U.S. patent application number 17/094718 was filed with the patent office on 2021-10-07 for methods and compositions for nucleoside triphosphate and ribonucleic acid production.
This patent application is currently assigned to GreenLight Biosciences, Inc.. The applicant listed for this patent is GreenLight Biosciences, Inc.. Invention is credited to James Robbins Abshire, Drew S. Cunningham, Himanshu Dhamankar, Mehak Gupta, Ifeyinwa Iwuchukwu, Rachit Jain, Daniel MacEachran, Matthew Eduardo Moura, Karthikeyan Ramachandriya, Nicholas Skizim, Naveen Sudharsan.
Application Number | 20210309691 17/094718 |
Document ID | / |
Family ID | 1000005653026 |
Filed Date | 2021-10-07 |
United States Patent
Application |
20210309691 |
Kind Code |
A1 |
Cunningham; Drew S. ; et
al. |
October 7, 2021 |
METHODS AND COMPOSITIONS FOR NUCLEOSIDE TRIPHOSPHATE AND
RIBONUCLEIC ACID PRODUCTION
Abstract
Provided herein, in some embodiments, are methods and
composition for the production of nucleoside triphosphates and
ribonucleic acids.
Inventors: |
Cunningham; Drew S.;
(Winchester, MA) ; MacEachran; Daniel; (Medford,
MA) ; Abshire; James Robbins; (Cambridge, MA)
; Dhamankar; Himanshu; (Arlington, MA) ;
Iwuchukwu; Ifeyinwa; (Billerica, MA) ; Gupta;
Mehak; (Medford, MA) ; Moura; Matthew Eduardo;
(Cambridge, MA) ; Sudharsan; Naveen; (Malden,
MA) ; Skizim; Nicholas; (Dedham, MA) ; Jain;
Rachit; (Medford, MA) ; Ramachandriya;
Karthikeyan; (Winchester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GreenLight Biosciences, Inc. |
Medford |
MA |
US |
|
|
Assignee: |
GreenLight Biosciences,
Inc.
Medford
MA
|
Family ID: |
1000005653026 |
Appl. No.: |
17/094718 |
Filed: |
November 10, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16196059 |
Nov 20, 2018 |
10858385 |
|
|
17094718 |
|
|
|
|
PCT/US2018/055353 |
Oct 11, 2018 |
|
|
|
16196059 |
|
|
|
|
62571071 |
Oct 11, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 207/0104 20130101;
C12P 19/34 20130101; C12Y 207/04014 20130101; C12Y 207/0401
20130101; C07H 21/02 20130101; C12Y 207/07004 20130101; C12Y
207/04003 20130101; C12N 9/1229 20130101; C12Y 204/02 20130101;
C12P 19/30 20130101; C12N 9/90 20130101; C12Y 207/04006 20130101;
C12Y 108/99002 20130101; C12P 19/32 20130101; C12Y 207/07005
20130101; C12Y 207/07001 20130101; C12N 15/11 20130101; C12N 9/22
20130101; C12Y 207/04004 20130101 |
International
Class: |
C07H 21/02 20060101
C07H021/02; C12P 19/32 20060101 C12P019/32; C12P 19/30 20060101
C12P019/30; C12P 19/34 20060101 C12P019/34; C12N 9/12 20060101
C12N009/12; C12N 9/22 20060101 C12N009/22; C12N 9/90 20060101
C12N009/90; C12N 15/11 20060101 C12N015/11 |
Claims
1. A method for producing one or more nucleoside triphosphates
(NTPs), comprising: incubating in a reaction mixture one or more
nucleoside monophosphates (NMPs), at least one polyphosphate kinase
(PPK), and polyphosphate.
2. (canceled)
3. The method of claim 1 further comprising: incubating in a
reaction mixture cellular ribonucleic acid (RNA) and (a) a
polynucleotide phosphorylase (PNPase) and inorganic phosphate;
and/or (b) a ribonuclease.
4.-8. (canceled)
9. The method of claim 1, wherein the polyphosphate kinase is
selected from PPK1 and PPK2 family enzymes.
10-26. (canceled)
27. The method of claim 1 further comprising at least one NMP
kinase and/or NDP kinase.
28. The method of claim 1, wherein the at least one PPK is a Class
III PPK2 enzyme from Deinococcus geothermalis (SEQ ID NO: 1).
29. The method of claim 1, wherein the method is a cell-free
method.
30. The method of claim 1, wherein the one or more NMPs comprise
AMP, GMP, CMP, and/or UMP.
31. The method of claim 1, wherein the one or more NMPs comprise
modified nucleoside monophosphates.
32. The method of claim 1, wherein the polyphosphate is selected
from the group consisting of tetrapolyphosphates,
pentapolyphosphates, and hexametaphosphates.
33. The method of claim 27, wherein: (i) the at least one NMP
kinase is an AMP kinase, a GMP kinase, a CMP kinase, and/or a UMP
kinase; and/or (ii) the at least one NDP kinase is an ADP kinase, a
GDP kinase, a CDP kinase, or a UDP kinase.
34. The method of claim 27, wherein: (i) the at least one NMP
kinase is AMP kinase from Thermus thermophilus (SEQ ID NO: 12), CMP
kinase from Thermus thermophilus (SEQ ID NO: 13), UMP kinase from
Pyrococcus furiosus (SEQ ID NO: 14), and/or GMP kinase from
Thermotoga maritima (SEQ ID NO: 15); and/or (ii) the at least one
NDP kinase is from Aquifex aeolicus (SEQ ID NO: 16).
35. A method for producing one or more nucleoside triphosphates
(NTPs), comprising: incubating in a reaction mixture one or more
nucleoside diphosphates (NDPs), at least one polyphosphate kinase,
and polyphosphate.
36. A method for producing nucleoside triphosphates (NTPs)
comprising: (a) incubating (i) cellular RNA and (ii) a ribonuclease
to produce 5' nucleoside monophosphates (NMPs); (b) eliminating the
ribonuclease; and (c) incubating the 5' NMPs with at least one
polyphosphate kinase (PPK), and polyphosphate to produce NTPs.
37. The method of claim 36, wherein step (c) further comprises at
least one NMP kinase and/or at least one NDP kinase.
38. The method of claim 36, wherein the cellular RNA comprises
ribosomal RNA, messenger RNA, and/or transfer RNA.
39. The method of claim 36, wherein the ribonuclease is Nuclease P1
or RNase R.
40. The method of claim 36, wherein the ribonuclease is eliminated
via temperature, pH, salt, detergent, alcohol, chemical inhibitors,
separation, precipitation, filtration, capture, and/or
chromatography.
41. The method of claim 37, wherein: (i) the at least one NMP
kinase is an AMP kinase, a GMP kinase, a CMP kinase, and/or a UMP
kinase; and/or (ii) the at least one NDP kinase is an ADP kinase, a
GDP kinase, a CDP kinase, or a UDP kinase.
42. The method of claim 37, wherein: (i) the at least one NMP
kinase is AMP kinase from Thermus thermophilus (SEQ ID NO: 12), CMP
kinase from Thermus thermophilus (SEQ ID NO: 13), UMP kinase from
Pyrococcus furiosus (SEQ ID NO: 14), and/or GMP kinase from
Thermotoga maritima (SEQ ID NO: 15); and/or (ii) the at least one
NDP kinase is from Aquifex aeolicus (SEQ ID NO: 16).
43. The method of claim 36, wherein the at least one PPK is a PPK1
family enzyme or a PPK2 family enzyme.
44. The method of claim 36, wherein the at least one PPK is a Class
III PPK2 enzyme from Deinococcus geothermalis (SEQ ID NO: 1).
45. The method of claim 36, wherein the polyphosphate is selected
from the group consisting of tertapolyphosphates,
pentapolyphosphates, and hexametaphosphates.
46. The method of claim 37, wherein step (c) comprises an enzyme
preparation or cell lysate obtained from cells that produce the
PPK, the NMP kinase, and/or the NDP kinase.
47. The method of claim 46, wherein the activity of native enzymes
in the cells has been eliminated via genetic modification, enzyme
secretion from a cell, protease targeting, temperature, pH, salt,
detergent, alcohol, chemical inhibitors, separation, precipitation,
filtration, capture, and/or chromatography.
48. The method of claim 47, wherein the native enzymes are selected
from the group consisting of phosphatases, nucleases, proteases,
deaminases, oxidoreductases, and hydrolases.
49. The method of claim 1, further comprising: incubating in a
reaction mixture cellular ribonucleic acid (RNA), a polynucleotide
phosphorylase (PNPase), inorganic phosphate, and a helicase.
Description
RELATED APPLICATION
[0001] This application is a continuation of international
application number PCT/US2018/055353 filed Oct. 11, 2018, which
claims the benefit under 35 U.S.C. .sctn. 119(e) of U.S.
provisional application No. 62/571,071, filed Oct. 11, 2017, each
of which is incorporated by reference herein in its entirety.
BACKGROUND
[0002] Ribonucleic acid (RNA) comprises repeating units of
ribonucleotides and plays a role in key cellular processes,
including gene expression and protein synthesis. Thus, RNA is an
attractive target for modulating fundamental processes of the cell,
for example, RNA vaccines that induce a cellular immune response.
Low-cost production of RNA on a commercial scale (e.g., grams to
kilograms), however, is challenging due in part to the cost of the
starting material (e.g., nucleoside triphosphates (NTPs)) and
reaction components (e.g., DNA template and polymerase). Providing
high-quality RNA at commercially relevant scales requires cost
efficient production of both NTPs and RNA.
