Development of genetic tools for heterologous protein expression in a pentose-utilizing environmental isolate of Pseudomonas putida

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.     

Factors affecting antagonism of the growth of Phanerochaete chrysosporium by bacteria isolated from soils

Bacteria from polluted and agricultural soils antagonize the growth of Phanerochaete chrysosporium on solid media. The antagonistic bacteria in a soil contaminated with trinitrotoluene included fluorescent pseudomonads. Antagonism by fluorescent pseudomonads was variable according to the pH, and carbon and nitrogen sources used in the growth medium. A fluorescent siderophore produced by a Pseudomonas putida strain did not inhibit the growth of Phanerochaete chrysosporium but pseudomonad isolates capable of producing phenazine derivatives were strongly inhibitory.Utah Agricultural Experiment Station paper no. 4416     

Pyrimidine base catabolism in Pseudomonas putida biotype B

Reductive catabolism of the pyrimidine bases uracil and thymine was found to occur in Pseudomonas putida biotype B. The pyrimidine reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl--alanine amidohydrolase activities were detected in this pseudomonad. The initial reductive pathway enzyme dihydropyrimidine dehydrogenase utilized NADH or NADPH as its nicotinamide cofactor. The source of nitrogen in the culture medium influenced the reductive pathway enzyme activities and, in particular, dihydropyrimidinase activity was highly affected by nitrogen source. The reductive pathway enzyme activities in succinate-grown P. putida biotype B cells were induced when uracil served as the nitrogen source.     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida

We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.     

Substrate-dependent autoaggregation of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1 during the degradation of mono-chlorophenols and phenol

A bacterium, CP1, identified as Pseudomonas putida strain, was investigated for its ability to grow on and degrade mono-chlorophenols and phenols as sole carbon sources in aerobic shaking batch culture. The organism degraded up to 1.56 mM 2- and 3-chlorophenol, 2.34 mM 4-chlorophenol and 8.5 mM phenol using an ortho-cleavage pathway. P. putida CP1, acclimated to degrade 2-chlorophenol, was capable of 3-chlorocatechol degradation, while P. putida, acclimated to 4-chlorophenol degradation, degraded 4-chlorocatechol. Growth of P. putida CP1 on higher concentrations of the mono-chlorophenols, ≥1.56 mM 4-chlorophenol and ≥0.78 mM 2- and 3-chlorophenol, resulted in decreases in cell biomass despite metabolism of the substrates, and the formation of large aggregates of cells in the culture medium. Increases in cell biomass with no clumping of the cells resulted from growth of P. putida CP1 on phenol or on lower concentrations of mono-chlorophenol. Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of mono-chlorophenol. The results suggested that increased hydrophobicity and autoaggregation of P. putida CP1 were a response to toxicity of the added substrates. Journal of Industrial Microbiology & Biotechnology (2002) 28, 316–324 DOI: 10.1038/sj/jim/7000249 Received 27 June 2001/ Accepted in revised form 09 February 2002     

Peroxyl Radical Scavenging Activity of Caffeic Acid and Its Related Phenolic Compounds in Solution

The esterase gene (est) of Pseudomonas putida MR-2068 was cloned into Escherichia coli JM109. An 8-kb inserted DNA directed synthesis of an esterase in E. coli. The esterase gene was in a 1.1-kb PstI-ClaI fragment within the insert DNA. The complete nucleotides of the DNA fragment containing the esterase gene were sequenced and found to include a single open reading frame of 828 bp coding for a protein of 276 amino acid residues. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the purified esterase. A potential Shine-Dalgarno sequence is followed by the open reading frame. The esterase activity of the recombinant E. coli was more than 200 times higher than that of parental strain, P. putida MR-2068.     

Measurement of the Growth of a Floc Forming Bacterium <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1

Pseudomonas putida CP1 formed clumps of cells when grown on mono-chlorophenols but not on phenol or glucose. An increase in cell numbers for the organism grown on mono-chlorophenols was accompanied by a decrease in the dry weight. The change in shape of the bacterium from rod shape to coccus shape coupled with a reduction in cell size when the organism was grown under nutritional stress was found. This result together with cell aggregation affected the measurement of growth parameters in the system by conventional methods (optical density measurements, dry weight measurements and the plate count technique). Monitoring growth of Pseudomonas putida CP1 by a direct microscopic count technique was found to be more representative than conventional methods including optical density measurements, dry weight measurements and the plate count technique when grown on phenolics.     

Engineering alternative butanol production platforms in heterologous bacteria

Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.     

Characterization of cis-4-hydroxy-D-proline dehydrogenase from Sinorhizobium meliloti

The hypO gene from Sinorhizobium meliloti, located within the trans-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host Pseudomonas putida. Purified HypO protein functioned as a FAD-containing cis-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.     

