Researchers Demonstrate Paradigm Shift in Engineering Enzyme-Derived Products
Research teams at the Center for Biorenewable Chemicals (CBiRC), an NSF-funded Engineering Research Center (ERC) headquartered at Iowa State University (ISU), working in tandem, have created substantially more stable versions of several polyketide synthases (a class of enzymes). The focus was on increasing the enzymes' kinetic activity as well as in vivo lifetime during multi-day fermentation runs.
This advance demonstrates a new paradigm shift in the engineering of enzyme-derived products in vivo (see figure). With regard to the pyrone testbed (one of CBiRC's mechanisms to evaluate concepts for production potential), this engineering approach has greatly increased the economic value of CBiRC's current production platforms.
Longer term, the strategy of enhancing the oxidation resistance of polyketide synthases will be employed generally during the engineering of additional polyketide and fatty acid synthases; the stabilized enzymes are being commercialized through Pareto Biotechnologies, Inc. (www.paretobio.com), a CBiRC member company. The work has also resulted in filing a Patent Cooperation Treaty (PCT) application (PCTUS1519058) that includes all members of the two CBiRC teams focused on pyrone-synthase engineering.
Through a combination of in vitro enzyme assays and in vivo fermentation runs, the researchers demonstrated substantial improvements in production of TAL (triacetic acid lactone, a platform molecule) due to the enhanced lifetime of 2-pyrone synthase in yeast production strains. This approach is now being implemented with additional polyketide synthases as a complementary method to active site engineering and the discovery of new polyketide synthases.
A library of mutant 2-pyrone synthases was constructed with multiple combinations of cysteine to serine mutations, and the resultant mutants screened for in vivo TAL production in yeast production strains. Combinations of active site mutants and cysteine replacements have resulted in synthetic pyrone synthases that now produce TAL in excess of 7 g/L during fed-batch culture with engineered strains