01 April 2023 | Saturday | Analysis
Source : Twist BioScience
With its resplendent flowers, the Madagascar periwinkle plant (Catharanthus roseus) easily draws attention to itself, both in a natural setting and in pharmacological textbooks. Embedded in the plant’s eye-catching flowers are some of the world’s most used anticancer compounds, known broadly as the vinca alkaloids1.
For more than half a century, the vinca alkaloids have served as a mainstay in the treatment of leukemias, lymphomas, and many other cancer types2. In spite of their prominence, sourcing these compounds remains a logistical challenge. Like other drugs derived from natural products, the vinca alkaloids are structurally complex compounds, which makes them exceedingly difficult to produce through chemical synthesis methods. As such, every therapeutic dose starts in a plant, and that reality can be a problem for both the sustainability and reliability of supply chains.
Consider the logistics of producing one of the vinca alkaloids’ most well-known members, vinblastine, a widely used chemotherapeutic. Extraction and purification of the compound requires more than 2,000 kg of the periwinkle’s colorful flowers to produce just 1 g of pure vinblastine3,4.
Having a heavy reliance on natural sources for compounds like vinblastine creates a shaky foundation for the global supply chain. Plant pathogens, natural disasters, and the fluctuating economics of drug extraction can suddenly disconnect drug developers from their resources5. Unfortunately, this is more than theory: Such a disconnect has happened several times for the world’s supply of vinblastine, forcing patients and clinicians that need this essential chemotherapeutic to make difficult, life-altering decisions6.
In the hopes of preventing future shortages, researchers are turning to synthetic biology and metabolic engineering to build a new kind of supply chain, one derived from microbes.
Beer is perhaps the best-known product from a microbial supply chain. Breweries cultivate special strains of yeast (Saccharomyces cerevisiae) that, according to their strain-specific genetics, convert sugar, hops, and other ingredients into beer on a grand scale. In this example, the yeast serve as a sort of microbial biofactory by using their internal proteins to manufacture a desired compound (beer).
Synthetic biologists and metabolic engineers aim to use this same concept for sustainable drug production. The strategy involves manipulating the genome of an easy-to-grow microbe so that it can express a complement of foreign proteins. These proteins then allow the microbe to synthesize complex chemical structures that are normally only found in plants, including essential therapeutic agents.
? For the Love of Beer
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Such an approach could greatly improve the world’s supply of drugs like vinblastine. Unlike plants, microbes can be sustainably grown in large bioreactors that are insulated from environmental influence, enabling the consistent, year-round production of therapeutics.
Already, advances in metabolic engineering have led to the development of yeast strains that are capable of producing cannabinoids, artemisinic acid, opioids, and many more compounds. However, for any one microbial supply chain to be successful, researchers have to overcome several challenges7-9.
To build natural products like vinblastine, plants have evolved a complex, cascading network of proteins that coordinate drug synthesis. These networks are known as biosynthetic pathways and are often spatially and temporally sensitive, meaning the success of any one step in the pathway depends on the timing of protein expression and the cellular compartment in which it takes place10. Put simply, biosynthetic pathways can be very complex.
To emulate this complexity, researchers need detailed characterization of the desired natural biosynthetic pathway; they need to select the proper microbe host; and they need to endure considerable trial and error.
Detailing natural pathways
It is difficult to mimic biosynthetic pathways if we don’t understand them. While tools like next-generation sequencing (NGS) can help researchers find and characterize these molecular relays, many sources for plant-based natural products have yet to be fully sequenced and characterized at the genomic, transcriptomic, or metabolomic level. Therefore, identifying the genes and regulatory systems that are involved in natural biosynthetic pathways can be a challenging and lengthy discovery process.
Chassis Organisms
At the heart of the microbial supply chain is the microbe, and deciding which species to use is a critical choice. While many options exist, Escherichia coli and Saccharomyces cerevisiae are commonly used11. However, genetic and biological differences can lead to species-specific outcomes when biosynthetic pathways are introduced. Choosing one species over the other often requires several design, build, test, and learn (DBTL) cycles which can be both time and resource-intensive12,13.
Optimizing pathway parts
Finally, biosynthetic pathways involve several proteins and genetic regulators that help guide the reaction. Synthetic biology treats these various components as “parts” that can be easily transferred from one system to another (like moving a wheel from one car to another)14. However, biological systems can get messy, and predicting quantitative gene expression using well-validated parts is still challenging. Therefore researchers will have to go through a lengthy process of optimization to ensure that each part in the biosynthetic pathway is expressed at the right time, in the right quantity, and in the right place.
Though difficult, researchers are getting better at engineering microbial biofactories, thanks in part to advancements in synthetic DNA technology.
In an effort to prevent future shortages of essential drugs in the vinca alkaloid family, Jay Keasling's group at the University of California, Berkeley engineered a biosynthetic pathway that, for the first time, enables the synthesis of vinblastine15.
"This is a significant step towards stabilizing the world’s supply of complex drugs"
Previous work had established that vinblastine’s natural biosynthetic pathway involves 31 steps and a large number of genes16. Building on this work, Keasling’s group edited the genome of Saccharomyces cerevisiae, introducing 34 heterologous plant genes and tweaking ten yeast genes via deletion, knock-down, or overexpression. A total of 56 genes were edited in this tour-de-force metabolic engineering project, resulting in the production of 2 vinblastine precursors, catharanthine and vindoline. These precursors were then converted to vinblastine using a final, well-established synthetic chemistry step.
The work reported by Keasling’s group represents a significant step towards stabilizing the world’s supply of complex drugs by demonstrating that this yeast-based platform can provide scalable synthesis of vinblastine and other vinca alkaloids.
Critical to their success was the use of synthetic DNA, which enabled sequencing and annotation of the Catharanthus roseus genome; the large-scale synthesis of heterologous gene variants; and the precise manipulation of the yeast genome. Twist helped enable this achievement in metabolic engineering by supplying the team with tools to support this effort.
Twist has developed a suite of synthetic DNA tools that can help enable metabolic engineering, from custom NGS library preparation kits and target enrichment panels to the scalable synthesis of combinatorial gene variants. Such tools have been successfully used to design, test, build, and optimize biosynthetic pathways, including a carotenoid synthesis pathway in E. coli and, most recently, Keasling’s efforts to engineer the vinblastine biosynthetic pathway in yeast.
The use of Twist synbio and NGS tools in these projects is not surprising. Twist’s silicon-based DNA synthesis platform delivers oligonucleotides with industry-leading precision and uniformity that form the backbone of all their products, from genes and libraries to antibodies and NGS panels. This precision and uniformity enables efficient and thorough exploration of biosynthetic pathways, empowering researchers to forge a path towards more sustainable therapeutic supply chains.
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