U.S. patent application number 12/705170 was filed with the patent office on 2010-07-22 for recycling system for manipulation of intracellular nadh availability.
This patent application is currently assigned to Rice University. Invention is credited to George N. Bennett, Susana J. Berrios-Rivera, Ka-Yiu San.
Application Number | 20100184195 12/705170 |
Document ID | / |
Family ID | 23311497 |
Filed Date | 2010-07-22 |
United States Patent
Application |
20100184195 |
Kind Code |
A1 |
San; Ka-Yiu ; et
al. |
July 22, 2010 |
Recycling System for Manipulation of Intracellular NADH
Availability
Abstract
The present invention describes a novel recombinant NADH
recycling system that is used as a process for producing reduced
compounds. In a specific embodiment, the reduced compounds include
ethanol, succinate, lactate, a vitamin, a pharmaceutical and a
biodegraded organic molecule. The NADH recycling system effects
metabolic flux of reductive pathways in aerobic and anaerobic
environments.
Inventors: |
San; Ka-Yiu; (Houston,
TX) ; Berrios-Rivera; Susana J.; (Pearland, TX)
; Bennett; George N.; (Houston, TX) |
Correspondence
Address: |
BAKER & MCKENZIE LLP
711 Louisiana, Suite 3400
HOUSTON
TX
77002
US
|
Assignee: |
Rice University
Houston
TX
|
Family ID: |
23311497 |
Appl. No.: |
12/705170 |
Filed: |
February 12, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11773408 |
Jul 4, 2007 |
7709261 |
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12705170 |
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10286326 |
Nov 1, 2002 |
7256016 |
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11773408 |
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60335371 |
Nov 2, 2001 |
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Current U.S.
Class: |
435/252.33 ;
435/252.3 |
Current CPC
Class: |
C12P 7/625 20130101;
C12P 1/00 20130101; C12N 9/0036 20130101 |
Class at
Publication: |
435/252.33 ;
435/252.3 |
International
Class: |
C12N 1/21 20060101
C12N001/21 |
Goverment Interests
GOVERNMENT INTEREST
[0002] The present invention was developed with funds from the
United States Government. Therefore, the United States Government
may have certain rights in the invention.
Claims
1-10. (canceled)
11. A cell comprising a recombinant NADH-recycling system, wherein
said system comprises a heterologous nucleotide sequence encoding
an NAH+-dependent dehydrogenase.
12. The cell of claim 11, wherein said heterologous nucleotide
sequence encoding an NAH+-dependent dehydrogenase is a nucleotide
sequence encoding an NAH+-dependent formate dehydrogenase.
13. The cell of claim 11, wherein said heterologous nucleotide
sequence encoding an NAH+-dependent dehydrogenase is a yeast
nucleotide sequence encoding an NAH+-dependent dehydrogenase.
14. The cell of claim 11, wherein said heterologous nucleotide
sequence encoding an NAH+-dependent dehydrogenase is a yeast
nucleotide sequence encoding an NAH+-dependent formate
dehydrogenase.
15. The cell of claim 11, wherein said heterologous nucleotide
sequence encoding an NAH+-dependent dehydrogenase is a nucleotide
sequence from Candida boidinii.
16. The cell of claim 11, wherein said heterologous nucleotide
sequence encoding an NAH+-dependent dehydrogenase is a nucleotide
sequence from Candida boidinii comprising SEQ ID NO: 1.
17. The cell of claim 11, wherein said cell is a bacterium.
18. The cell of claim 11, wherein said cell is E. coli.
19. The cell of claim 11, wherein said cell is a member of the
genus Rhodococcus.
20-47. (canceled)
Description
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/335,371, filed Nov. 2, 2001, which is
incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to the fields of microbiology,
molecular biology, cell biology and biochemistry. More
specifically, the present invention relates to manipulating
reductive metabolic processes in vivo using genetic and metabolic
engineering, thereby allowing external control of intracellular
nicotinamide adenine dinucleotide (NADH) availability. Further, the
present invention relates to a method of producing increased
reduced metabolites such as ethanol through aerobic or anaerobic
growth of a living system comprised of a recombinant NADH recycling
system.
[0005] 2. Related Art
[0006] The metabolic pathways leading to the production of most
industrially important compounds involve oxidation-reduction
(redox) reactions. Biosynthetic transformations involving redox
reactions offer a significant economic and environmental advantage
for the production of fine chemicals over conventional chemical
processes, in particular those redox reactions requiring
stereospecificity. Furthermore, biodegradation of toxic chemicals
often also involves redox reactions.
[0007] Nicotinamide adenine dinucleotide (NAD) functions as a
cofactor in over 300 redox reactions and regulates various enzymes
and genetic processes (Foster et al., 1990). The NADH/NAD+ cofactor
pair plays a major role in microbial catabolism in which a carbon
source, such as glucose, is oxidized using NAD+ producing reducing
equivalents in the form of NADH. It is crucially important for
continued cell growth that this reduced NADH be oxidized to NAD+
and a redox balance be achieved. Under aerobic growth, oxygen
achieves this recycling by acting as the oxidizing agent. While
under anaerobic growth, and in the absence of an alternate
oxidizing agent, the regeneration of NAD+ is achieved through
fermentation by using NADH to reduce metabolic intermediates.
[0008] The metabolic pathways leading to the production of most
industrially important compounds involve redox reactions.
Biosynthetic transformations involving redox reactions also offer a
considerable potential for the production of fine chemicals over
conventional chemical processes, especially those requiring
stereospecificity.
[0009] Enzymes referred to in general as oxidoreductases, or more
specifically as oxidases, reductases or dehydrogenases, catalyze
these biological redox reactions. These enzymes require a donor
and/or an acceptor of reducing equivalents in the form of
electrons, hydrogen or oxygen atoms. Cofactor pairs that are
transformed reversibly between their reduced and oxidized states,
nucleotide cofactors such as NADH/NAD+ and NADPH/NADP+ among
others, serve as donors and/or acceptors of reducing equivalents
very effectively in a living cell.
[0010] The NADH/NAD+ cofactor pair has demonstrated a regulatory
effect on gene expression and enzymatic activity. Examples include,
among others, the induction by NADH of adhE expression, which
encodes an alcohol dehydrogenase (Leonardo et al., 1993; Leonardo
et al., 1996) and catalyzes the production of ethanol during
fermentation, the inhibition by high NADH/NAD+ ratios on the
pyruvate dehydrogenase complex (Graef et al., 1999), and the
regulation by the NADH/NAD+ ratio on the shift between oxidation or
reduction of L-lactaldehyde (Baldoma and Aguilar, 1988).
[0011] The ratio of the reduced to oxidized form of this cofactor,
the NADH/NAD+ ratio, is critical for the cell. The NAD(H/+)
cofactor pair is very important in microbial catabolism, where a
carbon source, such as glucose, is oxidized through a series of
reactions utilizing NAD+ as a cofactor and producing reducing
equivalents in the form of NADH. It is crucially important for the
continued growth of the cell that this reduced NADH be oxidized to
NAD+, thus achieving a redox balance. Under aerobic growth, oxygen
achieves this by acting as the oxidizing agent. While under
anaerobic growth and in the absence of an alternate oxidizing
agent, this process occurs through fermentation, where NADH is used
to reduce metabolic intermediates and regenerate NAD+ (FIG. 1).
[0012] The high influence of cofactors in metabolic networks has
been evidenced by studies in which the NADH/NAD+ ratio has been
altered by feeding carbon sources possessing different oxidation
states (Alam and Clark, 1989; Leonardo et al., 1996), by
supplementing anaerobic growth with different electron acceptors,
such as fumarate and nitrate (Graef et al., 1999) and by expressing
an enzyme like NADH oxidase (Lopez de Felipe et al., 1998). Other
previous efforts to manipulate NADH levels have included the
addition of electron dye carriers (Park and Zeikus, 1999) and the
variation of oxidoreduction potential conditions (Riondet et al.,
2000).
[0013] The effective regeneration of used cofactors is critical in
industrial cofactor-dependent production systems because of the
impeding high cost of cofactors such as NAD. The cofactors, also
referred to as co-enzymes, NAD+ and NADP+ are expensive chemicals,
thereby making their regeneration by reoxidation to the original
state imperative if they are to be used economically in low cost,
chemical production systems. Efforts to do such have been
described. U.S. Pat. No. 4,766,071 describes in vitro regeneration
of NADH using a cell lysate of Clostridium kluyveri as a
biocatalyst and an aldehyde as an oxidizing agent. U.S. Pat. No.
5,393,615 describes electrochemical regeneration of NADH using an
electrode characterized by a mediator function. Similarly, U.S.
Pat. No. 5,264,092 discloses mediators covalently attached to a
polymeric backbone wherein the polymeric backbone coats the surface
of an electrode. U.S. Pat. No. 5,302,520 discloses a NAD
regeneration system and an adenosine phosphate regeneration system
that, in the presence of pyruvate, yields a labeled
carbohydrate.
[0014] In enzyme bioreactors, NAD+-dependent formate dehydrogenase
(FDH) from methylotrophic yeast and bacteria is extensively used to
regenerate NADH from NAD+ in vitro. FDH catalyzes the practically
irreversible oxidation of formate to CO.sub.2 and the simultaneous
reduction of NAD+ to NADH. This system of cofactor regeneration has
been successfully applied in the production of optically active
amino acids (Galkin et al., 1997), chiral hydroxy acids, esters,
alcohols, and other fine chemicals synthesized by different
dehydrogenases (Hummel and Kula, 1989), (Tishkov et al., 1999).
Purified FDH has also been used to regenerate NADH in vitro for the
industrial production of non-natural amino acids that cannot be
obtained by fermentation, such as L-tert-leucine which has
important applications when used in pharmaceuticals (Kragl et al.,
1996).
[0015] In spite of these advances, biotransformation with whole
cells remains the preferred industrial method for the synthesis of
most cofactor-dependent products. In these systems, the cell
naturally regenerates the cofactor; however, the enzyme of interest
has to compete for the required cofactor with a large number of
other enzymes within the cell. For this reason, in
cofactor-dependent production systems utilizing whole cells, after
the enzymes of interest have been overexpressed, cofactor levels
and the availability of the required form of the cofactor (reduced
or oxidized) become crucial for optimal production.
[0016] Furthermore, one of the long-sought goals in recombinant
polypeptide production processes is to achieve a high cloned gene
expression level and high cell density. Unfortunately, under these
demanding conditions, the amount of acetate accumulated in the
reactor increases precipitously. Acetate accumulation is associated
with decreased recombinant polypeptide productivity (Aristidou et
al., 1995). Methods of controlling acetate production would be
beneficial in increasing recombinant polypeptide yield in
large-scale industrial synthesis of polypeptides. Additionally, the
sort of metabolic manipulation used to increase recombinant
polypeptide yields could also he applied to the production of any
biomolecule in a large-scale system in which the stress of
biomolecule production normally leads to acetate accumulation, such
as biopolymers.
[0017] Catalytic hydrodesulfurization has the potential to remove
sulfur from various fuels. However, this technology is associated
with high costs due to hydrogen consumption and heavy metal
deactivation of the catalyst. A lower cost treatment is
microbiological biodesulfurization. U.S. Pat. No. 6,337,204
describes a Rhodococcus bacterial culture capable of
biodesulfurization. One obstacle in this method is that these
reactions require NADH as a cofactor, the availability of which is
a limiting factor.
[0018] Although it is generally known that cofactors play a major
role in the production of different fermentation products, their
role has not been studied thoroughly and systematically in
engineered systems. Instead, metabolic engineering studies have
focused on manipulating enzyme levels through the amplification,
addition or deletion of a particular pathway. Such steps relegate
cofactor manipulations as a powerful tool for metabolic
engineering, as many enzymes require them. The dehydrogenases are
but one example of selective catalysis requiring the
energy-transferring redox couple, NADH/NAD+.
[0019] Prior to the present invention, a genetic means of
manipulating the availability of intracellular NADH in vivo by
regenerating NADH through the heterologous expression of an
NAD+-dependent formate dehydrogenase was not known. By way of the
present invention, the effect of manipulating intracellular NADH on
the metabolic patterns in Escherichia coli under anaerobic and
aerobic conditions by substituting the native cofactor-independent
formate dehydrogenase (FDH) by an NAD+-dependent FDH such as from
Candida boidinii is described. This manipulation provoked a
significant change in the final metabolite concentration pattern
both anaerobically and aerobically. Under anaerobic conditions, the
production of more reduced metabolites was favored, as evidenced by
a dramatic increase in the ethanol to acetate ratio. Unexpectedly
during aerobic growth, the increased availability of NADH induced a
shift to fermentation even in the presence of oxygen by stimulating
pathways that are normally inactive under these conditions.
SUMMARY OF THE INVENTION
[0020] The present invention is directed to a method for increasing
the intracellular availability of NADH, comprising the
transformation of a cell with a nucleic acid encoding an
NAD+-dependent dehydrogenase and growth of said cell under
conditions in which said NAD+-dependent dehydrogenase increases the
intracellular availability of NADH. In a specific embodiment, the
NAD+-dependent dehydrogcnase is a formate dehydrogenase. In a
further specific embodiment, the formate dehydrogenase is Candida
boidinii formate dehydrogenase.
[0021] The present invention is directed to methods of utilizing a
recombinant NADH recycling system to produce NADH and other
metabolites in vivo. One embodiment of the present invention is a
method to produce NADH in vivo comprising growing a culture of
cells that comprises at least one cell, comprising a recombinant
NADH recycling system. In a specific embodiment of the invention,
the cell which comprises the recombinant NADH recycling system is a
bacterium, including E. coli. In further specific embodiments of
the invention, the recombinant NADH recycling system comprises a
nucleotide sequence encoding a NAD+-dependent formate
dehydrogenase, which may be from, but is not limited to, yeast, or
Candida boidinii, operatively linked to a promoter
[0022] In another embodiment of the present invention, there is a
cell comprising a recombinant NADH recycling system. In specific
embodiments, the recombinant NADH recycling system of the cell
comprises a nucleotide sequence encoding a NAD+-dependent formate
dehydrogenase operatively linked to a promoter. In a specific
embodiment, the sequence is heterologous.
[0023] Yet another embodiment of the invention is a method to
produce a reduced compound in vivo comprising growing a culture of
cells that comprises at least one cell comprising a recombinant
NADH recycling system. In a specific embodiment, the reduced
compound produced is ethanol, lactate, succinate, a vitamin, a
pharmaceutical or a biodegraded organic molecule. In a further
specific embodiment, the pharmaceutical compound is an antibiotic.
In another specific embodiment, the growing of cell culture takes
place in an oxygen-deficient atmosphere. In another specific
embodiment, the growing is in an oxygen-rich atmosphere. In another
specific embodiment, formate is added to the culture of cells. In a
further specific embodiment, the amount of formate added is at
least about 100 mM.
[0024] In an additional embodiment of the present invention there
is a method to produce ethanol comprising growing a culture of
cells wherein the culture comprises at least one cell comprising a
recombinant NADH recycling system.
[0025] Yet another embodiment of the invention is a method of
altering metabolic flux of a reduction pathway comprising growing a
culture of cells, wherein the culture comprises at least one cell
comprising a recombinant NADH recycling system, and the flux of the
metabolic pathway is redistributed as compared to a normal
metabolic flux of the pathway.
[0026] Another embodiment of the invention is a method of
biodegradation in vivo comprising growing a culture of cells,
wherein the culture comprises at least one cell comprising a
recombinant NADH recycling system.
[0027] Yet another embodiment of the present invention is a method
for biodesulfurization in vivo comprising growing a culture of
cells, wherein the culture comprises at least one cell comprising a
recombinant NADH recycling system. In a further embodiment, the
cells are bacteria cells. In a specific embodiment, the bacterial
cells are Rhodococcus bacteria.
[0028] Another embodiment of the present invention is a method of
biopolymer production in vivo comprising growing a culture of
cells, wherein the culture comprises at least one cell comprising a
recombinant NADH recycling system.
[0029] One embodiment of the present invention is a method for
polypeptide production in vivo comprising growing a culture of
cells, wherein the culture comprises at least one cell comprising a
recombinant NADH recycling system. A specific embodiment is the
production of heterologous recombinant protein.
[0030] Other embodiments, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF SUMMARY OF THE DRAWINGS
[0031] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein:
[0032] FIG. 1. Schematic representation of Escherichia coli central
anaerobic metabolic pathways illustrating involvement of the
NADH/NAD+ cofactor pair.
[0033] FIG. 2. Diagram illustrating the native cofactor-independent
formate degradation pathway and the newly introduced NAD+-dependent
pathway.
[0034] FIG. 3A to 3F. Graphical illustrations of results of
anaerobic tube experiments of strains after 72 hours.
[0035] FIGS. 4A and 4B. Graphical illustrations of (A) formate
consumed and ethanol/acetate ratio (B) of strains grown in
anaerobic tube experiments.
[0036] FIG. 5A to 5F. Graphical illustrations of aerobic shake
flask experiment after 24 hours.
[0037] FIGS. 6A and 6B. Graphical illustrations of (A) lactate and
(B) succinate concentrations from aerobic growth in various
concentrations of supplemented formate.
[0038] FIG. 7. Central aerobic metabolic pathway of Escherichia
coli showing generation of NADH and regeneration of NAD+ and
metabolic flux of each contributing pathway.
[0039] FIG. 8. Diagram illustrating the native cofactor-independent
formate degradation pathway and the recombinant NADH recycling
system.
[0040] FIG. 9. Diagram showing the uptake of 1 C-mole of glucose in
a cell together with yields of reduced products obtained in an
anaerobic chemostatic experiment.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0041] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one. As used herein "another" may mean at least a second
or more.
[0042] As used herein, the expressions "cell", "cell line" and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
[0043] As used herein, the term "recombinant" cells or host cells
are intended to refer to a cell into which an exogenous nucleic
acid sequence, such as, for example, a vector, has been introduced.
Therefore, recombinant cells are distinguishable from naturally
occurring cells which do not contain a recombinantly introduced
nucleic acid. Recombinant DNA refers to DNA which has been modified
by joining genetic material from two different sources, which may
be different species or the same species. Recombinant polypeptides
may be the gene products of recombinant DNA, or polypeptides
produced in recombinant cells. The term "recombinant NADH recycling
system" refers to an engineered system for the recycling of NADH.
It can refer to cells that comprise this system, or recombinant DNA
or polypeptide sequences that comprise such a system.
[0044] The terms "modified" or "modification" as used herein refer
to the state of a metabolic pathway being altered in which a step
or process in the pathway is increased or upregulated, such as in
activity of an enzyme or expression of a nucleic acid sequence,
respectively. In a specific embodiment, the modification is the
result of an alteration in a nucleic acid sequence which encodes an
enzyme in the pathway, an alteration in expression of a nucleic
acid sequence which encodes an enzyme in the pathway, or an
alteration in translation or proteolysis of an enzyme in the
pathway (i.e., formate dehydrogenase), or a combination thereof. A
skilled artisan recognizes that there are commonly used standard
methods in the art to obtain the alterations, such as by
mutation.
