Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system

Faizal, Irvan; Dozen, Kana; Hong, Chia Swee; Kuroda, Akio; Takiguchi, Noboru; Ohtake, Hisao; Takeda, Koji; Tsunekawa, Hiroshi; Kato, Junichi

doi:10.1007/s10295-005-0253-y

Cited by 37 publications

(21 citation statements)

References 14 publications

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“…Similarly, P. putida strains IH-2000, S12, DOT-T1E, and T-57 and Pseudomonas sp. strain BCNU 171 have been shown to be capable to thrive in the presence of a second phase of toluene (28,29,32,66,67).…”

Section: Discussionmentioning

confidence: 99%

“…Some solvent-tolerant bacteria, often Pseudomonads, are able to survive sudden exposure to toxic organic solvents (24)(25)(26)(27), and some are even able to grow in the presence of a second phase of toxic solvents in complex media (28)(29)(30)(31)(32). Such solvent tolerance is based on a combination of several adaptive mechanisms, which have been intensively investigated and reviewed for Pseudomonas putida S12 and DOT-T1E (14,33,34).…”

mentioning

confidence: 99%

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Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

Volmer

Neumann

Bühler

et al. 2014

Appl Environ Microbiol

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The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120⌬C. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120⌬C parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120⌬C⌬ttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

Volmer

Neumann

Bühler

et al. 2014

Appl Environ Microbiol

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“…A limitation associated with organic solvents is the potential toxicity that they may possess towards the biocatalyst. In order to minimize the toxic effects of a solvent, researchers have utilized solvent tolerant strains of bacteria (Faizal et al, 2005;Neumann et al, 2005;Wery et al, 2000;Wierckx et al, 2005), alternative bioreactor designs (Husken et al, 2002b) and have implemented intelligent solvent selection based on the octanol-water partitioning coefficient (log P) of solvents (Brink and Tramper, 1985;Bruce and Daugulis, 1991;Laane and Tramper, 1990). Of the techniques attempted to reduce solvent toxicity, rational solvent selection represents the most direct approach.…”

Section: Introductionmentioning

confidence: 99%

Solvent selection for enhanced bioproduction of 3‐methylcatechol in a two‐phase partitioning bioreactor

Prpich

Daugulis

2006

Biotech & Bioengineering

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The biotransformation of toluene to 3-methycatechol (3MC) via Pseudomonas putida MC2 was used as a model system for the development of a biphasic process offering enhanced overall volumetric productivity. Three factors were investigated for the identification of an appropriate organic solvent and they included solvent toxicity, bioavailability of the solvent as well as solvent affinity for 3MC. The critical log P (log P(crit)) of the biocatalyst was found to be 3.1 and log P values were used to predict a solvent's toxicity. The presence of various functional groups of candidate solvents were used to predict the absorption of 3MC and it was found that solvents possessing polarity showed an affinity towards 3MC. Bis (2-ethylhexyl) sebecate was selected for use in the biphasic system as it fulfilled all selection criteria. A two-phase biotransformation with BES and a 50% phase volume ratio, achieved an overall volumetric productivity of 440 mg 3MC/L-h, which was an improvement by a factor of approximately 4 over previously operated systems. Additional work focused on reducing the toluene feed in order to minimize possible toxicity and decrease loss of substrate (toluene), a result of volatilization. Toluene losses were reduced by a factor of 4, compared to previously operated systems, without suffering an appreciable loss in overall volumetric productivity.

