Integrated bioproduction and extraction of 3-methylcatechol

Hüsken, Leonie E.; Dalm, M.C.F.; Tramper, J.; Wery, Jan; Bont, J.A.M. de; Beeftink, Rik

doi:10.1016/s0168-1656(01)00252-8

Cited by 44 publications

(60 citation statements)

References 27 publications

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“…5B). This is in agreement with previous reports that 3MC is unstable in aqueous phase at a relatively basic pH and either undergoes oxidation or chemical degradation (Husken et al, 2001(Husken et al, , 2002 or polymerization to form o-benzoquinone (Borraccino et al, 2001;Hüsken et al, 2001;Mijangos et al, 2006;Wery et al, 2000), while it was more stable in organic phase (Fig. 5B).…”

Section: Fed-batch Production Of 3mc In a 2-l Bioreactorsupporting

confidence: 93%

“…The production of 3MC has been reported to be pHdependent (Husken et al, 2001). Therefore, the effect of the pH on the fed-batch production of 3MC by TODE1 was evaluated at three controlled pH values and compared to that of an uncontrolled pH (Fig.…”

Section: Fed-batch Production Of 3mc In a 2-l Bioreactormentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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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Section: Fed-batch Production Of 3mc In a 2-l Bioreactorsupporting

confidence: 93%

Section: Fed-batch Production Of 3mc In a 2-l Bioreactormentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…putida DOT-T1E and related strains such as P. putida S12 set an example for bacteria which can tolerate solvents and are able to produce fine chemicals of interest in economically sound concentrations (14,38). These bacteria contain at least three different solvent efflux pumps that play a major role in adaptation (32) and are therefore the most effective potential biocatalysts among all P. putida strains.…”

Section: Fig 1 Effect Of 1-octanol (A) and 1-decanol (B) On Growth mentioning

confidence: 99%

Prediction of the Adaptability of Pseudomonas putida DOT-T1E to a Second Phase of a Solvent for Economically Sound Two-Phase Biotransformations

Neumann

Kabelitz

Zehnsdorf

et al. 2005

Appl Environ Microbiol

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The strain Pseudomonas putida DOT-T1E was tested for its ability to tolerate second phases of different alkanols for their use as solvents in two-liquid-phase biotransformations. Although 1-decanol showed an about 10-fold higher toxicity to the cells than 1-octanol, the cells were able to adapt completely to 1-decanol only and could not be adapted in order to grow stably in the presence of a second phase of 1-octanol. The main explanation for this observation can be seen in the higher water and membrane solubility of 1-octanol. The hydrophobicity (log P) of a substance correlates with a certain partitioning of that compound into the membrane. Combining the log P value with the water solubility, the maximum membrane concentration of a compound can be calculated. With this simple calculation, it is possible to predict the property of an organic chemical for its potential applicability as a solvent for two-liquid-phase biotransformations with solventtolerant P. putida strains. Only compounds that show a maximum membrane concentration of less than 400 mM, such as 1-decanol, seem to be tolerated by these bacterial strains when applied in supersaturating concentrations to the medium. Taking into consideration that a solvent for a two-liquid-phase system should possess partitioning properties for potential substrates and products of a fine chemical synthesis, it can be seen that 1-decanol is a suitable solvent for such biotransformation processes. This was also demonstrated in shake cultures, where increasing amounts of a second phase of 1-decanol led to bacteria tolerating higher concentrations of the model substrate 3-nitrotoluene. Transferring this example to a 5-liter-scale bioreactor with 10% (vol/vol) 1-decanol, the amount of 3-nitrotoluene tolerated by the cells is up to 200-fold higher than in pure aqueous medium. The system demonstrates the usefulness of two-phase biotransformations utilizing solventtolerant bacteria.Biocatalysis using whole cells promised to play an important role in the industrial synthesis of fine chemicals, pharmaceuticals, and precursors for chemical syntheses. However, the number of successful processes using whole-cell biotransformations is very small so far because several factors limit the number of applications (29,30). One important limitation of a successful application of whole-cell biotechnological processes toward classical chemical synthesis is that many reactions of interest involve substrates or products that are extremely toxic for the bacteria (35). This problem can be solved by the application of an organic solvent phase that functions as a source and a sink for toxic organic substrates and products, respectively (36). For biotransformations with whole cells, the major advantage of an addition of a second organic phase lies within its ability to act as a sink for the substrates as well as in the continuous removal of products (19,21,36). In both cases, the presence of a solvent phase keeps the concentrations of substrates and toxins at a level that does not lead to toxic e...

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“…Yet, microbial cells, especially in a nongrowing state, often lose biooxidation activity over time, which may be due to oxygenase inactivation and/or the metabolic burden imposed by redox-coenzyme withdrawal and reactive oxygen species formed via uncoupled oxygen reduction. This led to the use of growing cells as biocatalysts, the metabolism of which supports not only coenzyme regeneration but also continuous oxygenase synthesis and cell reproduction (5,19,27,32,49). However, during cell growth, various reactions-especially oxidative phosphorylation-consume high amounts of reduction equivalents (59).…”

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confidence: 99%

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Bühler

Park

Blank

et al. 2008

Appl Environ Microbiol

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Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 mol per min per g cells (dry weight), the absence of limitations by O 2 and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.Oxygenases catalyze regio-and enantioselective oxyfunctionalization reactions and have a considerable potential in the area of asymmetric organic synthesis (2, 35). These enzymes typically depend on coenzymes such as NAD(P)H and are unstable outside cells, and they often consist of multiple components. Thus, for synthetic applications, their use in whole cells is favored over the use of isolated enzymes (7,18).The productivity of oxygenase-based whole-cell biocatalysts is influenced by various factors, such as maximal enzyme activity, enzyme level, substrate mass transfer, substrate and/or product inhibition/toxicity, and cofactor regeneration (7,13,54,68). Enzyme synthesis and cofactor regeneration are related to host metabolism, which in turn may be affected by the toxicity of organic substrates and products. Such toxicity can efficiently be attenuated by regulated substrate feeding (1, 11) and in situ product removal (7,38,61,66,74). Yet, microbial cells, especially in a nongrowing state, often lose biooxidation activity over time, which may be due to oxygenase inactivation and/or the metabolic burden imposed by redox-coenzyme withdrawal and reactive oxygen species formed via...

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Integrated bioproduction and extraction of 3-methylcatechol

Cited by 44 publications

References 27 publications

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

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

Prediction of the Adaptability of Pseudomonas putida DOT-T1E to a Second Phase of a Solvent for Economically Sound Two-Phase Biotransformations

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

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