SUMMARY
[0003] Provided herein are systems, methods, compositions (e.g.,
cells, cell lysates, reagents, and reaction mixtures), and kits for
low-cost production (biosynthesis) of NTPs and/or RNAs, using
biosynthetic pathways developed to utilize low-cost substrates
(e.g., cellular RNA, nucleobases, nucleosides, nucleoside
monophosphates (NMPs), and/or nucleoside diphosphates (NDPs)),
recombinant and/or endogenous enzymes (e.g., kinases and/or
polymerases), and energy sources (e.g., NTPs, polyphosphate, and/or
pyrophosphate). The production of NTPs and/or RNA, in some
embodiments, is achieved using in vitro and/or cell-free lysate
systems designed to minimize (e.g., reduce, inhibit, and/or remove)
undesired enzymatic activities, thus increasing efficiency of the
process and yield of the desired end product.
[0004] The biosynthetic pathways described herein typically utilize
polyphosphate kinase and polyphosphate as an alternative to
endogenous pathway enzymes and phosphate sources.
[0005] Thus, some aspects of the present disclosure provide methods
and compositions for producing NTPs that comprise incubating in a
reaction mixture NDPs (e.g., ADP, CDP, GDP, and/or UDP), a
polyphosphate kinase (e.g., PPK2), and a polyphosphate (e.g.,
hexametaphosphate) under conditions suitable for the production of
NTPs. As shown in FIG. 2A, PPK transfers a phosphate from
polyphosphate to ADP, CDP, GDP, and UDP, resulting in production of
ATP, CTP, GDP, and UTP. In some embodiments, this reaction mixture
further comprises a NDP kinase (e.g., ndk).
[0006] Other aspects of the present disclosure provide systems,
methods, compositions, and kits for producing NTPs that comprise
incubating in a reaction mixture NMPs (e.g., 5'-NMPs, such as
5'-AMP, 5'-GMP, 5'-GMP, and/or 5'-UMP), a polyphosphate kinase, and
a polyphosphate under conditions suitable for the production of
NTPs. In some embodiments, the reaction mixture further comprises a
NMP kinase or a NDP kinase (e.g., ndk). In some embodiments, the
reaction mixture further comprises a NMP kinase (e.g., adk, cmk,
gmk, and/or pyrH) and a NDP kinase (e.g., ndk).
[0007] Still other aspects of the present disclosure provide
systems, methods, compositions, and kits for producing NTPs that
comprise incubating in a reaction mixture nucleosides (e.g.,
adenosine, cytidine, guanosine, and/or uridine), a polyphosphate
kinase, and a polyphosphate under conditions suitable for the
production of NTPs. In some embodiments, the reaction mixture
further comprises a nucleoside kinase, a NMP kinase, or a NDP
kinase. In some embodiments, the reaction mixture further comprises
a nucleoside kinase, a NMP kinase, and a NDP kinase.
[0008] Further aspects of the present disclosure provide systems,
methods, compositions, and kits for producing NTPs that comprise
incubating in a reaction mixture nucleobases (e.g., adenine,
cytosine, guanine, and/or uracil), a phosphoribosyltransferase, a
phosphoribosylpyrophosphate, a polyphosphate kinase, and a
polyphosphate under conditions suitable for the production of NTPs.
In some embodiments, the reaction mixture further comprises a
nucleoside kinase, a NMP kinase, or a NDP kinase. In some
embodiments, the reaction mixture further comprises a nucleoside
kinase, a NMP kinase, and a NDP kinase.
[0009] In some embodiments, the starting material (e.g., NMPs,
NDPs, and/or nucleosides) for the biosynthesis of NTPs is produced
from cellular RNA. Thus, some aspects of the present disclosure
provide systems, methods, compositions, and kits for producing NTPs
that comprise (a) incubating in a reaction mixture cellular RNA
(e.g., obtained from unicellular or multicellular organisms), a
polynucleotide phosphorylase (PNPase), and inorganic phosphate
under conditions suitable for the production of nucleoside
diphosphates (NDPs); (b) eliminating the PNPase (and optionally
eliminating other undesired enzymatic activities); and (c)
incubating in the resulting reaction mixture the NDPs, a
polyphosphate kinase, and a polyphosphate under conditions suitable
for the production of NTPs. In some embodiments, the reaction
mixture of step (c) further comprises a NDP kinase. Alternatively,
the methods may comprise (a) incubating in a reaction mixture
cellular ribonucleic acid (RNA), a PNPase, inorganic phosphate, a
polyphosphate kinase, and a polyphosphate under conditions suitable
for the production of nucleoside diphosphates (optionally wherein
the reaction mixture further comprises a NDP kinase); (b)
eliminating the PNPase; and (c) incubating the reaction mixture
under conditions suitable for the production of NTPs. In some
embodiments, the required pathway enzymes (e.g., polyphosphate
kinase and/or NDP kinase) can withstand the elimination conditions
(e.g., exposure to high temperature or a chemical inhibitor) such
that they retain their activity (for example, at least 50% of their
activity) following exposure to the conditions used to eliminate
(e.g., reduce, inhibit and/or remove) the PNPase.
[0010] Other aspects of the present disclosure provide systems,
methods, compositions, and kits for producing NTPs that comprise
(a) incubating in a first reaction mixture cellular RNA and a
ribonuclease (RNase, for example RNase R or Nuclease P1) under
conditions suitable for the production of NMPs (e.g., 5'-NMPs); (b)
eliminating the RNase (and optionally v other undesired enzymatic
activities); and (c) incubating in the resulting reaction mixture
the NMPs, a polyphosphate kinase, and a polyphosphate under
conditions suitable for the production of NTPs. In some
embodiments, the reaction mixture of step (c) further comprises a
NMP kinase, a NDP kinase, or both a NMP kinase and a NDP kinase.
Alternatively, the methods may comprise (a) incubating in a
reaction mixture cellular RNA, a RNase, a polyphosphate kinase, and
a polyphosphate under conditions suitable for the production of
NMPs (e.g., 5'-NMPs); (b) eliminating the RNase; and (c) incubating
the reaction mixture under conditions suitable for the production
of NTPs.
[0011] The NTPs produced herein are used, in some embodiments, for
the production of RNA (e.g., mRNA or double-stranded RNA). This may
be achieved, for example, by adding DNA template and polymerase
(e.g., T7 RNA polymerase) to any of the reaction mixtures used for
the production of NTP, as described herein. Alternatively, the NTPs
may be isolated and combined with DNA template and polymerase in a
separate reaction mixture to produce RNA. Thus, the present
disclosure also provides methods and compositions for the
production of RNA.
[0012] In any of the biosynthetic pathways described herein the
nucleobases, nucleosides, NMPs, NDPs, or NTPs, when used as the
starting substrate, may be chemically synthesized, a product of
fermentation, or produced by other means.
[0013] The polyphosphate kinase used in the systems, reaction
mixtures, and methods described herein may be selected from any of
the polyphosphate kinases listed in Table 2 or 12. In some
embodiments, the polyphosphate kinase comprises a Class III
polyphosphate kinase 2 from Deinococcus geothermalis.
[0014] The polyphosphate may be any polyphosphate that serves as a
substrate for a pathway enzymes. In some embodiments, the
polyphosphate is hexametaphosphate.
[0015] In embodiments where cellular RNA is used, the cellular RNA
comprises, for example, ribosomal RNA, messenger RNA, and/or
transfer RNA. The cellular RNA may be from a unicellular organism
(e.g., bacteria or yeast) or a multicellular organism (e.g.,
plants).
[0016] Enzymes of the biosynthetic pathways useful in the present
disclosure may be obtained from (isolated and/or purified) a (at
least one) cell lysate prepared from, for example, cells (e.g.,
engineered cells) that express enzymes of the pathway (e.g.,
nucleases (such as RNases and/or PNPases), polyphosphate kinases,
NMP kinases, NDP kinase, and/or polymerases). Exemplary methods for
preparing these cell lysates are described herein. Alternatively,
the reaction mixture may comprise a cell lysate (a single cell
lysate or a mixture of cell lysates) prepared from cells (e.g.,
engineered cells) that express enzymes of the pathway. That is, a
complete reaction may be performed in a cell lysate or a mixture of
cell lysates containing recombinant enzymes and/or endogenous
enzymes of the pathway as well as other reaction components (e.g.,
polyphosphate) required for the production of NTPs. In some
embodiments, a (at least one) purified pathway enzyme is added to a
reaction mixture.
[0017] For reaction mixtures that include cell lysate(s) or enzymes
obtained from cell lysate(s), it may be advantageous to eliminate
undesired native enzymatic activities using any of the elimination
methods described herein. Undesired native enzymatic activities
include, for example, phosphatases, nucleases, proteases,
deaminases, oxidoreductases, and hydrolases. In some embodiments,
native enzymatic activity is eliminated via genetic modification,
enzyme secretion from a cell, localization (e.g., periplasmic
targeting), and/or protease targeting. In other embodiments, native
enzymatic activity is eliminated via temperature, pH, salt,
detergent, alcohol or other solvents, and/or chemical inhibitors.
In yet other embodiments, native enzymatic activity is eliminated
via separation, precipitation, filtration, capture, and/or
chromatography.