[0045] Nucleic acid is "operatively linked" when it is placed into
a functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operatively
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operatively linked to a coding sequence if it
affects the transcription of the sequence; or a ribosome binding
site is operatively linked to a coding sequence if it is positioned
so as to facilitate translation. Generally, "operatively linked"
means that the DNA sequences being linked are contiguous and, in
the case of a secretory leader, contiguous and in reading phase.
However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist, then synthetic oligonucleotide adaptors or
linkers are used in accord with conventional practice.
[0046] "Plasmids" are designated by lower case p preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are commercially available, are publicly available on an
unrestricted basis, or can be constructed from such available
plasmids in accord with published procedures. In addition, other
equivalent plasmids are known in the art and will be apparent to
the ordinary artisan.
[0047] One embodiment of the present invention is to provide a
method to produce NADH in vivo comprising growing a culture of
cells that comprises at least one cell comprising a recombinant
NADH recycling system, under conditions to produce NADH. The NADH
recycling system comprises a nucleic acid sequence encoding a
dehydrogenase that is NAD+-dependent formate, such as FDH. The
recombinant NADH recycling system increases the intracellular
availability of NADH. FDH catalyzes the practically irreversible
oxidation of formate to CO.sub.2 and the simultaneous reduction of
NAD+ to NADH. A skilled artisan is aware that NADH and NAD+ refer
to nicotinamide adenine dinucleotide in two distinct oxidation
states, respectively, and are cofactors that mediate a large number
of biological oxidations and reductions, which generally provide a
step or steps in catabolic metabolic pathways. When a substrate is
hydrolyzed (in the instant case, formate is the substrate) hydride
(H.sup.-) is transferred to the C-4 of the nicotinamide ring of
NAD+, and the H.sup.+ is lost to the medium. Further, a skilled
artisan recognizes that a dehydrogenase such as formate
dehydrogenase catalyzes a reversible hydride transfer, generally a
stereospecific reaction owing to the two distinct domains that the
dehydrogenase protein conforms, in which each domain is specific
for the binding of cofactor or substrate.
[0048] A skilled artisan recognizes that sequences useful in the
present invention may be obtained in a database such as the
National Center for Biotechnology's GenBank database. For example,
a NAD+-dependent FDH1 of Candida boidinii (SEQ ID NO: 1, GenBank
Accession NO: AF004096) and is a non-limiting example of a suitable
FDH of the present invention. SEQ ID NO: 1 and any other nucleotide
sequences encoding the polypeptide SEQ ID NO: 2 (GenBank Accession
NO: AAC49766) are suitable. Other suitable FDHs are from Candida
methylica (SEQ ID NO: 3, GenBank Accession NO: CAA57036),
Pseodomonas sp. 101 (SEQ ID NO: 4, GenBank Accession NO: P33160),
Arabidopsis thaliana (SEQ ID NO: 5, GenBank Accession NO:
AAF19436), and Staphylococcus aureus (SEQ ID NO: 6, GenBank
Accession NO: BAB94016). Any nucleic acids encoding SEQ ID NOS: 3,
4, 5, and 6 are also appropriate. Other species in embodiments of
the invention are contemplated, such as Saccharomyces bayanus,
Saccharomyces exiguus, Saccharomyces servazzii, Zygosaccharomyces
rouxii, Saccharomyces kluyveri, Kluyveromyces thermotolerans,
Kluyveromyces lactis, Kluyveromyces marxianus, Pichia angusta,
Debaryomyces hansenii, Pichia sorbitophila, Candida tropicalis and
Yarrowia lipolytica. Standard methods and reagents in the field of
molecular biology are well known in the art. A reference for such
methods includes Current Protocols in Molecular Biology, Chapter 13
(Ausubel et al., 1994), herein incorporated by reference.
[0049] In a preferred embodiment, the nucleic acid sequence
encoding the non-native FDH is inserted into a vector such that the
expression of the non-native FDH is controlled by a promoter that
is operatively linked to the non-native FDH. A skilled artisan is
aware of appropriate vectors and promoters, not excluding the
native promoter of the non-native FDH gene, for expression of a
recombinant gene in a host organism and methods to develop a
resulting recombinant plasmid. The recombinant plasmid comprising
the non-native FDH and promoter are then transformed into a host
cell by methods well known in the art.
[0050] In a specific embodiment the host organism is an anaerobe,
such as, for example, Escherichia coli, a facultative anaerobe that
grows either in the presence or the absence of oxygen, or any
aerotolerant organism that is capable of fermentation. In another
specific embodiment, the wild-type FDH gene is inactivated, meaning
that the nucleic acid sequence encoding for the native FDH gene is
inoperative. For example, the fdhF (SEQ ID NO:7, GenBank Accession
NO: M13563) of E. coli is replaced by homologous recombination
using methods well known in the art. Engineering a host cell such
that an enzymatic activity is removed can be screened, in one
manner, by confirming the lack of the enzymatic activity in the
recombinant host cell. Upon expression of the plasmid, the
recombinant FDH gene assumes the responsibility of providing the
respective enzymatic activity (e.g. dehydrogenation) for the host
cell.
[0051] For example, the nucleic acid sequence of the formate
dehydrogenase is regulated by an inducible promoter.
[0052] The recombinant cell is grown in an oxygen-rich atmosphere
(aerobic) or in an oxygen-deficient atmosphere (anaerobic) to
produce NADH, thereby increasing intracellular availability of
NADH.
[0053] By increasing intracellular NADH availability, the present
invention provides a method to produce a reduced metabolite
comprising growing a culture of cells that comprises at least one
cell comprising a recombinant NADH recycling system, and removing
the reduced metabolite from the culture. The NADH recycling system
effects increased intracellular NADH availability as compared to a
control cell and consequently accumulates reduced products and
metabolites such as, for example, reduced metabolites of
glucose.
[0054] The method to produce reduced metabolites of the present
invention includes metabolites not originally synthesized by the
host cell. For example, in vivo reduction provided by the present
invention is applied to biodegradation of toxic chemicals and/or
semi-synthesis of a compound, wherein the compound is, for example,
a vitamin or a pharmaceutical or a medicament. The pharmaceutical
compound may be an antibiotic, such as tetracycline, amoxicillin,
erythromycin, or zithromycin. Often the syntheses of such compounds
to an appropriate oxidation state is required to, for example,
ensure solubility, and such requirements include a reduction
reaction. In such cases, the method of the present invention is
contemplated especially involving a reduction reaction where
stereospecificity is desired.
[0055] In yet another object of the present invention is a method
to produce ethanol comprising growing a culture of cells that
comprises at least one cell comprising a recombinant NADH recycling
system, and removing the ethanol from the culture.
[0056] Another object is a method to produce lactate comprising
growing a culture of cells, wherein the culture comprises at least
one cell comprising a NADH recycling system, wherein the lactate
may be removed from the culture.
[0057] It is another object of the present invention to provide a
method to produce succinate comprising growing a culture of cells,
wherein the culture comprises at least one cell comprising a NADH
recycling system, wherein the succinate may be removed from the
culture.
[0058] One object is a method of altering metabolic flux of a
metabolic pathway comprising growing a culture of cells, wherein
the culture comprises at least one cell comprising a NADH recycling
system. The present invention enables the altering of metabolic
flux of a reduction pathway to produce a reduced metabolite or
reduced compound.
[0059] In a specific embodiment, the compound to be degraded is an
environmental toxin, such as a toxic organic or inorganic compound.
A skilled artisan recognizes that a toxic organic pollutant (also
referred to as a xenobiotic) includes but is not limited to
benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene,
phenol, o-cresol, m-cresol, p-cresol, or styrene, as well as
halogenated organic compounds such as pentachlorophenol. Examples
of other environmental toxins include petroleum hydrocarbons (such
as fuel oil or gasoline), insecticides (such as polychlorinated
biphenyls (PCBs) or DDT), halogenated hydrocarbons, chlorinated
benzenes, chlorophenols, chloroquaiacols, chloroveratroles,
chlorocatechols, chlorinated aliphatics, perchlorates, nitrates,
hydrolysates, or polycyclic aromatic hydrocarbons (PAHs, such as
phenanthrene).
[0060] Another object of the present invention is a method of
biodesulfurization in order to remove sulfur from fossil fuels,
such as crude oil. Such an embodiment comprises at least one cell
comprising an NADH recycling system where the NADH produced is a
necessary cofactor for the enzymes involved in the
biodesulfurization pathway. Dibenzothiophene is a model compound
for organic sulfur in fossil fuels. Known members of the
dibenzothiophene desulfurization pathway include dibenzothiophene
monooxygenase, dibenzothipohene-5,5-dioxide monooxygenase, and
2'-hydroxybiphenly-2-sulfinate sulfinoylase. Known bacterial
strains which are capable of breaking down dibenzothiophene using
this pathway include Rhodococcus strains, including IGTS8, T09, and
RA-18, and Gordonia desulfuricans 213E. Also capable of
biodesulfurization are E. coli that express recombinant genes from
Rhodococcus, and Pseudomonas putida that express recombinant genes
from Rhodococcus. Gordonia rubropertinctus strain T08 is capable of
biodesulfurization using a novel pathway. Rhodococcus strain IGTS8,
Gordonia rubropertinctus strain T08, E. coli, and Pseudomonas
putida are available from the American Type Culture Collection
(ATCC). In one embodiment, a cell or cells comprising the NADH
recycling system are transformed with vectors that are capable of
expressing the gene products of the biodesulfurization pathway
genes. In another embodiment, cells capable of biodesulfurization
are transformed with a recombinant NADH recycling system. In such
an embodiment, the cells capable of biodesulfurization may be
Rhodococcus or recombinant E. coli.
[0061] It is an object of the present invention to create a method
for the production of biopolymers in bacteria. Such an embodiment
comprises at least one cell comprising an NADH recycling system
where the NADH produced is a necessary cofactor for the enzymes
involved in the biopolymer production pathway. The enzymes involved
in the biopolymer production pathway may be host cell enzymes or
recombinant enzymes. Biopolymers are polymers that are either
naturally occurring or can be produced through engineering of a
host organism. Examples of biopolymers are polysaccharides,
polythioesters, polyhydroxybutyrates, polyhydroxyalkanoates. Other
examples are chitins, starch, lignin, glycogen, cellulose, and
xanthan gum. In some embodiments, biopolymers can also include
polypeptides and amino acid polymers.
[0062] Another object of the present invention is a method of
producing polypeptides in bacteria. The polypeptides produced may
be under the transcriptional control of the host cell, or may be
encoded by nucleic acids operatively linked to a promoter. The
polypeptide may be of host cell origin or heterologous, and may be
recombinant. Heterologous refers to polypeptides not naturally
occurring in the host. Heterologous peptides may be from another
species. Heterologous polypeptides may be encoded for by
heterologous nucleic acids. Such an embodiment comprises at least
one cell comprising an NADH recycling system where the NADH
produced is able to shift the metabolic pattern of the cell to
cause decreased acetate levels. Decreased acetate levels are
associated with increased yields of recombinant polypeptide.
Nucleic Acid-Based Expression Systems
[0063] 1. Vectors
[0064] The term "vector" is used to refer to a carrier nucleic acid
molecule into which a nucleic acid sequence can be inserted for
introduction into a cell where it can he replicated. A nucleic acid
sequence can be "exogenous," which means that it is foreign to the
cell into which the vector is being introduced or that the sequence
is homologous to a sequence in the cell but in a position within
the host cell nucleic acid in which the sequence is ordinarily not
found. Vectors include plasmids, cosmids, viruses (bacteriophage,
animal viruses, and plant viruses), and artificial chromosomes
(e.g., YACs). One of skill in the art would be well equipped to
construct a vector through standard recombinant techniques, which
are described in Maniatis et al., 1988 and Ausubel et al., 1994,
both incorporated herein by reference.
[0065] The term "expression vector" refers to a vector containing a
nucleic acid sequence coding for at least part of a gene product
capable of being transcribed. In some cases, RNA molecules are then
translated into a protein, polypeptide, or peptide. In other cases,
these sequences are not translated, for example, in the production
of antisense molecules or ribozymes. Expression vectors can contain
a variety of "control sequences," which refer to nucleic acid
sequences necessary for the transcription and possibly translation
of an operatively linked coding sequence in a particular host
organism. In addition to control sequences that govern
transcription and translation, vectors and expression vectors may
contain nucleic acid sequences that serve other functions as well
and are described infra.
[0066] a. Promoters and Enhancers
[0067] A "promoter" is a control sequence that is a region of a
nucleic acid sequence at which initiation and rate of transcription
are controlled. It may contain genetic elements at which regulatory
proteins and molecules may bind such as RNA polymerase and other
transcription factors. The phrases "operatively positioned,"
"operatively linked," "under control," and "under transcriptional
control" mean that a promoter is in a correct functional location
and/or orientation in relation to a nucleic acid sequence to
control transcriptional initiation and/or expression of that
sequence. A promoter may or may not be used in conjunction with an
"enhancer," which refers to a cis-acting regulatory sequence
involved in the transcriptional activation of a nucleic acid
sequence. In specific embodiments, the promoter functions in a
prokaryotic cell.
[0068] A promoter may be one naturally associated with a gene or
sequence, as may be obtained by isolating the 5' non-coding
sequences located upstream of the coding segment and/or exon. Such
a promoter can be referred to as "endogenous." Similarly, an
enhancer may be one naturally associated with a nucleic acid
sequence, located either downstream or upstream of that sequence.
Alternatively, certain advantages will be gained by positioning the
coding nucleic acid segment under the control of a recombinant or
heterologous promoter, which refers to a promoter that is not
normally associated with a nucleic acid sequence in its natural
environment. A recombinant or heterologous enhancer refers also to
an enhancer not normally associated with a nucleic acid sequence in
its natural environment. Such promoters or enhancers may include
promoters or enhancers of other genes, and promoters or enhancers
isolated from any other prokaryotic, viral, or eukaryotic cell, and
promoters or enhancers not "naturally occurring," i.e., containing
different elements of different transcriptional regulatory regions,
and/or mutations that alter expression. In addition to producing
nucleic acid sequences of promoters and enhancers synthetically,
sequences may be produced using recombinant cloning and/or nucleic
acid amplification technology, including PCR.TM., in connection
with the compositions disclosed herein (see U.S. Pat. No.
4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by
reference). Furthermore, it is contemplated the control sequences
that direct transcription and/or expression of sequences within
non-nuclear organelles such as mitochondria, chloroplasts, and the
like, can be employed as well.
[0069] Naturally, it will be important to employ a promoter and/or
enhancer that effectively directs the expression of the DNA segment
in the cell type, organelle, and organism chosen for expression.
Those of skill in the art of molecular biology generally know the
use of promoters, enhancers, and cell type combinations for protein
expression, for example, see Sambrook et al. (1989), incorporated
herein by reference. The promoters employed may be constitutive,
tissue-specific, inducible, and/or useful under the appropriate
conditions to direct high level expression of the introduced DNA
segment, such as is advantageous in the large-scale production of
recombinant proteins and/or peptides. The promoter may be
heterologous or endogenous.
[0070] b. Multiple Cloning Sites
[0071] Vectors can include a multiple cloning site (MCS), which is
a nucleic acid region that contains multiple restriction enzyme
sites, any of which can be used in conjunction with standard
recombinant technology to digest the vector. (See Carbonclli et
al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated
herein by reference.) "Restriction enzyme digestion" refers to
catalytic cleavage of a nucleic acid molecule with an enzyme that
functions only at specific locations in a nucleic acid molecule.
Many of these restriction enzymes are commercially available. Use
of such enzymes is widely understood by those of skill in the art.
Frequently, a vector is linearized or fragmented using a
restriction enzyme that cuts within the MCS to enable exogenous
sequences to be ligated to the vector. "Ligation" refers to the
process of forming phosphodiester bonds between two nucleic acid
fragments, which may or may not be contiguous with each other.
Techniques involving restriction enzymes and ligation reactions are
well known to those of skill in the art of recombinant
technology.
[0072] c. Origins of Replication
[0073] In order to propagate a vector in a host cell, it may
contain one or more origins of replication sites (often termed
"ori"), which is a specific nucleic acid sequence at which
replication is initiated. In specific embodiments, the origin of
replication functions in a prokaryotic cell.
[0074] d. Selectable and Screenable Markers
[0075] In certain embodiments of the invention, the cells contain
nucleic acid construct of the present invention, a cell may be
identified in vitro or in vivo by including a marker in the
expression vector. Such markers would confer an identifiable change
to the cell permitting easy identification of cells containing the
expression vector. Generally, a selectable marker is one that
confers a property that allows for selection. A positive selectable
marker is one in which the presence of the marker allows for its
selection, while a negative selectable marker is one in which its
presence prevents its selection. An example of a positive
selectable marker is a drug resistance marker.
[0076] Usually the inclusion of a drug selection marker aids in the
cloning and identification of transformants, for example, genes
that confer resistance to neomycin, puromycin, hygromycin, DHFR,
GPT, zeocin and histidinol are useful selectable markers. In
addition to markers conferring a phenotype that allows for the
discrimination of transformants based on the implementation of
conditions, other types of markers including screenable markers
such as GFP, whose basis is colorimetric analysis, are also
contemplated. Further examples of selectable and screenable markers
are well known to one of skill in the art.
[0077] 2. Host Cells
[0078] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these term also
include their progeny, which is any and all subsequent generations.
It is understood that all progeny may not be identical due to
deliberate or inadvertent mutations. In the context of expressing a
heterologous nucleic acid sequence, "host cell" refers to a
prokaryotic or eukaryotic cell, and it includes any transformable
organisms that is capable of replicating a vector and/or expressing
a heterologous gene encoded by a vector. A host cell can, and has
been, used as a recipient for vectors. A host cell may be
"transfected" or "transformed," which refers to a process by which
exogenous nucleic acid is transferred or introduced into the host
cell. A transformed cell includes the primary subject cell and its
progeny.
[0079] Host cells may be prokaryotic, depending upon whether the
desired result is replication of the vector or expression of part
or all of the vector-encoded nucleic acid sequences. Numerous cell
lines and cultures are available for use as a host cell, and they
can be obtained through the American Type Culture Collection
(ATCC), which is an organization that serves as an archive for
living cultures and genetic materials, which is readily accessible
on the world wide web. An appropriate host can be determined by one
of skill in the art based on the vector backbone and the desired
result. A plasmid or cosmid, for example, can be introduced into a
prokaryote host cell for replication of many vectors. Bacterial
cells used as host cells for vector replication and/or expression
include DH5.alpha., JM109, and KC8, as well as a number of
commercially available bacterial hosts such as SURE.RTM. Competent
Cells and SOLOPACK.TM. Gold Cells (STRATAGENE.RTM., La Jolla).
Alternatively, bacterial cells such as E. coli LE392 could be used
as host cells for phage viruses.
[0080] Similarly, a viral vector may be used in conjunction with a
prokaryotic host cell, particularly one that is permissive for
replication or expression of the vector.
[0081] Some vectors may employ control sequences that allow it to
be replicated and/or expressed in prokaryotic cells. One of skill
in the art would further understand the conditions under which to
incubate all of the above described host cells to maintain them and
to permit replication of a vector. Also understood and known are
techniques and conditions that would allow large-scale production
of vectors, as well as production of the nucleic acids encoded by
vectors and their cognate polypeptides, proteins, or peptides.
