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Reversing Evolution Promises Cheaper Pharmaceuticals

May 1, 2014


Biosynthesis, in which microbes are genetically engineered to produce a compound, has become an effective way to reduce the cost and time it takes to manufacture several blockbuster drugs. So far, the biosynthetic approach has been primarily limited to compounds found in nature. Designing routes within microbes to produce unnatural compounds, on the other hand, has faced challenges, until now.

Led by Brian Bachmann, an associate professor of chemistry at Vanderbilt Univ., a team of scientists has come up with a novel way to apply biosynthesis to the production of unnatural chemicals and demonstrated its use for the synthesis of the antiretroviral drug didanosine. Called bioretrosynthesis, the new technique builds a genetic pathway for the production of a particular drug molecule by evolving enzymeencoding genes in the reverse direction, i.e., starting with the last enzyme in the reaction sequence and moving backwards to the first enzyme.

Biosynthesis involves designing a pathway, or series of reactions, using natural selection to evolve the optimal enzyme for each step. In the conventional technique (i.e., forward evolution), a simple molecule is chosen and an enzyme is evolved to produce a more-complex molecule. A second enzyme is then evolved to convert this first intermediate product into a morecomplex compound, and so on until the final product is obtained. In backwards evolution, the last enzyme is evolved first, followed by the second to last, and so on along the pathway to the starting material.

"These days, synthetic chemists can make almost any molecule imaginable in an academic laboratory setting. But they can't always make them cheaply or in large quantities," Bachmann says. "Using bioretrosynthesis, it is theoretically possible to make almost any organic molecule out of simple sugars."

Bachmann's idea for bioretrosynthesis is based on a hypothesis put. forth by the late geneticist Norman Horowitz in 1945 to explain the evolution of biosynthetic pathways. Horowitz proposed that in the early stages of the development of life, organisms developed pathways to make vital compounds via backwards evolution. Organisms in the primordial soup had a need for Compound A, which gave those organisms a fitness advantage. As time passed, the organisms that consumed Compound A multiplied, consuming more of Compound A until it was depleted. At that point, according to Horowitz, those organisms found a way to convert another available compound (e.g., Compound B) into Compound A. The oiganisms that evolved a pathway to convert Compound B into Compound A multiplied, consuming more of Compound B until it was depleted. The process continued and the early life forms developed a pathway to convert a readily available precursor, e.g., Compound C, into Compound B and then Compound B into Compound A.

To demonstrate the use of this theory to produce unnatural drugs, Bachmann and his team developed a biosynthetic pathway in Escherichia coli to produce didanosine. The scientists first identified the following steps for the production of didanosine:

1. Convert 2,3-dideoxyribose to 2,3-dideoxyribose 5-phosphate in the presence of the enzyme ribokinase (RK)

2. Convert 2,3-dideoxyribose 5-phosphate to 2,3-dideoxyribose 1-phosphate using the enzyme 1,5-phosphopentomutase (PPM)

3. Add 2,3-dideoxyribose 1 -phosphate to the naturally occurring compound hypoxanthine in the presence of the enzyme purine nucleoside phosphorylase (PNP) to produce didanosine.

They then considered these conversions in the reverse direction, and first optimized the last step in the process (Step A in the figure), then the next-to-last step (Step B), and then the first step (Step C). They first sequenced DNA that codes for PNP, third-step enzyme, and copied it - creating thousands of mutated DN A sequences, each containing a slightly different mutation. Next, they inserted the gene copies into E. coli and filled tiny test tubes with these colonies of bacteria. To test the enzymes produced in the bacteria, they broke open the E. coli cells and mixed their contents with the precursor molecule, and then screened for the presence of didanosine.

Once the researchers identified the best PNP enzyme mutant, they went back one step to evolve the best PPM enzyme in the presence of the optimized PNP enzyme. Then they repeated this process to evolve the best first-step enzyme, RK, in the presence of the optimized PNP and optimized PPM.

One of the benefits of this technique is that it requires only one screening test to characterize the ability of each enzyme to produce a desired chemical. In traditional biosynthesis, a separate screening method must be developed to identify the presence of each intermediate in the process. Bachmann and his colleagues were able to identify the presence of the product didanosine with only one test.

The Vanderbilt team successfully produced didanosine via bioretrosynthesis. They are now working on building the pathway back even further, to a simpler starting material. thetic nanomaterials to enhance as well as add new functions to the already-robust systems.

"Plants are very attractive as a technology platform," Strano says. "They repair themselves, they're environmentally stable outside, they survive in harsh environments, and they provide their own power source and water distribution."

The MIT team has demonstrated the potential of plant nanobionics by embedding nanomaterials into the chloroplasts of plants. In one demonstration, the scientists boosted a plant's ability to absorb light by inserting semiconducting carbon nanotubes into its chloroplasts. They also showed that equipping a plant with a different type of carbon nanotube enables the now-bionic leaves to detect nitric oxide.

"We could someday use these carbon nanotubes to make sensors that detect, in real time, at the single-particle level, free radicals or signaling molecules that are at very low concentration and difficult to detect," says postdoctoral researcher and plant biologist Juan Pablo Giraldo.

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Source: Chemical Engineering Progress

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