Polyketides are natural products produced by plants, fungi, and bacteria that represent a diverse class of compounds with a broad range of activities useful for many applications ranging from anticancer agents to antibacterials. However, these small molecules are so sufficiently complex that they cannot be easily synthesized through organic chemistry. To produce them recombinantly has also presented significant gene-expression challenges.
Blaine A. Pfeifer, Ph.D., assistant professor of chemical and biological engineering at Tufts University, is working to overcome these hurdles. “In the last 15 years, there has been a steady effort to produce polyketides in engineering-friendly organisms such as E. coli. The problem is that many of the polyketide synthetases are large proteins (>300 kD) that have unique assembly characteristics.”
Dr. Pfeifer says pathways to express polyketides are often very complicated. “There can be up to 20 coordinately expressed genes that are required for complete biosynthesis. Some of the larger protein products may be dysfunctional when produced, further complicating the issue. We are finding that the typical rules of molecular biology need to be ‘bent’ to succeed.”
As an example, Dr. Pfeifer has targeted production of a polyketide that is normally purified from specific strains of soil-dwelling bacteria. “Our case study features introduction of 17 genes into an E. coli expression system. To do this requires a number of optimization steps such as engineering different promoters, optimizing codons, adding a chaperonin, and even adjusting the temperature for E. coli growth. Despite these challenges, we succeeded in producing our compound of interest.”
Aside from being able to produce polyketides recombinantly, another important advantage of heterologous production is the ability to engineer new derivatives. “By modifying and re-engineering the products, we may be able to produce new compounds, such as modified antibiotics, that have increased potency. A key goal would be to leverage our recombinant production platform to produce new compounds against, for example, antibiotic-resistant bacterial pathogens.”