[0082] Cells may be grown in culture medium. Culture medium may be
liquid or solid. Liquid culture medium may be a broth. Solid medium
may be molded into a plate. Liquid media are used for growth of
pure batch cultures while solidified media are used widely for the
isolation of pure cultures, for estimating viable bacterial
populations, and a variety of other purposes. The usual gelling
agent for solid or semisolid medium is agar, a hydrocolloid derived
from red algae. Agar is used because of its unique physical
properties (it melts at 100 degrees and remains liquid until cooled
to 40 degrees, the temperature at which it gels) and because it
cannot be metabolized by most bacteria. Hence as a medium component
it is relatively inert; it simply holds (gels) nutrients that are
in aquaeous solution. Types of culture medium include differential,
selective, minimal, and enrichment.
[0083] 3. Expression Systems
[0084] Numerous expression systems exist that comprise at least a
part or all of the compositions discussed above. Prokaryote- and/or
eukaryote-based systems can be employed for use with the present
invention to produce nucleic acid sequences, or their cognate
polypeptides, proteins and peptides. Many such systems are
commercially and widely available.
[0085] An expression system from STRATAGENE.RTM. (La Jolla, Calif.)
is the pET E. COLI EXPRESSION SYSTEM is a widely used in vivo
bacterial expression system due to the strong selectivity of the
bacteriophage T7 RNA polymerase, the high level of activity of the
polymerase and the high efficiency of translation. One of skill in
the art would know how to express a vector, such as an expression
construct, to produce a nucleic acid sequence or its cognate
polypeptide, protein, or peptide.
[0086] 4. Derivatives of Promoter Sequences
[0087] One aspect of the invention provides derivatives of specific
promoters. One means for preparing derivatives of such promoters
comprises introducing mutations into the promoter sequences. Such
mutants may potentially have enhanced, reduced, or altered function
relative to the native sequence or alternatively, may be silent
with regard to function.
[0088] Mutagenesis may be carried out at random and the mutagenized
sequences screened for function. Alternatively, particular
sequences which provide the promoter region with desirable
expression characteristics could be identified and these or similar
sequences introduced into other related or non-related sequences
via mutation. Similarly, non-essential elements may be deleted
without significantly altering the function of the promoter. It is
further contemplated that one could mutagenize these sequences in
order to enhance their utility in expressing transgenes, especially
in a gene therapy construct in humans.
[0089] The means for mutagenizing a DNA segment comprising a
specific promoter sequence are well-known to those of skill in the
art. Mutagenesis may be performed in accordance with any of the
techniques known in the art, such as, and not limited to,
synthesizing an oligonucleotide having one or more mutations within
the sequence of a particular regulatory region. In particular,
site-specific mutagenesis is a technique useful in the preparation
of promoter mutants, through specific mutagenesis of the underlying
DNA. The technique further provides a ready ability to prepare and
test sequence variants, by introducing one or more nucleotide
sequence changes into the DNA.
[0090] Site-specific mutagenesis allows the production of mutants
through the use of specific oligonucleotide sequences which encode
the DNA sequence of the desired mutation, as well as a sufficient
number of adjacent nucleotides, to provide a primer sequence of
sufficient size and sequence complexity to form a stable duplex on
both sides of the deletion junction being traversed. Typically, a
primer of about 17 to about 75 nucleotides or more in length is
preferred, with about 10 to about 25 or more residues on both sides
of the junction of the sequence being altered.
[0091] In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by various publications. As
will be appreciated, the technique typically employs a phage vector
which exists in both a single stranded and double stranded form.
Typical vectors useful in site-directed mutagenesis include vectors
such as the M13 phage. These phage are readily commercially
available and their use is generally well known to those skilled in
the art. Double stranded plasmids also are routinely employed in
site directed mutagenesis to eliminate the step of transferring the
gene of interest from a plasmid to a phage.
[0092] Alternatively, the use of PCR.TM. with commercially
available thermostable enzymes such as Taq polymerase may be used
to incorporate a mutagenic oligonucleotide primer into an amplified
DNA fragment that can then be cloned into an appropriate cloning or
expression vector. The PCR.TM.-mediated mutagenesis procedures of
Tomic et al. (1990) and Upender et al. (1995) provide two examples
of such protocols.
[0093] The preparation of sequence variants of the selected
promoter or intron-encoding DNA segments using site-directed
mutagenesis is provided as a means of producing potentially useful
species and is not meant to be limiting as there are other ways in
which sequence variants of DNA sequences may be obtained. For
example, recombinant vectors encoding the desired promoter sequence
may be treated with mutagenic agents, such as hydroxylamine, to
obtain sequence variants.
[0094] Typically, vector mediated methodologies involve the
introduction of the nucleic acid fragment into a DNA or RNA vector,
the clonal amplification of the vector, and the recovery of the
amplified nucleic acid fragment. Examples of such methodologies are
provided by U.S. Pat. No. 4,237,224, incorporated herein by
reference. A number of template dependent processes are available
to amplify the target sequences of interest present in a sample,
such methods being well known in the art and specifically disclosed
herein.
[0095] One efficient, targeted means for preparing mutagenized
promoters or enhancers relies upon the identification of putative
regulatory elements within the target sequence. These can be
identified, for example, by comparison with known promoter
sequences. Sequences which are shared among genes with similar
functions or expression patterns are likely candidates for the
binding of transcription factors and are likely elements to confer
tissue specific expression patterns.
[0096] Other assays may be used to identify responsive elements in
a promoter region or gene. Such assays will be known to those of
skill in the art (see for example, Sambrook et al., 1989; Zhang et
al, 1997; Shan et al., 1997; Dai and Burnstein, 1996; Cleutjens et
al., 1997; Ng et al., 1994; Shida et al., 1993), and include DNase
I footprinting studies, Elecromobility Shift Assay patterns (EMSA),
the binding pattern of purified transcription factors, effects of
specific transcription factor antibodies in inhibiting the binding
of a transcription factor to a putative responsive element, Western
analysis, nuclear run-on assays, and DNA methylation interference
analysis.
[0097] Preferred promoter constructs may be identified that retain
the desired, or even enhanced, activity. The smallest segment
required for activity may be identified through comparison of the
selected deletion or mutation constructs. Once identified, such
segments may be duplicated, mutated, or combined with other known
or regulatory elements and assayed for activity or regulatory
properties. Promoter region sequences used to identify regulatory
elements can also be used to identify and isolate transcription
factors that bind a putative regulatory sequence or element,
according to standard methods of protein purification, such as
affinity chromatography, as discussed above.
[0098] Preferably, identified promoter region sequences, whether
used alone or combined with additional promoters, enhancers, or
regulatory elements, will be induced and/or regulated by an
external agent, such as a hormone, transcription factor, enzyme, or
pharmaceutical agent, to express operatively linked genes or
sequences (Zhang et al., 1997; Shan et al., 1997). Alternatively,
such a construct may be designed to cease expression upon exposure
to an external agent.
[0099] Following selection of a range of deletion mutants of
varying size, the activities of the deleted promoters for
expression of the linked CAT gene may be determined according to
standard protocols.
[0100] The precise nature of the deleted portion of the promoter
may be determined using standard DNA sequencing, such as Sanger
dideoxy termination sequencing, to identify which promoter
sequences have been removed in each of the assayed deletion
mutants. Thus, a correlation may be obtained between the presence
or absence of specific elements within the promoter sequence and
changes in activity of the linked reporter gene.
[0101] 5. FDH Nucleic Acids
[0102] a. Nucleic Acids and Uses Thereof
[0103] Certain aspects of the present invention concern at least
one FDH nucleic acid. In certain aspects, the at least one FDH
nucleic acid comprises a wild-type or mutant FDH or nucleic acid.
In particular aspects, the FDH or nucleic acid encodes for at least
one transcribed nucleic acid. In certain aspects, the FDH or
nucleic acid comprises at least one transcribed nucleic acid. in
particular aspects, the FDH or nucleic acid encodes at least one
FDH or protein, polypeptide or peptide, or biologically functional
equivalent thereof. In other aspects, the FDH or nucleic acid
comprises at least one nucleic acid segment of the exemplary SEQ ID
NO:1, or at least one biologically functional equivalent
thereof.
[0104] The present invention also concerns the isolation or
creation of at least one recombinant construct or at least one
recombinant host cell through the application of recombinant
nucleic acid technology known to those of skill in the art or as
described herein. The recombinant construct or host cell may
comprise at least one FDH or nucleic acid, and may express at least
one FDH or protein, peptide or peptide, or at least one
biologically functional equivalent thereof.
[0105] As used herein "wild-type" refers to the naturally occurring
sequence of a nucleic acid at a genetic locus in the genome of an
organism, and sequences transcribed or translated from such a
nucleic acid. Thus, the term "wild-type" also may refer to the
amino acid sequence encoded by the nucleic acid. As a genetic locus
may have more than one sequence or alleles in a population of
individuals, the term "wild-type" encompasses all such naturally
occurring alleles. As used herein the term "polymorphic" means that
variation exists (i.e., two or more alleles exist) at a genetic
locus in the individuals of a population. As used herein "mutant"
refers to a change in the sequence of a nucleic acid or its encoded
protein, polypeptide or peptide that is the result of the hand of
man.
[0106] A nucleic acid may be made by any technique known to one of
ordinary skill in the art. Non-limiting examples of synthetic
nucleic acid, particularly a synthetic oligonucleotide, include a
nucleic acid made by in vitro chemically synthesis using
phosphotriester, phosphite or phosphoramidite chemistry and solid
phase techniques such as described in EP 266,032, incorporated
herein by reference, or via deoxynucleoside H-phosphonate
intermediates as described by Froehler et al., 1986, and U.S. Pat.
No. 5,705,629, each incorporated herein by reference. A
non-limiting example of enzymatically produced nucleic acid include
one produced by enzymes in amplification reactions such as PCR.TM.
(see for example, U.S. Pat. No. 4,683,202 and U.S. Pat. No.
4,682,195, each incorporated herein by reference), or the synthesis
of oligonucleotides described in U.S. Pat. No. 5,645,897,
incorporated herein by reference. A non-limiting example of a
biologically produced nucleic acid includes recombinant nucleic
acid production in living cells, such as recombinant DNA vector
production in bacteria (see for example, Sambrook et al. 1989,
incorporated herein by reference).
[0107] A nucleic acid may be purified on polyacrylamide gels,
cesium chloride centrifugation gradients, or by any other means
known to one of ordinary skill in the art (see for example,
Sambrook et al. 1989, incorporated herein by reference).
[0108] The term "nucleic acid" will generally refer to at least one
molecule or strand of DNA, RNA or a derivative or mimic thereof,
comprising at least one nucleobase, such as, for example, a
naturally occurring purine or pyrimidine base found in DNA (e.g.
adenine "A," guanine "G," thymine "T" and cytosine "C") or RNA
(e.g. A, G, uracil "U" and C). The term "nucleic acid" encompass
the terms "oligonucleotide" and "polynucleotide." The term
"oligonucleotide" refers to at least one molecule of between about
3 and about 100 nucleobases in length. The term "polynucleotide"
refers to at least one molecule of greater than about 100
nucleobases in length. These definitions generally refer to at
least one single-stranded molecule, but in specific embodiments
will also encompass at least one additional strand that is
partially, substantially or fully complementary to the at least one
single-stranded molecule. Thus, a nucleic acid may encompass at
least one double-stranded molecule or at least one triple-stranded
molecule that comprises one or more complementary strand(s) or
"complement(s)" of a particular sequence comprising a strand of the
molecule. As used herein, a single stranded nucleic acid may be
denoted by the prefix "ss", a double stranded nucleic acid by the
prefix "ds", and a triple stranded nucleic acid by the prefix
"ts."
[0109] Thus, the present invention also encompasses at least one
nucleic acid that is complementary to a FDH or nucleic acid. In
particular embodiments the invention encompasses at least one
nucleic acid or nucleic acid segment complementary to the sequence
set forth in, for example, SEQ ID NO:1. Nucleic acid(s) that are
"complementary" or "complement(s)" are those that are capable of
base-pairing according to the standard Watson-Crick, Hoogsteen or
reverse Hoogsteen binding complementarity rules. As used herein,
the term "complementary" or "complement(s)" also refers to nucleic
acid(s) that are substantially complementary, as may be assessed by
the same nucleotide comparison set forth above. The term
"substantially complementary" refers to a nucleic acid comprising
at least one sequence of consecutive nucleobases, or
semiconsecutive nucleobases if one or more nucleobase moieties are
not present in the molecule, are capable of hybridizing to at least
one nucleic acid strand or duplex even if less than all nucleobases
do not base pair with a counterpart nucleobase. In certain
embodiments, a "substantially complementary" nucleic acid contains
at least one sequence in which about 70%, about 71%, about 72%,
about 73%, about 74%, about 75%, about 76%, about 77%, about 77%,
about 78%, about 79%, about 80%, about 81%, about 82%, about 83%,
about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,
about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99%, to about 100%, and any
range therein, of the nucleobase sequence is capable of
base-pairing with at least one single or double stranded nucleic
acid molecule during hybridization. In certain embodiments, the
term "substantially complementary" refers to at least one nucleic
acid that may hybridize to at least one nucleic acid strand or
duplex in stringent conditions. In certain embodiments, a "partly
complementary" nucleic acid comprises at least one sequence that
may hybridize in low stringency conditions to at least one single
or double stranded nucleic acid, or contains at least one sequence
in which less than about 70% of the nucleobase sequence is capable
of base-pairing with at least one single or double stranded nucleic
acid molecule during hybridization.
[0110] 6. Assays of Gene Expression
[0111] Assays may be employed within the scope of the instant
invention for determination of the relative efficiency of gene
expression. For example, assays may be used to determine the
efficacy of deletion mutants of specific promoter regions in
directing expression of operatively linked genes. Similarly, one
could produce random or site-specific mutants of promoter regions
and assay the efficacy of the mutants in the expression of an
operatively linked gene. Alternatively, assays could be used to
determine the function of a promoter region in enhancing gene
expression when used in conjunction with various different
regulatory elements, enhancers, and exogenous genes.
[0112] Gene expression may be determined by measuring the
production of RNA, protein or both. The gene product (RNA or
protein) may be isolated and/or detected by methods well known in
the art. Following detection, one may compare the results seen in a
given cell line or individual with a statistically significant
reference group of non-transformed control cells. Alternatively,
one may compare production of RNA or protein products in cell lines
transformed with the same gene operatively linked to various
mutants of a promoter sequence. In this way, it is possible to
identify regulatory regions within a novel promoter sequence by
their effect on the expression of an operatively linked gene.
[0113] In certain embodiments, it will be desirable to use genes
whose expression is naturally linked to a given promoter or other
regulatory element. For example, a prostate specific promoter may
be operatively linked to a gene that is normally expressed in
prostate tissues. Alternatively, marker genes may be used for
assaying promoter activity. Using, for example, a selectable marker
gene, one could quantitatively determine the resistance conferred
upon a tissue culture cell line or animal cell by a construct
comprising the selectable marker gene operatively linked to the
promoter to be assayed. Alternatively, various tissue culture cell
line or animal parts could be exposed to a selective agent and the
relative resistance provided in these parts quantified, thereby
providing an estimate of the tissue specific expression of the
promoter.
[0114] Screenable markers constitute another efficient means for
quantifying the expression of a given gene. Potentially any
screenable marker could be expressed and the marker gene product
quantified, thereby providing an estimate of the efficiency with
which the promoter directs expression of the gene. Quantification
can readily be carried out using either visual means, or, for
example, a photon counting device.
[0115] A preferred screenable marker gene for use with the current
invention is .beta.-glucuronidase (GUS). Detection of GUS activity
can be performed histochemically using 5-bromo-4-chloro-3-indolyl
glucuronide (X-gluc) as the substrate for the GUS enzyme, yielding
a blue precipitate inside of cells containing GUS activity. This
assay has been described in detail (Jefferson, 1987). The blue
coloration can then be visually scored, and estimates of expression
efficiency thereby provided. GUS activity also can be determined by
immunoblot analysis or a fluorometric GUS specific activity assay
(Jefferson, 1987). Similarly, 5-bromo-4chloro-3-indolyl galactoside
(X-gal) is often used as a selectable marker, which confers a blue
color on those transformants that comprise .beta.-galactosidase
activity.
EXAMPLES
[0116] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those
skilled in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the concept, spirit and scope
of the invention. More specifically, it will be apparent that
certain agents that are both chemically and physiologically related
may be substituted for the agents described herein while the same
or similar results would be achieved. All such similar substitutes
and modifications apparent to those skilled in the art are deemed
to be within the spirit, scope and concept of the invention as
defined by the appended claims.
Example 1
Methods to Construct Bacterial Strain and Plasmid
[0117] The strain BS1 was constructed from the strain GJT001
(Tolentino et al., 1992) by inactivating the native formate
dehydrogenase. The exemplary plasmid pSBF2 contains the fdh1 gene
from the yeast Candida boidinii (SEQ ID NO:1) under the control of
the lac promoter. The fdh1 gene encodes an NAD+-dependent formate
dehydrogenase (FDH) that converts formate to CO.sub.2 with the
regeneration of NADH from NAD+. This is in contrast with the native
formate dehydrogenase that converts formate to CO.sub.2 and H.sub.2
with no cofactor involvement (FIG. 2). Also shown in FIG. 2 is the
conversion of pyruvate to acetyl-CoA and formate by pyruvate
formate lyase (PFL) under anaerobic conditions and to acetyl-CoA
and CO.sub.2 by pyruvate dehydrogenase (PDH) under aerobic
conditions.
[0118] Recombinant bacterial strains and plasmids used in this
study are listed in Table 3.
TABLE-US-00001 TABLE 3 Bacterial strains and plasmids Significant
genotype Strains GJT001 Spontaneous cadR mutant of MC4100, Sm.sup.R
DH10B Cloning host M9s MC4100 .phi.(fdhF'-'lacZ), Ap.sup.R BS1
GJT001 .phi.(fdhF'-'lacZ), Ap.sup.R Plasmids pUC18 Cloning vector,
Ap.sup.R PDHK29, Control, cloning vector, Km.sup.R pDHK30 pFDH1
fdh1 in pBluescriptII SK+ PUCFDH Intermediate plasmid, fdh1 in
pUC18, Ap.sup.R pSBF2 fdh1 in pDHK30, Km.sup.R
[0119] Strain BS1 was constructed by replacing the wild-type fdhF
gene with a fdhF'-'lacZ fusion by a P1 vir-mediated phage
transduction with E. coli M9s (Pecher et al., 1983) as donor and E.
coli GJT001 as recipient. The P1 phage transduction was performed
following standard protocols (Maniatis et al., 1989). Ampicillin
resistant transductants were selected for further analysis. The
lack of formate dehydrogenase activity was confirmed by a
previously described method with minor modifications
(Mandrand-Berthelot et al., 1978). Briefly, wild type and
transduced GJT001 were grown on glucose minimal media plates for
two days in an anaerobic chamber under an atmosphere of H.sub.2 and
CO.sub.2. An overlay solution composed of 0.6% agar, 2 mg/ml benzyl
viologen, 0.25M sodium formate and 25 mM KH.sub.2PO.sub.4 (pH 7.0)
was poured over the plates. The presence of formate dehydrogenase
activity in the wild type GJT001 was evidenced by a change in color
of the colonies, which turned purple. The colonies of the
transductants remained white, thus indicating the lack of formate
dehydrogenase activity. The presence of the mutation of fdhF in the
transductants was also confirmed by PCR. Primers complementary to
the ends of the fdhF gene (forward primer SEQ ID NO:8,
5'-GATTAACTGGAGCGAGACC-3'; reverse primer SEQ ID NO:9,
5'-TCCGAAAGGAGGCTGTAG-3') (Zinoni et al., 1986) were used to
amplify this gene in both wild type and transduced GJT001. The
disruption of the fdhF gene in the transduced strain was confirmed
by the absence of a PCR product as opposed to a 2.2-kb product
corresponding to the complete gene in the wild type strain.