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“…P. putida TODE1 is a 3MC-accumulating mutant derived from OST P. putida T-57 in which the methylcatechol 2,3-dioxygenase gene (todE) was disrupted by a kanamycin cassette insertion (Faizal et al, 2007). The bacterial cultivation medium was either Luria-Bertani (LB) medium or minimal salt basal medium (MSB) (Faizal et al, 2005) as indicated. When MSB was supplemented with glucose (11 mM), it was referred to as MSBG, and with glycerol (4 mM) and FeSO 4 (0.4 mM), it was referred to as the optimized medium (OM-1 or OM-2, as indicated).…”

Section: Methodsmentioning

confidence: 99%

“…The decreased TDO activity in the presence of Ca 2+ agreed with the low overall 3MC concentrations. Magnesium (Mg 2+ ) has been reported to improve cell tolerance towards organic solvents in Pseudomonas (Faizal et al, 2005;Isken and Bont, 1998). Although Mg 2+ supplementation promoted a higher TDO activity (~2 fold), an increase in the overall 3MC yield was only observed at high concentrations of Mg 2+ (10 and 20 mM).…”

Section: Factors Promoting Overall Production Of 3mc In a Two-phase Smentioning

confidence: 99%

Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system

Kongpol¹,

Kato²,

Tajima³

et al. 2014

J. Gen. Appl. Microbiol.

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In this study, genetically engineered Pseudomonas putida TODE1 served as a biocatalyst for the bioproduction of valuable 3-methylcatechol (3MC) from toluene in an aqueous-organic two-phase system. The two-phase system was used as an approach to increase the biocatalyst efficiency. Among the organic solvent tested, n-decanol offered several benefits including having the highest partitioning of 3MC, with a high 3MC yield and low cell toxicity. The effect of media supplementation with carbon/ energy sources (glucose, glycerol, acetate and succinate), divalent metal cations (Mg 2+ , Ca 2+, Mn 2+ and Fe 2+ ), and short-chain alcohols (ethanol, n-propanol and n-butanol) as a cofactor regeneration system on the toluene dioxygenase (TDO) activity, cell viability, and overall 3MC yield were evaluated. Along with the two-step cell preparation protocol, supplementation of the medium with 4 mM glycerol as a carbon/energy source, and 0.4 mM Fe 2+ as a cofactor for TDO significantly enhanced the 3MC production level. When in combination with the use of n-decanol and n-butanol as the organic phase, a maximum overall 3MC concentration of 31.8 mM (166 mM in the organic phase) was obtained in a small-scale production, while it was at 160.5 mM (333.2 mM in the organic phase) in a 2-L scale. To our knowledge, this is the highest 3MC yield obtained from a TDO-based system so far.Key words: an aqueous-organic two-phase fermentation; bioproduction; 3-methylcatechol; Pseudomonas putida TODE1; toluene; toluene dioxygenase Introduction 3-Methylcatechol (3MC) is an important precursor for the production of valuable pharmaceutical compounds such as barbatusol and L-DOPA analogues, as well as synthetic food flavors (Husken et al., 2002;Shirai, 1986). Due to difficulties and relatively low overall production yield from chemical synthesis (Held et al., 1999), 3MC production mainly relies on an economic and efficient biotechnological process, in which toluene is employed as a substrate for a bioconversion to 3MC using bacterial whole-cells as biocatalysts (Faizal et al., 2007;Wery et al., 2000). However, since toluene and 3MC, even at concentrations as low as 1% (v/v), detrimentally affect microbial survival and their catalytic activity, their toxicity, especially to 3MC generated and accumulated in the fermentation system, is the main constraint of the production process (de Smet et al., 1978;Fillet et al., 2012;Husken et al., 2001). Two common approaches to overcoming the toxicity limitation are 1) the use of an organic solvent-tolerant (OST) bacteria, which can thrive in relative high concentrations of organic compounds, as a biocatalyst, and 2) the use of a two-liquid (organicaqueous) phase biotransformation system (referred to as a two-phase system hereafter), where the immiscible organic phase with microbial compatibility acts effectively as a source and a sink for the particular organic substrate and product (Heipieper et al., 2007).3MC is typically produced by a genetically engineered bacterial host from a direct bioconversion of tolue...

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Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system

Cited by 37 publications

References 14 publications

Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

Engineering of Pseudomonas taiwanensis VLB120 for Constitutive Solvent Tolerance and Increased Specific Styrene Epoxidation Activity

Solvent selection for enhanced bioproduction of 3‐methylcatechol in a two‐phase partitioning bioreactor

Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system

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