[0018] The details of several embodiments of the invention are set
forth in the accompanying Examples, Figures and the Detailed
Description. Other features, objects, and advantages of the
invention will be apparent from the description and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A shows biosynthetic pathways for the production of
nucleoside triphosphates (NTPs), and downstream ribonucleic acid
(RNA) using nucleotide starting materials. FIG. 1B shows examples
of high-energy phosphate strategies where polyphosphate is fed to
the reaction mixture. FIG. 1C shows examples of additional
high-energy phosphate strategies.
[0020] FIG. 2A shows a biosynthetic pathway for the production of
NTPs, and downstream RNA, using nucleoside diphosphates (NDPs) as
the starting materials. FIG. 2B shows a biosynthetic pathway for
the production of NTPs, and downstream RNA, using 5' nucleoside
monophosphates (5'-NMPs) as the starting materials. FIG. 2C shows a
biosynthetic pathway for the production of NTPs, and downstream
RNA, using nucleosides as the starting materials. FIG. 2D shows a
biosynthetic pathway for the production of NTPs, and downstream
RNA, using nucleobases as the starting materials. FIG. 2E shows a
biosynthetic pathway for the production of NTPs, and downstream
RNA, using nucleobases and ribose as the starting materials.
[0021] FIG. 3A shows a biosynthetic pathway for the production of
NTPs, and downstream RNA, using cellular RNA as the starting
material. In this pathway, a polynucleotide phosphorylase is used
to degrade cellular RNA into NDPs. FIG. 3B shows a biosynthetic
pathway for the production of NTPs, and downstream RNA, using
cellular RNA as the starting material. In this pathway, a
ribonuclease is used to degrade cellular RNA into NMPs.
[0022] FIG. 4A shows a biosynthetic pathway for the production of
NTPs, and downstream RNA, using only polyphosphate kinases. FIG. 4B
shows a biosynthetic pathway for the production of NTPs, and
downstream RNA, using both polyphosphate kinases (e.g., PPK2) and
ATP/ADP-dependent kinases (e.g., NMP kinases such as adk, cmk, gmk,
and/or pyrH, and/or NDP kinases such as ndk).
[0023] FIG. 5 shows a biosynthetic pathway for the production of
RNA starting from 5'-NMPs.
[0024] FIG. 6 shows a biosynthetic pathway for the production of
RNA starting from cellular RNA. The schematic shows an example in
which the template, the kinase, and the polymerase are added during
a RNA production reaction.
[0025] FIG. 7 shows a biosynthetic pathway for the production of
RNA starting from cellular RNA. The schematic shows an example in
which the template may be added during the depolymerization phase
or the RNA production phase, the kinase may be added during the
depolymerization phase or the RNA production phase, and the
polymerase may added during the depolymerization phase or the RNA
production phase.
[0026] FIG. 8A shows a graph of acid-soluble nucleotides (mM)
produced over time during depolymerization of RNA from E. coli
lysates using overexpressed RNase R. Acid-soluble nucleotides were
measured by UV absorbance.
[0027] FIG. 8B shows an agarose gel of RNA products produced in
reactions comprising RNA polymerase and NMPs produced by
depolymerization (-NMPs) or purified NMPs (+NMPs, 4 mM each).
Abbreviations: -2 log: 2-log DNA ladder (New England Biolabs),
NMPs: equimolar mix of 5-NMPs, RNA Pol: thermostable T7 RNA
polymerase, Template 1: Linear DNA template, Template 2: Plasmid
DNA template.
[0028] FIG. 9A shows a graph of acid-soluble nucleotides (mM)
produced over time during depolymerization of purified RNA using 1
mg/mL purified RNase R. Acid-soluble nucleotides were measured by
UV absorbance.
[0029] FIG. 9B shows an agarose gel of RNA products produced in
reactions comprising RNA polymerase and NMPs produced by
depolymerization of purified RNA. As a negative control, reaction
were performed in the absence of RNA polymerase. Abbreviations: -2
log: 2-log DNA ladder (New England Biolabs), NMPs: equimolar mix of
5'-nucleoside monophosphates, RNA Pol: thermostable T7 RNA
polymerase, Template 1: Linear DNA template, Template 2: Plasmid
DNA template.
[0030] FIG. 10 shows an agarose gel of RNA products produced by
cell-free RNA synthesis using a wild-type polymerase (W) or a
thermostable polymerase mutant (T) at 37.degree. C. Abbreviations:
-2 log: 2-log DNA ladder (New England Biolabs), W: wild-type T7 RNA
polymerase (New England Biolabs), T: thermostable T7 RNA
polymerase, Template 1: Linear DNA template, Template 2: Plasmid
DNA template.
[0031] FIG. 11A shows a plot of the response factor (calculated as
ratio of area of the dsRNA of interest to that of a
commercially-available dsRNA internal standard) of the reactions
comprising either DgPPK2 as a sole kinase or the 5-enzyme lysate
system.
[0032] FIG. 11B shows HPLC chromatograms of the dsRNA product
produced in reactions comprising DgPPK2 lysate, 5-enzyme lysate
system, and the negative controls without T7 RNA polymerase.
[0033] FIG. 12A shows a graph of acid-soluble nucleotides (mM)
produced over time during depolymerization of various sources of
RNA using purified RNase R or Nuclease P1. Acid-soluble nucleotides
were measured by UV absorbance.
[0034] FIG. 12B shows a graph of the percent of available 5'-NMPs
produced over time during depolymerization of RNA from E. coli or
yeast using Nuclease P1. Percent of available 5'-NMPs was
determined by LC-MS.
[0035] FIG. 13 shows nucleomic profile plots for RNA
depolymerization across different temperatures of the lysate from
GL17-109. Cumulative concentrations of the 20 analytes are shown.
Nucleosides are shown in a white-speckled pattern, and were
minimally produced. Data for 50.degree. C. was collected but is not
shown.
[0036] FIG. 14 is a schematic of an enzymatic pathway for
production of ATP from pyrophosphate through the cyclical
phosphorylation of acetate. The meaning of the abbreviations is as
follows: AcK1=first acetate kinase, AcK2=second acetate kinase,
PP.sub.i=inorganic pyrophosphate, P.sub.i=inorganic phosphate,
ATP=adenosine triphosphate, ADP=adenosine diphosphate, and
Acetyl-P=acetyl-phosphate.
[0037] FIG. 15A-15B is a schematic of an enzymatic pathway for
production of ATP from citrate. FIG. 15A presents the three
enzymatic reactions for ATP production from citrate and
pyrophosphate. FIG. 15B presents the overall chemical reaction. The
meaning of the abbreviations is as follows: PP.sub.i=inorganic
pyrophosphate, PEP=phosphoenolpyruvate, CO.sub.2=carbon dioxide,
P.sub.i=inorganic phosphate, ATP=adenosine triphosphate, and
AMP=adenosine monophosphate.
[0038] FIG. 16 is a schematic of an enzymatic pathway for
production of ATP from sulfite. The meaning of the abbreviations is
as follows: ATP=adenosine triphosphate, AMP=adenosine
monophosphate, APS=adenosine 5'-phosphosulfate, and
PP.sub.i=inorganic pyrophosphate.
[0039] FIG. 17 is a graph showing that cell-free synthesis of dsRNA
produces similar product titers regardless of nucleotide
source.
[0040] FIG. 18 is a graph showing that cell-free synthesis of dsRNA
results in comparable product titers with wild-type and
thermostable mutant RNA polymerases at mesophilic reaction
temperature.
[0041] FIG. 19 is a graph showing that cell-free synthesis of NTPs
results in similar NTP titers regardless of nucleotide source after
a 1 hour incubation at 48.degree. C. For each source of nucleotides
(cellular RNA, purified NMPs, or purified NDPs), a quantity of
substrate sufficient to provide approximately 4 mM of each
nucleotide was added to the reaction. For example, reactions with
NDPs comprised 4 mM each ADP, CDP, GDP, and UDP.
DETAILED DESCRIPTION
[0042] The present disclosure provides, in some aspects,
biosynthetic pathways for the production of NTPs and/or RNA that
utilize cost-effective reaction components, such as cellular RNA
substrates or monomeric substrates such as nucleobases,
nucleosides, NMPs, or NDPs, recombinant and/or purified pathway
enzymes (e.g., phosphoribosyltransferases, nucleoside
phosphorylases, ribokinases, phosphopentomutaes, nucleases,
polyphosphate kinases, NMP kinases, NDP kinases, nucleoside
kinases, RNA polymerases), sources of high energy phosphate (e.g.,
polyphosphate), and/or DNA templates.
Reaction Components
[0043] Cellular RNA. Cellular RNA includes, for example, messenger
RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA) obtained
from cellular material (biomass). Cellular RNA may be obtained from
any source of cellular material including, but not limited to,
unicellular organisms (e.g., bacteria and yeast) and multicellular
organisms (e.g., plants and animals), either from fermentation or
from a process waste stream, for example, cellular RNA obtained
from a lysate expressing an enzyme (e.g., a kinase).
[0044] Nucleobases. A nucleobase is a nitrogenous base component of
a nucleoside or nucleotide. Nucleobases function as fundamental
units of the genetic code. Nucleobases include adenine (A),
cytosine (C), guanine (G), thymine (T), and uracil (U). Nucleobases
include modified nucleobases including, but not limited to,
pseudouridine (.PSI.), dihydrouridine (D), and 7-methylguanosine
(m.sup.7G).
[0045] Nucleosides. A nucleoside is a nucleobase linked to a
five-carbon sugar (e.g., a ribose). Examples of nucleosides include
adenosine, cytidine, guanosine, thymidine and uridine.
