An enhanced toluene dioxygenase platform for the production of cis-1,2-dihydrocatechol in Escherichia coli BW25113 lacking glycerol dehydrogenase activity

Chapter 8 - Production of fine chemicals from food wastes

The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons. Toluene dioxygenase was shown to oxidize 6-fluoroindole to a meta-stable fluorescent product, 6-fluoroindoxyl. The fluorescent output allowed monitoring relative levels of tod operon induction in whole cells using microtiter well plates. Mono- and polyfluorinated aromatic compounds were shown to induce toluene dioxygenase, in some cases to a greater extent than compounds serving as growth substrates. Compounds that are oxidized by toluene dioxygenase and undergoing defluorination were shown to induce their own metabolism. 1,2,4-Trifluorobenzene caused significant induction and computational modelling indicated productive binding to the TodS sensor domain of the TodST regulator. Toluene dioxygenase also showed preferential binding of 1,2,4-trifluorobenzene such that defluorination was favoured. Fluorinated aliphatic compounds were shown to induce toluene dioxygenase. An aliphatic ether with seven fluorine atoms, 1,1,1,2-tetrafluoro-2-trifluoromethoxy-4-iodobutane (TTIB), was an excellent inducer of toluene dioxygenase activity and shown to undergo transformation in cultures of P. putida F1.

Cytochrome P450 monooxygenases in whole-cell format: Application notes from a biotechnological perspective.

Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water.

Eleutherococcus koreanum Nakai is an endemic medicinal plant grown in Jeju Island, South Korea. Extracts from this plant have traditionally been used in Korea as a tonic and for treating rheumatism, diabetes and hepatitis. These extracts contain many useful bioactive substances, particularly eleutherosides, chlorogenic acid and other phenolic compounds. The quality and quantity of major bioactive compounds from naturally grown plants are greatly affected by harvest time and environmental conditions. Therefore, determination of suitable growing and harvesting conditions is necessary to achieve reliable supply of E. koreanum-based bioactive compounds for commercial use. To establish an efficient method for the year-round production of bioactive compounds, adventitious roots of E. koreanum were tested with various physical and chemical factors (inoculum density, aeration volume, salt strength, nitrogen source, and sucrose concentration) that affect root biomass and production of target bioactive compounds. Root biomass, concentrations of five target bioactive compounds (eleutherosides B and E, chlorogenic acid, total phenolics, and flavonoids), physiological responses of the adventitious roots and other environmental conditions in the culture vessels were determined. In addition, responses of roots subjected to chemical elicitors (methyl jasmonate and salicylic acid) were evaluated to determine a strategy for enhancing the final production of bioactive compounds. Finally, we compared the contents of bioactive compounds and typical DNA histograms for the adventitious roots and naturally grown plants to verify the competitive ability and genetic stability of cultured E. koreanum adventitious roots. The development of in vitro culture protocol, which controls the quality and quantity of elicited bioactive compounds, will be beneficial for the pilot-scale production of E. koreanum-based bioactive compounds for commercial use.

Rieske dioxygenases are multi-component enzyme systems, naturally found in many soil bacteria, that have been widely applied in the production of fine chemicals, owing to the unique and valuable oxidative dearomatization reactions they catalyze. The range of practical applications for these enzymes in this context has historically been limited, however, due to their limited substrate scope and strict selectivity. In an attempt to overcome these limitations, our research group has employed the tools of enzyme engineering to expand the substrate scope or improve the reactivity of these enzyme systems in specific contexts. Traditionally, enzyme engineering campaigns targeting metalloenzymes have avoided mutations to metal-coordinating residues, based on the assumption that these residues are essential for enzyme activity. Inspired by the success of other recent enzyme engineering reports, our research group investigated the potential to alter or improve the reactivity of Rieske dioxygenases by altering or eliminating iron coordination in the active site of these enzymes. Herein, we report the modification of all three iron-coordinating residues in the active site of toluene dioxygenase both to alternate residues capable of coordinating iron, and to a residue that would eliminate iron coordination. The enzyme variants produced in this way were tested for their activity in the cis-dihydroxylation of a small library of potential aromatic substrates. The results of these studies demonstrated that all three iron-coordinating residues, in their natural state, are essential for enzyme activity in toluene dioxygenase, as the introduction of any mutations at these sites resulted in a complete loss of cis-dihydroxylation activity.

