Grantee Research Project Results

Final Report: Engineering the Biosynthesis of Styrene in Yeast

EPA Grant Number: SU833519
Title: Engineering the Biosynthesis of Styrene in Yeast
Investigators: Washburn, Newell , Domach, Michael M. , Clark, Darin , Chandra, Divyam
Institution: Carnegie Mellon University
EPA Project Officer: Page, Angela
Phase: I
Project Period: August 31, 2007 through July 31, 2008
Project Amount: $10,000
RFA: P3 Awards: A National Student Design Competition for Sustainability Focusing on People, Prosperity and the Planet (2007) RFA Text |  Recipients Lists
Research Category: Pollution Prevention/Sustainable Development , P3 Awards , P3 Challenge Area - Chemical Safety , Sustainable and Healthy Communities

Objective:

In 2006, over 6 billion pounds of styrene were produced by U.S. manufacturers [1]. Styrene is derived from petroleum and is currently the most energy-intensive commodity chemical, requiring 200 trillion BTU of steam used per year in its production [2]. The goal of this research is to engineer yeast genetically to produce styrene biosynthetically. Yeast naturally produce high levels of aromatic compounds, such as folic acid, and have a robust, multi-organelle structure that could make them suitable producers of this reactive, toxic chemical.

Summary/Accomplishments (Outputs/Outcomes):

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].

Conclusions:

Current work is focused on using glycerol as a carbon source for yeast that are producing styrene. This would provide an option for the biosynthesis of styrene that would utilize a waste product from biodiesel production. However, significant research will be necessary to produce styrene at a cost that is competitive with current manufacturing techniques.

References:

[1] Plastics Industry Producers’ Statistics Group

[2] Department of Energy website (http://www.doe.gov Exit )

[3] Snell KD, Draths KM, Frost JW. J Amer Chem Soc 1996, 118, 5606-5614.