Amino Acid Racemization in Pseudomonas putida KT2440

d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k_cat/K_m values with l- and d-lysine were 3 orders of magnitude greater than the k_cat/K_m values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k_cat/K_m values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.     

Improved transformation of Pseudomonas putida KT2440 by electroporation

Summary An electric field-mediated transformation (i.e. electroporation) was performed to determine optimal conditions for P. putida transformation. The effects of culture age, electroporation buffer composition, electric field strength, pulse time constant and DNA concentration on transformation efficiency were examined. When plasmid DNA of 8 to 11 kb in size was used with an electroporation buffer containing 1 mM HEPES (pH 7.0), maximum transformation efficiency of 1.0 × 10⁷ transformants/g DNA was obtained at field strength of 12 kV/cm with pulse time of 2.5 millisecond. A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude. A linear relationship was observed between growth phase and transformation efficiency up to OD₆₀₀ = 2.0. This reliable and simple method should be useful for introduction of plasmid DNA into intact P. putida cells.     

Acetone extraction of mcl-PHA from Pseudomonas putida KT2440

A methodology was developed for the extraction of medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) from Pseudomonas putida. It was determined that if dry P. putida biomass containing mcl-PHA was washed in 20 volumes of methanol for 5 min followed by Soxhlet extraction in 10 volumes of acetone for 5 h, almost all of the PHA could be recovered with no detectable loss of molecular weight. Biomass containing higher amounts of PHA required less methanol during the pretreatment step but more acetone in the solvent extraction step than biomass containing less PHA. Further purification could be achieved by redissolving the PHA in acetone and reprecipitating in cold methanol. UV spectroscopy at 241 and 275 nm could be used as an indication of product purity.     

Biochemical characterization of ferredoxin-NADP(+) reductase interaction with flavodoxin in Pseudomonas putida

Flavodoxin (Fld) has been demonstrated to bind to ferredoxin- NADP(+) reductase A (FprA) in Pseudomonas putida. Two residues (Phe(256), Lys(259)) of FprA are likely to be important for interacting with Fld based on homology modeling. Sitedirected mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-Ala(259) and FprA-Asp(259) proteins were two-fold lower than those of the wild-type FprA. Homology modeling also strongly suggested that these two residues are important for electron transfer. Thermodynamic properties such as entropy, enthalpy, and heat capacity changes of FprA-Ala(259) and FprA-Asp(259) were examined by isothermal titration calorimetry. We demonstrated, for the first time, that Phe(256) and Lys(259) are critical residues for the interaction between FprA and Fld. Van der Waals interactions and hydrogen bonding were also more important than ionic interactions for forming the FprA-Fld complex. [BMB Reports 2012; 45(8): 476-481].     

Engineering Pseudomonas putida KT2440 for the production of isobutanol

We engineered P. putida for the production of isobutanol from glucose by preventing product and precursor degradation, inactivation of the soluble transhydrogenase SthA, overexpression of the native ilvC and ilvD genes, and implementation of the feedback‐resistant acetolactate synthase AlsS from Bacillus subtilis, ketoacid decarboxylase KivD from Lactococcus lactis, and aldehyde dehydrogenase YqhD from Escherichia coli. The resulting strain P. putida Iso2 produced isobutanol with a substrate specific product yield (Y_Iso/S) of 22 ± 2 mg per gram of glucose under aerobic conditions. Furthermore, we identified the ketoacid decarboxylase from Carnobacterium maltaromaticum to be a suitable alternative for isobutanol production, since replacement of kivD from L. lactis in P. putida Iso2 by the variant from C. maltaromaticum yielded an identical Y_Iso/S. Although P. putida is regarded as obligate aerobic, we show that under oxygen deprivation conditions this bacterium does not grow, remains metabolically active, and that engineered producer strains secreted isobutanol also under the non‐growing conditions.     

