METABOLICALLY ENGINEERED ESCHERICHIA COLI FOR DEGRADATION
OF TOXIC RESIDUES OF NITRO-PHENOLS AND OTHER AROMATIC
COMPOUNDS IN THE ENVIRONMENT
TECHNICAL FIELD OF THE INVENTION
[0001J This invention relates to production of metabolically engineered E coli with
plasmids that express a novel open reading frame, designated as orf243 of
a small RNA, designated as seco10054A both of which can be used for
biodegradation of p-nitrophenol and other nitro aromatic toxic residues
present in the environment.
2. BACKGROUND AND THE PRIOR ART
10002] Organophosphorus compounds are in widespread use as insecticides and
chemical .warfare agents. These are known as potent cholinesterase ;.
inhibitors exhibiting high level of toxicity to insects and other animals. This
toxicity is due to a decrease in the ability of acety/cholinesterase (an
enzyme and referred to as'cholinesterase'for brevity) to breakdown
acetylcholine, a neurotransmitter which results in excessively high
acetylcholine levels. The organophosphorus compounds have been shown
to effectively block the active sites of acetyl cholinesterase rendering it
... incapable of its normal function.
[0003] With their increased use in agriculture replacing more persistent chlorinated
pesticides as we)) as chemical warfare,'there is enhanced worldwide
interest and concern to find viable methods for bidremediation of these
toxicants in soil as well as in destroying the ever-increasing stockpiles of
chemical warfare agents1 due to their high toxicity after use and associated
risks to human beings
[0004] The organophosphates which shot into prominence and widespread use
during 1970s and 1980s in preference to recalcitrant chlorinated pesticides
are known to have the general structure of (I) as shown in Fig 1 wherein
groups represented by R1, R2 and R3 are alkyl or. aryl. The susceptibility of
O-P bond to hydrolysis in organophosphates by microbial enzymes leading
to separation of thio-phosphate (II) was shown to be the reason for their: •'
reduced persistence in soil environment. This understanding of their
metabolism also led to development of a number of microbial hydrolytic
enzymes for their detection as well as degradation and related waste
detoxification and remediation technologies2. «,«
[0005] A group of structurally unrelated enzymes that cleave the triester linkage
found in both organophosphate (OP) insecticides1 and OP nerve agents are
known as bacterial phosphodiesterases (PTEs) or organo phosphorus ;
hydrolases. Due to their broad substrate range and high catalytic efficiency .
they have been exploited for detection as well as decontamination of
organo phosphorus compounds. The PTEs have been classified into three ; '
main groups j) the organophosphate hydrolases (OPHs), ii) methyl
parathion hydrolases (MPHs), and iii) organophosphate acid anhydrases
(OPAAs)34.
[0006] The success of enzymatic hydrolysis of toxic organophosphates, however,
besides the ease with which the cleavage of O-P bond takes place also
depends on the completion of metabolic pathways including TCA cycle
ending in the release of innocuous molecules like carbon dioxide and water.
There are quite a few organophosphorus pesticides in which the group R1
is an aromatic ring substituted with various functional groups. For example,
parathion, methyl parathion, fenitrothion etc. have nitro benzene rings for
R1 in (I) which in the first step of hydrolysis release p-nitrophenols (PNPs)
as metabolites. PNRs.are known as potent toxic entities in themselves and
mayadopt different, pathways: fpc^ their further: degradation. Numerous..
studies on the biodegradation of 2- and 4-nitrophenol indicate the isomers
to be inherently biodegradable in water under aerobic conditions.
Mineralization, of nitrophenols under anaerobic conditions in subsoil, • ••:•••
however, may require exte,ndeci. adaptation of microbial communities4. .
[0007] Nitrophenols produced through the above referred metabolic pathways of
organophosphorus insecticides and nerve agents are not of only concern.
Large quantities of PNP and other nitraphenols are industrially produced for
use as intermediates for production of drugs, dyestuffs, pigments, rubber?
chemicals, insecticides and fungicides. p-Nitrophenol is also used
extensively in leather tanning. Safe disposal of nitrophenol residues from
these industries is also therefore a major concern.
[0008] Incineration under controlled conditions seems to be the method of choice
for disposal of wastes containing nitraphenols though there are also other
known methods, such as biological treatment with powdered activated
carbon and activated sludge, oxidation by passing ajr at 275°C through the
aqueous waste, bioremediation with oxygen releasing agents5 etc.
