“The technical field has made tremendous progress in overcoming what we once thought were insurmountable challenges,” said Blaine Pfeifer, Ph.D., assistant professor in chemical and biological engineering at Tufts University. At CHI’s recent “Protein Engineering Summit” Dr. Pfeifer talked about his lab’s efforts to implement an optimized expression system and reconstitute polyketide and isoprenoid natural pathways in E. coli as an expression host.
While significant efforts in the early days focused on producing a protein of interest in E. coli expression systems, the field is now taking a step further. “At present, we need to produce several proteins in their active form that can, at the same time, work together to engineer the small molecule formation in bacteria.”
Recently, Dr. Pfeifer and colleagues developed an E.coli expression system to synthesize erythromycin A, an antibiotic that is produced by the actinomycete bacterium Saccharopolyspora erythraea and requires over 20 enzymes.
In collaboration with Gregory Strephanopoulos from MIT, Dr. Pfeifer uses a similar approach to produce early-stage intermediates of taxol, a promising anticancer drug. Taxol was originally isolated from the Pacific Yew (Taxus brevifolia), but isolating it from its natural source is both economically challenging and ecologically destructive.
The plasmid copy number, the co-expression of chaperones, and the choice of promoters and specific strains are some of the major considerations when expressing foreign genes in E. coli. All these technical aspects are important for the coordinate expression of over 20 genes that are needed for an active natural product pathway.
“This resonates a lot with general challenges that we face during protein expression,” said Dr. Pfeifer. An additional consideration is that the total number of genes in these pathways exceeds 20, and some are larger than 10 kb, exceeding by far the average E. coli gene size.
Therefore, one of the biggest challenges is whether the new host can accommodate these genes, particularly when they are expressed together. “We often don’t pay enough attention to the optimal level of gene expression and to the metabolic burden on the cell. These are some of the next steps that we need to address, as we tune the level of protein expression and metabolites to maximize the enzyme activity and the flow of carbon through our products of interest.”