Biosynthesis of synthons in two-liquid-phase media

Wubbolts, Marcel; Favre-Bulle, Olivier; Witholt, Bernard

doi:10.1002/(sici)1097-0290(19961020)52:2<301::aid-bit10>3.0.co;2-m

Cited by 95 publications

(52 citation statements)

References 29 publications

(36 reference statements)

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“…Experiments were started by batch cultivation in 1 liter MS medium in a stirred tank reactor with a total volume of 3 liters (54,75). The dissolved oxygen tension (DOT) was determined with an autoclavable amperometric probe (Mettler Toledo, Greifensee, Switzerland), and the pH was automatically kept at 7.1 by feeding 25% NH 4 OH.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…Chiral alkane epoxides are synthons for a variety of different syntheses. The alkane hydroxylase and xylene oxygenases of P. putida are versatile monooxygenases for stereo-and regioselective oxidation of aliphatic and aromatic hydrocarbons (600,672). Epoxide hydrolases can transform the resulting epoxides into diols (353).…”

Section: Enzymatic Upgrading Of Petroleum Fractions and Pure Hydrocarmentioning

confidence: 99%

“…To overcome degradation of the products of the xylene monooxygenase from P. putida, the xyl genes from the TOL plasmid encoding this enzyme may be inserted in E. coli (672). P. oleovorans can convert octane to medium-chain poly(3-hydroxyalkanoates), with potential for use in biodegradable plastics (247), at projected large-scale manufacturing costs of less than US$10 per kg (348).…”

Section: Enzymatic Upgrading Of Petroleum Fractions and Pure Hydrocarmentioning

confidence: 99%

Recent Advances in Petroleum Microbiology

Hamme¹,

Singh

Ward

2003

Microbiol Mol Biol Rev

1,193

640

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Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times (<60 s) are being used effectively. Microbes are being injected into partially spent petroleum reservoirs to enhance oil recovery. However, these microbial processes have not exhibited consistent and effective performance, primarily because of our inability to control conditions in the subsurface environment. Microbes may be exploited to break stable oilfield emulsions to produce pipeline quality oil. There is interest in replacing physical oil desulfurization processes with biodesulfurization methods through promotion of selective sulfur removal without degradation of associated carbon moieties. However, since microbes require an environment containing some water, a two-phase oil-water system must be established to optimize contact between the microbes and the hydrocarbon, and such an emulsion is not easily created with viscous crude oil. This challenge may be circumvented by application of the technology to more refined gasoline and diesel substrates, where aqueous-hydrocarbon emulsions are more easily generated. Molecular approaches are being used to broaden the substrate specificity and increase the rates and extents of desulfurization. Bacterial processes are being commercialized for removal of H2S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments

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“…Nonconventional reaction media such as an aqueousorganic two-liquid-phase system are promising alternatives (8,40). A second immiscible phase can act as a reservoir for substrate and products, regulating the concentration of such compounds in the biocatalyst microenvironment, minimizing toxicity and simplifying product recovery (24,60,63).…”

mentioning

confidence: 99%

Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis

Bühler

Witholt

Hauer

et al. 2002

Appl Environ Microbiol

Self Cite

100

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Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275: 10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based twoliquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.During the last few decades, the microbial degradation pathways of aromatic and aliphatic hydrocarbons have received a lot of scientific interest because of the high potential of the enzyme systems involved for environmental (43) and preparative applications (38, 59). These pathways are usually initiated by an oxygenase-catalyzed chemo-, regio-, and stereoselective hydroxylation of the hydrocarbons, a reaction for which often no organic chemical counterpart is known (9, 13).The xylene degradation pathway of Pseudomonas putida mt-2 and its initiating oxygenase, the xylene monooxygenase (XMO), are among the best-studied examples of aromatic hydrocarbon degradation (5,36,57,61). The enzymes for xylene degradation are encoded on a catabolic plasmid, the TOL plasmid pWW0. XMO is the first enzyme in the upper degradation pathway for toluene and xylenes, in which a carboxylic acid is formed (1,16,58). The upper pathway also involves benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, which catalyze the oxidation of benzyl alcohols via benzaldehydes to benzoic acids (46-48). The carboxylic acid is then transformed to substrates of the Krebs cycle through the meta cleavage pathway (10,14,36,55).XMO consists of two polypeptide subunits, encoded by xylM and xylA (16,52). XylA, the NADH:acceptor reductase component, is an electron transport protein transferring reducing equivalents from NADH to XylM (45). XylM, the hydroxylase component, is located in the membrane, and its activity depends on phospholipids and ferrous ion, with a pH optimum of 7 (44, 62).The substrate spectrum of XMO was investigated, wi...

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Biosynthesis of synthons in two-liquid-phase media

Cited by 95 publications

References 29 publications

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

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

Recent Advances in Petroleum Microbiology

Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis

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