Microbial production of epoxides

Details Microbial production of epoxides Microbial production of epoxides

2001-03-12

2003-06-10

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S252100, C435S822000

Reexamination Certificate

active

06576449

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to microbial production of chemicals. More particularly, the invention relates to methods for microbial production of epoxides and hydroxylated chemical feedstocks in a multiphase reactor using gaseous inputs as precursors and for regeneration of reducing equivalents.
Methane monooxygenase (EC 1.14.13.25) is a multicomponent enzyme, produced by methanotrophic bacteria, which catalyzes the incorporation of atmospheric oxygen into methane to form methanol. This is the first in a series of reactions that ultimately provide energy and carbon to the cell. Methanol dehydrogenase further oxidizes methanol to form formaldehyde. Although reducing equivalents are consumed initially by the monooxygenase, the methanotrophic bacteria regenerate NADH by subsequent oxidations mediated by formaldehyde and formate dehydrogenases, respectively, the terminal product being CO
2
(for an excellent review, see J. D. Lipscomb, Biochemistry of the Soluble Methane Monooxygenase, 48 Annu. Rev. Microbiol. 371-399 (1994)).
Studies of methane monooxygenase refer to both a soluble (sMMO) and membrane-bound or particulate (pMMO) enzyme. The prevalent form in type II methanotrophs, such as
Methylosinus trichosporium
OB3b, and type X methanotrophs, such as
Methyloccus capsulatus
, is regulated in some fashion by the concentration of copper in the growth medium. K. J. Burrows et al., Substrate Specificities of the Soluble and Particulate Methane Mono-oxygenases of
Methylosinus trichosporium
OB3b, 130 J. Gen Microbiol. 3327-3333 (1984); S. H. Stanley et al., Copper Stress Underlies the Fundamental Change in Intracellular Location of Methane Monooxygenase in Methane-oxidizing Microorganisms: Studies in Batch and Continuous Culture, 5 Biotechnol. Lett. 487-492 (1983). Type I methanotrophs such as
Methylomonas methanica
typically express only pMMO, although one recent isolate has been found to be an exception to that generalization. S. -C. Koh et al., Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph,
Methylomonas methanica
68-1, 59 Appl. Environ. Microbiol. 960-967 (1993).
Components of sMMO from
Methylosinus trichosporium
OB3b that retained a high specific activity were initially purified by B. G. Fox et al., Methane Monooxygenase from
Methylosinus trichosporium
OB3b. Purification and Properties of a Three-component System with High Specific Activity from a Type II Methanotroph, 264 J. Biol. Chem. 10023-10033 (1989). The enzyme comprises a 245 kDa hydroxylase containing a hydroxo-bridged dinuclear iron cluster at the active site of methane oxidation, a 15.8-kDa component B, and a 38.4-kDa iron-sulfur reductase with a flavin prosthetic group.
Although not supporting growth, sMMO and pMMO will, in addition to methane, adventitiously oxidize a variety of alkanes and alkenes. C. T. Hou et al., Microbial Oxidation of Gaseous Hydrocarbons: Epoxidation of C
2
to C
4
n-alkenes by Methylotrophic Bacteria, 38 Appl. Environ. Microbiol. 127-134 (1979); D. I. Stirling et al., A Comparison of the Substrate and Electron-donor Specificities of the Methane Monooxygenases from Three Strains of Methane-oxidizing Bacteria, 177 J. Biochem. 361-364 (1979); H. Dalton, Oxidation of Hydrocarbons by Methane Monooxygenases from a Variety of Microbes, 26 Adv. Appl. Microbiol. 71-87 (1980). The substrate range of sMMO, however, also includes aromatic and alicyclic compounds, K. J. Burrows et al., supra; J. Colby et al., The Soluble Methane Monooxygenase of
Methylococcus capsulatus
(Bath). Its Ability to Oxygenate n-Alkanes, n-Alkenes, Ethers, and Alicyclic, Aromatic, and Heterocyclic Compounds, 165 J. Biochem. 395-402 (1977), ethers and heterocyclic compounds, J. Colby et al., supra, and halogenated aromatics and alkenes, J. Green & H. Dalton, Substrate Specificity of Soluble Methane Monooxygenase. Mechanistic Implications. 264 J. Biol. Chem. 17698-17703 (1989).
