Natural products are very important because they have significant medicinal properties such as antiviral, antimicrobial and anticancer activity. The latest breakthrough in DNA sequencing shows that many microbial genomes (such as Streptomyces species) encode many hidden natural product biosynthetic gene clusters. However, obtaining compounds from these clusters is still challenging, because during the laboratory cultivation process, many source organisms cannot be cultured or gene-silenced. Researchers have been stepping up the production of a promising drug to effectively kill the SARS-CoV virus in cell culture. Today, this drug may also be effective against SARS-CoV-2, a close relative of SARS-CoV virus.
In a recent study published in the international journal Metabolic Engineering, scientists from northwestern university in the us and Shanghai university of science and technology in China developed a potential molecule called valinomycin in a cell-free system. Based on the cell-free system, the researchers hope to increase the production of valinomycin by more than 5, 000 times in just a few quick design cycles, while also achieving higher concentrations of the molecule in cells than before.
The researchers have developed an effective cell-free platform for the rapid, in vitro total biosynthesis of the non-ribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (> 19 kb added in a single-pot reaction), and the optimized valinomycin produced about 37μg / L. Second, we combined CFPS with a cell-free metabolic engineering system by mixing two enzyme-rich cell lysates to perform a two-stage biosynthesis. This strategy has increased the production of valinomycin by 5,000 times, reaching nearly 30 mg / L. We expect that the cell-free biosynthesis system will provide a new way for in vitro expression, discovery and characterization of natural product gene clusters of interest.
Researcher said that because we use this cell-free system, we can optimize the speed of production in the cells, thereby further increasing the production of valinomycin. For example, the route optimization cycle takes several days instead of weeks or months. This speed may be particularly important when dealing with epidemics such as the COVID-19 epidemic.
The researchers believe that their results have several key characteristics. First, the use of cell-free systems allows for fine-tuning of reaction conditions, ease of monitoring, and avoids mechanisms that have been developed to promote species survival. Second, the cell-free system can achieve high titer natural products. We can use the flexibility of a cell-free system to adjust the way in which enzymes are enriched in lysates (from cell-free protein synthesis to heterologous overexpression) to promote the increase of natural product potency. Third, our cell-free method is fast. It takes only a few hours to obtain the target compound. Conversely, it may take days or weeks to cultivate the strains used for product production. Therefore, the cell-free biosynthesis system provides a fast and cost-effective method for synthesizing the target molecule.
Researchers are currently conducting multiple research projects, they aim to use cell-free biotechnology to accelerate the development of COVID-19 therapy. Researchers can take out cellular “molecular machines” from cells, and then use these “molecular machines” to manufacture products, such as therapeutic drugs, in a safe, cheap, and fast way. This idea is like opening the hood of a car. The engine is disassembled, so that researchers can use the engine for unused purposes without being restrained and constrained by the car.
As a natural peptide drug, valinomycin can potentially be used as an antiviral therapy to treat SARS infection. Researchers now want to try to use cell-free synthetic biology to find similar molecules, or by modifying valinomycin to make it safer and more effective.
- Lei Zhuang，Shuhui Huang，Wan-Qiu Liu,et al. Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin, Metabolic Engineering (2020). DOI: 10.1016/j.ymben.2020.03.009