Guidelines for maximum daily effluent discharge of these toxic nitraphenols
per liter of waste water are also operational which concerned industries are
bound to adhere to6. US4197198 claims a highly efficacious method of
decomposition of nitraphenols and nitrocresols in an aqueous solution of
chlorine dioxide which may not be quite suitable for remediating rhet
nitraphenols dispersed in environment jn low concentrations.
[0009] Thus, there is continuous need for evolving an efficient and cost-effective
method of elimination of toxic residues of nitraphenols in industrial wastes
and the soil environment which this invention discloses. The key to the
present invention lies in the discovery of a novel 'open reading frame',
5 Heitkamp, M., Bioremediation J. (1997), 1(2), 105-114. '
8 Hazardous Substances Data Bank. National Library of Medicine, National Toxicology Information
Program, Bethesda, MD. September 11, 1989.
designated as orf243 as part of hydrolytic enzymes of various unrelated soil
bacteria capable of countering the toxic effects of OP insecticide.
[00010] There are a number of genes involved in organophosphorus pesticide
degradation that have been identified, cloned and characterized. The
associated proteins are easily expressed and upstream and downstream
processes for protein production are well characterized in the literature7,8.
In previous studies of an organophosphate degradation gene cluster (opd
island), the inventors discovered this unique open reading frame (orf243)
which was successfully cloned and, expressed in E.: coli. It was found that
the recombinant E coli cells could grow ori p-nitrophenol (PNP) as sole
carbon source, an unexpected phenomenon that prompted the inventors to
develop this invention. r+.
[00011] It was further found that this phenomenon in recombinant E coli is due to
the induced expression of a novel small RNA, secol0054A, when orf243,
an esterase, is expressed. The experimental evidence of the existence of
orf243 and the rationale of its role in the metabolic pathways in expressing
a small RNA in the recombinant microorganism that regulates the carbon
catabolism in favour of p-nitrophenol will be fully understood as disclosed
and described subsequently in this invention.
7 Singh, B.K Organophosphorus-degrading bacteria: ecology and industrial applications. Nat
Rev Microbiol 7, 156-64(2009).
Theriot, CM. and A.M. Grunden. "Hydrolysis of organophosphorus compounds by microbial enzymes."
Applied Microbiology and Biotechnology Mini 100012] Itmust, however, be appreciated that this invention is absolutely hovel
describes for the first time a use of a recombinant microorganism for
.. elimination of toxic residues of p-nitrophenol from contaminated ecosystem.
Thus, a recombinant microorganism that is easily grown and capable of
overexpressing orf243 or seco10054A is the product of this invention along
-with a process of its preparation.
2. OBJECT OF INVENTION
[00013] In order to resolve the aforementioned problems associated with handling
the toxic residues of industrially produced nitrophenols and other nitro
aromatics ending up in the soil environment and water streams as well as
organophosphorus pesticides apd nerve agents; generating the metabolic
residues of nitrophenols, it is the principal object and advantage of the
present invention to provide a metabolically engineered E coli cell that is
capable of surviving on nitrophenols and other nitro aromatics as a source
of carbon, thereby eliminating these toxic residues.
[00014] It is an additional object and advantage of the present invention to provide a
method for making a metabolicaUy engineered E. coli cell, which meets the ,
above objective In a cost-effective manner.
3. SUMMARY OF INVENTION
[00015] Having discovered a novel open reading frame, orf243m opd gene art&ce
getting an insight about its role in degradation of organophosphate
insecticides with hydrolytic enzymes, metaboVically engineered £ coli
.. MG1655 cells were prepared that were capable, pf expressing orf243. it has
been demonstrated experimentally that these cells can grow exclusively on
PNP as a source of carbon consuming molar requirement of oxygen and
releasing corresponding amount of nitrite into the culture.
