Enzymatic Assembly Line Overview
Earlier this year, research at the Kee-Hong Kim lab of Purdue University had preliminary evidence showing that a trans-stilbene compound, Piceatannol, had an ability to inhibit the development of human adipose cells. The mechanism is based around the idea that Piceatannol interacts with a preadipocyte's (immature fat cell) insulin receptors in such a way that surpresses it's growth into a mature adipose cell. Piceatannol is a metabolite of resveratrol, a compoud currently under investigation for possible anti-cancer properites. Piceatannol differs from resveratrol by one hydroxyl group on one of the aromatic rings.
Piceatannol is currently very costly to synthesize. On the advent of such a discovery, we felt that if we were to engineer a pathway to optimize the production of Piceatannol from cheaper substrates through the utilization of our PUF and RNA scaffold projects, we could show the versatility of our PUF toolkit working with an RNA scaffold.
However, before researchers can begin using our PUF toolkit to produce piceatannol in vivo, we took steps to acquire and characterize the necessary genes for our:
Enzymatic Assembly Line Overview
Earlier this year, research at the Kee-Hong Kim lab of Purdue University had preliminary evidence showing that a trans-stilbene compound, Piceatannol, had an ability to inhibit the development of human adipose cells. The mechanism is based around the idea that Piceatannol interacts with a preadipocyte's (immature fat cell) insulin receptors in such a way that surpresses it's growth into a mature adipose cell. Piceatannol is a metabolite of resveratrol, a compoud currently under investigation for possible anti-cancer properites. Piceatannol differs from resveratrol by one hydroxyl group on one of the aromatic rings.
Piceatannol is currently very costly to synthesize. On the advent of such a discovery, we felt that if we were to engineer a pathway to optimize the production of Piceatannol from cheaper substrates through the utilization of our PUF and RNA scaffold projects, we could show the versatility of our PUF toolkit working with an RNA scaffold.
However, before researchers can begin using our PUF toolkit to produce piceatannol in vivo, we took steps to acquire and characterize the necessary genes for our:
Design and Theory
The above is our theoretical construct involving the four aforementioned genes. As labeled, the sequence of enzymatic activity begins at TAL, which converts the naturally present amino acid in E. coli, tyrosine, into p-coumaric acid. P-coumaric acid along with malonyl-CoenzymeA are converted into resveratrol by the 4CL:STS fusion protein. Lastly, resveratrol is metabolized into piceatannol by BM3. A substrate could be hypothetically processed by these sequential proteins, resulting in a molecular, in vivo assembly line in E. coli. Such an assembly line could be optimized, as shown by data provided by the
RNA scaffold section..
The following is a technical diagram of the stepwise modifications and production of piceatannol from tyrosine. The Malonyl-CoA must be added to supplement 4CL:STS as it processes p-coumaric acid..
Experimental Approach
To begin the intricate process of testing and building the enzymatic assembly line in vivo, we gathered the different enzymes and necessary reagents from multiple sources, making international connections in the process. We learned from members of the Chul-Ho Yun that group cytochrome P450 BM3 had been mutated at several points to create seventeen mutants of wild type BM3. In the paper “Generation of the Human Metabolite Piceatannol from the Anticancer-Preventive Agent Resveratrol by Bacterial Cytochrome P450 BM3”, they described the activity and longevity of wild type BM3 as well as seventeen mutants of the enzyme examined in in vitro assays.
According to their literature, BM3 mutant #13 created the most piceatannol per minute per nmol of enzyme, and BM3 mutant #10 had high stability during the in vitro testing. Given the reported differences in stability and vigor among the wild type and two mutant forms of the enzyme, we requested the wild type, mutant #10, and mutant #13 enzymes from the group. We planned to investigate the effects of stability and vigor of BM3 in a novel in vivo application; the enzymes had not been previously tested by the Chul-Ho Yun group in this way. We also hoped to investigate any effect the longevity and vigor of BM3 could have on the vitality of the enzymatic assembly line.
After gathering the four enzymes we would use in our assembly line, we began to tackle the simplest steps first. Since we planned to characterize BM3 while integrating it into the assembly line, we chose to begin testing its ability to synthesize piceatannol from resveratrol in vivo. We needed to discern whether piceatannol would be exported from the cell after production, so we devised an assay to differentiate piceatannol from resveratrol and other cellular products in cell growth medium. Using thin-layer liquid chromatography (TLC), we experimented with different procedures for separating control samples of resveratrol and piceatannol.
Fig. 1. From left to right, the plate lanes are marked Resveratrol, Piceatannol, Concentrated Biofermentation (WT, 10, or 13), and Reference (containing both Resveratrol and Piceatannol)
We were able to achieve separation of the controls on a normal phase TLC plate, but when we applied the concentrated supernatant of biofermentations of BM3 culture, we were unable to see piceatannol on the TLC plate. In the interest of the PUF project, we suspended work on this project but hope to work toward the realization of a enzymatic assembly line in the future.
Prospective Planning
In the future, we would like to focus more efforts on making enzymatic assembly line production of piceatannol a reality. Piceatannol may have potential health benefits, and we would like to determine a protocol that can be scaled up for mass production of the compound. Before the assembly line can become a reality, we must examine current issues with production in E. coli. Since we were not able to detect piceatannol on our TLC plates, we would first like to identify a more sensitive assay for detecting the compound as well as determine its true interaction with the bacteria after it is produced.
We must investigate whether or not the compound is being sequestered by the cells or if resveratrol fails to be taken up by the cells. These pressing issues must be resolved in order to move forward with our synthesis plans.
In a more broad sense, we must also determine if any of the substrates or products in our proposed assembly line are toxic to E. coli or exhibit any other behavior that would complicate in vivo synthesis. However, we have bright hopes and determination to explore this potentially valuable production method for piceatannol in the future.
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