[0120] Plasmid pFDH1 was kindly provided by Dr. Y. Sakai (Sakai et
al., 1997). It contains a 3kb EcoRI insert containing the fdh1 gene
from the yeast Candida boidinii in pBluescriptII SK+. The fdh1 gene
in this plasmid is under the control of its native promoter.
Preliminary experiments with this plasmid showed no FDH activity,
suggesting that fdh1 from the yeast was not properly expressed in
E. coli. For this reason, the open reading frame of the fdh gene
from C. boidinii was amplified by PCR and placed under the control
of the lac promoter for overexpression in E. coli.
[0121] XL-PCR was performed using the GeneAmp XL PCR kit from PE
Applied Biosystems following the manufacturer's protocol. This kit
was chosen because of the proofreading ability of the enzyme rTth
DNA Polymerase, Polymerase, which not only promotes efficient DNA
synthesis but also corrects nucleotide misincorporations. Plasmid
pFDH1 was used as a template and the following were used as forward
and reverse primers respectively: forward primer SEQ ID NO:10,
5'-GCGGAATTCAGGAGGAATTTAAAAAGATCGTTTTAGTCTTATATGATGCT-3'; reverse
primer SEQ ID NO:11, CGCGGATCCTTTCTTATCGTGTTTACCGTAAGC-3'. An EcoRI
and a BamHI site were inserted in the forward and reverse primers
respectively, as represented by the underlined regions.
[0122] The program used for the PCR reaction consisted of an
initial denaturation step at 94.degree. C. for 1 minute and 30
seconds followed by 18 cycles of denaturation at 94.degree. C. for
30 seconds and combined annealing/extension at 55-66.degree. C. for
5 minutes. This was followed by 12 cycles in which the
annealing/extension time was increased by 15 seconds in each cycle
until it reached 8 minutes. A final step at 72.degree. C. for 10
minutes concluded the PCR.
[0123] The PCR product was verified by agarose gel electrophoresis.
It was purified from the reaction mixture and concentrated
following the protocol of the StrataPrep.TM. PCR Purification Kit
(Stratagene--La Jolla, Calif.). The purified fdh PCR product and
the vector pUC18 were digested with EcoRI and BamHI. Both fragments
were ligated and the ligation product was transformed into E. coli
strain DH10B. White colonies from Ap/Xgal/IPTG plates were selected
for further analysis and minipreps were performed. Insertion of the
fdh gene was confirmed by agarose gel electrophoresis after
digestion with EcoRI/SalI.
[0124] This plasmid served as an intermediate vector to facilitate
the insertion of the fdh gene into pDHK30 (Phillips et al., 2000)
in the right orientation. It was ultimately desired to have the fdh
gene in the pDHK30 backbone because it is a high copy number
plasmid with kanamycin resistance, which will not interfere with
the ampicillin resistance of the BSI strain. An additional
advantage of this vector is that it can be co-transformed in a
two-plasmid system together with the most common high copy number
vectors bearing a ColE1 origin.
[0125] The intermediate plasmid containing fdh (pUCFDH), and pDHK30
were digested with EcoRI/XbaI and ligated to obtain plasmid pSBF2.
The ligation product was transformed into DH10B and white colonies
from Km/Xgal plates were analyzed. Minipreps were obtained and
analyzed by agarose gel electrophoresis after digestion with
EcoRI/XbaI. An appropriate plasmid was selected and transformed
both into GJT001 and the fdh.sup.- strain BS1. Strain GJT001 was
also transformed with pDHK29, and BS1 was transformed with pDHK30
to serve as negative controls.
Example 2
FDH Activity Assay
[0126] Determining FDH activity of strains GJT001 (pSBF2) and BS1
(pSBF2) comprised growing a culture of cells overnight in LB media
supplemented with 20 g/L glucose and 100 mg/L kanamycin (Km) under
anaerobic conditions. The cultures were inoculated with 100 .mu.l
of a 5 ml overnight LB culture and grown in a shaker at 37.degree.
C. and 250 rpm. Cells were harvested by centrifugation of 20 ml of
culture at 4,000 g and 4.degree. C. for 10 minutes. The pellet was
suspended in 10 ml of 10-mM sodium phosphate buffer (refrigerated)
at pH 7.5 with 0.1M .beta.-mercaptoethanol and centrifuged as
described above. The cells were resuspended in 10 ml of 10-mM
sodium phosphate buffer (refrigerated) at pH 7.5 with 0.1M
.beta.-mercaptoethanol and sonicated for 6 minutes in an ice bath
(Sonicator: Heat System Ultrasonics, Inc. Model W-255; Settings:
60% cycle, max power=8). The sonicated cells were centrifuged at
1,500 g and 4.degree. C. for 60 min to remove cell debris and
reduce the NAD background. The formate dehydrogenase activity was
assayed at 30.degree. C. by adding 100 .mu.l of cell extract to 1
ml of a reaction mixture containing 1.67 mM NAD+, 167 mM sodium
formate and 100 mM .beta.-mercaptoethanol in phosphate buffer pH
7.5 and measuring the increase in absorbance of NADH at 340 nm
(Schutte et al., 1976) modified). One unit was defined as the
amount of enzyme that produced 1 .mu.mol of NADH per minute at
30.degree. C. Total protein concentration in cell extracts was
measured by Lowry's method (Sigma Kit) using bovine serum albumin
as standard.
Example 3
Growth Experiments: Anaerobic and Aerobic Conditions
[0127] Growth experiments were conducted on strains GJT001 (pDHK29)
and BS1 (pSBF2) by growing aerobically triplicate cultures in a
rotary shaker at 37.degree. C. and 250 rpm. The cultures were grown
in 250-ml shake flasks containing 50 ml of LB media supplemented
with 10 g/L glucose, 100 mg/L kanamycin, and 0 or 100 mM formate.
The O.D. at 600 nm was measured every 30 minutes during the
exponential growth phase.
[0128] The anaerobic tube experiments were performed using 40-ml or
45-ml glass vials with open top caps and PTFE/silicone rubber
septa. Each vial was filled with 35 ml (40-ml vials) or 40 ml
(45-ml vials) of LB media supplemented with 20 g/L glucose, 100
mg/L kanamycin, 0 or 50 mM formate, and 1 g/L NaHCO.sub.3 to reduce
the initial lag time that occurs under anaerobic conditions. The
triplicate cultures were inoculated with 100 .mu.l of a 5 ml LB
overnight culture. After inoculation, air (6 ml) was removed with a
syringe from the headspace to ensure anaerobic conditions. The
cultures were grown in a rotary shaker at 37.degree. C. and 250
rpm. A sample of the initial media was saved for analysis and
samples were withdrawn with a syringe at 24 hour intervals (24, 48,
and 72 hrs).
[0129] The aerobic experiment was performed by growing triplicate
cultures aerobically using either 125-ml shake flasks containing 25
ml of LB media or 250-ml shake flasks containing 50 ml of LB media.
The LB media was supplemented with about 10 g/L glucose, 100 mg/L
kanamycin, and different amounts of formate. The cultures were
inoculated with 50 .mu.l or 100 .mu.l of a 5 ml LB overnight
culture and grown in a rotary shaker at 37.degree. C. and 250 rpm.
A sample of the initial media was saved for HPLC analysis and
samples were collected after 24 hours of growth.
Example 4
Methods of Analysis
[0130] Cell density (OD) was measured at 600 nm in a Spectronic
1001 spectrophotometer (Bausch & Lomb, Rochester, N.Y.).
Fermentation samples were centrifuged for 5 minutes in a
microcentrifuge. The supernatant was filtered through a 0.45-micron
syringe filter and stored chilled for HPLC analysis. The
fermentation products and glucose concentrations were quantified
using an HPLC system (Thermo Separation Products, Allschwil,
Switzerland) equipped with a cation-exchange column (HPX-87H,
BioRad Labs, Hercules, Calif.) and a differential refractive index
detector. A mobile phase of 2.5 mM H.sub.2SO.sub.4 solution at a
0.6 ml/min flow rate was used, and the column was operated at
55.degree. C.
Example 5
FDH Activity
[0131] The effect of increasing intracellular NADH availability by
genetic engineering on the metabolic patterns of Escherichia coli
under anaerobic and aerobic conditions was determined. More
specifically, the effect of regenerating NADH by substituting the
native cofactor-independent formate dehydrogenase in E. coli by the
NAD+-dependent FDH from Candida boidinii, as well as the effect of
supplementing the culture media with formate was demonstrated
herein.
[0132] Plasmid pSBF2, containing the fdh1 gene from Candida
boidinii under the control of the lac promoter, was constructed and
characterized by determining the activity of the new FDH. Table 4
shows the specific FDH activity of strains BS1 (pSBF2) and GJT001
(pSBF2) in Units/mg of total protein. One unit is defined as the
amount of enzyme that produced 1 .mu.mol of NADH per minute at
30.degree. C. Values shown are average of triplicates from
anaerobic tube cultures. N.D.: not detected (less than 0.001 U/mg).
The FDH activity of strain GJT001 (pSBF2) was 46% higher (0.416
U/mg) than the activity of BS1 (pSBF2) (0.284 U/mg). Control
strains GJT001 (pDHK29) and BS1 (pDHK30) showed no detectable FDH
activity.
TABLE-US-00002 TABLE 4 Specific FDH activity Strain Activity (U/mg)
BS1 (pSBF2) 0.284 .+-. 0.002 GJT001 (pSBF2) 0.416 .+-. 0.004 GJT001
(pDHK29) N.D. BS1 (pDHK30) N.D.
[0133] The effect of substituting the native FDH with the
NAD+-dependent pathway was characterized by calculating the
specific growth rate (.mu.) of strains BS1 (pSBF2) and GJT001
(pDHK29) in aerobic shake flask experiments. Table 5 presents the
results of these experiments with and without 100 mM formate. The
specific growth rate of strain BS1 (pSBF2) was 35% lower
(0.986.+-.0.002) than that of GJT001 (pDHK29) (1.511.+-.0.016)
without formate supplementation. However, by the end of the
fermentation the cell density of BS1 (pSBF2) was comparable to or
even higher than that of GJT001 (pDHK29).
[0134] In addition, the effect on the specific growth rate of
formate supplementation at the level of 100 mM was examined.
Formate addition to the media lengthened the duration of the lag
phase for both strains, but more for BS1 (pSBF2). The difference in
the specific growth rate between BS1 (pSBF2) and GJT001 (pDHK29)
decreases with addition of formate. Under these conditions, the
specific growth rate of GJT001 (pDHK29) is only 10% higher.
Addition of formate did not affect significantly the specific
growth rate of BS1 (pSBF2), however; it decreased that of GJT001
(pDHK29) by 28%. As in the case without formate supplementation,
the final cell density of BS1 (pSBF2) was comparable to that of
GJT001 (pDHK29).
TABLE-US-00003 TABLE 5 Specific aerobic growth rate (.mu.) of
strains BS1 (pSBF2) and GJT001 (pDHK29). Strains: 0 mM Formate 100
mM Formate BS1(pSBF2) 0.986 .+-. 0.002 0.972 .+-. 0.014
GJT001(pDHK29) 1.511 .+-. 0.016 1.086 .+-. 0.043 Values shown are
average of triplicates.
Example 6
Increased Intracellular NADH Availability and Alcohol Production
During Anaerobiosis
[0135] Anaerobic tube experiments were performed with strains
GJT001 (pDHK29), GJT001 (pSBF2), BS1 (pSBF2), and BS1 (pDHK30) to
investigate the effect on the metabolic patterns of the elimination
of the native FDH and the addition or substitution of the new FDH.
FIG. 3A to 3F illustrate the results of these experiments,
including the final cell density (FIG. 3A), the amount of glucose
consumed in millimolar (mM) (FIG. 3B), and the concentrations of
different metabolites produced (mM) after 72 hours of culture (FIG.
3C to 3F). Values shown are the average of triplicate cultures.
[0136] A comparison of the results for the control strains GJT001
(pDHK29) and BS1 (pDHK30) shows the effect of eliminating the
native FDH on the metabolic patterns of E. coli. An increase in
residual formate was observed for the strain lacking FDH activity.
As shown in FIG. 3B, glucose consumption for BS1 (pDHK30) decreased
by 47% relative to GJT001 (pDHK29). This led to a decrease in final
cell density (29%; FIG. 3A), as well as, in succinate (39%; FIG.
3E), lactate (66%; FIG. 3F), and ethanol (22%; FIG. 3C) production.
However, the level of acetate (FIG. 3D) was very similar to that of
GJT001 (pDHK29). This translates into a decrease (24%) in the
ethanol to acetate (Et/Ac) ratio. This decrease in the Et/Ac ratio
together with the decrease in other reduced metabolites (lactate
and succinate) indicates the presence of a more oxidized
environment for the strain lacking formate dehydrogenase activity.
These results suggest that under normal conditions GJT001 (pDHK29)
recaptures a portion of the H.sub.2 produced from the degradation
of formate by the native FDH possibly by means of some hydrogenase,
and this recapture accounts for the slightly more reduced
intracellular environment observed for this strain relative to BS1
(pDHK30).
[0137] Table 6 gives the quantitative amounts of NADH in terms of
(NADH).sub.U/G1=moles of NADH available for reduced product
formation per mole of glucose consumed, where (NADH).sub.U=Total
NADH used for product formation per unit volume at the end of
fermentation (mmol/L) and was estimated from the concentrations of
reduced metabolites by calculating the NADH used for their
production according to the pathways shown on FIG. 1, with 50 mM
initial formate supplementation. Values shown are from average of
triplicate cultures.
TABLE-US-00004 TABLE 6 NADH availability of various strains under
anaerobic conditions. (NADH).sub.U/Gl Strain (mol/mol) GJT001
(pDHK29) 2.40 GJT001 (pSBF2) 4.34 BS1 (pSBF2) 4.35 BS1 (pDHK30)
2.38 GJT001 (pDHK29) + F 2.33 BS1 (pSBF2) + F 4.39
[0138] An analysis of the results for BS1 (pSBF2) relative to BS1
(pDHK30) and for GJT001 (pSBF2) relative to GJT001 (pDHK29)
provides an understanding of the effect of overexpressing the
NAD+-dependent FDH both alone or in conjunction with the native
FDH, respectively. In both cases the trend is similar, but the
effect is more pronounced for the BS1 strains due to the decrease
in the metabolites observed for BS1 (pDHK30) relative to GJT001
(pDHK29). Both strains containing the new FDH present a significant
increase in glucose consumption, cell density, ethanol, and
succinate formation, accompanied by a decrease in lactate and
acetate relative to the control strains. This translates into a
dramatic increase in the ethanol to acetate (Et/Ac) ratio of
22-fold for GJT001 (pSBF2) and 35 to 36-fold for BS1 (pSBF2).
[0139] The results for GJT001 (pSBF2) and BS1 (pSBF2) show the
effect of having both the native and new FDH active in the same
strain or just the new FDH, respectively. A comparison of these
results shows that these strains behave very similarly. The largest
difference between these two strains is a 16% decrease in acetate,
and consequently a 21% increase in Et/Ac ratio for BS1 (pSBF2)
relative to GJT001 (pSBF2). This means that the NAD+-dependent FDH
is competing effectively with the native FDH for available formate.
This finding is supported by the fact that the Km value for formate
of the native FDH is twice (26 mM) that of the NAD+-dependent FDH
(13 mM) according to the literature (Schutte et al., 1976; Axley
and Grahame, 1991). Although these results suggest that the
fdh.sup.- mutation is not necessary to observe the effect of
overexpressing the NAD+-dependent FDH, the decrease in acetate
levels observed for the fdh.sup.- strain suggests that this
mutation is be slightly beneficial in some cases.
[0140] Analyzing the results of BS1 (pSBF2) relative to GJT001
(pDHK29) can better elucidate the effect of substituting the
cofactor-independent native formate-degradation pathway in E. coli
by the NAD+-dependent pathway. Substitution of the native FDH by
the new FDH increased glucose consumption (3-fold), final cell
density (59%), as well as the production of ethanol (15-fold) and
succinate (55%), while it decreased lactate (91%) and acetate (43%)
production (see FIG. 3C to 3F). This translates into a dramatic
increase in the ethanol to acetate (Et/Ac) ratio of 27-fold (FIG.
4B).
[0141] These results suggest that overexpression of the
NAD+-dependent FDH increases intracellular NADH availability, and
this in turn leads to a drastic shift in the metabolic patterns of
E. coli. An increase in NADH availability favored the production of
more reduced metabolites, particularly, those requiring 2NADH
molecules per molecule of product formed, like ethanol and
succinate. The preferred product was ethanol, with a final
concentration reaching as high as 175 mM for BS1 (pSBF2), as
compared to 11.5 mM for the wild type control, GJT001 (pDHK29).
This makes ethanol the major fermentation product for BS1 (pSBF2)
anaerobic cultures, accounting for 91% of the metabolites produced
based on mM concentrations, as opposed to 18% for GJT001 (pDHK29).
Simultaneously, lactate was converted from a major product,
representing 57% of the produced metabolites in the wild type
strain to only a minor product, accounting for less than 2% of the
metabolites. This shift towards the production of ethanol as a
major product is comparable to that obtained with overexpression of
the ethanologenic enzymes from Zymomonas mobilis in the pet operon
in E. coli (Ingram and Conway, 1988). Remarkably, these results
indicate a significant production of ethanol despite the lack of
overexpression of enzymes specifically directed towards ethanol
production.
[0142] The dramatic increase in ethanol production combined with a
decrease in acetate levels led to the drastic increase in the Et/Ac
ratio observed, which reached as high as 27 for BS1 (pSBF2), as
compared to 1.0 for GJT001 (pDHK29). It is evident from these
results that the cell adjusts its partitioning at the acetyl-CoA
node by changing the ethanol (consumes 2 NADH) to acetate (consumes
no NADH) ratio to achieve a redox balance, as was previously
observed in experiments utilizing carbon sources with different
oxidation states (San et al., 2001). These findings also support
the idea that NADH induces expression of alcohol dehydrogenase
(adhE) (Leonardo et al., 1996).
