Fed-batch production of unsaturated medium-chain-length polyhydroxyalkanoates with controlled composition by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

Unsaturated medium-chain-length polyhydroxyalkanoates (MCL-PHA) were produced at a productivity of 0.63–1.09 g PHA l⁻¹ h⁻¹ with final PHA content ranging from 42.6 to 55.8% in single-stage, carbon-limited, fed-batch fermentations of Pseudomonas putida KT2440. A mixture of nonanoic acid (NA) and 10-undecenoic acid (UDA⁼) was fed exponentially to control growth rate. Varying the specific growth rate (0.14 h⁻¹ vs. 0.23 h⁻¹) at similar substrate feed ratios (NA:UDA⁼ = 5:1) had little effect on the final PHA content and relative composition. However, decreasing the NA:UDA⁼ ratio decreased the final amount of PHA produced from 56% with NA:UDA⁼ = 5.07:1 to only 42% at NA:UDA⁼ = 2.47:1. The molar fraction of all 3-hydroxyalkanoate monomers in the PHA product was relatively constant throughout each fermentation, indicating that the final product was homogeneous rather than a mixture of different copolymers. A linear relationship between unsaturation of the PHA produced and unsaturation of the carbon feed was found, which demonstrates the feasibility of producing unsaturated MCL-PHAs with controlled polymeric composition in a fed-batch process.     

Enhanced yield of medium-chain-length polyhydroxyalkanoates from nonanoic acid by co-feeding glucose in carbon-limited, fed-batch culture

Medium-chain-length polyhydroxyalkanoates (MCL-PHAs) were produced in carbon-limited, single-stage, fed-batch fermentations of Pseudomonas putida KT2440 by co-feeding nonanoic acid (NA) and glucose (G) to enhance the yield of PHA from NA. An exponential (μ = 0.25 h⁻¹) followed by a linear feeding strategy at a NA:G ratio of 1:1 (w/w) achieved 71 g l⁻¹ biomass containing 56% PHA. Although the same overall PHA productivity (1.44 g l⁻¹ h⁻¹) was obtained when NA alone was fed at the same specific growth rate, the overall yield of PHA from NA increased by 25% (0.66 g PHA g NA⁻¹ versus 0.53 g g⁻¹) with glucose co-feeding. Further increasing glucose in the feed (NA:G = 1:1.5) resulted in a slightly higher yield (0.69 g PHA g NA⁻¹) but lower PHA content (48%) and productivity (1.16 g l⁻¹ h⁻¹). There was very little change in the PHA composition.     

Cloning,expression and characterization of a Baeyer-Villiger monooxygenase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

The gene encoding a Baeyer-Villiger monooxygenase and identified in Pseudomonas putida KT2440 was cloned and functionally expressed in Escherichia coli. The highest yield of soluble protein could be achieved by co-expression of molecular chaperones. In order to determine the substrate specificity, biocatalyses were performed using crude cell extract, growing and resting cells. Examination of aromatic, cyclic and aliphatic ketones revealed a high specificity towards short-chain aliphatic ketones. Interestingly, some open-chain ketones were converted to the alkylacetates, while for others formation of the ester products with oxygen on the other side of the keto group could also be detected yielding the corresponding methyl or ethyl esters.     

Kinetics of BTEX biodegradation by a coculture of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> and<Emphasis Type="Italic"> Pseudomonas fluorescens</Emphasis> under hypoxic conditions

Pseudomonas putida and Pseudomonas fluorescens present as a coculture were studied for their abilities to degrade benzene, toluene, ethylbenzene, and xylenes (collectively known as BTEX) under various growth conditions. The coculture effectively degraded various concentrations of BTEX as sole carbon sources. However, all BTEX compounds showed substrate inhibition to the bacteria, in terms of specific growth, degradation rate, and cell net yield. Cell growth was completely inhibited at 500mgl^–1 of benzene, 600mgl^–1 of o-xylene, and 1000mgl^–1 of toluene. Without aeration, aerobic biodegradation of BTEX required additional oxygen provided as hydrogen peroxide in the medium. Under hypoxic conditions, however, nitrate could be used as an alternative electron acceptor for BTEX biodegradation when oxygen was limited and denitrification took place in the culture. The carbon mass balance study confirmed that benzene and toluene were completely mineralized to CO₂ and H₂O without producing any identifiable intermediate metabolites.     

