Researchers from North Carolina State University have developed an artificial enzymatic pathway for synthesizing isoprenoids, or terpenes, in E. coli. This shorter, more efficient, cost-effective and customizable pathway transforms the bacterium into a factory that can produce terpenes for use in everything from cancer drugs to biofuels. A paper on their work is published in the journal ACS Synthetic Biology.

Terpenes are a large class of naturally occurring molecules that are useful in a range of industries. In nature, terpenes are found in plants and microbes; for example, lycopene—which gives tomatoes their color—is a terpene.

Since it isn’t practical to extract these molecules directly from their natural sources, scientists can use biosynthesis to produce them. However, biosynthesizing terpenes has traditionally proven challenging.

Terpenes are difficult to biosynthesize because nature’s methods for making the building blocks of these molecules are lengthy, complicated and involve enzymes that are difficult to engineer. These difficulties in turn make it hard to engineer microbes to manufacture these molecules in large amounts.

—Gavin Williams, associate professor of chemistry at NC State and lead author

Williams works with E. coli, inserting enzymatic pathways into the bacteria that transform them into tiny molecular production factories. With former Ph.D. student Sean Lund, and current graduate student Rachael Hall, Williams designed an artificial pathway for terpene synthesis that utilizes only two enzymes, rather than the six or seven that occur in natural pathways.

Nature uses approximately two routes for terpene synthesis, and each consists of six or seven enzymes. We created a third route—a shortcut—with two enzymes that occur in nature, but that aren’t normally involved in this pathway.

—Gavin Williams

One of the key enzymes Williams and his team used—an acid phosphatase (PhoN)—normally removes phosphates. But in the artificial pathway, this enzyme cleverly performs the reverse reaction.

PhoN is particularly useful here, due to its promiscuous nature. Promiscuity in enzymes means that they can carry out the same transformation on many different molecules.

—Gavin Williams

The team engineered E. coli to produce several different varieties of terpene with the simplified pathway, including lycopene. They found that the new pathway was equally as productive as longer, more difficult-to-engineer pathways currently in use. Because the pathway is promiscuous, it’s customizable, Williams said.

… the prototype chemo-enzymatic pathway is a critical first step towards the construction of engineered microbial strains for bioconversion of simple scalable building blocks into complex isoprenoid scaffolds.

—Lund et al.

Next steps for the researchers include using the pathway to make terpenes that are new to nature for use in compounds that are too expensive to manufacture with current methods.

The work was supported in part by the National Institutes of Health (grant GM104258).

Resources

• Sean Lund, Rachael Hall, and Gavin J Williams (2019) “An Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism” ACS Synthetic Biology doi: 10.1021/acssynbio.8b00383