High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system

Hüsken, Leonie E.; Beeftink, Rik; Bont, J.A.M. de; Wery, Jan

doi:10.1007/s002530000566

Cited by 49 publications

(38 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: 83%

“…It was previously reported that OST P. putida could tolerate 3MC up to 5.5 mM (Hüsken et al, 2001), while this study showed that the P. putida T57 wild-type strain as well as the TODE1 mutant strain could tolerate up to 7 mM 3MC when tested under growing-cell conditions. In this study, as the production goal was to produce 3MC at higher concentrations, the use of a high-density OST resting-cell-based bioproduction was employed instead of a growing-cell based one.…”

Section: Resultsmentioning

confidence: 71%

Section: +mentioning

confidence: 99%

“…Since these genes are present in most, though not all, Pseudomonas putida strains (Faizal et al, 2007;Hüsken et al, 2001;Wery et al, 2000), genetic modifications of OST P. putida such as strain MC2 and strain MC1 were conducted to increase gene dosage or gene expression level in order to enhance 3MC production yield (Hüsken et al, 2001;Wery et al, 2000). In addition, the 3MC production system could be improved by optimization of two-phase bioconversion conditions, e.g.…”

Section: Introductionmentioning

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: 83%

Section: Resultsmentioning

confidence: 71%

Section: +mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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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“…Alternatively, solvent-tolerant strains can be used to express the enzyme to improve biocatalyst stability in the presence of solvents that are generally toxic to microorganisms (14). Solvent-tolerant strains have been used previously for production of toxic products, such as 3-methyl catechol, in the presence of toxic organic solvents (14,15,32). More work is being done to improve 1-naphthol production using solvent-tolerant strains and biphasic systems.…”

Section: Discussionmentioning

confidence: 99%

Whole-Cell Biocatalysis for 1-Naphthol Production in Liquid-Liquid Biphasic Systems

Garikipati

McIver

Peeples

2009

Appl Environ Microbiol

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Whole-cell biocatalysis to oxidize naphthalene to 1-naphthol in liquid-liquid biphasic systems was performed. Escherichia coli expressing TOM-Green, a variant of toluene ortho-monooxygenase (TOM), was used for this oxidation. Three different solvents, dodecane, dioctyl phthalate, and lauryl acetate, were screened for biotransformations in biphasic media. Of the solvents tested, lauryl acetate gave the best results, producing 0.72 ؎ 0.03 g/liter 1-naphthol with a productivity of 0.46 ؎ 0.02 g/g (dry weight) cells after 48 h. The effects of the organic phase ratio and the naphthalene concentration in the organic phase were investigated. The highest 1-naphthol concentration (1.43 g/liter) and the highest 1-naphthol productivity (0.55 g/g [dry weight] cells) were achieved by optimization of the organic phase. The ability to recycle both free cells and cells immobilized in calcium alginate was tested. Both free and immobilized cells lost more than ϳ60% of their activity after the first run, which could be attributed to product toxicity. On a constant-volume basis, an eightfold improvement in 1-naphthol production was achieved using biphasic media compared to biotransformation in aqueous media.Biocatalysis has emerged as an important technology in industrial organic synthesis for the production of chemical synthons and high-value products (29,34,37). Biocatalysis offers the advantage of performing reactions under mild conditions and provides an environmentally benign approach for chemical reactions (1,38). Oxygenases are a class of enzymes that have great potential and versatility for catalyzing reactions that are generally not accessible by chemical routes with high regio-, stereo-, and enantioselectivities (6,27,42,43). Oxygenases introduce either one or two atoms of molecular oxygen into organic molecules using NADH or NADPH as a cofactor. To eliminate the addition of a costly cofactor, whole cells expressing oxygenases are generally used (34,43).One of the potential applications of biocatalysis utilizing oxygenases is the oxidation of naphthalene to 1-naphthol. 1-Naphthol has wide applications in the manufacture of dyes, drugs, insecticides, perfumes, and surfactants (2, 7, 17). Tao et al. (39) have compared the reaction rates and regioselectivities of various wild-type and modified monooxygenases for the oxidation of naphthalene to 1-naphthol. Of the monooxygenases tested, the best enzyme for the oxidation of naphthalene to 1-naphthol was a toluene ortho-monooxygenase (TOM) variant, TomA3(V106A), also known as TOM-Green. TOM was isolated from Burkholderia cepacia G4 and consists of an ␣ 2 ␤ 2 ␥ 2 hydroxylase (encoded by tomA1, tomA3, and tomA4) with two catalytic oxygen-bridged binuclear iron centers, an NADH-oxidoreductase (encoded by tomA5), a protein (encoded by tomA2) involved in electron transfer between oxidoreductase and hydroxylase, and a relatively unknown subunit (encoded by tomA0) (26,36). TOM-Green was produced by directed evolution of TOM with one amino acid change in the alpha-subunit of the hydro...

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Asymmetric Dihydroxylation of Cinnamonitrile to trans‐3‐[(5S,6R)‐5,6‐Dihydroxycyclohexa‐1,3‐dienyl]‐acrylonitrile using Chlorobenzene Dioxygenase in Escherichia coli (pTEZ30)

et al. 2004

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Asymmetric cis-dihydroxylations of aromatic compounds are catalyzed by bacterial dioxygenases. In order to prevent through conversion, either dihydrodiol dehydrogenase blocked mutant strains or recombinant bacterial cells are used as biocatalysts for synthetic purposes. We characterized the cis-dihydroxylation of cinnamonitrile by chlorobenzene dioxygenase (CDO) in recombinant E. coli on different reaction scales. The absolute stereochemistry of the product was determined to be trans-3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-acrylonitrile. The cells showed a maximum specific activity of 3.76 U/g cdw in shake-flask experiments. Stable expression of the dioxygenase genes in E. coli JM101 (pTEZ30) resulted in increasing volumetric productivities. The maximum volumetric productivities of 80 and 92 mg product/L/h were achieved on 2-L and 30-L scales, respectively. The specific growth rate correlated with the volumetric productivity during the biotransformations. An average volumetric productivity of 40 mg product/L/h in reactors on 2-L and 30-L scales resulted in 0.96 and 16.4 g of isolated product at the end of the biotransformations. This points out the need for metabolically active cells and controllable expression systems for achieving high volumetric productivities for cofactor dependent biooxidations. We have now applied this concept for the asymmetric dihydroxylation of the non-natural substrate cinnamonitrile using multicomponent CDO in tailored E. coli JM101 in long-term reactions.

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High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system

Cited by 49 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

Whole-Cell Biocatalysis for 1-Naphthol Production in Liquid-Liquid Biphasic Systems

Asymmetric Dihydroxylation of Cinnamonitrile to trans‐3‐[(5S,6R)‐5,6‐Dihydroxycyclohexa‐1,3‐dienyl]‐acrylonitrile using Chlorobenzene Dioxygenase in Escherichia coli (pTEZ30)

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