3. Strategies to modulate expression using synthetic biology
Synthetic biology encompasses a broad curation of methods aimed at reprogramming different cell types including microbial and mammalian cells for the purposes of producing compounds used in biofuels, pharmaceuticals, and materials-based applications. The synthetic processes necessary to produce these compounds requires the introduction of metabolic pathways not native to the original host species in many cases. One challenge synthetic biology has the potential to address is producing high-quality MPs for structural characterization and other biochemical analyses. Progress in MP research is often stymied by a lack of high-throughput, robust methods to produce high-quality material. The following summarizes and highlights some of the most effective tools available that can be used to tune MP expression and production inS. cerevisiae , Much of the progress made in synthetic biology has been possible using E. coli and S. cerevisiae – two of the best studied microbes. Application of these tools is slowly being introduced into other microbes. Processes in yeast will be the focus for the remainder of the review. The intracellular processes involved in protein production include transcriptional regulation through adaptation of genetic elements such as gene promoters, terminator regions, and other mechanisms of transcriptional control including transcription factors. A brief discussion on the use of fusion tags to boost protein yields follows along with whole genome editing strategies to generate selective knockout (KO) strains and down-regulate undesired proteolytic pathways. Conversely, metabolic engineering methods are discussed as a way of introducing novel pathways and genes identified to support MP production along with future insights into how this technology can be used to tune processes involved in expression. Innovative culturing methods that include comparative insights into differential protein induction methods, constitutive versus inductive, are discussed. Lastly, the latest developments in cell-free (CF) expression, a field that has seen significant advances in protein production applications, is presented with an emphasis on advantages over whole cell expression and future directions.