Synthetic Fungal Biology for Human and Environmental Health through Enhanced Degradation of Benzo(a)pyrene

EPA Grant Number: FP917791
Title: Synthetic Fungal Biology for Human and Environmental Health through Enhanced Degradation of Benzo(a)pyrene
Investigators: Ostrem, Erin M
Institution: University of Wisconsin - Madison
EPA Project Officer: Lee, Sonja
Project Period: September 1, 2015 through August 31, 2018
Project Amount: $132,000
RFA: STAR Graduate Fellowships (2015) RFA Text |  Recipients Lists
Research Category: Academic Fellowships

Objective:

Benzo(a)pyrene (BaP) is a persistent and ubiquitous soil contaminant which is formed by the partial combustion of organic matter. The EPA lists BaP in the top ten Superfund site contaminants of greatest concern, and is also listed by IARC as a group I carcinogen, making a significant threat to human health. Filamentous fungi are a group of organisms which live mostly as saprophytes, and hence have the capacity to utilize a diverse range of carbon sources. They are relatively understudied when compared to bacteria, yet the possibility of utilizing them to decompose organic contaminants is more promising because their ability to live in arid and nutrient poor soils is enhanced by their filamentous growth. This makes them excellent candidates for remediation of contaminated sites. My research uses Aspergillus nidulans, a soil dwelling filamentous fungus, which is a model laboratory system used to study molecular mechanisms.

Approach:

I plan to use a variety of complementary techniques to investigate the ability of Aspergillus nidulans to metabolize BaP. First I will confirm that the initial oxidation step is mediated by a particular cytochrome p450 (CYP) enzyme by incubating protein fractions isolated from wild-type and gene deletion cells with BaP. I will additionally identify the final metabolite by incubating isotopically labeled BaP with whole cells and detect metabolites using LC/MS on full scan mode. I will further identify the other genes necessary for metabolism by analyzing RNA sequencing data and distinguishing genes which are upregulated in BaP treated cells but not in cells receiving no treatment. This set of experiments will give me the information necessary to engineer a strain of Aspergillus that has an enhanced ability to degrade BaP.

Expected Results:

The Aspergillus nidulans genome contains many predicted cytochrome p450 enzymes, with three of them showing high homology with another known BaP metabolizing CYP. It is likely that because of its ecological niche as a saprophytic soil dweller, A. nidulans is capable of quickly adapting to new carbon sources by differentially regulating specific CYP encoding genes. Because gene manipulation is relatively easy with this organism, it is likely gene deletion of one of these CYPs will show a loss of BaP metabolizing capability. Overexpression of this CYP is hence likely to enhance metabolism of BaP. Information obtained by metabolite identification via LC/MS and RNA sequencing data will help in understanding other key steps in the metabolism of BaP. Their predicted role can be supported by subsequent gene deletion and overexpression.

Supplemental Keywords:

benzo(a)pyrene, soil contamination, Aspergillus, fungi, degradation, PAHs

Progress and Final Reports:

  • 2016
  • 2017
  • Final