[00016] Further since Orf243 shows no direct activity on PNP, it was apparent that
its influence on PNP catapolism takes place through induction of novel
proteins or pathways. From further work on the engineered cells through
proteome map and expression profiling, it was found that there was a
significant up regulation of phenyl propionate (PP) and hydroxyl phenyl
propionate (HPP) pathway enzymes. The heat map generated from micro
array data revealed that i) there is a significant decrease in transcription of
genes for glycolysis and TCA cycle enzymes, and ii) there is a significant
a increase in the expressionof genes codingifor.alternative carbon catabolic
pathways e.g., propionate cataboJic pathway, methyl citrate cycle (MCC)
pathway and glyoxylate pathway. These changes in metabolic pathways are !'•
extremely significant since the end products generated from PNP
degradation, such as succiqyl-CoA.ancl.acetylrGpA, gain direct entry into
the TCA and glyoxylate pathways
[00017] With the above insight into the changes in metabolic pathways for PNP
degradation with engineered cell expressing 6rf243, identification of 'master
switch' in catabblic pathway regulation was the next task. Expression
. profiling of small RNAs through heat map led to the discovery of a unique
sRNA, designated as seco10054A playing this crucial role.
[00018] Hypothesizing that sRNA, seco10054A is indeed a key regulator, '''"•"
metabolically engineered E coli MG1655 expressing seco10054A has been
prepared and it has been demonstrated that the these cells can be grown
using PNP as sole source of carbon and can therefore be used as a tool for
detoxification of PNP residues.
4. STATEMENT OF INVENTION
[00019] The new sRNA discovered in the present invention specifically promotes
nitrophenol metabolism by inhibiting conventional catabplic pathways like
glycolysis and TCA cycle. The invention describes the constitutive
expression of sRNA and.meta
tool for elimination of nrtrophenols and other aromatic compounds.
5. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING(S)
[00020] Having thus described the invention in general terms, reference will now be
made to the accompanying drawings, wherein:
FIG. 1 illustrates the reaction sequence of metabolic process of enzymatic
hydrolysis of organo phosphates (as described in the 'background').
FIG. 2 shows the PNP catabolic pathway in E. coli MG1655 (pSDP10);
heat maps represent differential expression of genes encoding
glycolysis, glyoxylate and TCA cycle enzymes. The qPCF* data and
a portion of 2-D gel images are also shown to authenticate down
/up regulation of pathway-specific enzymes.
FIG. 3 illustrates the experimental evidence of the growth of metabolically .
engineered E coli (of this invention) along with depletion of -
nttrophenol concentration in the culture.
FIG. 4 illustrates the experimental evidence of the re/ease and growth of
nitrite along with depletion of nitrophenoi concentration in the
culture.
6. DETAILED DESCRIPTION OF THE INVENTION
[00021] The details of the inventive steps; along with preferred embodiments of this
invention as well as the definitions of key terms for their intended meaning
wilt now be described that address the issues raised with risks and hazards
of toxic residues of nitrophenols and other aromatics in the environment that
need to be eliminated efficiently and cost-effectively not yet known in the
prior art; There could be several other possible embodiments of this
invention using varied forms of the essential components of this invention in
terms of the key inventive steps described herein and well understood by
those skilled in the art. Therefore, the scope and objective of the patent are
not limited;to the embodiments described as examples.
[00022] Definitions,
"Catabolism" - generally referring to breakdown of complex molecules of
carbon compounds by living organisms to form simpler ones tooetlw with
the release of energy specifically refers in this invention to similar processes .
initiated by engineered or reference cells with respect to breakdown of p-nitrophenol and other related aromatic compounds..
"Cloning"- refers to the production of a new nucleic acid molecule through
the ligation of previously unlinked nucleic acid pieces to one anothen A
molecule produced by such ligation is considered a "clone" for the purposes
of the present application, even before it has been replicated.
"Degradation" - is meant here as breaking of macromolecules into small.
molecule; the term "microbial degradation" is used to emphasize breaking of
macromolecules into small molecules involving microorganisms."
"Down,/ up regulated" - The term 'down regulated' in this invention is . .;i
referred to a situation when the cell decreases the expression of specific .
proteins or transcripts in response to an external variable; conversely the
term "up regulation" refers to the increase of specific proteins or transcripts
in similar situations,
"Expression"- means that one or more of (i) production of an RNA template
from a DNA sequence;; (ij^pjpcessjna; (e.g., splicing| and/or; 3' end formation)
of a pre-mRNA to produce ah mRNA; and (iii) translation of ah mRNA into a
protein has occurred. A related term, "induced expression" is the expression .
that is possible only in presence of a specific organic molecule or a protein.
