October 1, 2014 (Vol. 34, No. 17)

Utilizing Synthetic Biology Techniques in the Production of Novel Biotherapeutics

Now that synthetic biology (synbio) has become an official bio-buzzword, it is time to take a step back and ask, “What’s in it for biomanufacturing?” At least one expert is not shy about answering the question.

“Synthetic biology’s most immediate potential commercial benefits will be in the biomanufacturing of therapeutic proteins and vaccines,” says Klaus Lindpaintner, M.D., chief science officer, analytical instruments at Thermo Fisher Scientific. “And more broadly, it will affect many areas of chemical engineering, specifically in petrochemicals and plastics, as well as environmental remediation, crop science, and animal breeding. There are almost no limits to the promise of synthetic biology.”

Yet the definition of the term “synthetic biology” has been obfuscated by practicalities and a lack of clear boundaries between classical molecular biology and the “real thing,” that is, the really synthetic thing. Dean Griffiths, Ph.D. medical technology expert at Cambridge Consultants, wryly notes that researchers add the term to a grant application because it “helps them get funding.”

But Dr. Griffiths’ view of synbio’s potential in biomanufacturing is anything but cynical. He foresees a future for bioprocessors in which modified organisms simplify and add efficiencies that are inaccessible today through conventional molecular biology.

“Synthetic biology will improve efficiency in biomanufacturing, for example, by expressing relevant genes at higher levels or secreting proteins more efficiently,” Dr. Griffiths asserts, adding that these gains may come “with built-in mechanisms for suppressing degradation pathways or with a higher affinity for purification media.”

Unnatural Genes, Amino Acids

Synbio possibilities extend beyond the creation of new, improved expression systems to the introduction of novel genes into organisms and, further downstream, to the synthesis of proteins containing unnatural amino acids. Based on work at The Scripps Research Institute by Floyd E. Romesberg, Ph.D., a startup company named Synthorx has expanded the genetic code to include two additional nucleotides.

Court Turner, chief excecutive officer at the company, explains that its goals are to make the new genes functional, and with them to create Escherichia coli strains that produce novel proteins for catalysis, protein expression, purification, and synthesis of novel drugs.

Synthorx first transforms the organisms with a plasmid containing instructions for synthesis of the synthetic nucleotides, then expresses an exogenous transporter that brings the unnatural triphosphate into the cell, where the synthetic nucleotides are then replicated.

“The only real change was transporter expression,” Turner says. “Everything else is replicated using the cell’s endogenous machinery. As long as you continue to feed cells the unnatural triphosphates, they produce them as if they were natural bases.”

“Conventional” synbio, Turner explains, looks for new applications of the standard 64 codons. Synthorx has 256 codons to work with, which allows incorporation of up to 172 unnatural amino acids into proteins that do not exist naturally. Drug discovery is the obvious application, but through application of a similar process, one could envision super-enzymes used in the manufacture of both protein and small molecule pharmaceuticals.

The limited combinations of the DNA bases, A, T, G, and C, have restricted the types of new proteins, RNA, and DNA that can be made. Adding two new synthetic bases, termed X and Y, to the genetic alphabet, Synthorx can now employ an expanded vocabulary to improve the discovery and development of new therapeutics, diagnostics, and vaccines, as well as create innovative products and processes. [Synthorx]

What Mol-Bio Can’t Do

“Everyone has their own definition of synthetic biology,” says Mike Mendez, co-founder and CTO of Pareto Biotechnologies. According to Mendez, who in 1994 created the Xenomouse, the first animal to have a fully human immune system, synbio is more about systems and pathways than the one-off manipulations achievable through molecular biology. Differences are both qualitative and quantitative, as synbio is not just molecular biology (mol-bio) on steroids.

Molecular biology allows cutting, amplifying, and pasting DNA with exquisite precision, but inducing radical changes to a protein or dropping an entire system into an organism involves much greater complexity.

“To me, synthetic biology is the ability to leverage cheap synthesis and cheap sequencing of DNA,” Mendez says. “If you can achieve something without synthesizing and sequencing large quantities of DNA, it’s simply molecular biology. But if the only way you can do an experiment is through a huge synthesis and sequencing effort, then it’s synthetic biology.”

Synbio is also about numbers, about creating genetic diversity at a level that is impractical through PCR point mutations, for example, the systematic replacement of every amino acid in a protein with every other amino acid in a quest for higher therapeutic efficacy or improved manufacturability.

Mendez predicts that synbio will have a “profound” effect on biomanufacturing. Optimizing an expression system’s carbon fluxes through pathway engineering requires tinkering with the entire pathway, not just a few genes.

“Synthetic biology enables us to synthesize an entire pathway on a few pieces of DNA, splice megabases together, and insert them into cells,” Mendez says, adding that achieving this 20 years ago in the Xenomouse was a “major undertaking.” Now it is achievable in a few weeks.

“For the first time, you can create whole pathways at the flick of a switch, which allows you to control carbon flux in and out of cell lines to maximize productivity,” he explains.

Mendez envisions production of a range of proteins in yeast or E. coli, with tight control over expression levels and inherent toxicity. “And you don’t have to be a Pfizer or a Merck. A grad student can do it. When synthetic biology is that cheap and flexible, its application will explode,” he predicts.

Perhaps as significant as optimizing “carbon flux” will be the ability to improve almost any molecule through the systematic replacement or forced evolution of a protein’s amino acid sequence. This will require creating huge libraries of as many as 10 billion possible substitions, and screening across every sequence. This approach has been used to improve RuBisCO, the enzyme involved in the first step of carbon fixation in plants.

“Without the ability to synthesize and sequence at large scale, we could never do that project,” continues Mendez. “Without synthetic biology, you could never ask that question or do that experiment.”

By analogy, Mendez sees similar efforts that will give life to old molecules or therapeutic protein projects that have been shelved due to suboptimal efficacy or toxicity. “These molecules are still in the freezer. Their sequences are known, as are the assays. Synthetic biology will bring new life to pipelines that were all but dead,” he emphasizes.

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