Comparative transcriptomics and proteomics of p-hydroxybenzoate producing Pseudomonas putida S12: novel responses and implications for strain improvement

A transcriptomics and proteomics approach was employed to study the expression changes associated with p-hydroxybenzoate production by the engineered Pseudomonas putida strain S12palB1. To establish p-hydroxybenzoate production, phenylalanine-tyrosine ammonia lyase (pal/tal) was introduced to connect the tyrosine biosynthetic and p-coumarate degradation pathways. In agreement with the efficient p-hydroxybenzoate production, the tyrosine biosynthetic and p-coumarate catabolic pathways were upregulated. Also many transporters were differentially expressed, one of which—a previously uncharacterized multidrug efflux transporter with locus tags PP1271-PP1273—was found to be associated with p-hydroxybenzoate export. In addition to tyrosine biosynthesis, also tyrosine degradative pathways were upregulated. Eliminating the most prominent of these resulted in a 22% p-hydroxybenzoate yield improvement. Remarkably, the upregulation of genes contributing to p-hydroxybenzoate formation was much higher in glucose than in glycerol-cultured cells.

[1]  Eduardo Díaz,et al.  Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. , 2002, Environmental microbiology.

[2]  John E. Hallsworth,et al.  Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies , 2007, Applied Microbiology and Biotechnology.

[3]  M. Hynes,et al.  Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. , 1993, Gene.

[4]  K. Oh,et al.  Cellular Responses of Pseudomonas sp. DJ-12 to the Stresses of Several Aromatic Pollutants , 1998 .

[5]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[6]  N. Henderson,et al.  Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[7]  B. Okeke,et al.  Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358. , 1998, Microbiology.

[8]  J. D. de Bont,et al.  Chemostat-based proteomic analysis of toluene-affected Pseudomonas putida S12. , 2006, Environmental microbiology.

[9]  D. Roop,et al.  Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. , 1995, Gene.

[10]  M. J. van der Werf,et al.  Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase , 1990, Applied and environmental microbiology.

[11]  N. Sternberg,et al.  Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. , 1981, Journal of molecular biology.

[12]  J. Bont,et al.  The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose , 2005, Applied Microbiology and Biotechnology.

[13]  K. Oh,et al.  Adaptive and Cross-Protective Responses of Pseudomonas sp. DJ-12 to Several Aromatics and Other Stress Shocks , 2001, Current Microbiology.

[14]  N. Wierckx,et al.  Engineering of Solvent-Tolerant Pseudomonas putida S12 for Bioproduction of Phenol from Glucose , 2005, Applied and Environmental Microbiology.

[15]  M. Gasson,et al.  4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL)--An enzyme of phenylpropanoid chain cleavage from Pseudomonas. , 1999, Archives of biochemistry and biophysics.

[16]  M. Ashburner.  A Laboratory manual , 1989 .

[17]  A. Shevchenko,et al.  Fast-response proteomics by accelerated in-gel digestion of proteins. , 2003, Analytical chemistry.

[18]  H. Ballerstedt,et al.  TrgI, toluene repressed gene I, a novel gene involved in toluene-tolerance in Pseudomonas putida S12 , 2008, Extremophiles.

[19]  J. Hoheisel,et al.  Functional Genomics of Stress Response in Pseudomonas putida KT2440 , 2006, Journal of bacteriology.

[20]  A. Heck,et al.  ProteomIQ blue, a potent post-stain for the visualization and subsequent mass spectrometry based identification of fluorescent stained proteins on 2D-gels. , 2006, Journal of proteome research.

[21]  Johannes H. de Winde,et al.  Transcriptome Analysis of a Phenol-Producing Pseudomonas putida S 12 Construct : Genetic and Physiological Basis for Improved Production † , 2007 .

[22]  J. D. de Winde,et al.  Engineering Pseudomonas putida S12 for Efficient Utilization of d-Xylose and l-Arabinose , 2008, Applied and Environmental Microbiology.

[23]  Nick Wierckx,et al.  Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12: verification and complementation of hypotheses derived from transcriptomics. , 2009, Journal of biotechnology.

[24]  N. Wierckx,et al.  Bioproduction of p-Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium Pseudomonas putida S12 in a Two-Phase Water-Decanol Fermentation , 2008, Applied and Environmental Microbiology.

[25]  P. Barghini,et al.  Regulation of ferulic catabolic genes in Pseudomonas fluorescens BF13: involvement of a MarR family regulator , 2008, Applied Microbiology and Biotechnology.

[26]  T. V. Van Dyk,et al.  Characterization of the Escherichia coli AaeAB Efflux Pump: a Metabolic Relief Valve? , 2004, Journal of bacteriology.

[27]  H. Ballerstedt,et al.  Optimization of the solvent-tolerant Pseudomonas putida S12 as host for the production of p-coumarate from glucose , 2007, Applied Microbiology and Biotechnology.

[28]  H. Ruijssenaars,et al.  Bioproduction of p-hydroxybenzoate from renewable feedstock by solvent-tolerant Pseudomonas putida S12. , 2007, Journal of biotechnology.