Metabolic flux analysis of oleaginous algae
Abstract
Algae are used as a source for numerous products including food, cosmetics, and pharmaceuticals. Currently algae are being examined as a means of producing biofuels. In particular, oleaginous algae such as Chlorella protothecoides have received a large amount of scrutiny in an effort to maximize their lipid production for use in biodiesel. Within metabolic engineering – the improvement of cellular activities using recombinant DNA technology – it is critical to not only understand the effect of various carbon sources on growth, but also to understand what is occurring within cellular metabolism so specific genes or pathways can be targeted. There are multiple techniques to study metabolism, among them flux balance analysis and 13C metabolic flux analysis. However, because C. protothecoides is a eukaryotic alga, the compartmental separation of its metabolism is a complicating factor. To overcome this we adapted the technique of nonaqueous fractionation which was used for the first time in an alga. For a two compartment separation we selected the enzyme phospho enolpyruvate carboxylase as the cytosolic marker enzyme and the enzyme prephenate aminotransferase was used as a plastid marker enzyme. We successfully fractionated the homogenized algal extract along a nonaqueous density gradient, as shown by enzyme analyses specific for the subcellular compartments and the correlation of a large number of metabolites to these enzymatic markers as found in our metabolic profiling work. Metabolic studies on cultures of C. protothecoides grown on either glucose or glycerol show that glycerol is energetically a better substrate based on the additional reducing power generated by the conversion of glycerol-3-phosphate into dihydroxyacetone phosphate. This impacts metabolic fluxes in multiple different ways. In flux balance analysis the difference between the two substrates is apparent based on the metabolic fluxes predicted in the TCA cycle, with a branched TCA cycle predicted to be active during growth on glycerol. Uncompartmented metabolic flux analysis shows that the reduction in reducing power generation during growth on glycerol is actually by the oxidative pentose phosphate pathway rather than the TCA cycle. Comparing our metabolic flux analysis results to that predicted by flux balance analysis there appears to be significant room for improvement during the exponential growth phase. The biomass yield for glycerol with respect to the enthalpy of combustion of its substrate is only 31.3 mg/kJ, while for glucose it is 33.2 mg/kJ, indicating that cultures grown on glucose are more efficient with respect to the total available enthalpy of the substrate. This also implies that there remains room for further improvement of C. protothecoides growth on glycerol.
Degree
Ph.D.
Advisors
Morgan, Purdue University.
Subject Area
Molecular biology|Chemical engineering
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