Integrated analysis of gene expression and metabolic fluxes in PHA-producing Pseudomonas putida grown on glycerol

Microbial Cell Factories

Research

Ignacio Poblete-Castro¹  Christoph Wittmann²  Veronique Beckers²  Jürgen Tomasch³ 

[1] Center for Bioinformatics and Integrative Biology, Biosystems Engineering Laboratory, Faculty of Biological Sciences, Universidad Andrés Bello, 8340176, Santiago, Chile;Institute of Systems Biotechnology, Saarland University, Campus A1.5, 66123, Saarbrücken, Germany;Research Group Microbial Communication, Helmholtz Centre for Infection Research, Brunswick, Germany;

关键词: Pseudomonas putida;    Glycerol metabolism;    Transcriptome;    Metabolic flux analysis;    Flux balance analysis;    Elementary flux modes;    Polyhydroxyalkanoates;    Nitrogen and carbon limitation;   

DOI  :  10.1186/s12934-016-0470-2

 received in 2016-01-19, accepted in 2016-04-24,  发布年份 2016

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Beckers et al. 2016

【 图 表 】

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