Plants are surrounded by complex set of environmental conditions which are categories into abiotic and biotic factors. The growth, development and overall survival of plants are regulated by the intensity of stresses exerts by biotic and abiotic factors of the environment. Stresses act individually or co-occurrence of different stresses at a time on plants and its study is challenging and complex process. However, the response of plants to the stresses is equally complex. Plant produces bioactive compounds in response of stresses as a stress tolerance. These bioactive compounds are also called secondary metabolites which plays significant role in the adaptation of plants to the stress condition and changing environment.In the present chapter emphasis was given on the study of production of different bioactive compounds in response to different abiotic stresses. The subtitles, types of bioactive compounds studied in plants showed wide variety of bioactive compound produce by plants in response to stress. Plant encounter number of abiotic stresses in plants like cold, heat drought, salinity, temperature and flood etc. Another subtitle, the impacts of different abiotic stresses on production of bioactive compounds in plants showed, the species of the families like Asteraceae, Papvaraceae, Apocynaceae, Lamiaceae, Brassicaceae, Malvaceaeetc shows responses to abiotic stresses and effect of abiotic stresses on productivity of agricultural crop plants. Overall study concluded that in this technological era several changes in environmental condition exert tremendous pressure of abiotic stresses on plants especially interfering with the productivity of agricultural land plants. Review shows that many plants cope up with the abiotic stresses by synthesizing bioactive compounds as a stress tolerance, but some plants fail to acclimatize and eventually die.

Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.

ABSTRACTPerfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising perfluorodiethers is the 2,2-fluoro-1,3-benzodioxole (DFBD) moiety. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. Pseudomonas putida F1 catalyzed defluorination of DFBD at an initial rate of 2,100 nmol/h per mg cellular protein. This is orders of magnitude higher than previously reported microbial defluorination rates with multiply fluorinated carbon atoms. Defluorination rates declined after several hours, and the medium darkened. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. Defluorination required only toluene dioxygenase. Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. The dihydrodiol could be oxidized to 4,5-dihydroxy-DFBD via the dihydrodiol dehydrogenase from P. putida F1. The dihydrodiol dehydrated with acid to yield a mixture of 4-hydroxy-DFBD and 5-hydroxy-DFBD. All those metabolites retained the difluoromethylene group; no fluoride or dark color was observed. The major route of DFBD-4,5-dihydrodiol decomposition produced fluoride and 1,2,3-trihydroxybenzene, or pyrogallol, and that was shown to be the source of the dark colors in the medium. A mechanism for DFBD-4,5-dihydrodiol transformation to two fluoride ions and pyrogallol is proposed. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment.

Rosa rugosa Thunb. is a popular ornamental and medicinal plant native to eastern Asia. In this study, a successful bioreactor culture system was established for the production of secondary metabolites of rugosa roses. We tested different concentrations and combinations of plant hormones in growth media for maximum shoot proliferation and production of bioactive compounds, different bioreactor systems for maximum biomass production and production of bioactive compounds, and different ratios of nitrogen sources for maximum shoot growth and accumulation of bioactive compounds. For multiple shoot proliferation, Murashige and Skoog (MS) medium was used, supplemented with different concentrations and combinations of plant hormones: 6-benzylaminopurine (BA; 0-13.2 µM), thidiazuron (TDZ; 0-13.5 µM), and indole butyric acid (IBA) at 2.5 µM, used alone or in a combination of IBA with BA or TDZ. Rapid micropropagation of multiple shoots of rugosa roses was successfully achieved using shoot tips explanted in semisolid MS medium supplemented with 4.4 µM BA. The average number of shoots grown was 15.6 per explant and the maximum shoot length was 2.7 cm at 8 weeks of culture. To investigate the effect of nitrogen sources on shoot growth and bioactive compound accumulation, shoots were treated with different ratios of nitrogen sources (NH4+:NO3-) for 1 week after 7 weeks of shoot culture. Next, to scale up biomass production for the generation of useful phytochemicals, multiple-shoot cultures were developed in large-scale bioreactors. Four bioreactor systems were used: continuous immersion bioreactor (CIB), continuous immersion bioreactor with net (CIB-N), temporary immersion bioreactor (TIB), and temporary immersion bioreactor with net (TIB-N). Solid and liquid media were used as controls. Of the different bioreactor types, the CIB system produced the highest biomass, followed by the TIB system. Multiple shoots grown in the CIB system resulted in the accumulation of 39.21 mg·g-1 dry weight (DW) of total phenolics and 13.28 mg·g-1 DW of total flavonoids. The productivity of total phenolics and flavonoids was highest in the shoots harvested from the CIB system. The results of this study suggest that multiple shoots of rugosa roses can be used in commercial-scale bioreactors to produce useful bioactive compounds for the pharmaceutical and cosmetic industries.

