GRADUATE STUDENTS
Avantika Shastri ashastri@ecn.purdue.edu
Photosynthesis is the major process that uses electromagnetic energy from the sun to fix inorganic carbon (CO2) and convert it to glucose. Because of the difficulty in quantification analysis of metabolic flux in plants, especially eukaryotic photosynthetic tissue, as well as the progress in genomics with the unicellular cyanobacteria, Synechocystis PCC 6803 is an ideal model system to analyze metabolic fluxes in photosynthetic organisms. Metabolic flux analysis (MFA) reveals the degree of pathway engagement in the overall metabolic process, however, it has limited potential in elucidating aspects of metabolic networks when relying on extracellular measurements only. This limitation can be overcome via the combination of isotopically labeled compounds with NMR or gas chromatography – mass spectrometry (GC-MS) and the stoichiometry of the metabolic map. From these measurements it is possible to determine a metabolic flux map indicating the engagement of individual pathways. The application of MFA techniques developed herein can be used to measure changes in flux in mutants as well as recombinant photosynthetic unicellular organisms. By a comparison of the changes in flux resulting from genetic changes, the iterative process of metabolic engineering can proceed in an expedient and rational manner.
Hao Chen chen90@purdue.edu
Cytochrome
P450 monooxygenases are found in almost all living organisms, and they play a
very important role in diverse reactions such as secondary metabolism or drug detoxification. P450s have a great potential to be used as the
biocatalysts for oxidation processes in industry, since they can introduce
oxygen at non-activated carbon–hydrogen bonds to yield pure compounds, and their efficiency is higher than most chemical
catalysts. However, some obstacles remain when applying P450s for industrial
biotransformations: low
activity, poor stability in isolated form, and the necessity of cofactors in
oxidation. Although the structures of P450s are well characterized, it remains
very hard to improve their character through rational design in practice due to
the lack of an exact knowledge of the complex reaction mechanism.
Based on the mimic of natural
evolution in test tubes, directed evolution is a method to
overcome these drawbacks by getting beneficial functions of enzymes through the
accumulation of small mutations. Through
the iterative process of creating DNA libraries of random mutants, expression in
recombinant cells, and high throughput screening of suitable enzymes, we can
shorten the process of evolution to several months or even weeks, compared with
natural evolution for millions of generations.
Directed evolution makes it possible to obtain improved
P450 enzymes with higher activity, more tolerance to harsh environments, and
even adaptability to different substrates. The “evolved” P450 enzymes are
potentially attractive catalysts for the synthesis of fine chemicals,
pharmaceuticals, and for the remediation of waste materials.
Nanette Boyle nboyle@purdue.edu
Metabolomic analysis of Chlamydomonas reinhardtii.
Sean Werner srwerner@purdue.edu