(188ay) Optimization of PHA Production by Pseudomonas using 13C-Metabolic Flux Analysis

Polyhydroxyalkanoates (PHA) are biopolymers that are biodegradable and can be produced from renewable resources, therefore it is a sustainable substitute to conventional ploymeric materials. In order to achieve this goal economically, a microorganism platform with high yield of PHA production is needed. Pseudomonas sp. LFM046 is a promising candidate platform as a Medium-Chain-Length Polyhydroxyalkanoate producer, but its PHA production yield must be optimized in order to make the process competitive. In this context, ¹³C-Metabolic Flux Analysis (¹³C-MFA) can be used in order to understand the generation of precursors and cofactors. The aim of this work is to estimate the flux distribution in Pseudomonas sp. LFM046 applying ¹³C-MFA with data obtained from PHA mass isotopomer measurements. In order to achieve this objective, experiments with labeled substrate were performed and the mass isotopomers of PHA were measured by Gas Chromatography–Mass Spectrometry (GC-MS).

A program was developed in MATLAB platform that performs ¹³C-MFA simulations, applying metabolite and isotopomer balances, in order to estimate the fluxes. The metabolic network used here is originated from a draft genome sequence of Pseudomonas sp. LFM046. It contains Entner-Doudoroff pathway (ED), Pentose Phosphate Pathway (non-oxidative phase) and a pathway to PHA biosynthesis.

Initial results were obtained with experiments using 20% of [U-¹³C]glucose and 80% of [U-¹²C]glucose, the estimated fluxes indicate that the Pentose Phosphate Pathway is active even without the oxidative phase, and operate in equilibrium. It was not possible to estimate the flux in the ED cycle, since it was non identifiable. A Design Optimal Experiment (DOE) was performed based on the D-criterion to obtain the pattern of labeled substrate that allow the estimation of ED cycle flux with high precision. The DOE evaluated substrates with only one labeled carbon in each of the six atoms of glucose, and the results indicate that [6-¹³C]glucose leads to the highest D-criterion. Various mixtures of this substrate with [U-¹²C]glucose were simulated in order to reduce experimental cost, and the effect of the mixture on the accuracy of estimation was analyzed. The analysis indicates that a mixture of at least 50% of [6-¹³C]glucose should be used. These experiments are being performed and the results will be presented in the Conference.

ACKNOWLEDGEMENT: J. G. C. Gomez would like to express his acknowledgement to FAPESP for its financial support through grant number 2013/50357-2