Global features of the Pseudomonas putida KT2440 genome sequence

The compositional bias of the G+C, di- and tetranucleotide contents in the 6 181 862 bp Pseudomonas putida KT2440 genome was analysed in sliding windows of 4000 bp in steps of 1000 bp. The genome has a low GC skew (mean 0.066) between the leading and lagging strand. The values of GC contents (mean 61.6%) and of dinucleotide relative abundance exhibit skewed Gaussian distributions. The variance of tetranucleotide frequencies, which increases linearly with increasing GC content, shows two overlapping Gaussian distributions of genome sections with low (minor fraction) or high variance (major fraction). Eighty per cent of the chromosome shares similar GC contents and oligonucleotide bias, but 105 islands of 4000 bp or more show atypical GC contents and/or oligonucleotide signature. Almost all islands provide added value to the metabolic proficiency of P. putida as a saprophytic omnivore. Major features are the uptake and degradation of organic chemicals, ion transport and the synthesis and secretion of secondary metabolites. Other islands endow P. putida with determinants of resistance and defenceor with constituents and appendages of the cell wall. A total of 29 islands carry the signature of mobile elements such as phage, transposons, insertion sequence (IS) elements and group II introns, indicating recent acquisition by horizontal gene transfer. The largest gene carries the most unusual sequence that encodes a multirepeat threonine-rich surface adhesion protein. Among the housekeeping genes, only genes of the translational apparatus were located in segments with an atypical signature, suggesting that the synthesis of ribosomal proteins is uncoupled from the rapidly changing translational demands of the cell by the separate utilization of tRNA pools.     

Tolerance of plastic-encapsulated Pseudomonas putida KT2440 to chemical stress

Pseudomonas putida dried in the presence of hydroxyectoine or trehalose can withstand exposure to organic solvents and therefore can be encapsulated inside plastics such as polystyrene. Here we show that P. putida in a plastic-encapsulated dried tablet exhibits remarkable tolerance to chemical stress, comparable to that of spores of Bacillus subtilis.     

Improving the autotransporter-based surface display of enzymes in Pseudomonas putida KT2440

Pseudomonas putida can be used as a host for the autotransporter-mediated surface display of enzymes (autodisplay), resulting in whole-cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter-mediated secretion depends on the N-terminal signal peptide as well as on the C-terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole-cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12-fold enhanced to 638 mU ml⁻¹ OD⁻¹ compared with the signal peptide of V. cholerae CtxB (52 mU ml⁻¹ OD⁻¹). This positive effect was confirmed with a β-glucosidase as a second example enzyme. Here, cells expressing the protein with N-terminal OprF signal peptide showed more than fourfold higher β-glucosidase activity (181 mU ml⁻¹ OD⁻¹) than with the CtxB signal peptide (42 mU ml⁻¹ OD⁻¹). SDS-PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.     

Regulation of the cyclopropane synthase cfaB gene in Pseudomonas putida KT2440

In Pseudomonas putida, as in many other eubacteria, cyclopropane fatty acids (CFAs) accumulate in the membrane during the stationary phase of growth. Here, we show that cfaB gene expression in P. putida KT2440 is dependent on the RpoS sigma factor that recognizes the sequence 5'-CTACTCT-3' between -8 and -14. We have carried out a mutational study of the cfa promoter and have determined that positions -9, -12, -13 and -14 are the most critical for maximal activity. In P. putida, the substrates of the CFA synthase, cis-unsaturated fatty acids (cis-UFAs), are also substrates for another stress-related enzyme, the cis-trans isomerase (CTI). Despite using the same substrates, we have found that the activity of the CTI is not limited by the CFA synthase activity and vice versa. For instance, in a cfaB knockout mutant, the amount of trans-UFAs synthesized after a specific stress was no higher than in the parental background despite the fact that there are more cis-UFAs available to be used by the CTI as substrates. In this regard, in a cti-deficient mutant background, the levels of CFAs were similar to those in the parental one under the same conditions.