The term, "expression profiling" has been used to mean as study of
expression pattern of all genes of an organism under a defined
physiological concentration, the term "heat map"- has been used to mean
as illustration of gene expression profile.
"Gene"- For the purposes of the present invention, the term "gene" has its "• ',
art understood meaning. However, it will be appreciated by those of
ordinary skillin the art that the term "gene" has a variety of meanings in the
art, some of which include gene regulatory sequences (e.g., promoters,
enhancers, etc.) and/or intron sequences, and others of which are limited to
coding sequences. It will further be appreciated that art definitions of "gene"
include references to nucleic acids that do not encode proteins but rather . .
encode functional RNA molecules, such as tRNAs, rRNAs and sRNAs etc!
For the purpose of clarity, we note that, as used in the present application,
the term "gene" generally.refers-,Jo a portion of,» nucleic acid that encodes a
protein; the term may optionally encompass.regulatory sequences. This
definition is not intended to exclude application of the term "gene" to fionprotein-
coding expression units, but rather to clarify that, in most cases, the
term as used in this documenthapp
nucleicacid -
"Metabolite" - is referred to the intermediate degradation product of a toxic
contaminant in this invention by enzymatic reaction within a cell; a series of
biochemical conversions leading to successive intermediate / final
degradation products is referred to as "metabolic pathways";
"Metabolic engineering" - refers to modification or diversion of one or^moje
metabolic pathways of an organism by ingenious techniques of genetic
engineering resulting into a commercially useful product or process; a
microorganism so engineered is referred to as 'metabolically engineered'.
"Operon" - refers to a functioning unit of genomic DNA containing a cluster
of genes under the control of a single promoter &W
uorf243' - is a gene that codes for a protein of 243 amino acids. .
"Plasmid" - is referred to as closed covalent circular DNA with the
capability to replicate and segregate into daughter cells.
.. gene expression.
"Transalation" - is referred to as the process of decoding genetic code to protein.
transposon" - is referred to a class of genetic elements that can "jump" to
different locations within a genome.
"Vector"- is referred to as a nucleic acid molecule that includes sequences
sufficient to direct invivo or in vitro replication of the molecule. Where the
vector includes in vivo replication sequences, these sequences may be selfreplication
sequences, or sequences sufficient to direct integration of the .
vector into another nucleic acid already present in the cell, so that the
, vector sequences are replicated during replication of the already-present
nucleic acid. Such already-present nucleic acitiImay be endogenous to the
cell, or may have been introduced into the cell through experimental
vectors include acloning site, at which foreig
nucleic acid molecules, preferably inventive product molecules, may be
introduced and ligated to the vectors. Particularly preferred vectors further
include control sequences selected for their ability to direct in vivo or in vitro
expression of nucleic acid sequences introduced into the vector. Vectors
may also include some coding sequence, so that transcription and
translation of sequences introduced into the vector results in production of a
fusion protein.
Inventive Steps and Conceptual Aspects
[00023] Several bacterial strains that can use organophosphate pesticides as a
source of carbon have, hitherto, been isolated from soil samples collected
fromrdiverse geographical regions,, AH ;these, organisms synthesize a
hydrolase enzyme of the PIE group, and in each case the enzyme is
encoded by a gen^ (opd) located on a large indigenous plasmid. These
plasmids show considerable genetic diversity, but the region containing
the opd gene is highly conserved. Two opd plasmids, pPDL2
from Flavobacterium sp. and pCMS1 from Pseudomonas diminuta, are well .
characterized, and in each of them a region of about 5.1 kb containing
the opc/gene shows an identical restriction pattern.
[00024] A complete sequence of the conserved region of plasmid pPDL2 was earlier
reported9 wherein it was shown that the opd gene is flanked upstream by an
14
insertion sequence, ISFIspl, that is a member of the IS21 family, and
downstream by a Th3-like element encoding a transposase and a
resolvase. Adjacent to opd but transcribed in the opposite direction is an ;
open reading frame (orf243) with the potential to encode an aromatic
hydrolase somewhat similar to Pseudomonas putida TodF. It was shown
that orf243 encodes a polypeptide of 27 kDa, which plays a role in the
degradation of p-nitrophenol and is likely to-act in concert with opd in the ,.-.
degradation of parathion. The linkage of opd and orf243, the organization pf
the genes flanking opd, and the wide geographical distribution of these
genes suggested that this DNA sequence may constitute a complex
catabolic transposon.
