Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol

In the Klebsiella pneumoniae reduction pathway for 1,3-propanediol (1,3-PD) synthesis, glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and then reduced to 1,3-PD with NADH consumption. Rapid conversion of 3-HPA to 1,3-PD is one of the ways to improve the yield of 1,3-PD from glycerol and to avoid 3-HPA accumulation, which depends on enzyme activity of the reaction and the amount of reducing equivalents available from the oxidative pathway of glycerol. In the present study, the yqhD gene, encoding 3-propanediol oxidoreductase isoenzyme from Escherichia coli and the dhaT gene, encoding 3-propanediol oxidoreductase from K. pneumoniae were expressed individually and co-expressed in K. pneumoniae using the double tac promoter expression plasmid pEtac-dhaT-tac-yqhD. The three resultant recombinant strains (K. pneumoniae/pEtac-yqhD, K. pneumoniae/pEtac-dhaT, and K. pneumoniae/pEtac-dhaT-tac-yqhD) were used for fermentation studies. Experimental results showed that the peak values for 3-HPA production in broth of the three recombinant strains were less than 25% of that of the parent strain. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth as NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased by-products and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-propanediol using two cofactors (NADH and NADPH). These results have important implications for further studies involving use of YqhD and DhaT for bioconversion of glycerol into 1,3-PD.     

Biotransformation of anisole and phenetole by aerobic hydrocarbonoxidizing bacteria

Wild type, mutant, and recombinant bacterial strains capable of oxidizing aromatic hydrocarbons were screened for their ability to oxidize anisole (methoxybenzene) and phenetole (ethoxybenzene). Toluene-induced cells ofPseudomonas putida F39/D transformed anisole to a compound tentatively identified ascis-1,2-dihydroxy-3-methoxyclohexa-3,5-diene (anisole-2,3-dihydrodiol), 2-methoxyphenol, catechol, and trace amounts of phenol while phenetole was converted primarily tocis-1,2-dihydroxy-3-ethoxycyclohexa-3,5-diene (phenetole-2,3-dihydrodiol) and 2-ethoxyphenol. Induced cells ofPseudomonas sp. NCIB 9816/11 andBeijerinckia sp. B8/36 transformed anisole to phenol, and phenetole to phenol and ethenyloxybenzene. Toluene-induced cells ofP. putida BG1 converted anisole to phenol but did not oxidize phenetole. In contrast, toluene-induced cells ofP. mendocina KR1, which oxidize toluene via monooxygenation at thepara position, transformed anisole to 4-methoxyphenol, and phenetole to 2-, 3- and 4-ethoxyphenol. The involvement of toluene and naphthalene dioxygenases in the reactions catalyzed by strains F39/D and NCIB 9816/11, respectively, was confirmed with recombinantE. coli strains expressing the cloned dioxygenase genes. The results show that the oxygenases from differentPseudomonas strains oxidize anisole and phenetole to different hydroxylated products.     

Microbial production of <Emphasis Type="Italic">cis</Emphasis>-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate by genetically modified <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L⁻¹ h⁻¹. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.     

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.     

Enhancement ofcis,cis-muconate productivity by overexpression of catechol 1,2-dioxygenase inPseudomonas putida BCM114

For enhancement ofcis,cis-muconate productivity from benzoate, catechol 1,2-dioxygenase (C12O) which catalyzes the rate-limiting step (catechol conversion tocis,cis-muconate) was cloned and expressed in recombinantPseudomonas putida BCM114. At higher benzoate concentrations (more than 15 mM),cis,cis-muconate productivity gradually decreased and unconverted catechol was accumulated up to 10 mM in the case of wildtypeP. putida BM014, whereascis,cis-muconate productivity continuously increased and catechol was completely transformed tocis,cis-muconate forP. putida BCM114. Specific C12O activity ofP. putida BCM114 was about three times higher than that ofP. putida BM014, and productivity was enhanced more than two times.     

Synthesis of trans unsaturated fatty acids in Pseudomonas putida P8 by direct isomerization of the double bond of lipids

The phospholipids of Pseudomonas putida P8 contain monounsaturated fatty acids in the cis and trans configuration. Cells of this phenol-degrading bacterium change the proportions of these isomers in response to the addition or elimination of a membrane active compound such as 4-chlorophenol. This study undoubtedly reveals that the cis unsaturated fatty acids are directly converted into trans isomers without involvement of de novo synthesis of fatty acids. Oleic acid, which cannot be synthesized by this bacterium, was incorporated as a cis unsaturated fatty acid marker in the membrane lipids of growing cells. The conversion of this fatty acid into the corresponding trans isomer was demonstrated by gas chromatographic-mass spectrometric analysis and use of ¹⁴C-labeled oleic acid. Separation and isolation of the cellular membranes showed that the fatty acid isomerase is located in the cytoplasmic membrane of P. putida P8.Abbreviation 4-CP 4-chlorophenol     