[0046] Nucleotides. A nucleotide includes a nucleoside and a
phosphate group. A nucleoside having one phosphate group is a
nucleoside monophosphate (NMP), which include adenosine
monophosphate (AMP), cytidine monophosphate (CMP), guanosine
monophosphate (GMP), thymidine monophosphate (TMP), and uridine
monophosphate (UMP). A nucleoside having two phosphate groups is a
nucleoside diphosphate (NDP), which include adenosine diphosphate
(ADP), cytidine diphosphate (CDP), guanosine diphosphate (GDP),
thymidine diphosphate (TDP), and uridine diphosphate (UDP). A
nucleoside having three phosphate groups is a nucleoside
triphosphate (NTP), which include adenosine triphosphate (ATP),
cytidine triphosphate (CTP), guanosine triphosphate (GTP),
thymidine triphosphate (TTP), and uridine triphosphate (UTP).
[0047] Phosphoribosyltransferases. Phosphoribosyltransferases, such
as adenine phosphoribosyltransferase (APRTase), is involved in the
nucleotide salvage pathway in cells, which provides an alternative
to nucleotide biosynthesis. APRTase catalyzes the following
reaction in the purine nucleotide salvage pathway:
Adenine+Phosphoribosyl Pyrophosphate (PRPP).fwdarw.Adenosine 5'
monophosphate (AMP)+Pyrophosphate (PPi).
[0048] Ribokinases. Ribokinase is an enzyme that transfers
phosphate from a high-energy phosphate source (e.g., ATP or
polyphosphate) to D-ribose forming D-ribose-5-phosphate. Examples
include the rbsK gene product of E. coli and the QT17_05185 gene
product of Thermus sp. 2.9.
[0049] Phosphopentomutases. Phosphopentomutase is an enzyme that
transfers phosphate within a ribose-phosphate molecule.
Specifically, phosphoribomutase catalyzes the reversible
interconversion of D-ribose-1-phosphate and D-ribose-5-phosphate.
Examples include the deoB gene product of E. coli and the TM0167
gene product of Thermotoga maritima.
[0050] Nucleoside phosphorylases. Nucleoside phosphorylases are
enzymes that catalyze the following reversible
reaction--nucleobase+D-ribose-1-phosphate.rarw..fwdarw.nucleoside+inorgan-
ic phosphate. Purine nucleoside phosphorylase catalyze such a
reaction with purine nucleobases (e.g., adenine, guanine) and
purine nucleosides (e.g., adenosine, guanosine). Pyrimidine
nucleoside phosphorylases catalyze such a reaction with pyrimidine
nucleobases (e.g., cytosine, uracil) and pyrimidine nucleosides
(e.g, cytidine, uridine). Examples of nucleoside phosphorylases
include the deoD, xapA, and udp gene products of E. coli, as well
as the TtPNPI, TtPNPII, and TtPyNP, enzymes of Thermus thermophilus
HB27.
[0051] Polynucleotide Phosphorylases. Polynucleotide phosphorylase
(PNPase) is a bifunctional enzyme with a phosphorolytic 3' to 5'
exoribonuclease activity and a 3'-terminal oligonucleotide
polymerase activity. PNPase is capable of catalyzing the
degradation of RNA into nucleoside 5' diphosphates (NDPs) using
inorganic phosphate as a co-substrate. Use of high concentrations
of inorganic phosphate while employing PNPase to degrade RNA may
simultaneously drive PNPase activity while reducing potential NDP
yield loss due to phosphatase activities that might be present in
the reaction mixture, as inorganic phosphate is known to inhibit
such undesirable activities. In some embodiments, a PNPase is used,
optionally in conjunction with one or more helicases, to catalyze
degradation of RNA into NDPs. Adding a helicase may improve
PNPase-meditated depolymerization of cellular RNA by improving
accessibility of structured RNA.
[0052] Nucleases. Nucleases are enzymes that cleave the
phosphodiester bonds in the backbone of DNA (DNases) or RNA
(RNases). Thus, ribonucleases (RNases) are capable of catalyzing
the degradation of RNA into nucleoside monophosphates (NMPs).
Non-limiting examples of enzymes that may be used to depolymerize
RNA, as provided herein, are provided in Table 1. In some
embodiments, more than one nuclease is used in a reaction mixture
to depolymerize RNA. In some embodiments, 2, 3, 4, or 5 different
nucleases are used in a reaction mixture.
TABLE-US-00001 TABLE 1 Examples of Enzymes for RNA Depolymerization
Enzyme Organism EC # UniProt Reference Nuclease P1 Penicillium
citrinum 3.1.30.1 P24289 1, 2, 3 (P1 Nuclease) RNase II Escherichia
coli 3.1.13.1 P30850 4, 5 RNase III Escherichia coli 3.1.26.3
P0A7Y0 6, 7, 8 RNase R Pseudomonas putida 3.1.13.-- R9V9M9 9 or
P21499 Escherichia coli RNase JI Bacillus subtilis 3.1.4.1 Q45493
10, 11 NucA Serratia marcescens 3.1.30.2 P13717 12, 13, 14 RNase T
Escherichia coli 3.1.27.3 P30014 15, 16, 17 RNase E Escherichia
coli 3.1.26.12 P21513 18, 19 PNPase Escherichia coli 2.7.7.8 P05055
55
[0053] Kinases. Kinases, generally, are enzymes that catalyze the
transfer of phosphate groups from a high-energy phosphate-donating
molecule (e.g., ATP, GTP, UTP, CTP, or polyphosphate containing n
phosphate groups in the polymer) to specific substrates/molecules.
This process produces a phosphorylated substrate and a
dephosphorylated form of the high-energy phosphate-donating
molecule (e.g., ADP, GDP, UDP, CDP, or polyphosphate containing n-1
phosphate groups in the polymer). Non-limiting examples of kinases
for use as provided herein include NMP kinases, NDP kinases,
nucleoside kinases, and polyphosphate kinases.
[0054] Polyphosphate Kinases. A polyphosphate kinase is an enzyme
that catalyzes the transfer of phosphate group(s) from high-energy,
phosphate-donating molecules, such as polyphosphate (PolyP.sub.n),
to specific substrates/molecules. This process is referred to as
phosphorylation, where the substrate gains a phosphate group and
the high-energy, phosphate-donating molecule donates a phosphate
group. This transesterification produces a phosphorylated substrate
and a phosphate-donating molecule lacking the donated phosphate
group, such as PolyP.sub.n-1. The polyphosphate kinases of the
present disclosure, in some embodiments, convert nucleosides to
NMPs, NMPs to NDPs, and/or NDPs to NTPs. Non-limiting examples of
polyphosphate kinases are provided in Table 2. In some embodiments,
more than one polyphosphate kinase is used in a reaction mixture.
In some embodiments, 2, 3, 4, or 5 different polyphosphate kinases
are used in a reaction mixture.
TABLE-US-00002 TABLE 2 Examples of Polyphosphate Kinases Sequence
GenBank # Identification Enzyme Organism UniProt # Number Reference
Thermophiles PPK2 Deinococcus WP_011531362.1 SEQ ID 20 geothermalis
NO: 1 DSM 11300 PPK2 Meiothermus ADD29239.1 SEQ ID 20 ruber NO: 2
DSM 1279 PPK2 Meiothermus WP_013159015.1 SEQ ID 20 silvanus NO: 3
DSM 9946 PPK2 Thermo- NP_682498.1 SEQ ID 20 synechococcus NO: 4
elongatus BP-1 PPK2 Anaerolinea WP_013558940 SEQ ID thermophila NO:
5 UNI-1 PPK2 Caldilinea WP_014433181 SEQ ID aerophila NO: 6 DSM
14535 PPK2 Chlorobaculum NP_661973.1 SEQ ID tepidum NO: 7 TLS PPK2
Oceanithermus WP_013458618 SEQ ID profundus NO: 8 DSM 14977 PPK2
Roseiflexus WP_012120763 SEQ ID castenholzii NO: 9 DSM 13941 PPK2
Roseiflexus WP_011956376 SEQ ID sp. RS-1 NO: 10 PPK2 Truepera
WP_013178933 SEQ ID radiovictrix NO: 11 DSM 17093 Solvent-tolerant
organisms PPK1 Pseudomonas AFO50238.1 42 putida I7BEV8 DOT-T1E PPK1
Escherichia AAC75554.1 coli P0A7B1 K-12 PPK1 Clostridium
NP_347259.1 43 acetobutylicum Q97LE0 ATCC 824 Acidophiles PPK1
Thermosynechococcus elongatus WP_011056068 PPK1 Acidithiobacillus
ferrooxidans WP_064219446 PPK1 Acidithiobacillus thiooxidans
WP_031572361 PPK1 Bacillus acidicola WP_066264350 PPK1 Acetobacter
aceti GAN58028 PPK2 Acetobacter aceti WP_077811826.1 PPK2
Acidithiobacillus thiooxidans WP_051690689.1 PPK2 Acidithiobacillus
ferrooxidans WP_064219816.1 Alkaliphiles PPK1 Thioalkalivibrio
denitrificans WP_077277945.1 Psychrophiles PPK1 Psychromonas
ingrahamii WP_041766473.1 PPK2 Psychrobacter arcticus
WP_083756052.1 PPK2 Psychroserpens jangbogonensis WP_033960485.1
PPK2 Cryobacterium psychrotolerans WP_092324020.1 PPK2 Nocardioides
psychrotolerans WP_091116082.1 PPK2 Pseudomonas psychrophila
WP_019411115.1
[0055] Nucleoside Kinases. Nucleoside kinases catalyze a phosphoryl
transfer from a high-energy phosphate-donating molecule (e.g., a
nucleotide triphosphate) to an R--OH acceptor, which is typically
the 5'-hydroxyl group of the sugar moiety of the nucleoside (e.g.,
adenosine, guanosine, cytidine, uridine). This process converts a
nucleoside to a NMP (e.g., AMP, CMP, GMP, UMP). In some
embodiments, the nucleoside kinase catalyzes the transfer of
phosphate from a phosphate-donating molecule to adenosine to
produce adenosine monophosphate (AMP). In some embodiments, the
nucleoside kinase catalyzes the transfer of phosphate from a
phosphate-donating molecule to cytidine to produce cytidine
monophosphate (CMP). In some embodiments, the nucleoside kinase
catalyzes the transfer of phosphate from a phosphate-donating
molecule to guanosine to produce guanosine monophosphate (GMP). In
some embodiments, the nucleoside kinase catalyzes the transfer of
phosphate from a phosphate-donating molecule to uridine to produce
uridine monophosphate (UMP). Non-limiting examples of nucleoside
kinases are provided in Table 3. In some embodiments, more than one
nucleoside kinase is used in a reaction mixture. In some
embodiments, 2, 3, 4, or 5 different nucleoside kinases are used in
a reaction mixture.