[0143] The significant decrease in lactate levels obtained with
overexpression of the NAD+-dependent FDH can be explained by noting
that although lactate formation also requires NADH, it only
consumes 1 NADH, while ethanol formation consumes 2 NADH. These
results suggest that when there is an excess of reducing
equivalents, ethanol formation (2 NADH) is preferred over lactate
formation (1 NADH) since it provides a faster route to NAD+
regeneration. These observations support previous findings in
experiments utilizing carbon sources with different oxidation
states (San et al., 2001).
[0144] FIG. 3A to 3F and FIGS. 4A and 4B illustrate the results of
anaerobic tube experiments performed with strains GJT001 (pDHK29)
and BS1 (pSBF2) in which the media was supplemented with 50 mM
formate. Addition of formate to both strains increased lactate
levels. A comparison of the results for BS1 (pSBF2) and GJT001
(pDHK29) indicates a 6-fold increase in ethanol accompanied by a
69% decrease in acetate levels. This leads to a 21-fold increase in
the Et/Ac ratio with the substitution of the native FDH for the
NAD+-dependent FDH. This means that anacrobically it is not
necessary to supplement the culture with formate.
[0145] The amounts of formate converted to CO.sub.2 for the
different strains, with and without formate addition under
anaerobic conditions, were calculated by subtracting the measured
residual formate concentration from the concentration of formate
produced plus the initial formate concentration in the media for
the experiments with formate supplementation. The amount of formate
produced was obtained based on the assumption that one mol of
formate is produced per mol of acetyl-CoA formed through the PFL
pathway (see FIG. 2). Therefore, the amount of formate produced was
calculated by adding the concentrations of ethanol and acetate
formed from acetyl-CoA.
[0146] The data indicate that overexpression of the NAD+-dependent
FDH drastically increases the conversion of formate almost equally
for both strains BS1 (pSBF2) and GJT001 (pSBF2) suggesting that
this new enzyme competes very effectively with the native FDH for
the available formate. These two strains as well as GJT001 (pDHK29)
converted all the formate produced during fermentation when there
was no external formate added to the media, while strain BS1
(pDHK30) converted only minimal amounts of formate as expected.
[0147] It is also interesting to note that external addition of
formate to the media had opposite effects on the native and new
FDH. Formate supplementation of GJT001 (pDHK29) cultures
significantly increased (2 to 3-fold) the amount of formate
converted by the native enzyme, although only 78% of the available
formate was converted. These results suggest that addition of extra
formate has a stimulatory effect on this pathway or that initially
the pathway was limited by the amount of formate, while after
formate supplementation it became limited by the enzyme activity
instead. In contrast, addition of formate to BS1 (pSBF2) anaerobic
cultures decreased the amount of formate converted, with only 69%
of the available formate being degraded, suggesting possible
inhibition of the new FDH at these levels of formate. Plausibly,
this decrease in formate conversion is the indirect consequence of
a lower glucose consumption and optical density. Although the total
levels of formate produced for this strain without external formate
addition were higher than with the 50 mM supplementation, the cells
did not experience high levels of formate at a given time because
it is being degraded as it is produced. In contrast, in the
supplementation experiment, the cell experienced a higher initial
formate concentration.
Example 7
Increased Intracellular NADH Availability During Aerobiosis
[0148] Shake flask experiments were performed with strains GJT001
(pDHK29) and BS1 (pSBF2) to investigate the effect of increasing
intracellular NADH availability by substituting the native FDH in
E. coli by the NAD+-dependent enzyme on the metabolic patterns
under aerobic conditions. These experiments were performed with and
without 100 mM formate supplementation. Addition of formate as a
substrate for the new FDH during aerobic growth was necessary
because under these conditions the cells normally do not produce
formate due to lack of activity of the pyruvate formate lyase (PFL)
enzyme. FIG. 5A to 5F presents the results of these experiments,
including the final cell density (FIG. 5A), glucose consumed (mM)
(FIG. 5B), and the concentrations of different metabolites produced
(mM) after 24 hours of culture (FIG. 5C to 5F). For both strains
only minimal amounts of residual formate (less than 6 mM) were
detected.
[0149] This data indicate that addition of formate to BS1 (pSBF2)
aerobic cultures induced the production of ethanol, lactate, and
succinate, metabolites that are normally produced only under
anaerobic conditions. The amount detected corresponds to a 36-fold
increase in ethanol (FIG. 5C), 7-fold increase in succinate (FIG.
5E), and the production of lactate (FIG. 6A). Glucose consumption
increased by 50% and acetate levels by 11%. The Et/Ac ratio
increased by 32-fold.
[0150] Also addressed by this data is the effect of formate
supplementation on the native FDH was also investigated. Addition
of formate to GJT001 (pDHK29) aerobic cultures caused an increase
of 50% in glucose consumption, the same percentage of increase
observed for BS1 (pSBF2). However, the increase in acetate levels
was much higher (47%) with formate supplementation, as well as the
increase in final cell density (48%). On the other hand, the
production of ethanol was much lower, only 5.15 mM after 24 hours,
and succinate levels increased only by 72% compared to a 7-fold
increase for BS1 (pSBF2).
[0151] The results obtained for both strains with formate
supplementation shows a 27-fold increase in lactate, 4-fold
increase in ethanol, 3-fold increase in succinate, accompanied by a
30% decrease in acetate (5-fold increase in Et/Ac) for the
NAD+-dependent FDH relative to the native FDH. The glucose
consumption was similar for both strains, while the final cell
density was slightly higher for BS1 (pSBF2).
[0152] These results demonstrate that it is possible to increase
the availability of intracellular NADH through the substitution of
the native FDH in E. coli by an NAD+-dependent FDH. The higher
intracellular NADH levels provide a more reduced environment even
under aerobic conditions. As a result, the cells utilize this extra
NADH to reduce metabolic intermediates leading to the formation of
fermentation products in order to achieve a redox balance.
Conversely, under normal aerobic conditions, the environment is so
oxidized that reduced fermentation products are not formed. Under
aerobic conditions, only acetate, a more oxidized metabolite that
does not require NADH, is normally produced. The results described
herein also suggest that although the native FDH is able to
indirectly recapture some of the extra reducing power in the
formate added, the new FDH is a lot more effective because it
recaptures this extra reducing power directly as NADH.
Example 8
Effect of Formate on Reduction Processes During Aerobiosis
[0153] In addition, the effect of supplementing the media with
different levels of formate (0, 50, 100, 150, and 200 mM) was
investigated in aerobic cultures of BS1 (pSBF2). It is interesting
to note that lactate was absent at 0 and 50 mM initial formate, but
it was produced at 100, 150, and 200 mM initial formate (FIGS. 6A
and 6B). The concentration of lactate increased with an increase in
the initial formate levels. The same trend was evident in succinate
production with the difference that similar levels were produced at
0 and 50 mM initial formate (FIG. 6B). On the other hand, the cells
produced ethanol only after formate supplementation, but the levels
did not significantly increase with an increase in formate levels.
Acetate production and final cell density did not follow any
notable trend with increasing levels of formate supplementation.
Glucose consumption increased with addition of formate and remained
constant with different formate levels because all the glucose was
consumed by 24 hours in all the formate supplemented cultures.
[0154] It was also observed that the concentration of residual
formate reached 63.5 mM for the 200 mM initial formate experiment,
a 10-fold increase from the residual levels in the 150 mM
experiment. The levels of residual formate were lower than 12 mM
for all other initial formate levels. These findings possibly
indicate that the culture is past saturation at this formate level.
In addition, based on the formate conversion levels observed, more
NADH is being generated by this pathway than that used to produce
reduced metabolites. The cells are possibly using this extra NADH
formed for ATP generation through the electron transport system
since they are growing aerobically.
[0155] The results of the formate supplementation experiment show
that different formate levels can be used to provide different
levels of reducing power. Higher levels of reducing power
aerobically mainly increased lactate production. In contrast, in
anaerobic cultures with no formate supplementation, where the
environment was a lot more reduced, ethanol production was highly
increased, while lactate levels decreased. However, formate
supplementation in anaerobic cultures provoked an increase in
lactate levels, which is consistent with the aerobic case.
Example 9
Increasing Reductive Capabilities In Vivo
[0156] The data indicates that it is possible to increase the
availability of intracellular NADH through metabolic engineering,
thereby providing enhanced reducing power under both anaerobic and
aerobic conditions.
[0157] The substitution of the native cofactor independent FDH
pathway by the NAD+-dependent FDH provoked a significant metabolic
redistribution both anaerobically and aerobically. Under anaerobic
conditions, the increased NADH availability favored the production
of more reduced metabolites, as evidenced by a dramatic increase in
the ethanol to acetate ratio for BS1 (pSBF2) as compared to the
GJT1 (pDHK29) control (FIG. 4B). This led to a shift towards the
production of ethanol as the major fermentation product (FIG.
3C).
[0158] Further during aerobic growth, the increased availability of
NADH induced a shift to fermentation even in the presence of oxygen
by stimulating pathways that are normally inactive under these
conditions. Because formate is not a normal product under aerobic
conditions, it was added to the media to increase NADH
availability. The addition of formate to BS1 (pSBF2) aerobic
cultures induced the production of ethanol, lactate, and succinate,
metabolites that are normally produced only under anaerobic
conditions.
Example 10
Chemostat Cultures
[0159] The novel approach to increasing availability of
intracellular NADH in vivo through a NADH recycling system is
applied to the production of commercially viable compounds, such as
ethanol. The NADH recycling system comprises a biologically active
NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii,
and overexpression thereof in Escherichia coli. The NADH recycling
system (e.g., recombinant formate dehydrogenase pathway) produces
one mole of NADH per one mole of formate converted to carbon
dioxide (FIG. 2). This recombinant system bears contrast with the
native formate dehydrogenase which converts formate to CO.sub.2 and
H.sub.2 with no cofactor involvement. The new NADH recycling system
allows the cells to retain the reducing power that are otherwise
lost by release of formate or hydrogen.
[0160] The functionality of this approach was further characterized
by evaluating anaerobic chemostat cultures in a controlled
bioreactor environment.
Example 11
Methods of Anaerobic Chemostat Experiments
[0161] Initially, the inoculum was grown as a 5-ml LB culture
supplemented with 100 mg/L ampicillin and/or kanamycin for 8-12
hours. Then, 100 .mu.l of the 5-ml culture was transferred to 50 ml
of LB in a 250-ml shake flask with the appropriate antibiotic, and
grown at 37.degree. C. and 250 rpm for 8-12 hours in a rotary
shaker. This culture was used to inoculate the bioreactor.
[0162] Luria-Bertani broth (LB) medium supplemented with 110 mM of
glucose, was used for the chemostat runs. To reduce the initial lag
time that occurs under anaerobic conditions, 1 g/L NaHCO.sub.3 was
added to the LB media. The media was also supplemented with 30
.mu.L/L antifoam 289 (Sigma), 100 mg/L ampicillin, and/or
kanamycin.
[0163] The fermentations were carried under anaerobic chemostat
conditions at a dilution rate of 0.2 hr.sup.-1. A 2.5 L bioreactor
(New Brunswick Scientific, Bioflo III) was used. It initially
contained 1.3 L of medium during the anaerobic batch stage and then
was maintained at 1.20 L working volume for the anaerobic chemostat
stage. The pH, temperature and agitation were maintained at 7.0,
32.degree. C., and 250 rpm, respectively. A constant flow of
nitrogen (10-12 ml/min) was maintained through the fermentor
headspace to establish anaerobic conditions. The continuous culture
reached steady state after 4 to 6 residence times. Samples were
taken during the steady state phase.
[0164] Cell dry weight was determined by collection of 100 ml of
culture in an ice bath. The samples were centrifuged at 4,000 g and
4.degree. C. for 10 minutes, washed with 0.15M sodium chloride
solution, and dried in an oven at 55.degree. C. until constant
weight. The final weight of the dried samples was corrected for the
weight of NaCl in the washing solution.
[0165] For chromatography, samples of the fermentation broth were
collected and centrifuged at 6000 g and 4.degree. C. for 10 minutes
in a Sorvall centrifuge (SS-34 rotor).
Example 12
FDH Activity in Anaerobic Chemostat Cultures
[0166] Experiments were performed under anaerobic chemostat
conditions with strains GJT001 and BS1 containing a control plasmid
to investigate the effect of eliminating the native formate
dehydrogenase activity. The results of those experiments indicated
that with inactivation of the native FDH, which converts formate to
CO.sub.2 and H.sub.2, reducing power is lost in the form of
formate. This resulted in a more oxidized intracellular environment
as reflected by a significant decrease in the NADH/NAD+ ratio (48%)
and a decrease in the Et/Ac ratio (19%). These observations arc
consistent with previously reported results under anaerobic tube
conditions with these two strains. These results imply that under
normal conditions when the native FDH is active, the cells are able
to recapture some of the reducing power in the hydrogen released
from the degradation of formate possibly by means of a native
hydrogenase. These findings suggest that substitution of the native
FDH by an NAD+-dependent FDH, which transfers the reducing
equivalents directly from formate to NADH, provides a more reduced
intracellular environment by recapturing more effectively the
reducing power that otherwise is lost.
[0167] Anaerobic chemostat experiments were performed with strains
GJT001 (pSBF2), BS1 (pSBF2), and GJT001 (pDHK29). Strain GJT001
(pDHK29) contains the native formate dehydrogenase (FDH) only,
while strain BS1 (pSBF2) has the C. boidinii FDH, and GJT001
(pSBF2) has both FDH enzymes active. A chemostat mode was chosen
because it allows the determination of the concentration of NADH
and NAD+ and the metabolic fluxes during steady state. It also
allows fixing of the specific growth rate for each strain by fixing
the dilution rate (0.2 h.sup.-1).
[0168] Table 7 presents the specific NAD+-dependent FDH activity of
strains GJT001 (pSBF2) and BS1 (pSBF2) obtained from the anaerobic
chemostat runs in units/mg of total protein. One unit is defined as
the amount of enzyme that produced 1 .mu.mol of NADH per minute at
30.degree. C. As this table shows, the specific FDH activity of
both strains was very similar. Strain GJT001 (pDHK29) showed no
detectable FDH activity. One unit is defined as the amount of
enzyme that produced 1 .mu.mol of NADH per minute at 30.degree. C.
Values shown are average of triplicates. N.D.: not detected (less
than 0.001 U/mg).
TABLE-US-00005 TABLE 7 Specific NAD+-dependent FDH activity. Strain
Activity (U/mgTP) GJT001 (pSBF2) 0.242 .+-. 0.009 BS1 (pSBF2) 0.231
.+-. 0.007 GJT001 (pDHK29) N.D.
Example 13
Metabolic Flux Redistribution in Anaerobic Chemostat Cultures
[0169] Steady state concentrations of metabolites are given in
Table 8 as millimolar (mM) units as measured by the HPLC, as well
as the percent of CO.sub.2 and H.sub.2 per volume in the off-gases
stream as measured by the GC. The concentrations are in anaerobic
chemostat (average of three samples) at D=0.2 h.sup.-1. CO.sub.2
and H.sub.2 in % per volume as measured from the off-gases by GC.
Dry weight (D.W.) in g/L.
[0170] Table 9 presents the results as calculated metabolic fluxes
in mmol/(g dry weight*h) represented as v.sub.1 to v.sub.12
according to the diagram illustrated in FIG. 8. Note that v.sub.12
represents the newly added NAD+-dependent FDH pathway. In addition,
v.sub.RF represents the flux of residual formate excreted to the
media based on HPLC measurements. The metabolic fluxes with an
asterisk were calculated based on measured metabolites, while the
other fluxes were derived from the measured metabolites based on
the relationships shown in FIG. 8, the law of mass conservation,
and the pseudo-steady-state hypothesis (PSSH) on the intracellular
intermediate metabolites as described previously (Aristidou et al.,
1999; Yang et al., 1999). Metabolic fluxes with an asterisk were
calculated based on measured metabolites, while the other fluxes
were derived from the measured metabolites based on the
relationships shown in FIG. 8. The percentages of increase (+) or
decrease (-) presented are relative to strain GJT001 (pDHK.29). The
"+" indicates that the culture comprised the newly added
NAD+-dependent FDH pathway.
[0171] Table 10 includes the NAD(H/+) concentrations in .mu.mol/g
dry weight (D.W.) in addition to the NADH formed through the
oxidation of glucose and the new FDH degradation pathway, as well
as the NADH utilized for the formation of reduced metabolites,
namely, succinate, lactate, and ethanol. The percentages of
increase (+) or decrease (-) presented on these tables are relative
to strain GJT001 (pDHK29), and are an average of three samples at a
dilution, D=0.2 h.sup.-1. (R.sub.NADH).sub.f=specific NADH
formation rate=v.sub.4+v.sub.12; (R.sub.NADH).sub.u=specific NADH
utilization rate=2v.sub.6+v.sub.7+2v.sub.10. Both rates are in
units of mmol/(gD.W.*h). The percentages of increase (+) or
decrease (-) are determined relative to strain GJT001 (pDHK29).
[0172] The overexpression of the NAD+-dependent FDH drastically
changed the distribution of metabolic fluxes in E. coli. The most
notable effect observed is the shift in the ethanol to acetate
ratio (Et/Ac), which indicates an increase in intracellular NADH
availability. This ratio increased from 1.06 for the control strain
to 3.47 for the strain with the new FDH and 3.82 for the strain
with both enzymes coexpressed. This represents a 3 to 4-fold
increase in the Et/Ac ratio relative to the control. These findings
are similar to the results obtained when sorbitol (Et/Ac=3.62), a
more reduced carbon source that can therefore produce more reducing
equivalents in the form of NADH, was used instead of glucose
(Et/Ac=1.00) in anaerobic chemostat experiments (San et al.,
2001).
TABLE-US-00006 TABLE 8 Metabolite concentrations of recombinant
strains. Strain GJT001 GJT001 BS1 (pDHK29) (pSBF2) (pSBF2) Glucose
113.36 .+-. 0.59 94.74 .+-. 3.99 64.43 .+-. 4.98 Consumed Succinate
13.50 .+-. 0.31 9.49 .+-. 1.33 5.05 .+-. 0.46 Lactate 37.37 .+-.
0.60 4.38 .+-. 0.41 1.96 .+-. 0.32 Residual 64.35 .+-. 0.96 36.89
.+-. 3.66 43.91 .+-. 2.04 Formate Acetate 74.26 .+-. 0.77 35.75
.+-. 2.74 25.88 .+-. 1.09 Ethanol 78.86 .+-. 0.97 136.54 .+-. 8.78
89.70 .+-. 6.62 Et/Ac 1.06 3.82 3.46 CO.sub.2 11.58 .+-. 0.38 16.25
.+-. 2.89 10.71 .+-. 0.93 H.sub.2 16.95 .+-. 0.05 7.01 .+-. 0.59
0.02 .+-. 0.02 D.W. 2.48 .+-. 0.03 1.31 .+-. 0.01 2.03 .+-.