The response of Pseudomonas putida CP1 cells to nutritional, chemical and environmental stresses

Summary The response of a pollutant-degrading bacterium P. putida CP1 to stresses was investigated. The growth on the mono-chlorophenols resulted in a decrease in dry weight of the organism, although there was an increase in cell number. There was a change of bacterial shape from rod to round as well as the reduction of cell size when grown on phenol and chlorophenols. Changes in cell shape and size were also evident in glucose-free medium, which suggested that alteration of cell shape from rod to round as well as reduction of cell size were due to nutritional stress. The increase in cell number but a drop in dry weight correlated with the reduction of cell size and shape. The organism flocculated with chlorophenols but not with phenol. The cause of flocculation was due to the toxicity of chlorophenol. Isomerization of cis to trans forms of the unsaturated fatty acids in P. putida CP1 occurred under conditions of environmental stress. Trace amounts of the polyunsaturated fatty acid linoleic acid (cis-9, cis-12-octadecadienoic acid) rarely found in bacterial membranes and oleic acid (cis-9-octadecanoic acid), which is a typical product of aerobic fatty acid synthesis, were found in P. putida CP1.     

Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes

The chemotactic responses of Pseudomonas putida F1, Burkholderia cepacia G4, and Pseudomonas stutzeri OX1 were investigated toward toluene, trichloroethylene (TCE), tetrachloroethylene (PCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC). P. stutzeri OX1 and P. putida F1 were chemotactic toward toluene, PCE, TCE, all DCEs, and VC. B. cepacia G4 was chemotactic toward toluene, PCE, TCE, cis-DCE, 1,1-DCE, and VC. Chemotaxis of P. stutzeri OX1 grown on o-xylene vapors was much stronger than when grown on o-cresol vapors toward some chlorinated ethenes. Expression of toluene-o-xylene monooxygenase (ToMO) from touABCDEF appears to be required for positive chemotaxis attraction, and the attraction is stronger with the touR (ToMO regulatory) gene.     

The Production of Catechols from Benzene and Toluene by Pseudomonas Putida in Glucose Fed-Batch Culture

Catechol and 3-methylcatechol were produced from benzene and toluene respectively using different mutants of Pseudomonas putida. P. putida 2313 lacked the extradiol cleavage enzyme, catechol 2,3-oxygenase, allowing overproduction of 3-methylcatechol from toluene to a level of 11.5 mM (1.27 g·1^-1) in glucose fed-batch culture. P. putida 6(12), a mutant of P. putida 2313, lacked both catechol-oxygenase and catechol 1,2-oxygenase, and accumulated catechol from benzene to a level of 27.5mM(3g·1^-1).

In both biotransformations product formation ceased within 10 hours of feeding the aromatic substrate, and this was due to product inhibition by the catechols. The primary site of catechol toxicity was inhibition of the aromatic dioxygenase. Neither cis-toluene dihydrodiol cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene), nor cis-benzene dihydrodiol (cis-l,2-dihydroxy-3-methylcyclohexa-3,5-diene) dehydrogenase was significantly inhibited by catechol overproduction whereas both ring activating dioxygenases were inhibited within 4-6 hours of the maximum product concentration being attained.

3-Methylcatechol overproduction from toluene was also studied using a continuous product removal system. Granular activated charcoal removed 3-methylcatechol efficiently and was easily regenerated by washing with ethyl acetate. Using P. putida 2313, it was shown that the final product concentration increased approximately fourfold. Additional products were formed and the significance of these are discussed.     

Synthesis of dihydroxynaphthalene isomers by microbial oxidation of 1- and 2-naphthol

The mutant strain Pseudomonas fluorescens TTC1 (NCIMB 40605), derived from the naphthalene-degrading Pseudomonas fluorescens N3 (NCIMB 40530), was used for the oxidation of 1- and 2-naphthols to give different isomers of dihydroxynaphthalene. The oxidation reactions proceed through the formation of dihydrodiol intermediates, which are too unstable to be isolated, since they spontaneously eliminate water to give the fully aromatic dihydroxynaphthalenes. The high regioselectivity of the dehydration reaction was confirmed by the study of the acid-catalysed aromatization of a series of stable monosubstituted naphthalene cis-1,2-dihydrodiols. Received: 24 March 1997 / Received revision: 6 June 1997 / Accepted: 7 June 1997     

3-Hydroxypropionaldehyde: applications and perspectives of biotechnological production