TABLE-US-00003 TABLE 3 Examples of Nucleoside Kinases GenBank #
Enzyme Organism UniProt # Reference Thermophiles Nucleoside
Thermoplasma CAC12009.1 21 kinase acidophilum Q9HJT3 Nucleoside
Methanocaldococcus AAB98396.1 22 kinase jannaschii Q57849
Nucleoside Burkholderia ABC38537.1 23 kinase thailandensis
AIP24308.1 Q2SZE4 Uridine- Thermus BAD70401.1 24 cytidine
thermophilus Q5SKR5 kinase (Y93H mutant) Uridine kinase Caldilinea
aerophila WP_014432899.1 Uridine kinase Geobacillus WP_043905564.1
stearothermophilus Uridine kinase Meiothermus ruber
WP_013014613.1
[0056] NMP Kinases. A nucleoside monophosphate kinase (NMP kinase)
is an enzyme that catalyzes the transfer of the terminal phosphoryl
group from a nucleoside triphosphate (NTP), usually ATP, to the
phosphoryl group on a nucleoside monophosphate (e.g., AMP, CMP,
GMP, UMP). This process converts a NMP to a NDP (e.g., ADP, CDP,
GDP, UDP). In some embodiments, the NMP kinase catalyzes the
transfer of phosphate from a phosphate-donating molecule to AMP to
produce adenosine diphosphate (ADP). In some embodiments, the NMP
kinase catalyzes the transfer of phosphate from a
phosphate-donating molecule to CMP to produce cytidine diphosphate
(CDP). In some embodiments, the NMP kinase catalyzes the transfer
of phosphate from a phosphate-donating molecule to GMP to produce
guanosine diphosphate (GDP). In some embodiments, the NMP kinase
catalyzes the transfer of phosphate from a phosphate-donating
molecule to UMP to produce uridine diphosphate (UDP). Non-limiting
examples of NMP kinases are provided in Table 4. In some
embodiments, more than one NMP kinases is used in a reaction
mixture. In some embodiments, 2, 3, 4, or 5 different NMP kinases
are used in a reaction mixture.
TABLE-US-00004 TABLE 4A Examples of AMP kinase enzymes Sequence
GenBank # Identification Enzyme Organism UniProt # Number Reference
Thermophiles Adk Thermus thermophilus Q72I25 SEQ ID 25, 26 NO: 12
Adk Pyrococcus furiosus Q8U207 27 Solvent-tolerant organisms Adk
Pseudomonas putida AFO48764.1 42 DOT-T1E I7CAA9 Adk Escherichia
coli BAE76253.1 44 K-12 W3110 P69441 Adk1 Aspergillus niger
CAK45139.1 45 CBS 513.88 A2QPN9 Adk1 Saccharomyces cerevisiae
AAC33143.1 46 ATCC 204508/S288c P07170 Adk Clostridium
acetobutylicum AAK81051.1 43 ATCC 824 Q97EJ9 Adk Halobacterium
salinarum AAG19963.1 32 ATCC 700922 Q9HPA7 Acidophiles Adk
Acidithiobacillus thiooxidans WP_024894015.1 Adk Acidithiobacillus
ferrooxidans WP_064218420.1 Adk Acetobacter aceti WP_077811596.1
Adk Bacillus acidicola WP_066267988.1 Adk Sulfolobus solfataricus
WP_009991241.1 Alkaliphiles Adk Thioalkalivibrio WP_019570706.1 Adk
Amphibacillus xylanus WP_015008883.1 Psychrophiles Adk Colwellia
psychrerythraea WP_033093471.1 28 (Vibrio psychroerythus) Q47XA8
Adk Psychromonas ingrahamii WP_011769361 A1STI3 Adk
Pseudoalteromonas haloplanktis CAI86283 29 Q3IKQ1 Adk Psychrobacter
arcticus WP_011280822 30 Adk Pseudomonas syringae WP_004406317.1 31
Q4ZWV2 Halophiles Adk Halobacterium halobium WP_010903261.1 32
Q9HPA7
TABLE-US-00005 TABLE 4B Examples of CMP kinase enzymes Sequence
GenBank # Identification Enzyme Organism UniProt # Number Reference
Thermophiles Cmk Thermus thermophilus Q5SL35 SEQ ID 33 NO: 13 Cmk
Pyrococcus furiosus Q8U2L4 27 Solvent-tolerant organisms Cmk
Pseudomonas putida AFO48857.1 42 DOT-T1E I7BXE2 Cmk Escherichia
coli AAC73996.1 47 K-12 MG1655 P0A6I0 Cmk Clostridium
acetobutylicum AAK79812.1 43 ATCC 824 Q97I08 Cmk Halobacterium
salinarum AAG19965.1 34 ATCC 700922 Q9HPA5 Acidophiles Cmk Bacillus
acidicola WP_066270173 Cmk Acetobacter aceti WP_010667744 Cmk
Acidithiobacillus thiooxidans WP_024892761.1 Cmk Acidithiobacillus
ferrooxidans WP_064220349.1 Cmk Metallosphaera sedula
WP_011921264.1 Alkaliphiles Cmk Amphibacillus xylanus
WP_015009966.1 Cmk Thioalkalivibrio denitrificans WP_077278466.1
Psychrophiles Cmk Colwellia psychrerythraea WP_011043148.1 28
(Vibrio psychroerythus) Q482G4 Cmk Pseudoalteromonas haloplanktis
CAI86499.1 29 Q3ILA1 Cmk Psychrobacter arcticus AAZ19343.1 30
Q4FRL5 Cmk Psychromonas ingrahamii ABM04716 A1SZ01 Cmk Pseudomonas
syringae YP_236713 31 Q4ZQ97 Halophiles Cmk Halobacterium salinarum
Q9HPA5 34
TABLE-US-00006 TABLE 4C Examples of UMP kinase enzymes Sequence
GenBank # Identification Enzyme Organism UniProt # Number Reference
Thermophiles PyrH Pyrococcus furiosus Q8U122 SEQ ID 35, 36 NO: 14
PyrH Thermus thermophilus P43891 33 Solvent-tolerant organisms PyrH
Pseudomonas putida AFO48412.1 42 DOT-T1E I7BW46 PyrH Escherichia
coli CAA55388.1 48 K-12 MG1655 P0A7E9 An13g00440 Aspergillus niger
CAK41445.1 45 CBS 513.88 A2R195 URA6 Saccharomyces cerevisiae
AAA35194.1 49 ATCC 204508/S288c P15700 PyrH Clostridium
acetobutylicum AAK79754.1 43 ATCC 824 Q97I64 PyrH Halobacterium
salinarum AAG20182.1 34 ATCC 700922 Q9HNN8 Acidophiles PyrH
Picrophilus torridus WP_048059653 PyrH Metallosphaera sedula
WP_012021705 PyrH Ferroplasma WP_009886950.1 PyrH Thermoplasma
acidophilum WP_010900913 PyrH Sulfolobus solfataricus WP_009992427
37 PyrH Acetobacter aceti WP_042788648 Alkaliphiles PyrH
Thioalkalivibrio WP_081759172.1 sp. HK1 PyrH Amphibacillus xylanus
WP_015010200.1 Psychrophiles PyrH Colwellia psychrerythraea
WP_011042391.1 28 (Vibrio psychroerythus) Q485G8 PyrH
Pseudoalteromonas haloplanktis CR954246.1 29 Q3IIX6 PyrH
Psychrobacter arcticus AAZ19383.1 30 Q4FRH5 PyrH Psychromonas
ingrahamii ABM04676.1 A1SYW1 PyrH Pseudomonas syringae YP_234434 31
Q4ZWS6 Halophiles PyrH Halobacterium salinarum WP_010903483.1 34
Q9HNN8
TABLE-US-00007 TABLE 4D Examples of GMP kinase enzymes Sequence
GenBank # Identification Enzyme Organism UniProt # Number Reference
Thermophiles Gmk Thermotoga maritima Q9X215 SEQ ID 38 NO: 15 Gmk
Thermus thermophilus Q5SI18 33 Solvent-tolerant organisms Gmk
Pseudomonas putida AFO49847.1 42 DOT-T1E I7C087 Gmk Escherichia
coli AAB88711.1 50 K-12 P60546 An08g00300 Aspergillus niger
CAK45182.1 45 CBS 513.88 A2QPV2 GUK1 Saccharomyces cerevisiae
AAA34657.1 51 ATCC 204508/S288c P15454 Gmk Clostridium
acetobutylicum AAK79684.1 43 ATCC 824 Q97ID0 Acidophiles Gmk
Acidithiobacillus ferrooxidans WP_064219869.1 Gmk Acidithiobacillus
thiooxidans WP_010637919.1 Gmk Bacillus acidicola WP_066264774.1
Gmk Acetobacter aceti WP_018308252.1 Alkaliphiles Gmk Amphibacillus
xylanus WP_015010280.1 Gmk Thioalkalivibrio sulfidiphilus
WP_018953989.1 Psychrophiles Gmk Colwellia psychrerythraea AAZ24463
28 (Vibrio psychroerythus) Q47UB3 Gmk Pseudoalteromonas
haloplanktis Q3IJH8 29 Gmk Psychrobacter arcticus WP_011280984.1 30
Q4FQY7 Gmk Psychromonas ingrahamii ABM05306 A1T0P1 Gmk Pseudomonas
syringae WP_003392601.1 31 Q4ZZY8
[0057] NDP Kinases. A nucleoside diphosphate kinase (NDP kinase) is
an enzyme that catalyzes the exchange of terminal phosphate between
different NDPs (e.g., ADP, CDP, GDP, UDP) and nucleoside
triphosphates (NTP) in a reversible manner to produce NTPs (e.g.,
ATP, CTP, GTP, UTP). In some embodiments, the NDP kinase catalyzes
the transfer of phosphate from a phosphate-donating molecule to ADP
to produce adenosine triphosphate (ATP). In some embodiments, the
NDP kinase catalyzes the transfer of phosphate from a
phosphate-donating molecule to CDP to produce cytidine triphosphate
(CTP). In some embodiments, the NDP kinase catalyzes the transfer
of phosphate from a phosphate-donating molecule to GDP to produce
guanosine triphosphate (GTP). In some embodiments, the NDP kinase
catalyzes the transfer of phosphate from a phosphate-donating
molecule to UDP to produce uridine triphosphate (UTP). Non-limiting
examples of NDP kinases are provided in Table 5. In some
embodiments, more than one NDP kinase is used in a reaction
mixture. In some embodiments, 2, 3, 4 or 5 different NDP kinases
are used in a reaction mixture.