Currently, there is considerable interest in enzyme function in non-aqueous solvents (for reviews, see J. S. Dordick, Enzymatic Catalysis in Monophasic Organic Solvents, 11 Enzyme Microb. Technol. 194-211 (1989); P. Nickolova & O. P. Ward, Whole Cell Biocatalysis in Nonconventional Media, 12 J. Ind. Microbiol. 76-86 (1993); G. J. Salter & D. B. Kell, Solvent Selection for Whole Cell Biotransformations in Organic Media, 15 Crit. Rev. Biotechnol. 139-177 (1995)). Several beneficial and unexpected modifications of typical enzyme behavior were noted in such systems. These include enhanced enzyme activity, R. Batra & M. N. Gupta, Enhancement of Enzyme Activity in Aqueous-organic Solvent Mixtures, 16 Biotechnol. Lett. 1059-1064 (1994), increased thermostability, and alterations in substrate specificity, A. Zaks & A. M. Klibanov, Enzymatic Catalysis in Organic Media at 100° C., 224 Science 1249-1251 (1984).
Recent efforts have concentrated on applications that may be less amenable to strictly aqueous approaches, since the substrates are largely insoluble in water. Examples of such an approach include degradation of sparingly soluble xenobiotics, M. Ascon-Cabrera & J. -M. Lebeault, Selection of Xenobiotic-degrading Microorganisms in a Biphasic Aqueous-organic System, 59 Appl. Environ. Microbiol. 1717-1724 (1993), petroleum fuel desulfurization, W. R. Finnerty, Organic Sulfur Biodesulfurization in Non-aqueous Media, 72 Fuel 1631-1634 (1993), and coal modification or solubilization, E. S. Olson et al., Non-aqueous Enzymatic Solubilization of Coal-derived Materials, 72 Fuel 1687-1693 (1993); C. D. Scott et al., The Chemical Modification of Enzymes to Enhance Solubilization in Organic Solvents for Interaction with Coal, 72 Fuel 1695-1700 (1993).
Production of propylene oxide from propylene for use as a chemical feedstock has been investigated using immobilized whole cells. L. E. S. Brink & J. Tramper, Production of Propene Oxide in an Organic Liquid-phase Immobilized Cell Reactor, 9 Enzyme Microb. Technol. 612-618 (1987); C. T. Hou, Propylene Oxide Production from Propylene by Immobilized Whole Cells of Methylosinus sp. CRL-31 in a Gas-solid Bioreactor, 19 Appl. Microbiol. Biotechnol. 1-4 (1984).
T. R. Clark & F. F. Roberto,
Methylosinus trichosporium
OB3b Whole-cell Methane Monooxygenase Activity in a Biphasic Matrix, 45 Appl. Microbiol. Biotechnol. 658-663 (1996), demonstrated soluble methane monooxygenase activity in a two-phase (biphasic) matrix comprising a buffered aqueous phase and 2,2,4-trimethylpentane (isooctane) using reconstituted whole-cell preparations of lyophilized
Methylosinus trichosporium
OB3b. The rate of conversion of gaseous propylene to propylene oxide, a non-metabolized liquid, was used as the primary measure of enzymatic activity. Appreciable soluble methane monooxygenase activity was detected when the volume of the aqueous phase represented at least 1% of the total volume, although the initial rate of product formation did increase as the volume of the aqueous phase increased. In comparison to the aqueous system, the specific rate and yields in the biphasic system were much less sensitive to increases in the concentrations of formate and protein (i.e., the methane monooxygenase). There was some evidence, however, that the enzyme system was more stable in the biphasic matrix, since the rate of propylene oxide formation remained linear for an extended period of time. V
(app.)
in the biphasic system decreased by a factor of 0.6 relative to the same parameter in the aqueous system. Conversely, K
m(app.)
for propylene was 1.6 times greater in the biphasic system. Hence, the apparent catalytic efficiency in the aqueous system was four times that in the biphasic system, as indicated by a decrease in the corresponding ratios of V
(app.)
to K
m(app.)
.
In view of the foregoing, it will be appreciated that providing a method for microbial production of epoxides and hydroxylated hydrocarbons would be a significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for microbial production of

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