3-Hydroxypropionaldehyde (3-HPA) forms, together with HPA-hydrate and HPA-dimer, a dynamic, multi-component system (HPA system) used in food preservation, as a precursor for many modern chemicals such as acrolein, acrylic acid, and 1,3-propanediol (1,3-PDO), and for polymer production. 3-HPA can be obtained both through traditional chemistry and bacterial fermentation. To date, 3-HPA has been produced from petrochemical resources as an intermediate in 1,3-PDO production. In vivo, glycerol is converted in one enzymatic step into 3-HPA. The 3-HPA-producing Lactobacillus reuteri is used as a probiotic in the health care of humans and animals. The biotechnological production of 3-HPA from renewable resources is desirable both for use of 3-HPA in foods and for the production of bulk chemicals. The main challenge will be the efficient production and recovery of pure 3-HPA.     

A genetically modified solvent-tolerant bacterium for optimized production of a toxic fine chemical

The aim of the study was to investigate whether toxic fine chemical production can be improved using the solvent-tolerant Pseudomonas putida S12 in a two-liquid-phase system consisting of aqueous media and a water-immiscible octanol phase with production of 3-methylcatechol from toluene as the model conversion. For this purpose the genes involved in this conversion, todC1C2BAD from P. putida F1, were introduced into P. putida S12 with high stable expression. Production of 3-methylcatechol was monitored in batch incubations with different media using a single medium and a two-liquid medium–octanol system. The maximum concentration of 3-methylcatechol increased two-fold using the two-liquid medium–octanol system, irrespective of the selected medium. Received: 29 December 1999 / Received revision: 29 February 2000 / Accepted: 6 March 2000     

Simultaneous degradation of chloro- and methyl-substituted aromatic compounds: competition between Pseudomonas strains using the ortho and meta pathway or the ortho pathway exclusively

Pseudomonas sp. D7-4 and Pseudomonas sp. B13 FR1(pFRC20P) degraded mixtures of chloro- and methyl-substituted benzoates exclusively via an extended ortho pathway, whereas in Pseudomonas putida WR201 both ortho and meta fission were induced by mixtures of 3-chloro- and 3-methylbenzoate or even by 3-chlorobenzoate alone. The competition behaviour of these strains was compared in batch and in chemostat cultures. Despite misrouting of metabolites, strain WR201 was competitive, in a lot of the competition experiments, with mixtures of these substrates. Only in a narrow range of the mixing ratio of chloro- and methylbenzoate was the presence of both the meta and ortho pathways a disadvantage for competitiveness. Outside these ranges other attributes, such as high growth rates or short lag periods, of a respective strain were even more essential for one strain to outcompete another. Received: 13 February 1998 / Received revision: 28 April 1998 / Accepted: 30 April 1998     

Evolutionary and Functional Relationships of the dha Regulon by Genomic Context Analysis

3-hydroxypropionaldehyde (3-HPA) and 1,3-propanediol (1,3-PD) are subproducts of glycerol degradation and of economical interest as they are used for polymers synthesis, such as polyesters and polyurethanes. Some few characterized bacterial species (mostly from Firmicutes and Gamma-proteobacteria groups) are able to catabolize these monomers from glycerol using the gene products from the dha regulon. To expand our knowledge and direct further experimental studies on the regulon and related genes for the anaerobic glycerol metabolism, an extensive genomic screening was performed to identify the presence of the dha genes in fully sequenced prokaryotic genomes. Interestingly, this work shows that although only few bacteria species are known to produce 3-HPA or 1,3-PD, the incomplete regulon is found in more than 100 prokaryotic genomes. However, the complete pathway is found only in a few dozen species belonging to five different taxonomic groups, including one Archaea species, Halalkalicoccus jeotgali. Phylogenetic analysis and conservation of both gene synteny and primary sequence similarity reinforce the idea that these genes have a common origin and were possibly acquired by lateral gene transfer (LGT). Besides the evolutionary aspect, the identification of homologs from several different organisms may predict potential alternative targets for faster or more efficient biological synthesis of 3-HPA or 1,3-PD.     

Directed Evolution of Toluene Dioxygenase from Pseudomonas putida for Improved Selectivity Toward cis-Indandiol during Indene Bioconversion

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S, 2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S, 2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.     