0.08
TABLE-US-00007 TABLE 9 Anaerobic chemostat results. GJT001 GJT001 %
Inc/ BS1 % Inc/ Flux To: (pDHK29) (pSBF2) Dec (pSBF2) Dec
.nu..sub.1 Glucose Uptake* 7.81 12.93 65.63 5.53 -29.20 .nu..sub.2
Biosynthesis 0.78 0.23 -71.06 0.27 -65.61 .nu..sub.3 Glyceraldehyde
3-P 7.03 12.71 80.87 5.26 -25.14 .nu..sub.4 PEP 14.05 25.42 80.87
10.52 -25.14 .nu..sub.5 Pyruvate 13.12 24.12 83.81 10.09 -23.13
.nu..sub.6 Succinate* 0.93 1.30 39.38 0.43 -53.41 .nu..sub.7
Lactate* 2.57 0.60 -76.76 0.17 -93.48 .nu..sub.8 Formate 10.55
23.52 123.00 9.92 -5.97 .nu..sub.9 H.sub.2* 6.12 1.55 -74.70 0.00
-99.95 .nu..sub.RF Residual Formate* 4.43 5.04 13.63 3.77 -15.00
.nu..sub.10 Ethanol* 5.43 18.64 243.15 7.70 41.71 .nu..sub.11
Acetate* 5.12 4.88 -4.59 2.22 -56.59 .nu..sub.12 New FDH
Pathway.sup.+ 0.00 16.94 -- 6.15 --
TABLE-US-00008 TABLE 10 Anaerobic chemostat results. Strain GJT001
GJT001 % Inc/ BS1 % Inc/ (pDHK29) (pSBF2) Dec (pSBF2) Dec NADH 6.64
6.40 -3.60 5.53 -16.70 NAD+ 6.27 5.90 -5.95 4.34 -30.74 NADH/NAD+
1.06 1.09 2.74 1.29 21.42 Total NAD(H/+) 12.90 12.29 -4.74 9.87
-23.52 (R.sub.NADH).sub.f 14.05 42.35 -- 16.67 --
(R.sub.NADH).sub.u 15.30 40.47 -- 16.43 -- (NADH).sub.U/Gl 1.96
3.13 59.69 2.97 51.53
Example 14
NADH/NAD+ Ratio
[0173] Importantly, the effect of the cofactor manipulations is
smaller under chemostat conditions as compared to previous findings
in anaerobic tube experiments (Et/Ac=27.0 for BS1 (pSBF2)). This is
explained by the difference in the growth environment and
conditions the cells are exposed to in a batch versus chemostat
cultivation. In a chemostat bioreactor the specific growth rate
equals the dilution rate, is fixed externally and is dependent on
the strain and media composition for a batch culture. In addition,
the transient nature of the batch cultivation implies that the
concentration of both substrates and metabolites varies constantly
with time, while at steady state these concentrations are
time-invariant for a chemostat culture. Specifically, the cells are
exposed to a very rich environment for most of the time during
batch cultivation, while they are always under limiting environment
under a chemostat setting. A similar behavior was observed
previously in experiments where a significant acetate reduction was
achieved under batch conditions by modulating glucose uptake using
a glucose analog supplementation strategy, however the effect was
greatly minimized under chemostat conditions (Chou et al.,
1994).
[0174] The current results support previous findings (San et al.,
2001) that the cell adjusts its partitioning at the acetyl-CoA node
by changing the ethanol (consumes 2 NADH) to acetate (consumes no
NADH) ratio to achieve a redox balance. Therefore, a change in the
ethanol to acetate ratio (Et/Ac) is used as an indirect indicator
of a change in the NADH/NAD+ ratio.
[0175] In the chemostat experiments, the NADH/NAD+ ratio increased
slightly in strain BS1 (pSBF2), and it remained relatively
unchanged for GJT001 (pSBF2) as compared to GJT001 (pDHK29). These
results suggest that the cells regenerate the extra reducing power
in the form of NADH that was available from the overexpression of
the new FDH by increasing the flux to ethanol, which consumes 2
NADH, instead of accumulating the NADH as such. These findings
might indicate that the NADH/NAD+ ratio is not always a good
indicator of the oxidation state of the cell because in an effort
to achieve a redox balance, the turnover is fast. This idea is
supported by the fact that more than 96% of the NADH formed through
the oxidation of glucose and the new FDH degradation pathway,
(R.sub.NADH).sub.f, can be accounted for as being utilized for the
formation of reduced metabolites, namely, succinate, lactate, and
ethanol, (R.sub.NADH).sub.u (Table 10). In addition, the specific
NADH formation and utilization rates for both strains containing
the new FDH are significantly higher than those of the control
strain (Table 10).
Example 15
Effect of Redistributing Metabolic Flux
[0176] An analysis of the metabolic fluxes of the two experimental
strains relative to the control strain shows a significant increase
in the flux to ethanol, accompanied by a decrease in the flux to
acetate and a marked decrease in the flux to lactate. The increase
in the ethanol flux (2 NADH) in combination with the decrease in
the flux to lactate (1 NADH) indicate that when there is an excess
of reducing equivalents, ethanol formation is preferred since it
provides a faster route to NAD+ regeneration. These results are in
agreement with our previous findings in chemostat experiments
utilizing carbon sources with different oxidation state (San et
al., 2001). In those experiments, the lactate flux was highest for
gluconate, a more oxidized carbon source, and lowest for sorbitol,
a more reduced carbon source relative to glucose.
[0177] Tn addition, Table 9 presents the flux of formate converted
to CO.sub.2 through both the native FDH pathway (v.sub.9) and the
new NAD+-dependent FDH pathway (v.sub.12) for the different
strains. The flux to formate was obtained based on the assumption
that one mole of formate is produced per mole of acetyl-CoA formed
through the PFL pathway (FIG. 9). Therefore, the flux to formate
(v.sub.8) was calculated by adding the fluxes to ethanol (v.sub.10)
and acetate (v.sub.11) from acetyl-CoA. The total formate converted
was calculated by subtracting the measured residual formate flux
(v.sub.RF) from the flux to formate (v.sub.8). The flux through the
new FDH pathway (v.sub.12) for strain GJT001 (pSBF2) was determined
by subtracting the flux to H.sub.2 (v.sub.9), determined from GC
measurements, from the total formate converted.
[0178] The absence of H.sub.2 production as determined by GC
analysis of the off-gases (Tables 7 and 8) confirmed the lack of
native formate dehydrogenase activity in strain BS1 (pSBF2). For
strain GJT001 (pSBF2), in which both FDH enzymes are active, 92% of
the total formate converted to CO.sub.2 was degraded through the
NAD+-dependent FDH pathway. This result indicates that the new FDH
enzyme competes very effectively with the native FDH for the
available formate. This finding is consistent with the reported Km
value for formate of the native FDH being twice (26 mM) that of the
NAD+-dependent FDH (13 mM) (Schutte et al., 1976; Axley and
Grahame, 1991).
[0179] Coexpression of both FDH enzymes in strain GJT001 (pSBF2)
increased glucose uptake under chemostat conditions relative to the
control strain. However, a decrease in glucose uptake was observed
under the same conditions for strain BS1 (pSBF2). Due to the
difference observed in glucose uptake, the yields in carbon-mole
produced per carbon-mole of glucose consumed were calculated for
the different metabolites. This allows a better understanding of
how one carbon-mole (C-mole) of glucose consumed by the cell is
distributed to the production of the different metabolites in each
of the strains studied.
Example 16
Effect on Fermentation Products
[0180] The calculated yields for the different fermentation
products are given in C-mole produced per C-mole of glucose
consumed on FIG. 9. Values shown are yields in C-mole produced per
C-mole of glucose consumed. The strains are identified as follows:
GC=GJT001 (pDHK29), GF=GJT001 (pSBF2), and BF=BS1 (pSBF2). Results
were obtained from anaerobic chemostat experiments at a dilution
rate of 0.2 hr.sup.-1. Unexpectedly, the percentage of carbon
recovery obtained without accounting for the biomass was 90% or
higher for all the strains.
[0181] For the control strain GJT001 (pDHK29), one C-mole of
glucose is distributed almost equally to ethanol (0.23), acetate
(0.22), and formate (0.23). The rest of it goes mostly to lactate
(0.16), with succinate (0.06) being only a minor product. In
contrast, for the strains containing the new FDH pathway, almost
half of each C-mole of glucose was directed towards ethanol
production (GJT001 (pSBF2): 0.48, BS1 (pSBF2): 0.46), while the
yield to acetate decreased to 0.13, and that of formate increased
(0.30) for both strains. At the same time, lactate proportion
decreased to that of a minor product with a yield as low as 0.02.
This yield is even lower than the yield of succinate, which
remained relatively unchanged. It is important to note that the
distribution of C-mole yields for strains GJT001 (pSBF2) and BS1
(pSBF2) is almost identical. This finding implies that under the
experimental conditions studied the native FDH does not interfere
with the action of the new FDH of redistributing the metabolic
fluxes on a C-mole basis.
[0182] FIG. 9 also shows the amount of formate produced that is
converted through either one or both of the FDH pathways. For the
control strain, 57% of the formate produced is converted, while 80%
is converted for GJT001 (pSBF2) and 63% for BS1 (pSBF2). These
results show an increase in the conversion of formate with the
overexpression of the new FDH, further suggesting that the new FDH
has higher activity or higher affinity for formate than the native
cofactor independent FDH.
Example 17
Recombinant FDH Competes with Native FDH
[0183] The reductive capabilities of the chemostat cultures further
demonstrate an increase in the availability of intracellular NADH
through metabolic engineering and therefore provide a more reduced
environment under anaerobic chemostat conditions. The substitution
of the native cofactor independent FDH pathway by the
NAD+-dependent FDH provoked a significant redistribution of both
metabolic fluxes and C-mole yields under anaerobic chemostat
conditions.
[0184] The increased NADH availability favored the production of
more reduced metabolites, as evidenced by a 3 to 4-fold increase in
the ethanol to acetate ratio for BS1 (pSBF2) and GJT001 (pSBF2) as
compared to the GJT1 (pDHK29) control. This was the result of an
increase in the ethanol yield combined with a decrease in the
acetate yield. It was also observed that the flux to lactate was
reduced significantly with the overexpression of the new FDH.
[0185] In addition, the chemostat results suggest that the new FDH
is able to compete very effectively with the native FDH; therefore,
it is not necessary to eliminate the native FDH activity in order
to achieve the desired results, making this approach easier to
implement in a variety of applications. It should also be noted
that the effect of this system was reduced under the current
experimental conditions as compared to the uncontrolled anaerobic
tube experiments reported previously, in which the Et/Ac ratio
represented a 27-fold increase with substitution of the native by
the NAD+-dependent FDH (see FIG. 4B).
[0186] Thus, the data demonstrate that NADH manipulations in a
system comprising a NADH recycling system achieve redirection of
carbon fluxes to produce reduced products. Based on this data,
effects on other reduced cofactors such as FADH or NADPH directly
are expected because of interconversions among the reduced
cofactors in the cell. This reasoning leads to a plausible
application of the present invention in terms of manipulating
intracellular availability of other reduced cofactors such as FADH,
a flavin coenzyme that is usually tightly bound to one particular
enzyme, and NADPH, a nicotinamide cofactor that like NADH acts as a
hydrogen carrier and is capable of diffusing from enzyme to
enzyme.
Example 18
NADH Recycling in Biodesulfurization
[0187] The usual model for the study of biodesulfurization is the
compound dibenzothiophene. It has been extensively studied in the
context of nonbiological and biological desulfurization.
Dibenzothiophene is a member of a class of polyaromatic sulfur
heterocycles (PASHs), and one of thousands of PASHs found in a
hydrotreated diesel sample. Alkylated dibenzothiophenes are also
target molecules for biodesulfurization technology.
[0188] Cells capable of biodesulfurization are transformed with a
recombinant NADH recycling system.
[0189] Known bacterial strains which are capable of breaking down
dibenzothiophene using this pathway include Rhodococcus strains
IGTS8, T09, and RA-18, and Gordonia desulfuricans 213E. Also
capable of biodesulfurization are E. coli that express recombinant
genes from Rhodococcus, and Pseudomonas putida that express
recombinant genes from Rhodococcus. Gordonia rubropertinctus strain
T08 is capable of biodesulfurization using a novel pathway.
[0190] The first step in the desulfurization pathway is the
transfer of the target molecules from oil into the cells.
Rhodococcus sp. and other bacteria have been shown to metabolize
many insoluble molecules through direct transfer from oil into the
cells.
[0191] Dibenzothiophene monooxygenase (SEQ ID NO:12, Accession NO:
P54995), the enzyme responsible for the first two oxidation in the
biodesulfurization pathway has been isolated and characterized, and
its gene has been cloned and sequenced. The enzyme catalyzes the
transfer of an electron from flavin mononucleotide to
dibenzothiophene, and catalyzes the oxidation of dibenzothiophene
to the sulfoxide and the oxidation of the sulfoxide to the sulfone.
The cleavage of the first carbon-sulfur linkage of dibenzothiophene
is catalyzed by dibenzothiophene sulfone monooxygenase (SEQ ID
NO:13, Accession NO: P54997). This enzyme and its gene have been
characterized. Production of sulfite is the last reaction in the
pathway. This is catalyzed by a desulfinase (SEQ ID NO:14,
Accession NO: P54998), whose gene has been cloned and sequenced.
Sulfite is released as well as an oil soluble product, hydroxyl
biphenyl.
[0192] NADH is required in this reaction system to keep the supply
of reduced flavin mononucleotide in balance.
[0193] Additionally, large-scale biodesulfurization in bacteria
utilizing recombinant, constitutively-expressed members of
biodesulfurization pathway (dsz class genes), requires NADH, which
can be limiting.
Example 19
NADH Recycling in Biopolymer Production
[0194] Polyhydroxyalkanoates (PHAs) are linear polyesters produced
in nature in bacteria. Bacteria accumulate PHAs when a carbon
source is abundant. The genes involved in PHA synthesis from well
over 20 different microorganisms have been characterized. These
recombinant genes are transformed into cells comprising the NADH
recycling system. The genes involved in PHA synthesis include
beta-ketothiolase, acetoacetyl-CoA-reductase, butyrate
dehydrogenase and poly-3-hydroxybutyrate synthase.
[0195] Bacterial cells capable of PHA synthesis include the carbon
monoxide (CO)-resistant strain of the hydrogen bacteria Ralstonia
eutropha B5786, Synechocystis sp. PCC6803, and Pseudomonas
corrugata. These bacteria are transformed with a recombinant NADH
recycling system.
[0196] NADH recycling allows increased polymer production.
Example 20
NADH Recycling in Polypeptide Production
[0197] Cells comprising the NADH recycling system are transformed
with a vector pSM552-545C-, containing the lacZ gene, which encodes
beta-galactosidase. The expression of the lacZ gene is regulated by
a powerful pH-inducible promoter. Experiments are conducted in a
well-controlled fermenter under optimal conditions for the
particular expression system. The expression of the lacZ gene is
induced by changing the pH from 7.5, which has minimal induction,
to a pH of 6.0, which is the optimal induction pH. NADH, acetate,
and beta-galactosidase production are monitored through standard
means in the art. Increased beta-galactosidase production is
associated with lower levels of acetate. Lower levels of acetate
production are associated with cells comprising the NADH recycling
system.
REFERENCES
[0198] All patents and publications mentioned in the specification
are indicative of the level of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
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[0205] U.S. Pat. No. 5,925,565
[0206] U.S. Pat. No. 5,935,819
[0207] U.S. Pat. No. 5,871,986
[0208] U.S. Pat. No. 4,879,236
[0209] U.S. Pat. No. 4,237,224
[0210] U.S. Pat. No. 5,783,681
[0211] U.S. Pat. No. 5,264,092
[0212] U.S. Pat. No. 5,705,629
[0213] U.S. Pat. No. 4,682,195
[0214] U.S. Pat. No. 5,645,897
[0215] U.S. Pat. No. 6,337,204
[0216] EP 266032
Publications
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[0224] Berrios-Rivera, S. J., Bennett, G. N. and San, K. -Y.
(2001). Metabolic Engineering of Escherichia coli Through Genetic
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[0225] Berrios-Rivera, S. J., Yang, Y. -T., San, K. -Y. and
Bennett, G. N. (2000). Effect of Glucose Analog Supplementation in
Anaerobic Chemostat Cultures of Escherichia coli. Metabolic Eng. 2,
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[0226] Chou, C. -H., Bennett, G. N. and San, K. -Y. (1994). Effect
of Modulated Glucose Uptake on High-Level Recombinant Protein
Production in a Dense Escherichia coli Culture. Biotech. Prog. 10,
644-647.
[0227] Foster, J. W., Park, Y. K., Penfound, T., Fenger, T. and
Spector, M. P. (1990). Regulation of NAD Metabolism in Salmonella
typhimurium: Molecular Sequence Analysis of the Bifunctional nadR
Regulator and the nadA-pnuC Operon. J. Bacteriol. 172,
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[0228] Galkin, A., Kulakova, L., Yoshimura, T., Soda, K. and Esaki,
N. (1997). Synthesis of Optically Active Amino Acids from
.alpha.-Keto Acids with Escherichia coli Cells Expressing
Heterologous Genes. App. Environ. Microbiol. 63, 4651-4656.
[0229] Graef, M. R., Alexeeva, S., DeSnoep, J. L. and Mattos, M. J.
T. d. (1999). The Steady-State Internal Redox State (NADH/NAD)
Reflects the External Redox State and Is Correlated with Catabolic
Adaptation in Escherichia coli. J. Bacteriol. 181, 2351-2357.
[0230] Hummel, H. and Kula, M. -R. (1989). Dehydrogenases for the
synthesis of chiral compounds. Eur. J. Biochem. 184, 1-13.
[0231] Ingram, L. O. and Conway, T. (1988). Expression of Different
Levels of Ethanologenic Enzymes from Zymomonas mobilis in
Recombinant Strains of Escherichia coli. App. Environ. Microbiol.
54, 397-404.
[0232] Kragl, U., Kruse, W., Hummel, W. and Wandrey, C. (1996).
Enzyme Engineering Aspects of Biocatalysis: Cofactor Regeneration
as Example. Biotech. Bioeng. 52, 309-319.
[0233] Leonardo, M. R., Cunningham, P. R. and Clark, D. P. (1993).
Anaerobic Regulation of the adhE Gene, Encoding the Fermentative
Alcohol Dehydrogenase of Escherichia coli. J. Bacteriol. 175,
870-878.
[0234] Leonardo, M. R., Dailly, Y. and Clark, D. P. (1996). Role of
NAD in regulating the adhE gene of Escherichia coli. J. Bacteriol.
178, 6013-6018.
[0235] Lopez de Felipe, F., Kleerebezem, M., Vos, W. M. d. and
Hugenholtz, J. (1998). Cofactor Engineering: a Novel Approach to
Metabolic Engineering in Lactococcus lactis by Controlled
Expression of NADH Oxidase. J. Bacteriol. 180, 3804-3808.
[0236] Mandrand-Berthelot, M. -A., Wee, M. Y. K. and Haddock, B. A.
(1978). An Improved Method for the Identification and
Characterization of Mutants of Escherichia coli Deficient in
Formate Dehydrogenase Activity. FEMS Microbiol. Let. 4, 37-40.
[0237] Maniatis, T., Fritsch, E. F. and Sambrook, J. (1989).
