Engineering robust yeasts for biorefinery applications

Yeasts are the industrial workhorses of the bioethanol industry and have proven their economic feasibility in large-scale facilities converting biomass into ethanol. However, fermentation production of biofuel molecules of higher energy density have been problematic due to the low toxic tolerance of the typical yeast strains in use currently. Lawrence Berkeley National Laboratory (LBNL) recently developed a suite of synthetic biology tools, collectively called the Artificial Positive Feedback Loop (APFL), which enables the engineering of microorganisms to amplify specific metabolic pathways without deleterious effects to the overall health of the organism. Consequently, precursors to many isoprenoid derivatives leading to high energy density biofuel molecules could be produced by non-conventional yeasts. For example, isoprene and sabinene have been produced by heterogonous synthetic pathways in engineered microorganisms, but the low toxic tolerance of conventional host organisms have resulted in low production yield of the target molecules. This SBIR Phase I project will apply LBNLs novel synthetic biology approach (APFL) to dramatically improve production yields of valuable high energy density compounds. Precursor production from yeast has already been demonstrated. The broader testing of the APFL technology in new constructs of engineered yeast strains is expected to enable a more benign and more efficient method to amplify production of high value target compounds. The researchers who developed the APFL at LBNL have founded Afingen, Inc. to pursue commercial development of this technology. Commercial application and benefit: The target advanced biofuel compounds, isoprene and sabinene, are produced from precursors derived from the well-known yeast terpene-isoprenoid-ergosterol biosynthesis pathway. In addition to their utilization as flavors and fragrances, both target terpenes have great potential in renewable energy since they can be rapidly converted into biofuels after a simple biofuel conversion. Renewable biofuels and biofuel precursor products are fast growing markets and demand will keep rising for decades. Upon further demonstration of the APFL technology in engineered yeast strains, a large variety of yeasts can be engineered to biologically synthesize biofuels and specialty biofuel precursor compounds. Using non-conventional yeasts and the new synthetic biology tools, we expect to increase yields of target compounds by 50x or better. Keyword: Synthetic Biology, Yeast Saccharomyces cerevisiae, APFL Technology, Transcriptionsfactors, Enzymes, Isoprenoids Synthetic Pathway, Carbon-neutral Biofuels, Combinatorial DNAAssembly Summary for member of congress: A new synthetic biology technique enables engineered yeast strains to increase production of advanced biofuel compounds by 50x to compete favorably with the cost of 2D diesel.