MIT chemical engineers have designed a novel genetic switch that allows them to dramatically boost bacteria's production of useful chemicals by shutting down competing metabolic pathways in the cells.
Researchers have been trying to engineer microbes to generate more complex products, including pharmaceuticals and biofuels. This usually requires adding several genes encoding the enzymes that catalyze each step of the overall synthesis.
In many cases, this approach also requires shutting down competing pathways that already exist in the cell. However, the timing of this shutdown is important because if the competing pathway is necessary for cell growth, turning it off limits the population size, and the bacteria won't produce enough of the desired compound.
A laboratory has engineered E. coli to produce glucaric acid by adding three genes -- one each from yeast, mice, and a strain of bacteria called Pseudomonas syringae. Using these three genes, bacteria can transform a compound called glucose-6-phosphate into glucaric acid. However, glucose-6-phosphate is also an intermediate in a critical metabolic pathway that breaks down glucose and converts it into the energy cells need to grow and reproduce.
To generate large quantities of glucaric acid, the researchers had to come up with a way to shut down the glucose-breakdown pathway, allowing glucose-6-phosphate to be diverted into their alternative metabolic pathway. However, they had to carefully time the shutdown so that the cell population would be large enough to produce a substantial amount of glucaric acid. More importantly, they wanted to do so without adding any new chemicals or changing the process conditions in any way.
In addition to adding the genes for glucaric acid production, the researchers engineered each cell to produce a protein that synthesizes a small molecule called AHL. The cells secrete this molecule into their environment, and when the concentration surrounding the cells gets to a certain point, it activates a switch that makes all of the cells stop producing an enzyme called phosphofructokinase (Pfk), which is part of the glucose breakdown pathway. With this enzyme turned off, glucose-6-phosphate accumulates and gets diverted into the alternative pathway that produces glucaric acid. By constructing a library of cells that produce AHL at different rates, the researchers could identify the best time to trigger shutdown of Pfk.
Using this switch, the researchers were able to generate about 0.8 grams of glucaric acid per liter of the bacterial mixture, while cells that were engineered to produce glucaric acid but did not have the metabolic switch produced hardly any.
To demonstrate this versatility, the researchers tested their approach with a metabolic pathway that produces a molecule called shikimate, which is a precursor to several different amino acids and is also an ingredient in some drugs including the influenza drug Tamiflu. They used the AHL quorum-sensing molecule to shut off an enzyme that moves shikimate further along in the amino acid synthesis pathway, allowing shikimate to build up in the cells. Without the switch, the cells could not accumulate any shikimate.
Posted by Dr. Tim Sandle