Abstract
BackgroundGiven its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. The soil bacterium Pseudomonas putida KT2440 can use glycerol to synthesize medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA), a class of biopolymers of industrial interest. Here, glycerol metabolism in P. putida KT2440 was studied on the level of gene expression (transcriptome) and metabolic fluxes (fluxome), using precisely adjusted chemostat cultures, growth kinetics and stoichiometry, to gain a systematic understanding of the underlying metabolic and regulatory network.ResultsGlycerol-grown P. putida KT2440 has a maintenance energy requirement [0.039 (mmolglycerol (gCDW h)−1)] that is about sixteen times lower than that of other bacteria, such as Escherichia coli, which provides a great advantage to use this substrate commercially. The shift from carbon (glycerol) to nitrogen (ammonium) limitation drives the modulation of specific genes involved in glycerol metabolism, transport electron chain, sensors to assess the energy level of the cell, and PHA synthesis, as well as changes in flux distribution to increase the precursor availability for PHA synthesis (Entner–Doudoroff pathway and pyruvate metabolism) and to reduce respiration (glyoxylate shunt). Under PHA-producing conditions (N-limitation), a higher PHA yield was achieved at low dilution rate (29.7 wt% of CDW) as compared to a high rate (12.8 wt% of CDW). By-product formation (succinate, malate) was specifically modulated under these regimes. On top of experimental data, elementary flux mode analysis revealed the metabolic potential of P. putida KT2440 to synthesize PHA and identified metabolic engineering targets towards improved production performance on glycerol.ConclusionThis study revealed the complex interplay of gene expression levels and metabolic fluxes under PHA- and non-PHA producing conditions using the attractive raw material glycerol as carbon substrate. This knowledge will form the basis for the development of future metabolically engineered hyper-PHA-producing strains derived from the versatile bacterium P. putida KT2440.
Highlights
Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals
We have shown that P. putida KT2440 is most suitable to synthesize PHA from raw glycerol among different P. putida strains due to reduced by-product formation under PHA-producing conditions [21]
As P. putida does not have a complete EMP pathway [61], we have previously proven that overexpression of genes of the PP pathway does not improve PHA synthesis in P. putida KT2440, when grown on glucose [15]
Summary
Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. When grown in batch culture on glycerol, the gene glpR (PP_1074) controls utilization of the substrate Inactivation of this regulator leads to increased synthesis of mcl-PHA in P. putida KT2440 [24]. To better understand the mechanisms in P. putida for the production of biopolymers from glycerol, it appears straightforward to further quantify and integrate metabolic function and regulation as well as physiological parameters in a systematic fashion Such systems biological approaches have proven valuable to understand cellular physiology [25,26,27] and enable metabolic engineering approaches, with the purpose of enhancing the synthesis of target chemicals in a rational manner [28,29,30]. We explored the effect of specific growth regimes on global gene expression and carbon flux distribution of pathways of P. putida
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