TABLE-US-00008 TABLE 5 Examples of NDP Kinases Sequence GenBank #
Identification Enzyme Organism UniProt # Number Reference
Thermophiles Ndk Aquifex aeolicus O67528 SEQ ID NO: 16
Solvent-tolerant organisms Ndk Pseudomonas putida AFO50002.1 42
DOT-T1E I7C0T7 Ndk Escherichia coli CAA40780.1 53 K-12 P0A763
An09g05870 Aspergillus niger CAK40394.1 45 CBS 513.88 A2QUJ6 YNK1
Saccharomyces cerevisiae AAS56589.1 ATCC 204508/S288c P36010 Ndk
Clostridium acetobutylicum ABR36342.1 43 ATCC 824 A6M162 Ndk
Halobacterium salinarum BAB17308.1 53 ATCC 700922 P61136
Acidophiles Ndk Acidithiobacillus thiooxidans WP_024892623.1 Ndk
Acetobacter aceti WP_042787791.1 Ndk Picrophilus WP_011178084.1 Ndk
Thermoplasma acidophilum WP_010901523.1 Ndk Sulfolobus solfataricus
WP_009990482.1 Ndk Bacillus acidicola WP_066262668.1 Ndk
Ferroplasma WP_009887649.1 Ndk Metallosphaera sedula WP_011921175.1
Psychrophiles Ndk Psychromonas ingrahamii WP_011771565.1 39 A1SZU8
Ndk Colwellia psychrerythraea WP_011044987.1 28 Q47WB6 Ndk
Psychrobacter arcticus WP_011279964.1 30 Q4FTX1 Ndk
Pseudoalteromonas haloplanktis CAI89189.1 Q3ID15 Halophiles Ndk
Halobacterium salinarum WP_010902835.1 40 P61136 Ndk Natrialba
magadii WP_004214013.1 41 D3SY02
[0058] Non-limiting examples of kinases that convert NDP to NTP
include nucleoside diphosphate kinase, polyphosphate kinase, and
pyruvate kinase. As discussed herein, thermostable variants of the
foregoing enzymes are encompassed by the present disclosure. In
some embodiments, the NDP kinase(s) is/are obtained from Aquifex
aeolicus.
[0059] Phosphorylation of NMPs to NTPs occurs, in some embodiments,
through the polyphosphate-dependent kinase pathway, where
high-energy phosphate is transferred from polyphosphate to ADP via
a polyphosphate kinase (PPK). In some embodiments, the
polyphosphate kinase belongs to the polyphosphate kinase 1 (PPK1)
family, which transfers high-energy phosphate from polyphosphate to
ADP to form ATP. This ATP is subsequently used by NMP kinases
(e.g., AMP kinase, UMP kinase, GMP kinase, and CMP kinase) to
convert NMPs to their cognate ribonucleotide diphosphates (NDPs).
Furthermore, ATP is subsequently used by nucleotide diphosphate
kinase to convert NDPs to NTPs.
[0060] In some embodiments, the polyphosphate kinase belongs to the
polyphosphate kinase 2 (PPK2) family. In some embodiments, the
polyphosphate kinase belongs to a Class I PPK2 family, which
transfers high-energy phosphate from polyphosphate to NDPs to form
NTPs. ATP produced by the system is used as a high-energy phosphate
donor to convert NMPs to NDPs. In some embodiments, the
polyphosphate kinase belongs to a Class III PPK2 family, which
transfers high-energy phosphate from polyphosphate to NMPs and NDPs
to form NTPs. In some embodiments, Class III PPK2 is used alone to
produce NTPs from NMPs. In other embodiments, Class III PPK2 is
used in combination with other kinases. Class III PPK2 produces ATP
from ADP, AMP, and polyphosphate, which is subsequently used by NMP
and NDP kinases to convert NMPs to NTPs.
[0061] Non-limiting examples of PPK2 enzymes for use as provided
herein are listed in Table 2. Thus, in some embodiments, the PPK2
enzymes are thermostable. For example, the PPK2 enzymes may be
thermostable Class III PPK2 enzymes, which favor ATP synthesis over
polyphosphate polymerization, and convert both ADP and AMP to ATP.
In some embodiments, the PPK2 enzymes are used to convert a
polyphosphate, such as hexametaphosphate to ATP, at rates ranging,
for example, from 10 to 800 mM per hour (e.g., 10, 15, 20, 25, 50,
75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, or 800 mM per hour).
[0062] Polyphosphate and Other High Energy Phosphates. High energy
phosphate molecules (phosphate-donating molecules) release energy
upon hydrolysis of high energy bond, thereby providing an energy
source for biochemical reactions. Polyphosphate (PolyP.sub.n) and
other high energy phosphate molecules may be used as phosphate
sources for production of NTPs, and downstream production of RNA,
as described herein. PolyP.sub.n, for example, comprises repeating
units of phosphate (PO.sub.4) linked together by shared oxygen
atoms. Phosphorylation of specific substrates/molecules by kinases
of the present disclosure involves donation of a phosphate group
from PolyP.sub.n, thereby producing PolyP.sub.n-1.
[0063] The present disclosure is not limited by the number of
phosphate groups in the polyphosphate. In some embodiments,
PolyP.sub.n comprises at least 3 phosphate groups (PolyP.sub.3). In
some embodiments, PolyP.sub.n comprises at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, or at least 10
phosphate groups. In some embodiments, PolyP.sub.n is
hexametaphosphate.
[0064] Other examples of high energy phosphate molecules include,
but are not limited to NTP (e.g., ATP), NDP (e.g., ADP), NMP (e.g.,
AMP), phosphoenolpyruvate, 1,3-bisphosphoglycerate,
phosphocreatine, phosphoenol pyruvate, glucose 1-phosphate,
fructose 6-phosphate, and glucose 6-phosphate. In some embodiments,
more than one high energy phosphate is used in a reaction mixture.
In some embodiments, 2, 3, 4, or 5 different high energy phosphates
are used in a reaction mixture.
[0065] Templates. A DNA template includes a promoter, optionally an
inducible promoter, operably linked to nucleotide sequence encoding
a desired RNA product and, optionally, a transcriptional
terminator. A DNA template is typically provided on a vector, such
as a plasmid, although other template formats may be used (e.g.,
linear DNA templates generated by polymerase chain reaction (PCR),
chemical synthesis, or other means known in the art). In some
embodiments, more than one DNA template is used in a reaction
mixture. In some embodiments, 2, 3, 4, or 5 different DNA templates
are used in a reaction mixture.
[0066] A promotor or a terminator may be a naturally-occurring
sequence or an engineered sequence. In some embodiments, an
engineered sequence is modified to enhance transcriptional
activity. In some embodiments, the promotor is a
naturally-occurring sequence. In other embodiments, the promoter is
an engineered sequence. In some embodiments, the terminator is a
naturally-occurring sequence. In other embodiments, the terminator
is an engineered sequence.
