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ENGINEERING THE BIOSYNTHESIS OF STYRENE IN YEAST
Impact/Purpose:
Replacing petroleum-derived commodity chemicals with those produced from sustainable resources requires a platform of molecules having a broad range of chemical properties. However, the top 12 sugar- or syngas-derived chemical building blocks recently identified by the Department of Energy are all aliphatic molecules. Aromatic compounds are critical components of the plastics and resins industry, and a robust biological source is needed to complement the development of feedstocks based on renewable resources. The goal of this research program is to engineer yeast capable of efficiently producing the resin styrene, which currently is produced from petroleum at billions of pounds per year. Because of their compartmentalized internal structure, yeast are well suited for multi-step biosynthesis of non-natural chemicals. Genetic manipulation of the shikimate pathway will be used to overproduce the amino acid phenylalanine, which can then be converted to styrene by incorporating the genes for the enzymes phenylalanine ammonia lysase and phenolic acid decarboxylase. Other important aromatic commodity chemicals, such as p-hydroxystyrene or phenol, could be produced biosynthetically using this basic approach, making progress in styrene biosynthesis more generally applicable. Optimization of yield and titer will be performed using metabolic pathway analyses, genetic modifications, and bioreactor design and will be benchmarked against current methods of commodity-chemical biosynthesis. Ultimately, development of biorefineries that produce a broad range of aromatics would provide a clean, sustainable source of chemicals currently missing from our portfolio of sustainable commodity chemicals. Multi-disciplinary undergraduate research and curriculum development are to be used to draw together molecular biologists, chemists, and engineers to help address the challenge of aromatics bioproduction. The results of this research progr
Replacing petroleum-derived commodity chemicals with those produced from sustainable resources requires a platform of molecules having a broad range of chemical properties. However, the top 12 sugar- or syngas-derived chemical building blocks recently identified by the Department of Energy are all aliphatic molecules. Aromatic compounds are critical components of the plastics and resins industry, and a robust biological source is needed to complement the development of feedstocks based on renewable resources. The goal of this research program is to engineer yeast capable of efficiently producing the resin styrene, which currently is produced from petroleum at billions of pounds per year. Because of their compartmentalized internal structure, yeast are well suited for multi-step biosynthesis of non-natural chemicals. Genetic manipulation of the shikimate pathway will be used to overproduce the amino acid phenylalanine, which can then be converted to styrene by incorporating the genes for the enzymes phenylalanine ammonia lysase and phenolic acid decarboxylase. Other important aromatic commodity chemicals, such as p-hydroxystyrene or phenol, could be produced biosynthetically using this basic approach, making progress in styrene biosynthesis more generally applicable. Optimization of yield and titer will be performed using metabolic pathway analyses, genetic modifications, and bioreactor design and will be benchmarked against current methods of commodity-chemical biosynthesis. Ultimately, development of biorefineries that produce a broad range of aromatics would provide a clean, sustainable source of chemicals currently missing from our portfolio of sustainable commodity chemicals. Multi-disciplinary undergraduate research and curriculum development are to be used to draw together molecular biologists, chemists, and engineers to help address the challenge of aromatics bioproduction. The results of this research prog
Description:
The strategy pursued was to insert genes for phenylalanine ammonia lysase (pal) and phenolic acid decarboxylase (pad) into the yeast that would convert phenylalanine to styrene through a cinnamic acid intermediate.
Figure 1. Strategy for producing styrene biosynthetically. The phenylalanine ammonia lysase (pal) gene converts phenylalanine to cinnamic acid, which is converted to styrene by the phenolic acid decarboxylase (pad) gene.
The genes for pal and pad are controlled by a galactose promoter. As shown in Figure 2, yeast growth on galactose is significantly slower with a doubling time of 8 h as opposed to 105 min for wild-type yeast grown on glucose.
Figure 2. Growth curves of wild-type (top) and mutant yeast strains (bottom) measured by recording optical density of the cultures at 600 nm.
Styrene production at 6 h was measured using high-performance liquid chromatography (HPLC). Spectral analysis and comparison to styrene standards was used to confirm that styrene was produced, which elutes at 22 min.
Figure 3. Spectral HPLC analysis of lysed cell products from mutant yeast strains. Samples were run on a C18 column in a 20/80-80/20 water/acetonitrile gradient. The top curve is the elution from the column recorded at 248 nm and the bottom two curves are the absorbance spectra of the elution peaks at 17 min and 22 min.
There is a significant amount of cinnamic acid left in the cells, which elutes at 17 min, suggesting that the pad enzyme may not be as active as pal. The concentration of styrene in these cultures was estimated to be 10 mg/l, which is consistent with that expected from microorganisms that have not been engineered to overproduce products from the Shikimate pathway [3].