Pseudomonas syringae pv. helianthi (Kawamura 1934) Dye, Wilkie et Young 1978, a Pathogen of Sunflower (Helianthus annuus L.)

Among the bacterial strains isolated from diseased sunflower leaves, eight were studied in some detail. A fluorescent pseudomonad isolated from necrotic tissues and its reisolates belong to group Ia of phytopathogenic pseudomonads which includes Pseudomonas syringae bacterium. A study of host range indicated that the pathogen infects only sunflower but not the other plant species. Based on the pathogenicity study and biochemical and physiological tests, it was concluded that the pathogen belongs to the bacterium Pseudomonas syringae pv. helianthi.     

Pseudomonas syringae pv. helianthi (Kawamura 1934) Dye,Wilkie et Young 1978, a Pathogen of Sunflower (Helianthus annuus L.)

Among the bacterial strains isolated from diseased sunflower leaves, eight were studied in some detail. A fluorescent pseudomonad isolated from necrotic tissues and its reisolates belong to group Ia of phytopathogenic pseudomonads which includes Pseudomonas syringae bacterium. A study of host range indicated that the pathogen infects only sunflower but not the other plant species. Based on the pathogenicity study and biochemical and physiological tests, it was concluded that the pathogen belongs to the bacterium Pseudomonas syringae pv. helianthi.     

Production of polyclonal antibodies to a recombinant coat protein of potato virus Y

The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Ω. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.     

The Influence of Glucose and Fructose on the Degradation of 2-Chlorophenol by Pseudomonas putida CP1

Summary Pseudomonas putida CP1 grew on 2-chlorophenol when supplied as the sole source of carbon. Chlorophenol degradation was stimulated in the presence of low concentrations of glucose (0.05–1%, w/v). Substrate removal was inhibited and there was a significant fall in pH with concentrations of glucose greater than 1.0% (w/v). When the pH was controlled at pH 7.0 inhibition of substrate removal was alleviated. The rate of removal of 2-chlorophenol was greater in the presence of fructose than in the presence of glucose. P. putida CP1 formed clumps of cells when grown on 2-chlorophenol and fructose but not on glucose. When the organism was grown on a combination of 2-chlorophenol and an additional carbon source clumping was present but to a lesser degree.     

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.     

Catechol 2,3-Dioxygenase from Pseudomonas sp. Strain ND6: Gene Sequence and Enzyme Characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5α using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

Revealing oxidative pentose metabolism in new Pseudomonas putida isolates

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.     

The Ssr protein (T1E_1405) from Pseudomonas putida DOT‐T1E enables oligonucleotide‐based recombineering in platform strain P. putida EM42

Some strains of the soil bacterium Pseudomonas putida have become in recent years platforms of choice for hosting biotransformations of industrial interest. Despite availability of many genetic tools for this microorganism, genomic editing of the cell factory P. putida EM42 (a derivative of reference strain KT2440) is still a time‐consuming endeavor. In this work we have investigated the in vivo activity of the Ssr protein encoded by the open reading frame T1E_1405 from Pseudomonas putida DOT‐T1E, a plausible functional homologue of the β protein of the Red recombination system of λ phage of Escherichia coli. A test based on the phenotypes of pyrF mutants of P. putida (the yeast's URA3 ortholog) was developed for quantifying the ability of Ssr to promote invasion of the genomic DNA replication fork by synthetic oligonucleotides. The efficiency of the process was measured by monitoring the inheritance of the changes entered into pyrF by oligonucleotides bearing mutated sequences. Ssr fostered short and long genomic deletions/insertions at considerable frequencies as well as single‐base swaps not affected by mismatch repair. These results not only demonstrate the feasibility of recombineering in P. putida, but they also enable a suite of multiplexed genomic manipulations in this biotechnologically important bacterium.     

Observations on the Effect of Inoculating Cassava (Manihot esculenta) Plantlets with Fluorescent Pseudomonads

Some 136 isolates of fluorescent pseudomonads were isolated from the rhizosphere of plants growing in 5 different ecosystems. Thirty-four percent of these isolates inhibited the causal agent of cassava stem rot, Erwinia carotovora pv. carotovora, in vitro. One month old plantlets, produced by rooting the shoots of 4 cultivars in distilled water, were inoculated with a suspension (1.1 × 10⁹ cells/ml) of each pseudomonad. Some isolates increased root weight by 95% over uninoculated controls two months after planting when inoculated at planting, and 15, and 30 days afterwards. Inoculated plants were free from symptoms of root pathogens and roots filled earlier than controls. Taxonomic studies showed that these bacterial isolates, were either Pseudomonas putida (90%) or P. fluorescens (10%).