[00025] The genetics of organophosphate degradation has attracted considerable
attention among soil microbiologists. Both the OPH-encoding
organophosphate degradation {opd) genes and the MPH-encoding methyl '.
parathion degradation (rfipd) genes have been shown to be part of mobile ,-••
genetic elements3'10,11 The lateral transfer of opd and mpd genes is
1 evidenced by the existence of identical opd and mpd genes among
taxonomically unrelated soil bacteria9,12. Even dissimilar indigenous
. plasmids found in bacteria collected from diverse geographical regions .
••... . contained identical opd gene clusters12. There are four indigenous pjas,mids
15 -
in OP-degrading Sphingobium fuliginis ATCC 27551. Of these four plasmids
the opd containing pPDL2 has been shown to be a mobilizable plasmid
• ' within which the opd region has unique organizational features1-. Along with
an operon that contributes for protocatechuate degradation, the opd gene.,
forms part of an active transposon10. In addition to the degradation module,
pPDL2 contains genes for plasmid mobility and site-specific integration, and
the plasmid has been shown to integrate site-specifically at an artificially
created attachment (attB) site11. Based on such experimental observations,
the opd island carried on the mobilizable plasmid pPDL2 has been
designated as an Integrative Mobilizable Element (JME)11. - :H •
[00026] A novel open reading frame (ORF); orf243 has been identified within the
opd island. It is found in between the opd gene and the truncated tnpA gene ;?
of a defective transposon, T/739. A canonical catalytic triad typically seen- in''? .
esterases and lipases was identified in Orf24313 and the esterase activity of
the protein has been demonstrated using phenyl acetate as a substrate. As
Orf243 shows very weak homology to the aromatichydrolases such as ;-'
TodF and CumD1, we tried to evaluate its role in degradation of aromatic
compounds and their meta-fission products13. p-Nitrophenol (PNP) being
. . the lone aromatic compound generated during the OPH/MPH-mediated
hydrolytic cleavage of OP insecticides such as methyl parathioh, parathjqp;v
16
or fenitrothion (sumjthion), it was of interest to elicit the role of Orf243 in pnitrophenol
degradation14. -:
[00027] These studies showed that E. coli MG1655 cells expressing Qrf243 were
unexpectedly capable of growth on p-nitrophenol as sole carbon source9,13.
While investigating the molecular basis for this unusual phenomenon, it was
observed that expression of Orf243 also induced a novel small RNA in • •
MG1655. This hitherto unknown small RNA, designated as secbl0054A,
inhibited expression of active pyruvate dehydrogenase and a-ketoglutarate
dehydrogenase, by inhibiting the translation of a critical Lpd subunit
common to both enzymes. In the absence of these two critical enzymes,
therefore, carbon catabolism proceeded via glyoxylate and methyl isocjtrate
cycle rather than via the TCA cycle as illustrated in Fig 2. Furthermore,
Orf243 expression also promoted catabolism pf aromatic compounds like
p-nltrophenol by inducing the hydroxy phenyl propionate (hca) and the
phenyl propionate (mhp) operons: Taken together these novel observations
offer a rationale for, the presence of orf243 within the opd island.
[00028] The above discovery of the expression of orf243 or the induced expression
of sRNA, seed 0054A which is responsible for preferential catabolism for pnitrophenol
forms the scientific basis for these inventors to design the new
inventive recombinant products that are capable of eliminating toxic
. residues of nitrophenols from the ecosystem and offers a most advanced
and cost-effective clean-up procedure for the same.
. Description of Preferred Embodiments .
Embodiment 1 : Metabolicallv engineered E coli MG1655 expressing orf243
[00029] The orf243 is a novel gene found in between mobile genetic elements IS21 ^ *r
: and Tn3 along with organophosphate degrading gene that codes for
phosphodiesterase. The entire genomic island is found on and indigenous
native plasmid pPDL2 isolated from Spingobium fuliginisATCC 2755110..
The Orf 243 has-canonical catalytic triad that is specifically found in
esterases. In one of the preferred embodiments, E coli was thus
metaboiically engineered that expresses orf243 and the cells thus
engineered were found capable of thriving oh PNP as a source of carbon.