[0067] Polymerases. Polymerases are enzymes that synthesize
polymers of nucleic acids. Polymerases of the present disclosure
include DNA-dependent RNA polymerases and RNA-dependent RNA
polymerases. Non-limiting examples of polymerases are provided in
Table 6. In some embodiments, a polymerase is a RNA polymerase,
such as a T7 RNA polymerase. In some embodiments, more than one
polymerase is used in a reaction mixture. In some embodiments, 2,
3, 4, or 5 different polymerases are used in a reaction
mixture.
TABLE-US-00009 TABLE 6 Examples of RNA Polymerases GenBank # Enzyme
Organism UniProt # T7 RNA Bacteriophage T7 NP_041960.1 Polymerase
P00573 .PHI.6 RdRP Bacteriophage .PHI.6 P11124 T3 RNA Bacteriophage
T3 NP_523301.1 polymerase Q778M8 SP6 Bacteriophage SP6 Y00105.1
Polymerase P06221 rpoA Escherichia coli - K12 MG1655 P0A7Z4 rpoB
Escherichia coli - K12 MG1655 P0A8V2 rpoC Escherichia coli - K12
MG1655 P0A8T7
[0068] RNA Products. RNA produced by the methods provided herein
may be any form of RNA, including single-stranded RNA (ssRNA) and
double-stranded RNA (dsRNA). Non-limiting examples of
single-stranded RNA include messenger RNA (mRNA), micro RNA
(miRNA), small interfering RNA (siRNA), and antisense RNA.
Double-stranded RNA herein includes wholly double-stranded
molecules that do not contain a single-stranded region (e.g., a
loop or overhang), as well as partially double-stranded molecules
that contain a double-stranded region and a single-stranded region
(e.g., a loop or overhang). Thus, short hairpin RNA (shRNA) may be
produced by the methods of the present disclosure.
[0069] RNA produced by the methods provided herein may be modified
as described herein. In some embodiments, RNA is produced according
to a method described herein and subsequently modified. In some
embodiments, RNA is produced according to a method described herein
using a modified starting material. In some embodiments, the
modified starting material is a modified nucleobase. In some
embodiments, the modified starting material is a modified
nucleoside. In some embodiments, the modified starting material is
a modified nucleotide.
[0070] In some embodiments, modified RNA comprises a backbone
modification. In some instances, backbone modification results in a
longer half-life for the RNA due to reduced nuclease-mediated
degradation. This is turn results in a longer half-life. Examples
of suitable backbone modifications include but are not limited to
phosphorothioate modifications, phosphorodithioate modifications,
p-ethoxy modifications, methylphosphonate modifications,
methylphosphorothioate modifications, alkyl- and aryl-phosphates
(in which the charged phosphonate oxygen is replaced by an alkyl or
aryl group), alkylphosphotriesters (in which the charged oxygen
moiety is alkylated), peptide nucleic acid (PNA) backbone
modifications, locked nucleic acid (LNA) backbone modifications,
and the like. These modifications may be used in combination with
each other and/or in combination with phosphodiester backbone
linkages.
[0071] Alternatively or additionally, RNA may comprise other
modifications, including modifications at the base or the sugar
moieties. Examples include RNA having sugars which are covalently
attached to low molecular weight organic groups other than a
hydroxyl group at the 3' position and other than a phosphate group
at the 5' position (e.g., a 2'-O-alkylated ribose), RNA having
sugars such as arabinose instead of ribose. RNA also embrace
substituted purines and pyrimidines such as C-5 propyne modified
bases (Wagner et al., Nature Biotechnology 14:840-844, 1996). Other
purines and pyrimidines include but are not limited to
5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,
2,6-diaminopurine, hypoxanthine. Other such modifications are well
known to those of skill in the art.
NTP Production Pathways
[0072] Provided herein are systems, methods, compositions, and kits
for the production of NTPs through various different enzymatic
pathways, each of which utilize energy sources as described herein,
and low-cost starting materials in the reaction mixture. These
enzymatic pathways can be extended, in some embodiments, to the
production of RNA (e.g., mRNA or double-stranded RNA) by adding a
DNA template and polymerase to the reaction mixture (see, e.g.,
FIG. 1).
[0073] It should be understood that any of the pathway enzymes
described herein (e.g., nucleases, kinases, and/or polymerases) may
be obtained from unmodified (native) or engineered cells. In some
embodiments, the pathway enzyme(s) are secreted from the cells
(e.g., the cells are engineered to secrete the enzyme(s)). In other
embodiments, the pathway enzymes are obtained from cell lysate(s)
of the cells. In some embodiments, the pathway enzymes are
components of cell lysate(s) of the cells, in which case, the cell
lysate(s) are added to or serve as the reaction mixture in a
biosynthetic reaction. In instances where cell lysate(s) is/are
used in or serve as the reaction mixture, the cell lysate may be
exposed to conditions to eliminate undesired enzymatic activities,
as described below, before producing the product (NTP and/or RNA)
of interest.
[0074] Conversion of NDP to NTP. In some aspects, NTPs are produced
using NDPs as substrates, as depicted in FIG. 2A. For example, NTP
production methods may include incubating in a reaction mixture
NDPs, a (e.g., 1, 2, 3, or 4) polyphosphate kinase, and a (e.g., 1,
2, 3, or 4) polyphosphate under conditions suitable for the
production of NTPs. In some embodiments, the reaction mixture for
NTP production includes a NDP kinase (see, e.g., Table 5). In some
embodiments, the NTP production reaction mixture may also include a
nucleoside kinase.
[0075] Conversion of NMP to NTP. In some aspects, NTPs are produced
using 5' NMPs as substrates, as depicted in FIG. 2B. For example,
NTP production methods may include incubating in a reaction mixture
5'-NMPs, a (e.g., 1, 2, 3, or 4) polyphosphate kinase, and a (e.g.,
1, 2, 3, or 4) polyphosphate under conditions suitable for the
production of NTPs. In some embodiments, the reaction mixture for
NTP production includes a NMP kinase (see, e.g., Table 4) and/or a
NDP kinase (see, e.g., Table 5). In some embodiments, the NTP
production reaction mixture may also include a nucleoside
kinase.
[0076] Conversion of Nucleosides to NTP. In some aspects, NTPs are
produced using nucleosides as substrates, as depicted in FIG. 2C.
For example, NTP production methods may include incubating in a
reaction nucleosides, a (e.g., 1, 2, 3, or 4) polyphosphate kinase,
and a (e.g., 1, 2, 3, or 4) polyphosphate under conditions suitable
for the production of NTPs. In some embodiments, the NTP production
reaction mixture may also include a nucleoside kinase (see, e.g.,
Table 3) and/or a NMP kinase (see, e.g., Table 4) and/or a NDP
kinase (see, e.g., Table 5).
[0077] Conversion of Nucleobases to NTP. In some aspects, NTPs are
produced using nucleobases as substrates, as depicted in FIG. 2D.
For example, NTP production methods may include incubating in a
reaction mixture nucleobases, a (e.g., 1, 2, 3, or 4)
phosphoribosyltransferase, a phosphoribosylpyrophosphate, a (e.g.,
1, 2, 3, or 4) polyphosphate kinase, and a (e.g., 1, 2, 3, or 4)
polyphosphate under conditions suitable for the production of NTPs.
In some embodiments, the NTP production reaction mixture may also
include a NMP kinase (see, e.g., Table 4) and/or a NDP kinase (see,
e.g., Table 5). In some embodiments, the NTP production reaction
mixture may also include a nucleoside kinase.
[0078] In some embodiments, a biosynthetic pathway for the
production of NTPs and/or RNA may use cellular RNA as the
substrate, by either first depolymerizing the cellular RNA into
NDPs or first depolymerizing the cellular RNA into NMPs.
[0079] Conversion of Nucleobases and Ribose to NTP. In some
aspects, NTPs are produced using nucleobases as substrates, as
depicted in FIG. 2E. For example, NTP production methods may
include incubating in a reaction nucleobases, D-ribose, ribokinase,
phosphopentomutase, at least one (e.g., 1, 2, 3, or 4) nucleoside
phosphorylase, at least one (e.g., 1, 2, 3, or 4) polyphosphate
kinase, and at least one (e.g., 1, 2, 3, or 4) polyphosphate under
conditions suitable for the production of NTPs. In some
embodiments, the NTP production reaction mixture may also include
at least one NMP kinase (see, e.g., Table 3) and/or at least one
NDP kinase (see, e.g., Table 4) and/or a nucleoside kinase.
[0080] Conversion of Cellular RNA into NTP via NDP. In some
aspects, NTP is produced using cellular RNA as a substrate by first
breaking down (degrading/depolymerizing) the cellular RNA into NDPs
and then converting NDPs into NTPs, as depicted in FIG. 3A. For
example, NTP production methods may include incubating in a
reaction mixture cellular RNA, a polynucleotide phosphorylase
(PNPase), and phosphate under conditions suitable for the
production of NDPs. To proceed to the production of NTPs, the
reaction mixture, in some embodiments, also comprises a
polyphosphate kinase and polyphosphate. Thus, the methods further
comprise incubating the reaction mixture under conditions suitable
for the production of NTPs. In some embodiments, the reaction
mixture further comprises a NDP kinase. In some embodiments, the
NTP production reaction mixture may also include a nucleoside
kinase.
