“As we engineer biological systems to drive the production of high-value products such as biofuels, we want to be able to solve control problems and avoid the buildup of toxic products and unbalanced metabolites,” says James M. Carothers, Ph.D., research fellow at the University of California Berkeley and the DOE Joint BioEnergy Institute.
Recently, Dr. Carothers and colleagues reported the design of a dynamic sensor-regulatory system to produce fatty-acid-based products in E. coli. This system incorporated biosensors for fatty acid/acyl-CoA and hybrid promoters that responded to changes in fatty acid levels and to an exogenous inducer.
“We found that if we engineer genetic elements that we combine into the system, and couple the production of the intermediates to regulation in the pathway, we can increase production to almost 30% of the theoretical maximum,” says Dr. Carothers.
In addition to increasing the production of fatty acid ethyl esters as compared to a previous study, the host strain harboring this system was stabilized, a finding with important implications for the large-scale implementation of this approach. This represents an advantage over genetically modified microorganisms, in which the loss of gene function as a result of toxic metabolite accumulation opens ongoing technical and practical challenges.
Previously, Dr. Carothers and colleagues reported techniques to design RNA-based genetic control systems that could process cellular information and program the expression of large numbers of genes, potentially enabling the model-driven construction of metabolic pathways in various systems.
“Synthetic biology presents a lot of relevance for industrial biotechnology, for producing low- and high-value chemicals, and I think that in the future we will see new technologies and new approaches that will allow us to build larger and better systems,” says Dr. Carothers.
Crossroads with Tumor Biology
Despite recent advances in tumor biology, molecular diagnostic and therapeutic approaches remain a challenging area. Recently, Yaakov Benenson, Ph.D., professor in the department of biosystems science and engineering at ETH Zürich, and colleagues designed a transcriptional/post-transcriptional synthetic regulatory circuit that can sense the expression pattern of a customizable battery of microRNAs, and illustrated its promise toward a new strategy with applicability in cancer diagnostics and therapeutics.
The investigators showed that this synthetic circuit can selectively trigger apoptosis in HeLa cells expressing specific combinations of markers expressed by cancer cells without affecting the surrounding, non-HeLa cell types.
“Our microRNA-based strategy provides a flexible approach that can use different marker combinations, and since different cancers may express very different combinations of markers, this approach can therefore be tailored to different types of tumors,” says Dr. Benenson.
This concept, enabling specific responses to be triggered based on defined and complex intracellular conditions, provides a therapeutic framework with applicability for other cellular states, and illustrates one of the applications of synthetic biology in cancer diagnosis and therapy. “Our long-term goal is to develop this into a therapy candidate but, like any potential treatment, it has to go first through all the stages, including cell culture and animal testing,” explains Dr. Benenson.
“Our expansion of optogenetics as a field involved an approach to convert electromagnetic waves from the visible range of blue light into a sustained transcription response, which allows genes to be controlled by shining blue light onto them,” says Martin Fussenegger, Ph.D., professor of biotechnology and bioengineering at ETH Zürich.
The strategy used by Dr. Fussenegger and colleagues takes advantage of the signal cascade of melanopsin, the photopigment found in the photosensitive ganglion cells from the retina, which is most sensitive to blue light (~480 nm). Upon light exposure, melanopsin activates a G protein, triggering a subsequent signal transduction cascade that leads to an intracellular calcium surge.
This calcium surge was rewired to the nuclear factor of activated T-cells, which can initiate gene transcription from specific promoters. After demonstrating the functionality of this synthetic device in mammalian cells, Dr. Fussenegger and colleagues subsequently illustrated its use in a mouse model, where it enabled the light-induced expression of a transgene.
In a type 2 diabetes mouse model harboring these transgenic synthetic devices in intraperitoneal hollow-fiber or subcutaneous implants, the investigators reported that, upon exposure to light, the animals showed increased glucagon-like peptide 1 expression, reduced glycemic excursions, and decreased glycemic levels.
“This system is ready for clinical applications,” emphasizes Dr. Fussenegger. A key feature, fundamental for the clinical use of this synthetic device, is that it is fully humanized, and it does not contain any components that would elicit an immune response.
As individual parts, such as promoters, open reading frames, terminators, and transcription factors are combined to generate pathways and circuits, one of the goals of synthetic biology is to generate synthetic chromosomes and genomes, some of which have never existed before.
An important milestone was reached less than a year ago, with the publication of the first partially synthetic eukaryotic chromosome—that of the budding yeast. This advancement, along with other developments in the field, are signaling the beginning of a new era, one that provides a new level of scientific inquiry and promises to reshape medicine, biomedical research, and biotechnology.