[00030] Construction of plasmid to express Orf243: The Orf243, originally present
as part of organophosphate degradation island of an indigenous plasmid
pPDL2 identified in natural soil bacteria, Spingobium fuliginis ATTC27551
was isolated by performing PCR. The isolated fragment is then cloned in
expression vectors to generate expression plasmids. These expression .
plasmids contain orf243 under the control of inducible promoters and hence
its product Orf243 can be induced when necessary by adding inducer to the
culture. Such induction stimulated metabolic shift in the host cell E. coli and
facilitates its growth on nirophenols as sole source of carbon.
[00031] The orf243 was amplified to clone in expression vectors (pMMB206 and -.
pET23b, p) - to express it as tagged protein. The purified protein was used
to assess its influence under in vitro conditions on p-nitrophenol. Though
the purified protein was seen to be active and exhibiting esterase activity,
yet it failed to act on p-nitrophenol. However, when p-nitrophenol was .-. •'
added to the culture medium, it quickly disappeared indicating that orf243 is
activating the innate ability of E. coli to degrade p-nitrophenol. These
experimental results led these inventors to discover the influence of 0^2-43
on metabolic flux of E. coli.
[00032] By performing DNA microarray, quantitative PCR experiments and
proteomics, the existence of this novel shift in metabolic flux of E. coli celts
expressing orf243 was further confirmed. In these engineered cells the
conventional glycolysis and TCA cycles are seen to be down regulated
even in the presence of glucose, instead of aromatic degrading operons,
hydroxy! phenyl propionate (mhp) and phenyl propionate (PP) degradation
pathways. These two operons, as revealed by mutational analysis, are seen
to be involved in degradation of p-nitrophenol. The orf243 induced
metabolic shift promotes degradation of aromatic compounds as against the
established carbon catabolic repression and is therefore highly useful for
preferential elimination of recalcitrant and toxic residues of nitrophenols and
other aromatic carbon compounds.
[00033] Experimental detail: E. coli strains were grown either in LB medium or in .
minimal salts medium at 37°C, when necessary 50mM PNP was added to
the culture medium as sole source of carbon. Ceil growth was recorded by
taking the culture OD at 600nm. PNP concentration in the culture medium
was determined spectrophotometrically. Nitrite estimation in the spent
medium of E. coli was done following protocols described elsewhere12. The
oxygen consumption in the resting cells of engineered E. coli was estimated
using a Gilson oxygraph. Extraction and separation of the PNP metabolites
in the culture medium of engineered MG1655 (pSPPiti) was done using
. liquid chromatography and masss spectrophotometry. The metabolites were
identified using a Bruker Daltonics mass spectrophotometer. Data
pertaining to the growth, nitrite estimation and oxygen consumption are the
average values of three independent experiments. All these experiments
have shown oxidation of p-nitrophenol to nitrocatchol, trihydroxy benzene
and finally to succinate and acetate that can be used as source of carbon
andenergy.
[00034] A typical experiment of the growth of engineered cell expressing orf243 and

surviving on PNP showed the depletion of PNP as in Fig 3 and the
corresponding release of nitrite was observed as in Fig 4. Thus, it is well
demonstrated that the or/243 induced metabolic shift is contributing for
complete mineralization ofp-nitrophenol.
[00035] Advantages: According to US environmental proteqtipn agency the pnitrophenpl
is a priority pollutant and is generated and released into
environment due to a variety of anthropogenic activities. In addition, it is
also generated as a hydrplytic product from OP insecticides. Therefore,
elimination of toxic residues of p-hitrophenols and other nitro aromatic v.
compounds through this invention is of great practical utility.
[00036] Specificity: This invention is npyel and quite different from other related
tools for decontamination of OP insecticides reported in the patent literature
which are based on enzymatic hydrolysis with OPH (organophosphatehydrolase). These tools of the prior art describe only their potential to cleave.;triester bond found in all OP insecticides and nerve agents but cannot ,r.;
degrade the aromatic compounds like p-nitrophenol generated due to OPH
mediated cleavage of OP compounds. Similarly the natural isolates
degrading PNP and other aromatic compounds fail to hydrolyze OP
compounds. Furthermore, since their degradation efficiency is governed by,
carbon cataboiic repression, they fail to degrade the aromatic compounds in
presence of preferred carbon source. Even when some of these natural
isolates tend have the ability to decontaminate nitrophenols, they actually
fail to use them .as a preferred source: of carbon in carbon rich environment.