Biotransformation of isoeugenol to vanillin by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27 cells

The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 °C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.     

Utilization of <Emphasis Type="Italic">fadA</Emphasis> knockout mutant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> for overproduction of medium chain-length-polyhydroxyalkanoate

The fadBA operon in the fatty acid β-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l⁻¹ stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l⁻¹ with a cell dry weight of 10.3 g l⁻¹.     

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.     

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.     

Purification,characterization and gene cloning of isoeugenol-degrading enzyme from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27

An isoeugenol-degrading enzyme was purified to homogeneity from Pseudomonas putida IE27, an isoeugenol-assimilating bacterium. The purified enzyme was a 55 kDa monomer and catalyzed the initial step of isoeugenol degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Another reaction product of isoeugenol degradation besides vanillin was identified to be acetaldehyde. The values of Km and k _cat for isoeugenol were 175 μM and 5.18 s^–1, respectively. The purified enzyme catalyzed the incorporation of an oxygen atom from either molecular oxygen or water into vanillin, suggesting that the isoeugenol-degrading enzyme is a kind of monooxygenase. The gene encoding the isoeugenol-degrading enzyme and its flanking regions were isolated from P. putida IE27. The amino acid sequence of the enzyme was similar to those of lignostilbene-α,β-dioxygenases, carotenoid monooxygenases and 9-cis-epoxycarotenoid dioxygenases.     

Conjugative transfer of preferential utilization of aromatic compounds from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CSV86

Pseudomonas putida CSV86 utilizes naphthalene (Nap), salicylate (Sal), benzyl alcohol (Balc), and methylnaphthalene (MN) preferentially over glucose. Methylnaphthalene is metabolized by ring-hydroxylation as well as side-chain hydroxylation pathway. Although the degradation property was found to be stable, the frequency of obtaining Nap⁻Sal⁻MN⁻Balc⁻ phenotype increased to 11% in the presence of curing agents. This property was transferred by conjugation to Stenotrophomonas maltophilia CSV89 with a frequency of 7 × 10⁻⁸ per donor cells. Transconjugants were Nap⁺Sal⁺MN⁺Balc⁺ and metabolized MN by ring- as well as side-chain hydroxylation pathway. Transconjugants also showed the preferential utilization of aromatic compounds over glucose indicating transfer of the preferential degradation property. The transferred properties were lost completely when transconjugants were grown on glucose or 2YT. Attempts to detect and isolate plasmid DNA from CSV86 and transconjugants were unsuccessful. Transfer of degradation genes and its subsequent loss from the transconjugants was confirmed by PCR using primers specific for 1,2-dihydroxynaphthalene dioxygenase and catechol 2,3-dioxygenase (C23O) as well as by DNA–DNA hybridizations using total DNA as template and C23O PCR fragment as a probe. These results indicate the involvement of a probable conjugative element in the: (i) metabolism of aromatic compounds, (ii) ring- and side-chain hydroxylation pathways for MN, and (iii) preferential utilization of aromatics over glucose.     

An efficient process for production and purification of hyaluronic acid from <Emphasis Type="Italic">Streptococcus </Emphasis><Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Growth of Streptococcus zooepidemicus in a 10 l bioreactor with 50 g sucrose/l and 10 g casein hydrolysate/l gave 5–6 g hyaluronic acid/l after 24–28 h. Purification of hyaluronic acid gave a recovery of 65% with the final material having an Mr of ∼4 × 10⁶ Da with less than 0.1% protein.     

