Graduate and Postdoctoral Studies
Chemical and Biomolecular Engineering
Development of de novo biosynthetic pathways
Thursday, July 27, 2017
to 3:00 PM
801 BioScience Research Collaborative
Microbial biorefineries and biomanufacturing, which harness biosynthetic pathways, are becoming economically viable alternatives to traditional petrochemical refineries and manufacturing due to their cleanness, flexibility, and usage of biorenewable or recycled feedstocks. However, the energy or carbon efficiencies and product diversity of biosynthetic pathways are not sufficient for scaled-up industrial processes. The goal of this thesis is to develop de novo biosynthetic pathways with improved energy or carbon efficiencies and/or product diversity as alternatives to three widely used biosynthetic pathways: ?-oxidation reversal, isoprenoid biosynthesis and polyketide biosynthesis.
A novel, iterative, modular, combinatorial and orthogonal carbon-carbon elongation platform composed of non-decarboxylative Claisen condensation reactions and susbsequent ?-reductions was developed. This platform follows a similar mechanism and exhibits equivalent high carbon and energy efficiencies to those of the ?-oxidation reversal, but in addition is able to accept various ?-, and ?-1-functionalized primers and ?-functionalized extender units and synthesize diverse chemicals with ?-, ?-, ?-, and ?-1-functionalities. During the study, the production of 18 compounds from 10 product classes was demonstrated with noticeable titers. Among them, seven compounds were produced for the first time via microbial fermentations.
This thesis also proposed four novel pathways for the biosynthesis of isoprenoid building blocks dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), which are more energy- or carbon-efficient than native mevalonate or non-mevalonate pathways. Though the search of some enzymes required for realizations of these pathways are still underway, many enzymatic components have been characterized in vitro and in vivo and utilization of confirmed components led to efficient microbial productions of 2-hydroxyisovalerate and prenol with titers of 8.46 g/L and 0.48 g/L respectively.
A novel, energy-efficient polyketide biosynthesis pathways has also been designed as an alternative to native, polyketide synthase (PKS)-based routes. Instead of using PKSs, the pathway is based on repetitive non-decarboxylative Claisen condensation reactions catalyzed by thiolases, which bypasses the ATP-consuming carboxylation step required for the generation of extender units used by PKSs. Among thiolases tested for polyketide synthesis, BktB, ScFadA, FadAx and DcaF showed promising signs of production of pyrone triacetic acid lactone (TAL) through condensation between acetoacetyl-CoA and acetyl-CoA with highest titer as 0.36 g/L. FadAx also promisingly exhibited in vitro condensation activity between two acetoacetyl-CoAs to generate another pyrone dehydroacetic acid.
Achievements of this thesis, in line with subsequent improvements, are expected to highly benefit the development of microbial biomanufacturing processes and biorefineries.