- The metabolically engineered strains of E: coli cells of this invention, have
uncompromising ability to use PNP and other aromatic compounds even in
presence of glucose and other carbon sources. Therefore, its application
facilitates definitive removal of toxic PNP residues from the environment
regardless of carbp.n status of the environment.
[00037] Industrial utility: E. coli used in this preferred embodiment is laboratory
organism and can be safely used for cleanup operations in the field. The
decontamination of dump yards of pesticide manufacturing units and other
industries using engineered E coli cells of this invention can be extremely
useful. PNP is often found in treated waste waters from the following
industries: iron and steel manufacturing (nitrophenols formed during the
coke making process); foundries (nitrophenols formed during the coke
making process); pharmaceutical manufacturing; rubber processing; and
electrical and electronic components. The engineered E. cpii cells of this
invention would be very useful in decontaminating these wastes containg
residues of nitrophenols.. •
Embodiment 2 : Metabolicallv engineered E coli MG1655 expressing sRNA.
seco10054A
[00038] It has been shown that while engineered cells expressing orf243 as
described above grow quite well on PNP as a sole source of carbon, pure
Orf243 taken as it is, shows,no direct activity on PNP thereby suggesting
that PNP catabolism with engineered cells takes place through induction of
novel proteins or pathways.15
[00039] It has been convincingly demonstrated in this study that there indeed exists
sRNA, designated as seco10054A, which acts as a 'master switch' to .
regulate catabolic pathways enabling the microorganism to survive on PNP
instead of glucose. While this entire work is incorporated in this patent by
way of reference, it was an object to isolate this sRNA and engineer the
cells that can directly express it whereby these can be used as an
alternative tool for decontaminating PNP residues.
[00040] Construction of plasmid to express seco10054A: The seco1'0054A was
isolated from E. coli by performing polymerase chain reaction as 500 base
pairs long EcoRI and BamHI fragment and cloned it in an expression vector
pMMB206 digested with similar enzymes as for embodiment 1. The
resulting recombinant plasmid designated as pDSP9 contains secotG054A
gene under the control of inducible lac promoter. Such cloning helped ..
induce expression of seco10054A when required. Before isolation, sRNA;
ted and validated using DNA microarray and performing southern
hybridization with a radioactive probe.
[00041] The metabolically engineered E. coli strains expressing seco10054A have
also been grown experimentally using PNP as source of carbon showing
similar curves of PNP depletion and nitrite release as shown with .
engineered cells expressing orf243 (Fig 3 and 4) thereby allowing us to ..'
conclude that this embodiment provides an alternative mode of using this
invention for decontaminating residues of p-nitrophenol and other aromatic . .
compounds.
[00042] Scope of this invention: The sRNA secoi0054A is present in many of
the Enterobacteriaceae members belonging to the Genus Shigella
(eg.Shigella sonnei), Escherichia (eg. Escherichia albertii, Escherichia coli)
and Citrobacter (Citrobacter rodentium). Therefore, the
sRNA seco10054A can be used for engineering any of
the Enterobacteriaceae members. For using orf243 approach, however, it
must be remembered thaUhe exact orf243 is nol present m all the
Enterobacteria members but there are other proteins which share the
• domain of orf243 (esterase/lipase). Thus, all these strains can be . • ;//.•
engineered in a similar way by using the proteins which are having these . . .
domains just as E coli can .be engineered with qrf243.
. 7 . FURTRHER POSSIBLE EMBODIMENTS
. [00043] Further possible embodiments of this invention for best mode of working
. " ' . . , " are:, _ • . • • • . ."
. 1 . Open reading frame, off243 isolated from E coli or other possible
organisms as described in this invention
; ••'" 2. Small RNA, secof0054A isolated from E coli or other possible"'
organisms as described in this invention
3. Metabolically engineered cells of B coli or other microorganisms
with orf243 expressing orf243 along with its method of preparation
4. Metabolically engineered cells of E coli or other microorganisms
with sFWA, seco10054A expressingseccii0054A.
5. System comprising of metabolically engineered cells of E coli or
other microorganisms expressing orf243 or inducing expression of
seco10054A designed for decontamination of p-nitrophenols and
other .nitre aromatic toxic residues