Microorganisms with ability to degrade indole and enzymes...

Details Microorganisms with ability to degrade indole and enzymes... Microorganisms with ability to degrade indole and enzymes...

2000-05-14

2003-11-04

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

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

Using a micro-organism to make a protein or polypeptide

C435S069100, C435S068100, C435S243000, C435S252100, C435S253300, C530S350000

Reexamination Certificate

active

06642031

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the isolation of a novel Pseudomonas species and identification of novel heterocyclic dioxygenases derived from this organism. The heterocyclic dioxygenase described herein is useful in the production of indigo in recombinant organisms.
BACKGROUND OF THE INVENTION
Pseudomonads are a diverse group of organisms capable of mineralizing biotic (e.g., camphor), xenobiotic (various chlorophenyl and biphenyl compounds) and fossil organic molecules (such as toluene or naphthalene). These capabilities are usually encoded by groups of genes collected in operons on extrachromosomal elements. A number of different enzyme classes are involved in the initial oxidation of these compounds. For example, camphor is degraded by a P450 monooxygenase while toluene, chlorobiphenyl and biphenyl, and naphthalene are oxidized by aromatic dioxygenases.
In the era of molecular biology, it was discovered that when certain aromatic dioxygenases were cloned into
Escherichia coli
and these were subsequently grown on a rich medium, colonies turned blue. It was later determined that the blue color was due to the conversion of tryptophan in the medium to indole by
E. coli
tryptophanase and its subsequent oxidation by the aromatic dioxygenases to indolediol, which in turn, in the presence of molecular oxygen, spontaneously further oxidized to indigo, leading to the observed blue coloration. Dioxygenases whose primary substrates are heterocyclic compounds, particularly where the substrate is indole, have not previously been characterized in detail. The heterocyclic dioxygenase reported here is capable of indole oxidation, leading to indigo formation.
The blue dye indigo is one of the oldest dyestuffs known to man. Its use as a textile dye dates back to at least 2000 BC. Until the late 1800s indigo, or indigotin, was principally obtained from plants of the genus Indigofera, which range widely in Africa, Asia, the East Indies and South America. As the industrial revolution swept through Europe and North America in the 1800s, demand for the dye's brilliant blue color led to its development as one of the main articles of trade between Europe and the Far East. In 1883, Adolph von Baeyer identified the formula of indigo: C
16
H
10
N
2
O
2
. In 1887, the first commercial chemical manufacturing process for indigo was developed. This process, still in use today, involves the fusion of sodium phenylglycinate in a mixture of caustic soda and sodamide to produce indoxyl. The process' final product, indoxyl, oxidized spontaneously to indigo by exposure to air.
Current commercial chemical processes for manufacturing indigo result in the generation of significant quantities of toxic waste products. Obviously, a method whereby indigo may be produced without the generation of toxic by-products is desirable. One such method which results in less toxic by-product generation involves indigo biosynthesis by microorganisms.
Ensley et al. ((1983)
Science
222:167-169) found that a DNA fragment from a transmissible plasmid isolated from the soil bacterium
Pseudomonas putida
enabled
Escherichia coli
that had been stably transformed with a plasmid harboring the fragment to synthesize indigo in the presence of indole or tryptophan. Ensley et al. postulated that indole, added either as a media supplement or produced as a result of enzymatic tryptophan catabolism, was converted to cis-indole-2,3-dihydrodiol by the previously identified multi-component-enzyme naphthalene dioxygenase (NDO) encoded by the
P. putida
DNA fragment. The indole-2,3-dihydrodiol so produced spontaneously lost water forming indoxyl and was then oxidized to indigo upon exposure to air.
NDO had previously been found to catalyze the oxidation of the aromatic hydrocarbon naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (Ensley et al. (1982)
J. Bacteriol
. 149:948-954). U.S. Pat. No. 4,520,103, incorporated by reference, describes the microbial production of indigo from indole by an aromatic dioxygenase enzyme such as NDO. The NDO enzyme is comprised of multiple components: a reductase polypeptide (Rd, molecular weight of approximately 37,000 daltons (37 kD)); an iron-sulfur ferredoxin polypeptide (Fd, molecular weight of approximately 13 kD); and a terminal oxygenase iron-sulfur protein (ISP). ISP itself is comprised of four subunits having an &agr;
2
&bgr;
2
subunit structure (approximate subunit molecular weights: &agr;, 55 kD; &bgr;, 21 kD). ISP is known to bind naphthalene, and in the presence of NADH, Rd, Fd and oxygen, to oxidize it to cis-naphthalene-dihydrodiol. Fd is believed to be the rate-limiting polypeptide in this naphthalene oxidation catalysis, (see U.S. Pat. No. 5,173,425, incorporated herein by reference, for a thorough discussion of the various NDO subunits and ways to improve them for purposes of indigo biosynthesis).
In addition, aromatic dioxygenases other than NDO may also be useful in the biosynthetic production of indigo, for example, a toluene monooxygenase (TMO) such as that from Pseudomonas (
P. mendocina
) capable of degrading toluene was also able to produce indigo when the culture medium was supplemented with indole. For details, see U.S. Pat. No. 5,017,495, incorporated herein by reference. In principle, any enzyme capable of introducing a hydroxyl moiety into the 3-position of indole to give indoxyl is a candidate for use in the biosynthetic production of indigo. This includes single component flavin containing monooxygenases.
Most, if not all, oxygenases described in the art for use in oxidation of the substrate indole, as for example in the production of indigo, are aromatic oxygenases. While these enzymes have been successfully employed in the synthesis of indigo, there is a need for an enzyme or class of enzymes which have as a primary substrate heterocyclic compounds such as indole. Such heterocyclic oxygenases are believed to be advantageous over aromatic oxygenases in oxidizing indole and ultimately in indigo production.
DEFINITION OF TERMS
The following terms will be understood as defined herein unless otherwise stated. Such definitions include without recitation those meanings associated with these terms known to those skilled in the art.
Tryptophan pathway genes useful in securing biosynthetic indole accumulation include a trp operon, isolated from a microorganism as a purified DNA molecule that encodes an enzymatic pathway capable of directing the biosynthesis of L-tryptophan from chorismic acid. (A. J. Pittard (1987)
Biosvnthesis of Aromatic Amino Acids in Escherichia coli and Salmonella typhimurium
, F. C. Neidhardt, ed., American Society for Microbiology, publisher, pp. 368-394.) Indole accumulation is enabled by modification of one or more of the pathway's structural elements and/or regulatory regions. This modified trp operon may then be introduced into a suitable host such as a microorganism, plant tissue culture system or other suitable expression system. It should be noted that the term “indole accumulation” does not necessarily indicate that indole actually accumulates intracellularly. Instead, this term can indicate that there is an increased flux of carbon to indole and indole is made available as a substrate for intracellular catalytic reactions such as indoxyl formation and other than the formation of L-tryptophan. In the context of this invention, the “accumulated” indole may be consumed in the conversion of indole to indoxyl by an oxygenase such as the aromatic dioxygenase NDO, or an aromatic monooxygenase such as TMO, or it may actually build up intracellularly and extracellularly, as would be the case when the desired end product is indole or one of its derivatives.
A suitable host microorganism or host cell is an autonomous single-celled organism useful for microbial indole and/or indigo production and includes both eucaryotic and procaryotic microorganisms. Such host microorganism contains all DNA, either endogenous or exogenous, required for the production of indole, indoxyl and/or indigo, from glucose, a

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