"Molecular cloning: a laboratory manual," pp. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
[0238] Park, D. H. and Zeikus, J. G. (1999). Utilization of
Electrically Reduced Neutral Red by Actinobacillus succinogenes:
Physiological Function of Neutral Red in Membrane-Driven Fumarate
Reduction and Energy Conservation. J. Bacteriol. 181,
2403-2410.
[0239] Pecher, A., Zinoni, F., Jatisatienr, C., Wirth, R.,
Hennecke, H. and Bock, A. (1983). On the redox control of synthesis
of anaerobically induced enzymes in enterobacteriaceae. Arch.
Microbiol. 136, 131-136.
[0240] Phillips, G. J., Park, S. -K. and Huber, D. (2000). High
Copy Number Plasmids Compatible with Commonly Used Cloning Vectors.
BioTechniques 28, 400-408.
[0241] Riondet, C., Cachon, R., Wache, Y., Alraraz, G. and Divies,
C. (2000). Extracellular Oxidoreduction Potential Modifies Carbon
and Electron Flow in Escherichia coli. J. Bacteriol. 182,
620-626.
[0242] Sakai, Y., Murdanoto, A. P., Konishi, T., Iwamatsu, A. and
Kato, N. (1997). Regulation of the Formate Dehydrogenase Gene,
FDH1, in the Methylotrophic Yeast Candida boidinii and Growth
Characteristics of an FDH1-Disrupted Strain on Methanol,
Methylamine and Choline. J. Bacteriol. 179, 4480-4485.
[0243] San, K. -Y., Bennett, G. N., Berrios-Rivera, S. J., Vadali,
R., Sariyar, B. and Blackwood, K. (2001). Metabolic engineering
through cofactor manipulation and its effects on metabolic flux
redistribution in Escherichia coli. (Submitted)
[0244] Schutte, H., Flossdorf, J., Salmi, H. and Kula, M. -R.
(1976). Purification and Properties of Formaldehyde Dehydrogenase
and Formate Dehydrogenase from Candida boidinii. Eur. J. Biochem.
62, 151-160.
[0245] Tishkov, V. I., Galkin, A. G., Fedorchuk, V. V., Savitsky,
P. A., Rojkova, A. M., Gieren, H. and Kula, M. R. (1999). Pilot
scale production and isolation of recombinant NAD+- and
NADP+-specific formate dehydrogenases. Biotech. Bioeng. 64,
187-93.
[0246] Tolentino, G. J., Meng, S. -Y., Bennett, G. N. and San, K.
-Y. (1992). A pH-regulated promoter for the expression of
recombinant proteins in Escherichia coli. Biotech. Let. 14,
157-162.
[0247] Wimpenny, J. W. T. and Firth, A. (1972). Levels of
Nicotinamide Adenine Dinucleotide and Reduced Nicotinamide Adenine
Dinucleotide in Facultative Bacteria and the Effect of Oxygen. J.
Bacteriol. 111, 24-32.
[0248] Yang, Y. -T., Aristidou, A. A., San, K. -Y. and Bennett, G.
N. (1999). Metabolic flux analysis of Escherichia coli deficient in
the acetate production pathway and expressing the Bacillus subtilis
acetolactate synthase. Metabolic Eng. 1, 26-34.
[0249] Zinoni, F., Birkmann, A., Stadtman, T. and Bock, A. (1986).
Nucleotide sequence and expression of the selenocysteine-containing
polypeptide of formate dehydrogenase
(formate-hydrogen-lyase-linked) from Escherichia coli. Proc. Nat.
Acad. Sci. 83, 4650-4.
[0250] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objectives and obtain
the ends and advantages mentioned as well as those inherent
therein. Systems, pharmaceutical compositions, treatments, methods,
procedures and techniques described herein are presently
representative of the preferred embodiments and are intended to be
exemplary and are not intended as limitations of the scope. Changes
therein and other uses will occur to those skilled in the art which
are encompassed within the spirit of the invention or defined by
the scope of the pending claims.
Sequence CWU 1
1
1411562DNACandida boidinii 1ttcaactaaa aattgaacta tttaaacact
atgatttcct tcaattatat taaaatcaat 60ttcatatttc cttacttctt tttgctttat
tatacatcaa taactcaatt aactcattga 120ttatttgaaa aaaaaaaaca
tttattaact taactccccg attatatatt atattattga 180ctttacaaaa
tgaagatcgt tttagtctta tatgatgctg gtaagcacgc tgctgatgaa
240gaaaaattat atggttgtac tgaaaataaa ttaggtattg ctaattggtt
aaaagatcaa 300ggtcatgaac taattactac ttctgataaa gaaggtgaaa
caagtgaatt ggataaacat 360atcccagatg ctgatattat catcaccact
cctttccatc ctgcttatat cactaaggaa 420agacttgaca aggctaagaa
cttaaaatta gtcgttgtcg ctggtgttgg ttctgatcac 480attgatttag
attatattaa tcaaacaggt aagaaaatct cagtcttgga agttacaggt
540tctaatgttg tctctgttgc tgaacacgtt gtcatgacca tgcttgtctt
ggttagaaat 600ttcgttccag cacatgaaca aattattaac cacgattggg
aggttgctgc tatcgctaag 660gatgcttacg atatcgaagg taaaactatt
gctaccattg gtgctggtag aattggttac 720agagtcttgg aaagattact
cccttttaat ccaaaagaat tattatacta cgattatcaa 780gctttaccaa
aagaagctga agaaaaagtt ggtgctagaa gagttgaaaa tattgaagaa
840ttagttgctc aagctgatat cgttacagtt aatgctccat tacacgcagg
tacaaaaggt 900ttaattaata aggaattatt atctaaattt aaaaaaggtg
cttggttagt caataccgca 960agaggtgcta tttgtgttgc tgaagatgtt
gcagcagctt tagaatctgg tcaattaaga 1020ggttacggtg gtgatgtttg
gttcccacaa ccagctccaa aggatcaccc atggagagat 1080atgagaaata
aatatggtgc tggtaatgcc atgactcctc actactctgg tactacttta
1140gatgctcaaa caagatacgc tgaaggtact aaaaatatct tggaatcatt
ctttactggt 1200aaatttgatt acagaccaca agatattatc ttattaaatg
gtgaatacgt tactaaagct 1260tacggtaaac acgataagaa ataaattttc
ttaacttgaa aactataatt gctataacaa 1320ttcttcaatt tctctttttc
ttcctttttt tgaagaattt ttaacaatca aaattttgac 1380tctttgattt
cccgcaatct ctgagctcag catactcatt attattttat tattattatt
1440attattactt ttattattat tatattttty cttctttaac gatatcgttt
gtgttttatc 1500ttttatgatt taaattttat acgaatttat gaatacaaca
aaatatttaa gtttacacaa 1560tg 15622364PRTCandida boidinii 2Met Lys
Ile Val Leu Val Leu Tyr Asp Ala Gly Lys His Ala Ala Asp1 5 10 15Glu
Glu Lys Leu Tyr Gly Cys Thr Glu Asn Lys Leu Gly Ile Ala Asn 20 25
30Trp Leu Lys Asp Gln Gly His Glu Leu Ile Thr Thr Ser Asp Lys Glu
35 40 45Gly Glu Thr Ser Glu Leu Asp Lys His Ile Pro Asp Ala Asp Ile
Ile 50 55 60Ile Thr Thr Pro Phe His Pro Ala Tyr Ile Thr Lys Glu Arg
Leu Asp65 70 75 80Lys Ala Lys Asn Leu Lys Leu Val Val Val Ala Gly
Val Gly Ser Asp 85 90 95His Ile Asp Leu Asp Tyr Ile Asn Gln Thr Gly
Lys Lys Ile Ser Val 100 105 110Leu Glu Val Thr Gly Ser Asn Val Val
Ser Val Ala Glu His Val Val 115 120 125Met Thr Met Leu Val Leu Val
Arg Asn Phe Val Pro Ala His Glu Gln 130 135 140Ile Ile Asn His Asp
Trp Glu Val Ala Ala Ile Ala Lys Asp Ala Tyr145 150 155 160Asp Ile
Glu Gly Lys Thr Ile Ala Thr Ile Gly Ala Gly Arg Ile Gly 165 170
175Tyr Arg Val Leu Glu Arg Leu Leu Pro Phe Asn Pro Lys Glu Leu Leu
180 185 190Tyr Tyr Asp Tyr Gln Ala Leu Pro Lys Glu Ala Glu Glu Lys
Val Gly 195 200 205Ala Arg Arg Val Glu Asn Ile Glu Glu Leu Val Ala
Gln Ala Asp Ile 210 215 220Val Thr Val Asn Ala Pro Leu His Ala Gly
Thr Lys Gly Leu Ile Asn225 230 235 240Lys Glu Leu Leu Ser Lys Phe
Lys Lys Gly Ala Trp Leu Val Asn Thr 245 250 255Ala Arg Gly Ala Ile
Cys Val Ala Glu Asp Val Ala Ala Ala Leu Glu 260 265 270Ser Gly Gln
Leu Arg Gly Tyr Gly Gly Asp Val Trp Phe Pro Gln Pro 275 280 285Ala
Pro Lys Asp His Pro Trp Arg Asp Met Arg Asn Lys Tyr Gly Ala 290 295
300Gly Asn Ala Met Thr Pro His Tyr Ser Gly Thr Thr Leu Asp Ala
Gln305 310 315 320Thr Arg Tyr Ala Glu Gly Thr Lys Asn Ile Leu Glu
Ser Phe Phe Thr 325 330 335Gly Lys Phe Asp Tyr Arg Pro Gln Asp Ile
Ile Leu Leu Asn Gly Glu 340 345 350Tyr Val Thr Lys Ala Tyr Gly Lys
His Asp Lys Lys 355 3603364PRTCandida methylica 3Met Lys Ile Val
Leu Val Leu Tyr Asp Ala Gly Lys His Ala Ala Asp1 5 10 15Glu Glu Lys
Leu Tyr Gly Cys Thr Glu Asn Lys Leu Gly Ile Ala Asn 20 25 30Trp Leu
Lys Asp Gln Gly His Glu Leu Ile Thr Thr Ser Asp Lys Glu 35 40 45Gly
Glu Thr Ser Glu Leu Asp Lys His Ile Pro Asp Ala Asp Ile Ile 50 55
60Ile Thr Thr Pro Phe His Pro Ala Tyr Ile Thr Lys Glu Arg Leu Asp65
70 75 80Lys Ala Lys Asn Leu Lys Ser Val Val Val Ala Gly Val Gly Ser
Asp 85 90 95His Ile Asp Leu Asp Tyr Ile Asn Gln Thr Gly Lys Lys Ile
Ser Val 100 105 110Leu Glu Val Thr Gly Ser Asn Val Val Ser Val Ala
Glu His Val Val 115 120 125Met Thr Met Leu Val Leu Val Arg Asn Phe
Val Pro Ala His Glu Gln 130 135 140Ile Ile Asn His Asp Trp Glu Val
Ala Ala Ile Ala Lys Asp Ala Tyr145 150 155 160Asp Ile Glu Gly Lys
Thr Ile Ala Thr Ile Gly Ala Gly Arg Ile Gly 165 170 175Tyr Arg Val
Leu Glu Arg Leu Leu Pro Phe Asn Pro Lys Glu Leu Leu 180 185 190Tyr
Tyr Asp Tyr Gln Ala Leu Pro Lys Glu Ala Glu Glu Lys Val Gly 195 200
205Ala Arg Arg Val Glu Asn Ile Glu Glu Leu Val Ala Gln Ala Asp Ile
210 215 220Val Thr Val Asn Ala Pro Leu His Ala Gly Thr Lys Gly Leu
Ile Asn225 230 235 240Lys Glu Leu Leu Ser Lys Phe Lys Lys Gly Ala
Trp Leu Val Asn Thr 245 250 255Ala Arg Gly Ala Ile Cys Val Ala Glu
Asp Val Ala Ala Ala Leu Glu 260 265 270Ser Gly Gln Leu Arg Gly Tyr
Gly Gly Asp Val Trp Phe Pro Gln Pro 275 280 285Ala Pro Lys Asp His
Pro Trp Arg Asp Met Arg Asn Lys Tyr Gly Ala 290 295 300Gly Asn Ala
Met Thr Pro His Tyr Ser Gly Thr Thr Leu Asp Ala Gln305 310 315
320Thr Arg Tyr Ala Glu Gly Thr Lys Asn Ile Leu Glu Ser Phe Phe Thr
325 330 335Gly Lys Phe Asp Tyr Arg Pro Gln Asp Ile Ile Leu Leu Asn
Gly Glu 340 345 350Tyr Val Thr Lys Ala Tyr Gly Lys His Asp Lys Lys
355 3604401PRTPseudomonas 4Met Ala Lys Val Leu Cys Val Leu Tyr Asp
Asp Pro Val Asp Gly Tyr1 5 10 15Pro Lys Thr Tyr Ala Arg Asp Asp Leu
Pro Lys Ile Asp His Tyr Pro 20 25 30Gly Gly Gln Thr Leu Pro Thr Pro
Lys Ala Ile Asp Phe Thr Pro Gly 35 40 45Gln Leu Leu Gly Ser Val Ser
Gly Glu Leu Gly Leu Arg Lys Tyr Leu 50 55 60Glu Ser Asn Gly His Thr
Leu Val Val Thr Ser Asp Lys Asp Gly Pro65 70 75 80Asp Ser Val Phe
Glu Arg Glu Leu Val Asp Ala Asp Val Val Ile Ser 85 90 95Gln Pro Phe
Trp Pro Ala Tyr Leu Thr Pro Glu Arg Ile Ala Lys Ala 100 105 110Lys
Asn Leu Lys Leu Ala Leu Thr Ala Gly Ile Gly Ser Asp His Val 115 120
125Asp Leu Gln Ser Ala Ile Asp Arg Asn Val Thr Val Ala Glu Val Thr
130 135 140Tyr Cys Asn Ser Ile Ser Val Ala Glu His Val Val Met Met
Ile Leu145 150 155 160Ser Leu Val Arg Asn Tyr Leu Pro Ser His Glu
Trp Ala Arg Lys Gly 165 170 175Gly Trp Asn Ile Ala Asp Cys Val Ser
His Ala Tyr Asp Leu Glu Ala 180 185 190Met His Val Gly Thr Val Ala
Ala Gly Arg Ile Gly Leu Ala Val Leu 195 200 205Arg Arg Leu Ala Pro
Phe Asp Val His Leu His Tyr Thr Asp Arg His 210 215 220Arg Leu Pro
Glu Ser Val Glu Lys Glu Leu Asn Leu Thr Trp His Ala225 230 235
240Thr Arg Glu Asp Met Tyr Pro Val Cys Asp Val Val Thr Leu Asn Cys
245 250 255Pro Leu His Pro Glu Thr Glu His Met Ile Asn Asp Glu Thr
Leu Lys 260 265 270Leu Phe Lys Arg Gly Ala Tyr Ile Val Asn Thr Ala
Arg Gly Lys Leu 275 280 285Cys Asp Arg Asp Ala Val Ala Arg Ala Leu
Glu Ser Gly Arg Leu Ala 290 295 300Gly Tyr Ala Gly Asp Val Trp Phe
Pro Gln Pro Ala Pro Lys Asp His305 310 315 320Pro Trp Arg Thr Met
Pro Tyr Asn Gly Met Thr Pro His Ile Ser Gly 325 330 335Thr Thr Leu
Thr Ala Gln Ala Arg Tyr Ala Ala Gly Thr Arg Glu Ile 340 345 350Leu
Glu Cys Phe Phe Glu Gly Arg Pro Ile Arg Asp Glu Tyr Leu Ile 355 360
365Val Gln Gly Gly Ala Leu Ala Gly Thr Gly Ala His Ser Tyr Ser Lys
370 375 380Gly Asn Ala Thr Gly Gly Ser Glu Glu Ala Ala Lys Phe Lys
Lys Ala385 390 395 400Val5384PRTArabidopsis thaliana 5Met Ala Met
Arg Gln Ala Ala Lys Ala Thr Ile Arg Ala Cys Ser Ser1 5 10 15Ser Ser
Ser Ser Gly Tyr Phe Ala Arg Arg Gln Phe Asn Ala Ser Ser 20 25 30Gly
Asp Ser Lys Lys Ile Val Gly Val Phe Tyr Lys Ala Asn Glu Tyr 35 40
45Ala Thr Lys Asn Pro Asn Phe Leu Gly Cys Val Glu Asn Ala Leu Gly
50 55 60Ile Arg Asp Trp Leu Glu Ser Gln Gly His Gln Tyr Ile Val Thr
Asp65 70 75 80Asp Lys Glu Gly Pro Asp Cys Glu Leu Glu Lys His Ile
Pro Asp Leu 85 90 95His Val Leu Ile Ser Thr Pro Phe His Pro Ala Tyr
Val Thr Ala Glu 100 105 110Arg Ile Lys Lys Ala Lys Asn Leu Lys Leu
Leu Leu Thr Ala Gly Ile 115 120 125Gly Ser Asp His Ile Asp Leu Gln
Ala Ala Ala Ala Ala Gly Leu Thr 130 135 140Val Ala Glu Val Thr Gly
Ser Asn Val Val Ser Val Ala Glu Asp Glu145 150 155 160Leu Met Arg
Ile Leu Ile Leu Met Arg Asn Phe Val Pro Gly Tyr Asn 165 170 175Gln
Val Val Lys Gly Glu Trp Asn Val Ala Gly Ile Ala Tyr Arg Ala 180 185
190Tyr Asp Leu Glu Gly Lys Thr Ile Gly Thr Val Gly Ala Gly Arg Ile
195 200 205Gly Lys Leu Leu Leu Gln Arg Leu Lys Pro Phe Gly Cys Asn
Leu Leu 210 215 220Tyr His Asp Arg Leu Gln Met Ala Pro Glu Leu Glu
Lys Glu Thr Gly225 230 235 240Ala Lys Phe Val Glu Asp Leu Asn Glu
Met Leu Pro Lys Cys Asp Val 245 250 255Ile Val Ile Asn Met Pro Leu
Thr Glu Lys Thr Arg Gly Met Phe Asn 260 265 270Lys Glu Leu Ile Gly
