Southern California Conferences for Undergraduate Research

Southern California Conferences for Undergraduate Research

Optimizing Heterologous Biosynthetic Pathways in Saccharomyces Cerevisiae


Sohini Khan


Christina Smolke, Associate Professor of Bioengineering, Stanford University

Optimizing Heterologous Biosynthetic Pathways in Saccharomyces Cerevisiae
Author: Sohini Khan, California Institute of Technology
Mentors: Stephanie Galanie & Christina Smolke, Bioengineering, Stanford University

A challenge now faced by many scientists is producing sufficient quantities of useful natural products via a biosynthetic platform to allow for microbes like Saccharomyces cerevisiae or Escherichia coli to produce such products in a more sustainable way. Microbes such as S. cerevisiae provide a more sustainable source for producing such plant products. Cytochrome P450 oxidoreductase enzymes (P450s) are common in plant biosynthetic pathways that produce these natural products. In heterologous hosts, coexpression of plant NADPH-cytochrome P450 oxidoreductases (CPRs) with plant P450s has resulted in enhanced P450 activity. The goal of my project is to be able to optimize CPR P450 partnering by first comparing ATR1 and ATR2, the two prototype CPRs from the model organism, Arabidopsis thaliana, and then studying Eschscholzia californica CPR (EcCPR) mutants from the ATR2-like family. The CPRs were characterized by Western blot analysis to determine the level of full-length expression, confocal microscopy to qualitatively observe localization of GFP-tagged CPRs, and in vivo substrate feeding assays analyzed by LC-MS to measure accumulation of products. Western blots show that there is more full-length ATR2 nascent protein than ATR1. However, confocal microscopy imaging indicates that ATR2 aggregates, while ATR1 expresses more uniformly in membranes. Finally, the in vivo assays demonstrate that less product accumulates with integrated ATR2 and EcCPR mutants than ATR1 in strains expressing the P450s. Further experiments are being conducted to determine whether the reduced function of ATR2 and the EcCPR mutants is a result of too high a level of expression of the CPR.

Presented by:

Sohini Khan


Saturday, November 17, 2012


9:00 AM — 9:15 AM


Bell Tower 1462

Presentation Type:

Oral Presentation