteria,” he says. “We can repurpose them, put them somewhere else, and they can be induced by all of these natural chemicals instead.”

The irreversible switch circuit could also help with another challenge to scaling up microbial bioproduction, the natural heterogeneity of large populations of bacteria.

“Nature doesn’t want to control that, because it gives advantages for evolutionary purposes,” Manan says, but for humans looking to optimize chemical production, bacterial heterogeneity is a hindrance. “We’re hypothesizing that this switch can actually help to homogenize that population further.”

But the immediate next step for Mannan and Bates will be experimental verification of their theoretical work—they are already discussing a collaboration with a synthetic biology lab at Warwick to test the ideas in an actual bacterial host.

“We have to genetically engineer the promotor sequences of the components of this circuit inside the cell, and we need to show it actually works like this,” he says. “Then it will become extremely attractive to take up in industry and actually to take up in other labs that want to scale up production.”

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