Orchids are considered to be the most highly differentiated and horticulturally important plants. Additionally, orchids have been used as traditional medicines in many countries since ancient times. Different organs of orchid plants, such as leaves, stems, and bulbs, contain various biologically active substances such as alkaloids, phenolics, terpenoids, and derivatives thereof. These bioactive compounds are secondary metabolites synthesized from primary metabolites of plants. To improve the utility of orchids, it is important to identify the pharmacological function of these plants. Moreover, the establishment of technologies for the large-scale production of a biomass of orchid plants using field cultivation or biotechnological methods is needed to prevent the overaccumulation of these plants in the natural state, which would ultimately result in these plants being enlisted as endangered species. Among various factors affecting the in vitro culture of medicinal orchids, growth regulators, light, sugar and activated charcoal are the most important. To establish a successful mass production system, it is necessary to determine the optimal concentration at which these factors maximize the production of biomass and bioactive compounds. In this chapter, we provide an overview of medicinal orchids and review recent studies on the in vitro production of biomass and bioactive compounds from these plants.

Chapter 9 - Mixing with a pfaudler type impeller; the effect of micromixing on reaction selectivity in the production of fine chemicals

The degradation of toluene by Pseudomonas putida F1 and of chlorobenzenes by Burkholderia sp. strain PS12 is initiated by incorporation of dioxygen into the aromatic nucleus to form cis-dihydrodihydroxybenzenes. Toluene-grown cells of P. putida F1 and 3-chlorobenzoate-grown cells of Burkholderia sp. strain PS12 were found to monooxygenate the side chain of 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols. Further metabolism of these products was slow, and the corresponding chlorobenzoates were usually observed as end products, whereas the 3-chlorobenzoate produced from 3-chlorotoluene in Burkholderia sp. strain PS12 was metabolized further. Escherichia coli cells containing the toluene dioxygenase genes from P. putida F1 oxidized 2- and 3-chlorotoluene to the corresponding chlorobenzyl alcohols as major products, demonstrating that this enzyme is responsible for the observed side chain monooxygenation. Two methyl- and chloro-substituted 1,2-dihydroxycyclohexadienes were formed as minor products from 2- and 3-chlorotoluene, whereas a chloro- and methyl-substituted cyclohexadiene was the only product formed from 4-chlorotoluene. The toluene dioxygenase of P. putida F1 and chlorobenzene dioxygenase from Burkholderia sp. strain PS12 are the first enzymes described that efficiently catalyze the oxidation of 2-chlorotoluene.

Toluene-induced cells of Pseudomonas putida F1 removed trichloroethylene from growth media at a significantly greater initial rate than the methanotroph Methylosinus trichosporium OB3b. With toluene-induced P. putida F1, the initial degradation rate varied linearly with trichloroethylene concentration over the range of 8 to 80 microM (1.05 to 10.5 ppm). At 80 microM (10.5 ppm) trichloroethylene and 30 degrees C, the initial rate was 1.8 nmol/min per mg of total cell protein, but the rate decreased rapidly with time. A series of mutant strains derived from P. putida F1 that are defective in the todC gene, which encodes the oxygenase component of toluene dioxygenase, failed to degrade trichloroethylene and to oxidize indole to indigo. A spontaneous revertant selected from a todC culture regained simultaneously the abilities to oxidize toluene, to form indigo, and to degrade trichloroethylene. The three isomeric dichloroethylenes were degraded by P. putida F1, but tetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures.

Chapter 11 - Production of fine chemicals from renewable feedstocks through the engineering of artificial enzyme cascades