Advance in Metabolic Engineering: Easier Way to Find Right Bacteria

Korean researchers find simpler way to discover which bacteria can produce the highest concentration of a valuable chemical intermediate.

by Menaka Wilhelm
February 11, 2019

Researchers at the Korea Advanced Institute of Science and Technology have developed a simpler way to discover which bacteria are capable of producing the highest concentrations of a valuable chemical intermediate.

The simplicity of the method is an important step forward in metabolic engineering, which involves optimizing cell genetics and regulatory processes to boost production of specific chemicals. Metabolic engineering provides an efficient yet sustainable route to often complex chemistries. Researchers typically randomly modify the genomes of bacteria, then use high-throughput screening to rapidly test all of the variations to find those with the highest yields of the target chemical.

Malonyl-CoA is one of those targets and it presents a quandary. The enzyme could be used to make everything from fragrances and food additives to polymers and biofuels, and it is an attractive candidate for drug discovery. Yet producing it in quantity is difficult, since cells—from bacteria to humans—use it to regulate fatty acids and food intake. As a result, researchers must compete with the metabolism of the very cells they want to harness for production.

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While several malonyl-CoA biosensors have been developed for high-throughput screening, they typically involve using microscopy to see fluorescent proteins. These biosensors also work only with Escherichia coli and Saccharomyces cerevisiae and require multiple signal transduction steps. As a result, the entire analytical process—from the sample preparation to the rapid quenching of cellular metabolism—is time consuming, labor intensive, and expensive.

The researchers at Korea Advanced Institute created a simpler way to manage this by enhancing the bacteria with an enzyme complex that creates a red pigment in the presence of malonyl-CoA.

“This system enables rapid and easy selection of malonyl-CoA overproducers even with the naked eye, without requiring expensive devices,” Sang Yup Lee, who led this work, said in an email.

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Equally important, the approach works with three industrially important bacteria, Escherichia coli, Pseudomonas putida, and Corynebacterium glutamicum.

By inserting the gene for this sensor-enzyme into a bacterium, Lee and his team could simply watch as different colonies turned various shades of light pinks and reds. Redder samples meant more malonyl-CoA.

As a proof-of-concept, Lee and his team used the biosensor to identify bacterial colonies capable of producing four different compounds. The chemicals ranged from antioxidants to antimicrobial chemicals, but all required malonyl-CoA for synthesis. The higher the level of malonyl-CoA, the more efficient the production and the higher the yield of these chemicals.

According to Lee, implementing this biosensor was fairly straightforward. Yet the work drew on years of working with a class of compounds called polyketides.

“While working on various polyketide compounds as antibiotics and functional natural compounds, we came up with an idea for a colorimetric sensor,” Lee said.

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That experience allowed the researchers to solve a tricky chemical detection problem.

Malonyl-CoA is a key ingredient to several cellular processes, so it can be hard to measure. This is because the cell shuttles it to an enzyme pathway, or a cellular compartment, almost as soon as it is synthesized. This makes it difficult to get an accurate read of the chemical’s concentration.

To quantify malonyl-CoA, Lee and his team had to measure malonyl-CoA concentrations within each bacterial cell. To do this, they chose a bacterial enzyme that converts malonyl-CoA into another chemical,1,3,6,8-tetrahydroxynaphthalene or THN. THN then naturally oxidizes to form a red-colored compound called flaviolin.

The sensor’s strength is that it provides a one-step readout of malonyl-CoA.

“Compared to previously reported biosensors that require multiple components, this biosensor is a much simpler system, with a single gene requirement,” Lee said.

The sensor-enzyme converts 5 molecules of malonyl-CoA to one molecule of THN, which becomes the red pigment. Without any intermediate steps to gum up the signal, the sensor works at very low levels of malonyl-CoA as well as higher concentrations.

After evaluating different forms of the sensor-enzyme, Lee and his team engineered the gene to produce it into three different bacterial species. In each case, some colonies tinted redder than others. Conventional chemical analysis tools confirmed that the red-tinted colonies were indeed rich in malonyl-CoA.

Initially, the goal was to create and test the biosensor platform, but Lee said he expects to use the approach to identify industrially-relevant microbial strains soon. Equipped with a quick readout, it’s possible to screen many more bacterial samples for productivity.

Likewise, Lee’s group has begun using an automated pipetting system to engineer different colonies with genetic variants thought to affect malonyl-CoA production.

Some aspects of this biosensor could also extend to other compounds. The enzyme used in this biosensor is specific to malonyl-CoA, but using other enzymes could lead to rapid detection of other chemicals, allowing companies to use more bacteria for sustainable production of complex molecules in the future.

Menaka Wilhelm is an independent writer.

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