Using a specialized technique to sift through genomic databases for a variety of bacteria, the researchers found 262 gene sequences that likely code for terpene synthases—enzymes that catalyze the production terpenes. The researchers then used several of those enzymes to isolate 13 previously unidentified bacterial terpenes.

We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes.

—Yamada et al.

The findings suggest that bacteria “represent a fertile source for discovery of new natural products,” the researchers write.

David Cane, a professor of chemistry at Brown and one of the authors on the new paper, began working about 15 years ago to understand how bacteria make terpenes.

At that time, the first genomic sequences of certain classes of bacteria were just beginning to come out. We had this idea that maybe you could find the enzymes responsible for making terpenes by looking at the sequences of the genes that were being discovered.

—David Cane

To do that, Cane searched through the genome data gathered for the bacteria Streptomyces, looking for sequences similar those known to produce terpene synthases in plants and fungi. Eventually, he found that Streptomyces did indeed have genes encoding terpene synthases and that those enzymes could be used to make terpenes.

Phylogenetic analysis of presumptive terpene synthases from bacterial databases. Monoterpene, sesquiterpene, and diterpene synthases are indicated in green-, blue-, and red-colored characters, respectively. The biochemical functions of terpene synthases written in bold and underlined were confirmed by these studies and published data, and they were found from gram-negative bacteria. The blue, orange, and green zones indicate geosmin, epi-isozizaene, and 2-methylisoborneol synthases, respectively. Yamada et al. Click to enlarge.

The verified bacterial sequences found by Cane and others enabled researchers to refine subsequent searches for additional terpene synthase genes.

Instead of using plant sequences or fungal sequences as your search query, we can now use bacterial sequences, which should yield a greater degree of similarity. So now we’re fishing in the right waters with the right kind of bait, and you can find more matches.

—David Cane

This latest paper made use of the third generation of iterative searches and a powerful search technique developed by Haruo Ikeda of Kitasato University in Japan. Previous work had identified 140 probable sequences for terpene synthases. This latest work expanded that to 262.

The next step was to verify that these sequences did indeed code for enzymes capable of making terpenes. Testing all 262 wasn’t practical, so the team chose a few they thought might give them the best chance of finding terpene compounds that hadn’t previously been identified. They looked for sequences that didn’t seem to fit clearly into previously known categories of terpenes.

After they had selected a few, the team made use of a genetically engineered Streptomyces bacterium as a bio-refinery to generate the terpene products.

By taking some of the gene sequences they found and splicing them into their test organism, the researchers could let the organisms generate the product using the instructions from the newly introduced gene. Using this method, they were able to make 13 previously unknown terpenes, their structures verified by mass spectrometry and nuclear magnetic resonance spectroscopy.

It’s a big step forward in the area in that it provides a paradigm for how one could go about discovering many new substances. It’s a good example of how one can use sequence analysis to identify genes of interest and then apply molecular genetic and microbiological techniques to produce the chemical substances of interest.

—David Cane

The work also suggests that there may be many new terpene products as yet undiscovered hiding in the genomes of bacteria.

The work was supported by a National Institutes of Health grant to David Cane (GM30301). Other authors on the paper, in addition to Cane and Ikeda, were Yuuki Yamada, Tomohisa Kuzuyama, Mamoru Komatsu, Kazuo Shin-ya, and Satoshi Omura.

Resources

• Yuuki Yamada, Tomohisa Kuzuyama, Mamoru Komatsu, Kazuo Shin-ya, Satoshi Omura, David E. Cane, and Haruo Ikeda (2014) “Terpene synthases are widely distributed in bacteria,” PNAS doi: 10.1073/pnas.1422108112