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

Probing the proteome response to toluene exposure in Pseudomonas putida

The toxic effects of organic solvents are a major drawback for their application in biotechnology and for the production of fine chemicals by whole-cell bioconversion and biotransformation. However, through the isolation of some microbial strains, like P. putida S12, which tolerate high concentrations of organic solvents, several limitations of organic solvents in biotechnology can be overcome. Although P. putida S12 has been studied for a long time with different functional genomics techniques, the molecular mechanisms that underlie the principles of solvent tolerance are not fully understood. The objective of this Ph.D. study was to identify and to characterize the proteins of P. putida S12 and P. putida KT2440 involved in tolerance towards toluene. The characterization of the P. putida proteome upon exposure to the organic solvent toluene revealed a well coordinated response, with an interplay between decreased permeability of the cell envelope and increased efflux pump activity

Bond slip behaviour of deep mounted carbon fibre reinforced polymer strops confined with a ductile adhesive in clay brick masonry

Retrofitting clay brick masonry using Deep Mounted (DM) Carbon Fibre Reinforced Polymer (CFRP) strips embedded in grooves filled with a ductile adhesive considerably increases the out-of-plane flexural capacity of slender unreinforced masonry walls. In order to investigate the bond-slip behaviour of CFRP-strips in a viscous-elastic adhesive, an extensive experimental program was initiated. Direct pull-out tests were conducted with clay brick masonry. Two parameters were investigated, namely, the type of adhesive (2 types) and the groove widths (10 and 15 mm). The second part of the experimental program focused on the pull-out capacity when surface treatment (primering or sandblasting) was applied to the CFRP-strips. In literature dealing with bond behaviour the critical bond length was found using masonry prisms of approximately 350 mm in height. In the current study with CFRP strip application in conjunction with ductile epoxy, the critical bond length was not reached for specimens of nearly 1000 mm in length. This finding was an indication of a significantly improved stress distribution over the length of the embedded CFRP-strip as the appearance of peak stresses was prevented. No premature brick splitting was initiated despite the depth of the groove being 65% of the specimen thickness, whereas in literature this phenomenon was reported for groove depths of only 30%

pH-stat fed-batch process to enhance the production of cis, cis-muconate from benzoate by Pseudomonas putida KT2440-JD1

Pseudomonas putida KT2440-JD1 is able to cometabolize benzoate to cis, cis-muconate in the presence of glucose as growth substrate. P. putida KT2440-JD1 was unable to grow in the presence of concentrations above 50 mM benzoate or 600 mM cis, cis-muconate. The inhibitory effects of both compounds were cumulative. The maximum specific uptake rate of benzoate was higher than the specific production rate of cis, cis-muconate during growth on glucose in the presence of benzoate, indicating that a benzoate derivative accumulated in the cells, which is likely to be catechol. Catechol was shown to reduce the expression level of the ben operon, which encodes the conversion of benzoate to cis, cis-muconate. To prevent overdoses of benzoate, a pH-stat fed-batch process for the production of cis, cis-muconate from benzoate was developed, in which the addition of benzoate was coupled to the acidification of the medium. The maximum specific production rate during the pH-stat fed-batch process was 0.6 g (4.3 mmol) g dry cell weight(-1) h(-1), whereas 18.5 g L(-1) cis, cis-muconate accumulated in the culture medium with a molar product yield of close to 100%. Proteome analysis revealed that the outer membrane protein H1 was upregulated during the pH-stat fed-batch process, whereas the expression of 10 other proteins was reduced. The identified proteins are involved in energy household, transport, translation of RNA, and motilit

Generation of a catR deficient mutant of P. putida KT2440 that produces cis, cis-muconate from benzoate at high rate and yield

Pseudomonas putida KT2440-JD1 was derived from P. putida KT2440 after N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-mutagenesis and exposure to 3-fluorobenzoate (3-FB). The mutant was no longer able to grow using benzoate as a sole carbon source, but co-metabolized benzoate to cis, cis-muconate during growth on glucose, which accumulated in the growth medium. The specific production rate (qpm) was 0.18 ± 0.03 g cis, cis-muconate/(gDCW h) in continuous cultures, and increased to 1.4 g cis, cis-muconate/(gDCW h) during wash-out cultivation. Transcriptome analysis showed that the cat operon was not induced in P. putida KT2440-JD1 in the presence of 5 mM benzoate, due to a point mutation in the highly conserved DNA binding domain of the transcriptional regulator (catR) of the cat operon. The ben operon was highly expressed in the presence of benzoate in the mutant and its parental strain. This operon contains PP_3166 (catA2), which was shown to be a second catechol 1,2-dioxygenase besides catA. P. putida KT2440-JD1 is the first cis, cis-muconate-accumulating mutant that was characterized at the genetic level. The specific production rate achieved is at least eight times higher than those reported for other cis, cis-muconate-producing strain