Lys Leu Lys Lys Gly Val Leu Ile Val Asn Asn 275 280 285Ala Arg Gly
Ala Ile Met Glu Arg Gln Ala Val Val Asp Ala Val Glu 290 295 300Ser
Gly His Ile Gly Gly Tyr Ser Gly Asp Val Trp Asp Pro Gln Pro305 310
315 320Ala Pro Lys Asp His Pro Trp Arg Tyr Met Pro Asn Gln Ala Met
Thr 325 330 335Pro His Thr Ser Gly Thr Thr Ile Asp Ala Gln Leu Arg
Tyr Ala Ala 340 345 350Gly Thr Lys Asp Met Leu Glu Arg Tyr Phe Lys
Gly Glu Asp Phe Pro 355 360 365Thr Glu Asn Tyr Ile Val Lys Asp Gly
Glu Leu Ala Pro Gln Tyr Arg 370 375 3806374PRTStaphylococcus aureus
6Met Ser Asn Gly Ala Val Phe Phe Val Ile Phe Leu Lys Gln Ala Thr1 5
10 15Cys Asn Thr Tyr Phe Lys Glu Val Lys Ile Tyr His Leu Gly Glu
Met 20 25 30Asp Met Lys Ile Val Ala Leu Phe Pro Glu Ala Val Glu Gly
Gln Glu 35 40 45Asn Gln Leu Leu Asn Thr Lys Lys Ala Leu Gly Leu Lys
Thr Phe Leu 50 55 60Glu Glu Arg Gly His Glu Phe Ile Ile Leu Ala Asp
Asn Gly Glu Asp65 70 75 80Leu Asp Lys His Leu Pro Asp Met Asp Val
Ile Ile Ser Ala Pro Phe 85 90 95Tyr Pro Ala Tyr Met Thr Arg Glu Arg
Ile Glu Lys Ala Pro Asn Leu 100 105 110Lys Leu Ala Ile Thr Ala Gly
Val Gly Ser Asp His Val Asp Leu Ala 115 120 125Ala Ala Ser Glu His
Asn Ile Gly Val Val Glu Val Thr Gly Ser Asn 130 135 140Thr Val Ser
Val Ala Glu His Ala Val Met Asp Leu Leu Ile Leu Leu145 150 155
160Arg Asn Tyr Glu Glu Gly His Arg Gln Ser Val Glu Gly Glu Trp Asn
165 170 175Leu Ser Gln Val Gly Asn His Ala His Glu Leu Gln His Lys
Thr Ile 180 185 190Gly Ile Phe Gly Phe Gly Arg Ile Gly Gln Leu Val
Ala Glu Arg Leu 195 200 205Ala Pro Phe Asn Val Thr Leu Gln His Tyr
Asp Pro Ile Asn Gln Gln 210 215 220Asp His Lys Leu Ser Lys Phe Val
Ser Phe Asp Glu Leu Val Ser Thr225 230 235 240Ser Asp Ala Ile Thr
Ile His Ala Pro Leu Thr Pro Glu Thr Asp Asn 245 250 255Leu Phe Asp
Lys Asp Val Leu Ser Arg Met Lys Lys His Ser Tyr Leu 260 265 270Val
Asn Thr Ala Arg Gly Lys Ile Val Asn Arg Asp Ala Leu Val Glu 275 280
285Ala Leu Ala Ser Glu His Leu Gln Gly Tyr Ala Gly Asp Val Trp Tyr
290 295 300Pro Gln Pro Ala Pro Ala Asp His Pro Trp Arg Thr Met Pro
Arg Asn305 310 315 320Ala Met Thr Val His Tyr Ser Gly Met Thr Leu
Glu Ala Gln Lys Arg 325 330 335Ile Glu Asp Gly Val Lys Asp Ile Leu
Glu Arg Phe Phe Asn His Glu 340 345 350Pro Phe Gln Asp Lys Asp Ile
Ile Val Ala Ser Gly Arg Ile Ala Ser 355 360 365Lys Ser Tyr Thr Ala
Lys 37072971DNAE. coli 7gggcgctgcc ggcacctgtc ctacgagttg catgataaag
aagacagtca taagtgcggc 60gacgatagtc atgccccgcg cccaccggaa ggagctaccg
gcagcggtgc ggactgttgt 120aactcagaat aagaaatgag gccgctcatg
gcgttggtct gaaattgccg ctgtttgacg 180gtggacggtt gaatgccaat
ctcgaaggca cgcgcgccgc cagcaacatg atgattgaac 240gttacaacca
gtcagtactg aacgcggtgc gtgacgttgc cgtcaacggc acgcgtctgc
300aaacgctcaa cgacgagcga gaaatgcagg ctgaacgcgt ggaagccacg
cgctttaccc 360agcgcgctgc cgaggccgcc tatcagcgcg gcttaaccag
ccgcttacag gccaccgaag 420cccggttgcc agtgcttgcc gaagagatgt
cattactgat gctggacagc cgccgggtga 480tccaaagcat tcagttgatg
aaatcgctgg gcggcgggta tcaggcaggt cccgtcgtcg 540agaaaaaata
aaatgtctgc cgcgtgatgg ctgtcacgcg gtatttcgtt tcgtcacgtc
600aaaactgacg acagcctgtt tttcgtcaga gttttgaata aatagtgccc
gtaatatcag 660ggaatgaccc cacataaaat gtggcataaa agatgcatac
tgtagtcgag agcgcgtatg 720cgtgatttga ttaactggag cgagaccgat
gaaaaaagtc gtcacggttt gcccctattg 780cgcatcaggt tgcaaaatca
acgtggtcgt cgataacggc aaaatcgtcc gggcggaggc 840agcgcagggg
aaaaccaacc agggtaccct gtgtctgaag ggttattatg gctgggactt
900cattaacgat acccagatcc tgaccccgcg cctgaaaacc cccatgatcc
gtcgccagcg 960tggcggcaaa ctcgaacctg tttcctggga tgaggcactg
aattacgttg ccgagcgcct 1020gagcgccatc aaagagaagt acggtccgga
tgccatccag acgaccggct cctcgcgtgg 1080tacgggtaac gaaaccaact
atgtaatgca aaaatttgcg cgcgccgtta ttggtaccaa 1140taacgttgac
tgctgcgctc gtgtctgaca cggcccatcg gttgcaggtc tgcaccaatc
1200ggtcggtaat ggcgcaatga gcaatgctat taacgaaatt gataataccg
atttagtgtt 1260cgttttcggg tacaacccgg cggattccca cccaatcgtg
gcgaatcacg taattaacgc 1320taaacgtaac ggggcgaaaa ttatcgtctg
cgatccgcgc aaaattgaaa ccgcgcgcat 1380tgctgacatg cacattgcac
tgaaaaacgg ctcgaacatc gcgctgttga atgcgatggg 1440ccatgtcatt
attgaagaaa atctgtacga caaagcgttc gtcgcttcac gtacagaagg
1500ctttgaagag tatcgtaaaa tcgttgaagg ctacacgccg gagtcggttg
aagatatcac 1560cggcgtcagc gccagtgaga ttcgtcaggc ggcacggatg
tatgcccagg cgaaaagcgc 1620cgccatcctg tggggcatgg gtgtaaccca
gttctaccag ggcgtggaaa ccgtgcgttc 1680tctgaccagc ctcgcgatgc
tgaccggtaa cctcggtaag ccgcatgcgg gtgttaaccc 1740ggttcgtggt
cagaacaacg ttcagggtgc ctgcgatatg ggcgcgctgc cggatacgta
1800tccgggatac cagtacgtga aagatccggc taaccgcgag aaattcgcca
aagcctgggg 1860cgtggaaagc ctgccagcgc
ataccggcta tcgcatcagc gagctgccgc accgcgcagc 1920gcatggcgaa
gtgcgtgccg cgtacattat gggcgaagat ccgctacaaa ctgacgcgga
1980gctgtcggca gtacgtaaag cctttgaaga tctggaactg gttatcgttc
aggacatctt 2040tatgaccaaa accgcgtcgg cggcggatgt tattttaccg
tcaacgtcgt ggggcgagca 2100tgaaggcgtg tttactgcgg ctgaccgtgg
cttccagcgt ttcttcaagg cggttgaacc 2160gaaatgggat ctgaaaacgg
actggcaaat catcagtgaa atcgccaccc gtatgggtta 2220tccgatgcac
tacaacaaca cccaggagat ctgggatgag ttgcgtcatc tgtgcccgga
2280tttctacggt gcgacttacg agaaaatggg cgaactgggc ttcattcagt
ggccttgccg 2340cgatacttca gatgccgatc aggggacttc ttatctgttt
aaagagaagt ttgatacccc 2400gaacggtctg gcgcagttct tcacctgcga
ctgggtagcg ccaatcgaca aactcaccga 2460cgagtacccg atggtactgt
caacggtgcg tgaagttggt cactactctt gccgttcgat 2520gaccggtaac
tgtgcggcac tggcggcgct ggctgatgaa cctggctacg cacaaatcaa
2580taccgaagac gccaaacgtc tgggtattga agatgaggca ttggtttggg
tgcactcgcg 2640taaaggcaaa attatcaccc gtgcgcaggt cagcgatcgt
ccgaacaaag gggcgattta 2700catgacctac cagtggtgga ttggtgcctg
taacgagctg gttaccgaaa acttaagccc 2760gattacgaaa acgccggagt
acaaatactg cgccgttcgc gtcgagccga tcgccgatca 2820gcgcgccgcc
gagcagtacg tgattgacga gtacaacaag ttgaaaactc gcctgcgcga
2880agcggcactg gcgtaatacc gtcctttcta cagcctcctt tcggaggctg
tttttttatc 2940cattcgaact ctttatactg gttacttccc g
2971819DNAArtificial SequencePRIMER 8gattaactgg agcgagacc
19918DNAArtificial SequencePRIMER 9tccgaaagga ggctgtag
181053DNAartificial sequencePRIMER 10gcggaattca ggaggaattt
aaaatgaaga tcgttttagt cttatatgat gct 531136DNAArtificial
SequencePRIMER 11cgcggatcct tatttcttat cgtgtttacc gtaagc
3612453PRTRhodococcus 12Met Thr Gln Gln Arg Gln Met His Leu Ala Gly
Phe Phe Ser Ala Gly1 5 10 15Asn Val Thr His Ala His Gly Ala Trp Arg
His Thr Asp Ala Ser Asn 20 25 30Asp Phe Leu Ser Gly Lys Tyr Tyr Gln
His Ile Ala Arg Thr Leu Glu 35 40 45Arg Gly Lys Phe Asp Leu Leu Phe
Leu Pro Asp Gly Leu Ala Val Glu 50 55 60Asp Ser Tyr Gly Asp Asn Leu
Asp Thr Gly Val Gly Leu Gly Gly Gln65 70 75 80Gly Ala Val Ala Leu
Glu Pro Ala Ser Val Val Ala Thr Met Ala Ala 85 90 95Val Thr Glu His
Leu Gly Leu Gly Ala Thr Ile Ser Ala Thr Tyr Tyr 100 105 110Pro Pro
Tyr His Val Ala Arg Val Phe Ala Thr Leu Asp Gln Leu Ser 115 120
125Gly Gly Arg Val Ser Trp Asn Val Val Thr Ser Leu Asn Asp Ala Glu
130 135 140Ala Arg Asn Phe Gly Ile Asn Gln His Leu Glu His Asp Ala
Arg Tyr145 150 155 160Asp Arg Ala Asp Glu Phe Leu Glu Ala Val Lys
Lys Leu Trp Asn Ser 165 170 175Trp Asp Glu Asp Ala Leu Val Leu Asp
Lys Ala Ala Gly Val Phe Ala 180 185 190Asp Pro Ala Lys Val His Tyr
Val Asp His His Gly Glu Trp Leu Asn 195 200 205Val Arg Gly Pro Leu
Gln Val Pro Arg Ser Pro Gln Gly Glu Pro Val 210 215 220Ile Leu Gln
Ala Gly Leu Ser Pro Arg Gly Arg Arg Phe Ala Gly Lys225 230 235
240Trp Ala Glu Ala Val Phe Ser Leu Ala Pro Asn Leu Glu Val Met Gln
245 250 255Ala Thr Tyr Gln Gly Ile Lys Ala Glu Val Asp Ala Ala Gly
Arg Asp 260 265 270Pro Asp Gln Thr Lys Ile Phe Thr Ala Val Met Pro
Val Leu Gly Glu 275 280 285Ser Gln Ala Val Ala Gln Glu Arg Leu Glu
Tyr Leu Asn Ser Leu Val 290 295 300His Pro Glu Val Gly Leu Ser Thr
Leu Ser Ser His Thr Gly Ile Asn305 310 315 320Leu Ala Ala Tyr Pro
Leu Asp Thr Pro Ile Lys Asp Ile Leu Arg Asp 325 330 335Leu Gln Asp
Arg Asn Val Pro Thr Gln Leu His Met Phe Ala Ala Ala 340 345 350Thr
His Ser Glu Glu Leu Thr Leu Ala Glu Met Gly Arg Arg Tyr Gly 355 360
365Thr Asn Val Gly Phe Val Pro Gln Trp Ala Gly Thr Gly Glu Gln Ile
370 375 380Ala Asp Glu Leu Ile Arg His Phe Glu Gly Gly Ala Ala Asp
Gly Phe385 390 395 400Ile Ile Ser Pro Ala Phe Leu Pro Gly Ser Tyr
Asp Glu Phe Val Asp 405 410 415Gln Val Val Pro Val Leu Gln Asp Arg
Gly Tyr Phe Arg Thr Glu Tyr 420 425 430Gln Gly Asn Thr Leu Arg Asp
His Leu Gly Leu Arg Val Pro Gln Leu 435 440 445Gln Gly Gln Pro Ser
45013365PRTRhodococcus 13Met Thr Ser Arg Val Asp Pro Ala Asn Pro
Gly Ser Glu Leu Asp Ser1 5 10 15Ala Ile Arg Asp Thr Leu Thr Tyr Ser
Asn Cys Pro Val Pro Asn Ala 20 25 30Leu Leu Thr Ala Ser Glu Ser Gly
Phe Leu Asp Ala Ala Gly Ile Glu 35 40 45Leu Asp Val Leu Ser Gly Gln
Gln Gly Thr Val His Phe Thr Tyr Asp 50 55 60Gln Pro Ala Tyr Thr Arg
Phe Gly Gly Glu Ile Pro Pro Leu Leu Ser65 70 75 80Glu Gly Leu Arg
Ala Pro Gly Arg Thr Arg Leu Leu Gly Ile Thr Pro 85 90 95Leu Leu Gly
Arg Gln Gly Phe Phe Val Arg Asp Asp Ser Pro Ile Thr 100 105 110Ala
Ala Ala Asp Leu Ala Gly Arg Arg Ile Gly Val Ser Ala Ser Ala 115 120
125Ile Arg Ile Leu Arg Gly Gln Leu Gly Asp Tyr Leu Glu Leu Asp Pro
130 135 140Trp Arg Gln Thr Leu Val Ala Leu Gly Ser Trp Glu Ala Arg
Ala Leu145 150 155 160Leu His Thr Leu Glu His Gly Glu Leu Gly Val
Asp Asp Val Glu Leu 165 170 175Val Pro Ile Ser Ser Pro Gly Val Asp
Val Pro Ala Glu Gln Leu Glu 180 185 190Glu Ser Ala Thr Val Lys Gly
Ala Asp Leu Phe Pro Asp Val Ala Arg 195 200 205Gly Gln Ala Ala Val
Leu Ala Ser Gly Asp Val Asp Ala Leu Tyr Ser 210 215 220Trp Leu Pro
Trp Ala Gly Glu Leu Gln Ala Thr Gly Ala Arg Pro Val225 230 235
240Val Asp Leu Gly Leu Asp Glu Arg Asn Ala Tyr Ala Ser Val Trp Thr
245 250 255Val Ser Ser Gly Leu Val Arg Gln Arg Pro Gly Leu Val Gln
Arg Leu 260 265 270Val Asp Ala Ala Val Asp Ala Gly Leu Trp Ala Arg
Asp His Ser Asp 275 280 285Ala Val Thr Ser Leu His Ala Ala Asn Leu
Gly Val Ser Thr Gly Ala 290 295 300Val Gly Gln Gly Phe Gly Ala Asp
Phe Gln Gln Arg Leu Val Pro Arg305 310 315 320Leu Asp His Asp Ala
Leu Ala Leu Leu Glu Arg Thr Gln Gln Phe Leu 325 330 335Leu Thr Asn
Asn Leu Leu Gln Glu Pro Val Ala Leu Asp Gln Trp Ala 340 345 350Ala
Pro Glu Phe Leu Asn Asn Ser Leu Asn Arg His Arg 355 360
36514417PRTRhodococcus 14Met Thr Leu Ser Pro Glu Lys Gln His Val
Arg Pro Arg Asp Ala Ala1 5 10 15Asp Asn Asp Pro Val Ala Val Ala Arg
Gly Leu Ala Glu Lys Trp Arg 20 25 30Ala Thr Ala Val Glu Arg Asp Arg
Ala Gly Gly Ser Ala Thr Ala Glu 35 40 45Arg Glu Asp Leu Arg Ala Ser
Gly Leu Leu Ser Leu Leu Val Pro Arg 50 55 60Glu Tyr Gly Gly Trp Gly
Ala Asp Trp Pro Thr Ala Ile Glu Val Val65 70 75 80Arg Glu Ile Ala
Ala Ala Asp Gly Ser Leu Gly His Leu Phe Gly Tyr 85 90 95His Leu Thr
Asn Ala Pro Met Ile Glu Leu Ile Gly Ser Gln Glu Gln 100 105 110Glu
Glu His Leu Tyr Thr Gln Ile Ala Gln Asn Asn Trp Trp Thr Gly 115 120
125Asn Ala Ser Ser Glu Asn Asn Ser His Val Leu Asp Trp Lys Val Ser
130 135 140Ala Thr Pro Thr Glu Asp Gly Gly Tyr Val Leu Asn Gly Thr
Lys His145 150 155 160Phe Cys Ser Gly Ala Lys Gly Ser Asp Leu Leu
Phe Val Phe Gly Val 165 170 175Val Gln Asp Asp Ser Pro Gln Gln Gly
Ala Ile Ile Ala Ala Ala Ile 180 185 190Pro Thr Ser Arg Ala Gly Val
Thr Pro Asn Asp Asp Trp Ala Ala Ile 195 200 205Gly Met Arg Gln Thr
Asp Ser Gly Ser Thr Asp Phe His Asn Val Lys 210 215 220Val Glu Pro
Asp Glu Val Leu Gly Ala Pro Asn Ala Phe Val Leu Ala225 230 235
240Phe Ile Gln Ser Glu Arg Gly Ser Leu Phe Ala Pro Ile Ala Gln Leu
245 250 255Ile Phe Ala Asn Val Tyr Leu Gly Ile Ala His Gly Ala Leu
Asp Ala 260 265 270Ala Arg Glu Tyr Thr Arg Thr Gln Ala Arg Pro Trp
Thr Pro Ala Gly 275 280 285Ile Gln Gln Ala Thr Glu Asp Pro Tyr Thr
Ile Arg Ser Tyr Gly Glu 290 295 300Phe Thr Ile Ala Leu Gln Gly Ala
Asp Ala Ala Ala Arg Glu Ala Ala305 310 315 320His Leu Leu Gln Thr
Val Trp Asp Lys Gly Asp Ala Leu Thr Pro Glu 325 330 335Asp Arg Gly
Glu Leu Met Val Lys Val Ser Gly Val Lys Ala Leu Ala 340 345 350Thr
Asn Ala Ala Leu Asn Ile Ser Ser Gly Val Phe Glu Val Ile Gly 355 360
365Ala Arg Gly Thr His Pro Arg Tyr Gly Phe Asp Arg Phe Trp Arg Asn
370 375 380Val Arg Thr His Ser Leu His Asp Pro Val Ser Tyr Lys Ile
Ala Asp385 390 395 400Val Gly Lys His Thr Leu Asn Gly Gln Tyr Pro
Ile Pro Gly Phe Thr 405 410 415Ser
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