Wednesday, 12 February 2020

A new way to control microbial metabolism


Microbes can be engineered to produce a variety of useful compounds, including plastics, biofuels, and pharmaceuticals. However, in many cases, these products compete with the metabolic pathways that the cells need to fuel themselves and grow.

To help optimize cells' ability to produce desired compounds but also maintain their own growth, MIT chemical engineers have devised a way to induce bacteria to switch between different metabolic pathways at different times. These switches are programmed into the cells and are triggered by changes in population density, with no need for human intervention.

This kind of switching allowed the researchers to boost the microbial yields of two different products by up to tenfold.

To make microbes synthesize useful compoundsv that they don't normally produce, engineers insert genes for enzymes involved in the metabolic pathway -- a chain of reactions that generate a specific product. This approach is now used to produce many complex products, such as pharmaceuticals and biofuels.

In some cases, intermediates produced during these reactions are also part of metabolic pathways that already exist in the cells. When cells divert these intermediates out of the engineered pathway, it lowers the overall yield of the end product.

Using a concept called dynamic metabolic engineering, Prather has previously built switches that help cells maintain the balance between their own metabolic needs and the pathway that produces the desired product. Her idea was to program the cells to autonomously switch between pathways, without the need for any intervention by the person operating the fermenter where the reactions take place.

The researchers this approach to program E. coli to produce glucaric acid, a precursor to products such as nylons and detergents. The researchers' strategy was based on quorum sensing, a phenomenon that bacterial cells normally use to communicate with each other. Each species of bacteria secretes particular molecules that help them sense nearby microbes and influence each other's behavior.

The MIT team engineered their E. coli cells to secrete a quorum sensing molecule called AHL. When AHL concentrations reach a certain level, the cells shut off an enzyme that diverts a glucaric acid precursor into one of the cells' own metabolic pathways. This allows the cells to grow and divide normally until the population is large enough to start producing large quantities of the desired product.

The researchers set out to engineer multiple switching points into their cells, giving them a greater degree of control over the production process. To achieve that, they used two quorum sensing systems from two different species of bacteria. They incorporated these systems into E. coli that were engineered to produce a compound called naringenin, a flavonoid that is naturally found in citrus fruits and has a variety of beneficial health effects.

Using these quorum sensing systems, the researchers engineered two switching points into the cells. One switch was designed to prevent bacteria from diverting a naringenin precursor called malonyl-CoA into the cells' own metabolic pathways. At the other switching point, the researchers delayed production of an enzyme in their engineered pathway, to avoid accumulating a precursor that normally inhibits the naringenin pathway if too much of the precursor accumulates.

The researchers created hundreds of E. coli variants that perform these two switches at different population densities, allowing them to identify which one was the most productive. The best-performing strain showed a tenfold increase in naringenin yield over strains that didn't have these control switches built in.


The researchers also demonstrated that the multiple-switch approach could be used to double E. coli production of salicylic acid, a building block of many drugs. This process could also help improve yields for any other type of product where the cells have to balance between using intermediates for product formation or their own growth. The researchers have not yet demonstrated that their method works on an industrial scale, but they are working on expanding the approach to more complex pathways and hope to test it at a larger scale in the future.

See:


Christina V. Dinh, Kristala L. J. Prather. Development of an autonomous and bifunctional quorum-sensing circuit for metabolic flux control in engineered Escherichia coli. Proceedings of the National Academy of Sciences, 2019; 201911144 DOI: 10.1073/pnas.1911144116

 Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

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