Toluene biodegradation by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1: targeting culture stability in long-term operation

The stability of Pseudomonas putida F1, a strain harbouring the genes responsible for toluene degradation in the chromosome was evaluated in a bioscrubber under high toluene loadings and nitrogen limiting conditions at two dilution rates (0.11 and 0.27 h⁻¹). Each experiment was run for 30 days, period long enough for microbial instability to occur considering previously reported studies carried out with bacterial strains encoding the catabolic genes in the TOL plasmid. At all tested conditions, P. putida F1 exhibited stable performance as shown by the constant values of the specific toluene degradation yield, CO₂ produced versus toluene degraded yield, and biomass concentration within each steady state. Benzyl alcohol, a curing agent causing TOL plasmid deletion in Pseudomonas strains, was present in the cultivation medium as a result of the monooxygenation of toluene by the diooxygenase system of P. putida F1. However, no mutant population growing at the expense of the extracellular excreted carbon or lysis products was established in the chemostat as confirmed by the constant dissolved total organic carbon (TOC) concentration and fraction of toluene degrading cells (approx. 100%). In addition, batch experiments conducted with both lysis substrate and toluene simultaneously confirmed that P. putida F1 preferentially consumed toluene rather than extracellular excreted carbon.     

Determination of copper binding in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CZ1 by chemical modifications and X-ray absorption spectroscopy

Previously performed studies have shown that Pseudomonas putida CZ1 biomass can bind an appreciable amount of Cu(II) and Zn(II) ions from aqueous solutions. The mechanisms of Cu- and Zn-binding by P. putida CZ1 were ascertained by chemical modifications of the biomass followed by Fourier transform infrared and X-ray absorption spectroscopic analyses of the living or nonliving cells. A dramatic decrease in Cu(II)- and Zn(II)-binding resulted after acidic methanol esterification of the nonliving cells, indicating that carboxyl functional groups play an important role in the binding of metal to the biomaterial. X-ray absorption spectroscopy was used to determine the speciation of Cu ions bound by living and nonliving cells, as well as to elucidate which functional groups were involved in binding of the Cu ions. The X-ray absorption near-edge structure spectra analysis showed that the majority of the Cu was bound in both samples as Cu(II). The fitting results of Cu K-edge extended X-ray absorption fine structure spectra showed that N/O ligands dominated in living and nonliving cells. Therefore, by combining different techniques, our results indicate that carboxyl functional groups are the major ligands responsible for the metal binding in P. putida CZ1.     

Enantioselective Nitrilase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis>: Cloning,Heterologous Expression,and Bioreactor Studies

Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(−)-mandelic acid.     

Psychrophilic <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> requires <Emphasis Type="Italic">trans</Emphasis>-monounsaturated fatty acid for growth at higher temperature

A psychrophilic bacterium, Pseudomonas syringae (Lz4W) from Antarctica, was used as a model system to establish a correlation, if any, between thermal adaptation, trans-fatty acid content and membrane fluidity. In addition, attempts were made to clone and sequence the cti gene of P. syringae (Lz4W) so as to establish its characteristics with respect to the cti of other Pseudomonas spp. and also to in vitro mutagenize the cti gene so as to generate a cti null mutant. The bacterium showed increased proportion of saturated and trans-monounsaturated fatty acids when grown at 28°C compared to cells grown at 5°C, and the membrane fluidity decreased with growth temperature. In the mutant, the trans-fatty acid was not synthesized, and the membrane fluidity also decreased with growth temperature, but the decrease was not to the extent that was observed in the wild-type cells. Thus, it would appear that synthesis of trans-fatty acid and modulation of membrane fluidity to levels comparable to the wild-type cells is essential for growth at higher temperatures since the mutant exhibits growth arrest at 28°C. In fact, the cti null mutant-complemented strain of P. syringae (Lz4W-C30b) that was capable of synthesizing the trans-fatty acid was indeed capable of growth at 28°C, thus confirming the above contention. The cti gene of P. syringae (Lz4W) that was cloned and sequenced exhibited high sequence identity with the cti of other Pseudomonas spp. and exhibited all the conserved features.     

High-yield production of erythritol from raw glycerol in fed-batch cultures of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

An acetate-negative mutant of Yarrowia lipolytica Wratislavia K1 was selected that, when grown with 300 g raw glycerol l⁻¹ at pH 3, produced 170 g erythritol l⁻¹ after 7 days, corresponding to a 56% yield and a productivity of 1 g l⁻¹ h⁻¹. The Wratislavia K1 strain did not produce citric acid.