Tetrameric structure and cellular location of catechol 2,3-dioxygenase

Catechol 2,3-dioxygenase from the meta-cleavage pathway encoded on the TOL plasmid of Pseudomonas putida (pWWO) was investigated by electron microscopy. Negatively stained samples of the purified catechol 2,3-dioxygenase revealed that the enzyme consists of four subunits arranged in a tetrahedral conformation. Monoclonal antibodies raised against catechol 2,3-dioxygenase showed highly specific reactions and were used to localize the enzyme in Escherichia coli (pAW31) and P. putida (pWWO), using the protein A-gold technique carried out as a post-embedding immunoelectron microscopy procedure. Our in situ labeling studies revealed a cytoplasmic location of the catechol 2,3-dioxygenase in both cell types.Abbreviations C23O Catechol 2,3-dioxygenase - 3MB 3 Methylbenzoate - AK1 Anti-C23O-IgG-antibody - G Gold particle     

Microbial transformation of benzene to <Emphasis Type="Italic">cis</Emphasis>-3,5-cyclohexadien-1,2-diols by recombinant bacteria harboring toluene dioxygenase gene <Emphasis Type="Italic">tod</Emphasis>

Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylation of aromatic compounds. To achieve high efficient biotransformation of benzene to benzene cis-diols, Pseudomonas putida KT2442, Pseudomonas stutzeri 1317, and Aeromonas hydrophila 4AK4 were used as hosts to express TDO gene tod. Plasmid pSPM01, a derivative of broad-host plasmid pBBR1MCS-2 harboring tod from plasmid pKST11, was constructed and introduced into the above three strains. Their abilities to catalyze the biotransformation of benzene to benzene cis-diols, namely, cis-3,5-cyclohexadien-1,2-diols abbreviated as DHCD, were examined. In shake-flask cultivation under optimized culture media and growth condition, benzene cis-diols production by recombinant P. putida KT2442 (pSPM01), P. stutzeri 1317 (pSPM01), and A. hydrophila 4AK4 (pSPM01) were 2.68, 2.13, and 1.17 g/l, respectively. In comparison, Escherichia coli JM109 (pSPM01) and E. coli JM109 (pKST11) produced 0.45 and 0.53 g/l of DHCD, respectively. When biotransformation was run in a 6-l fermenter, DHCD production in P. putida KT2442 (pSPM01) was approximately 60 g/l; this is the highest DHCD production yield reported so far.     

Regulation of 3-hydroxypropionaldehyde accumulation in Klebsiella pneumoniae by overexpression of dhaT and dhaD genes

3-Hydroxypropionaldehyde (3-HPA), an important intermediary metabolite of 1,3-propanediol (PDO) production, would be toxic to the cell growth and led to the abnormal cessation of the fermentation process. In this study, the dhaD gene encoding glycerol dehydrogenase (GDH) and dhaT gene encoding 1,3-propanediol oxidoreductase (PDOR) were overexpressed in Klebsiella pneumoniae ACCC 10082 to decrease the 3-HPA accumulation and increase the coenzyme NADH supply. By the construction of pTD plasmid, GDH and PDOR were both overexpressed and their enzyme activities were increased by 2.6- and 3.2-fold, respectively. The enzyme activity ratio of PDOR/GDHt (glycerol dehydratase) also was increased. On the other hand, NADH production was enhanced and the ratio of NADH/NAD⁺ exceeded 1 after the inducement of IPTG for the constructed strain. The two factors enhanced the transformation of 3-HPA to PDO. In the batch and fed-batch fermentation by the constructed strain, the peak of 3-HPA accumulation reduced by 52.2% and 33.3%, respectively, compared with the control. The PDO concentration and yield reached 59.2 g/L and 0.48 mol/mol, respectively. Furthermore, the fed-batch fermentation process appeared easier to be regulated. This work is considered helpful for the further understanding on the PDO metabolic mechanism of K. pneumoniae and also useful for the PDO fermentation in a large-scale bioreactor.     

Quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone influences siderophore biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa

Siderophore synthesis of Pseudomonas putida F1 was found to be regulated by quorum sensing since normalized siderophore production (per cell) increased 4.2-fold with cell density after the cells entered middle exponential phase; similarly, normalized siderophore concentrations in Pseudomonas aeruginosa JB2 increased 28-fold, and a 5.5-fold increase was seen for P. aeruginosa PAO1. Further evidence of the link between quorum sensing and siderophore synthesis of P. putida F1 was that the quorum-sensing-disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the marine red alga Delisea pulchra was found to inhibit the formation of the siderophore produced by P. putida F1 in a concentration-dependent manner, with 57% siderophore synthesis repressed by 100 g/ml furanone. In contrast, this furanone did not affect the siderophore synthesis of Burkholderia cepacia G4 at 20–40 g/ml, and stimulated siderophore synthesis of P. aeruginosa JB2 2.5- to 3.7-fold at 20–100 g/ml. Similarly, 100 g/ml furanone stimulated siderophore synthesis in P. aeruginosa PAO1 about 3.5-fold. The furanone appears to interact with the quorum-sensing machinery of P. aeruginosa PAO1 since it stimulates less siderophore synthesis in the P. aeruginosa qscR quorum-sensing mutant (QscR is a negative regulator of LasI, an acylated homoserine lactone synthase).