- Oct 7, 2019
Date & Location: October 7, 2019, at 4p; Room 1200 Molecular Plant Sciences Building
Subject: Stable isotope approaches to assess photosynthetic metabolism of algae and plants
Host: Berkley Walker
About the Speaker
University: Vanderbilt University
Abstract: Engineering cellular metabolism to promote high yield and productivity of target compounds is a major goal of the biotech industry. 13C metabolic flux analysis (MFA) provides a rigorous approach to quantify host metabolic phenotypes by applying isotope tracers to map the flow of carbon through intracellular biochemical pathways. In particular, dynamic measurements of isotope incorporation following a step change from unlabeled to labeled CO2 can be used to estimate photosynthetic carbon fluxes by applying isotopically nonstationary MFA (INST-MFA).
We have previously developed a package of MATLAB routines called INCA that automates the computational workflow of INST-MFA. INCA was the first publicly available software package capable of applying INST-MFA to biologically relevant metabolic networks. We have recently applied INCA to model the photoautotrophic metabolism of Synechococcus 7942 strains that have been engineered to produce the industrial biochemical isobutyraldehyde (IBA). The flux analysis identified a potential bottleneck at the pyruvate kinase (PK) reaction and indicated significant flux through an alternative three-step route from PEP to pyruvate involving PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Based on these results, we overexpressed PK or combinations of PEPC, MDH, and/or ME to engineer strains with significant improvements in IBA production. 13C flux analysis of the resulting strains identified further targets for debottlenecking IBA production in Synechococcus 7942, some of which are currently under investigation. We have also adapted our INST-MFA model to a terrestrial plant system. We performed in vivo isotopic labeling of Arabidopsis thaliana leaves with 13CO2, measured the transient labeling of 37 metabolite fragment ions using mass spectrometry, and estimated fluxes throughout leaf photosynthetic metabolism using INCA. Leaves were acclimated to either 200 (LL) or 500 (HL) μmol/m2/s light intensity. Approximately 1,400 independent mass isotopomer measurements were regressed to estimate 136 fluxes under each condition. Despite a doubling in the carboxylation rate, the photorespiratory flux increased from 17% to 28% of net CO2 assimilation with HL acclimation. Interestingly, the concentrations of multiple Calvin cycle intermediates were reduced during acclimation, indicating an inverse relationship between intermediate pool sizes and fluxes. These studies have established 13C INST-MFA and the INCA software package as a comprehensive platform to map carbon fluxes in algae, plants, and other photosynthetic organisms.