[0081] Conversion of Cellular RNA into NTP via NMP. In some
aspects, NTP is produced using cellular RNA as a substrate by first
breaking down the cellular RNA into 5'-NMPs and then converting
NMPs to NDPs and NDPs to NTPs, as depicted in FIG. 3B. For example,
NTP production methods may include incubating in a reaction mixture
cellular RNA, and a ribonuclease under conditions suitable for the
production of 5'-NMPs. To proceed to the production of NTPs, the
reaction mixture, in some embodiments, also comprises a
polyphosphate kinase, and a polyphosphate. Thus, the methods
further comprise incubating the reaction mixture under conditions
suitable for the production of NTPs. In some embodiments, the
reaction mixture further comprises a NDP kinase. In some
embodiments, the NTP production reaction mixture may also include a
nucleoside kinase. Alternatively, NTP production methods may
include incubating in a reaction mixture cellular RNA, a
ribonuclease that cleaves RNA into 3'-NMPs, and an appropriate
phosphatase (e.g. alkaline phosphatase or others) under conditions
suitable for the production of nucleosides. The phosphatase would
then be eliminated before proceeding to the production of NTPs.
RNA Production Pathways
[0082] As shown in FIG. 1, RNA (e.g., mRNA or double-stranded RNA)
may be produced through various different enzymatic pathways, each
of which utilize energy sources as described herein, and low-cost
starting materials in the reaction mixture(s). Thus, systems,
methods, compositions, and kits for the production of RNA are
provided herein.
[0083] Conversion of NDP to RNA. In some aspects, RNA is produced
using NDPs as substrates, as depicted in FIG. 2A. For examples, RNA
production methods may include incubating in a reaction mixture
NDPs, a polyphosphate kinase, a polyphosphate, a DNA template, and
a RNA polymerase under conditions suitable for the production of
RNA. In some embodiments, the RNA production reaction mixture may
also include a NDP kinase (see, e.g., Table 5). In some
embodiments, the RNA production reaction mixture may also include a
nucleoside kinase
[0084] Conversion of NMP to RNA. In some aspects, RNA is produced
using 5' NMPs as substrates, as depicted in FIG. 2B. For example,
RNA production methods may include incubating in a reaction mixture
5' NMPs, a polyphosphate kinase, a polyphosphate, a DNA template,
and a RNA polymerase under conditions suitable for the production
of RNA. In some embodiments, the RNA production reaction mixture
may also include a NMP kinase (see, e.g., Table 4) and/or a NDP
kinase (see, e.g., Table 5). In some embodiments, the RNA
production reaction mixture may also include a nucleoside kinase
Conversion of Nucleosides to RNA. In some aspects, RNA is produced
using nucleosides as substrates, as depicted in FIG. 2C. For
example, RNA production methods may include incubating in a
reaction mixture nucleosides, a polyphosphate kinase, a
polyphosphate, a DNA template, and a RNA polymerase under
conditions suitable for the production of RNA. In some embodiments,
the RNA production reaction mixture may also include a nucleoside
kinase (see, e.g., Table 3) and/or a NMP kinase (see, e.g., Table
4) and/or a NDP kinase (see, e.g., Table 5).
[0085] Conversion of Cellular RNA into RNA via NDP. In some
aspects, RNA is produced using cellular RNA as a substrate by first
breaking down the cellular RNA into NDPs, as depicted in FIG. 3A.
For example, RNA production methods may include incubating in a
reaction mixture cellular RNA, a polynucleotide phosphorylase
(PNPase), and phosphate under conditions suitable for the
production of NDPs. Before proceeding to the production of RNA, it
may be advantageous to eliminate the PNPase to avoid degrading the
end product. Thus, the methods may further comprise eliminating the
a PNPase, and incubating in the reaction mixture, or in a second
reaction mixture, the NDPs, a polyphosphate kinase, a
polyphosphate, a DNA template, and a polymerase under conditions
suitable for the production of RNA. In some embodiments, the
reaction mixture further comprises a NDP kinase. In some
embodiments, the RNA production reaction mixture may also include a
nucleoside kinase
[0086] In some embodiments, these pathway enzymes are capable of
withstanding elimination conditions, as discussed below, and, thus,
all reaction components are included in a single (one-step)
reaction mixture. For example, a RNA production method may comprise
(a) incubating in a reaction mixture cellular RNA, a PNPase,
phosphate, a polyphosphate kinase, a polyphosphate, a DNA template,
and a polymerase under conditions suitable for the production of
NDPs (optionally wherein the reaction mixture further comprises a
NDP kinase), (b) eliminating the a PNPase, and (c) incubating the
reaction mixture under conditions suitable for the production of
RNA.
[0087] Conversion of Cellular RNA into RNA via NMP. In some
aspects, RNA is produced using cellular RNA as a substrate by first
breaking down the cellular RNA into 5' NMPs, as depicted in FIG.
3B. For example, RNA production methods may include incubating in a
reaction mixture cellular RNA and a ribonuclease under conditions
suitable for the production of 5' NMPs. Before proceeding to the
production of RNA, it may be advantageous to eliminate the
ribonuclease to avoid degrading the end product. Thus, the methods
may further comprise eliminating the a ribonuclease, and incubating
in the reaction mixture, or in a second reaction mixture, the 5'
NMPs, a polyphosphate kinase, a polyphosphate, a DNA template, and
a polymerase under conditions suitable for the production of RNA.
In some embodiments, the reaction mixture further comprises a NMP
kinase and/or a NDP kinase. In some embodiments, the RNA production
reaction mixture may also include a nucleoside kinase
[0088] In some embodiments, these pathway enzymes are capable of
withstanding elimination conditions, as discussed below, and, thus,
all reaction components are included in a single (one-step)
reaction mixture. For example, a RNA production method may comprise
(a) incubating in a reaction mixture cellular RNA, a ribonuclease,
a polyphosphate kinase, a polyphosphate, a DNA template, and a
polymerase under conditions suitable for the production of NMPs
(optionally wherein the reaction mixture further comprises a NMP
kinase and/or a NDP kinase), (b) eliminating the a ribonuclease,
and (c) incubating the reaction mixture under conditions suitable
for the production of RNA.
[0089] Conversion of Nucleobases to RNA. In some aspects, RNA is
produced using nucleobases as substrates, as depicted in FIG. 2D.
For example, RNA production methods may include incubating in a
reaction mixture nucleobases, a (e.g., 1, 2, 3, or 4)
phosphoribosyltransferase, a phosphoribosylpyrophosphate, a
polyphosphate kinase, a polyphosphate, a DNA template, and a RNA
polymerase under conditions suitable for the production of RNA. In
some embodiments, the RNA production reaction mixture may also
include a NMP kinase (see, e.g., Table 4) and/or a NDP kinase (see,
e.g., Table 5). In some embodiments, the RNA production reaction
mixture may also include a nucleoside kinase
[0090] Conversion of Nucleobases and Ribose to RNA. In some
aspects, RNA is produced using nucleobases as substrates, as
depicted in FIG. 2E. For example, RNA production methods may
include incubating in a reaction mixture nucleobases, D-ribose,
ribokinase, phosphopentomutase, at least one (e.g., 1, 2, 3, or 4)
nucleoside phosphorylase, at least one polyphosphate kinase, at
least one polyphosphate, at least one DNA template, and at least
one RNA polymerase under conditions suitable for the production of
RNA. In some embodiments, the RNA production reaction mixture may
also include at least one NMP kinase (see, e.g., Table 3) and/or at
least one NDP kinase (see, e.g., Table 4) and/or a nucleoside
kinase.
Enzyme Sources
[0091] Any (e.g., one, two, three, or more) or all of the pathway
enzymes provided herein (e.g., nucleases, kinases, polymerases,
etc.) may be endogenous (unmodified) enzymes or recombinant enzymes
expressed by a cell. In some embodiments, the pathway enzymes are
provided as a component of a cell lysate, which is included in a
reaction mixture. In some embodiments, the pathway enzymes are
purified from a cell lysate an included in a reaction mixture. In
some embodiments, a pathway enzyme is provided as a component of a
cell lysate and a pathway enzyme is purified from a cell lysate. In
some embodiments, a pathway enzyme is secreted and optionally
purified from cell broth.
[0092] In some embodiments, a pathway enzyme (e.g., nucleases,
kinases, polymerases, etc.) is an endogenous enzyme purified from a
cell and included a reaction mixture as a purified enzyme. In some
embodiments, a pathway enzyme (e.g., nucleases, kinases,
polymerases, etc.) is an endogenous enzyme provided as a component
of a cell lysate that is included in a reaction mixture. In some
embodiments, a pathway enzyme (e.g., nucleases, kinases,
polymerases, etc.) is a recombinant enzyme purified from a cell and
included a reaction mixture as a purified enzyme. In some
embodiments, a pathway enzyme (e.g., nucleases, kinases,
polymerases, etc.) is a recombinant enzyme provided as a component
of a cell lysate that is included in a reaction mixture. In some
embodiments, a pathway enzyme is secreted and optionally purified
from cell broth.
[0093] The present disclosure also encompasses endogenous enzymes
and recombinant enzymes secreted by a cell. Thus, in some
embodiments, a pathway enzyme (e.g., nucleases, kinases,
polymerases, etc.) is an endogenous enzyme secreted from a cell. In
some embodiments, a pathway enzyme (e.g., nucleases, kinases,
polymerases, etc.) is a recombinant enzyme secreted from a
cell.
Elimination of Undesired Enzymatic Activities
[0094] In various embodiments provided herein, enzymes prepared
from cells or lysates of cells that express pathway enzymes are
used in a reaction mixture for the production of NTP and/or RNA. In
these cells or cell lysates, there are enzymes that may have
deleterious effects on NTP and/or RNA production. Non-limiting
examples of such enzymes include phosphatases, nucleases,
proteases, deaminases, oxidoreductases, and/or hydrolases, such as
those expressed by Escherichia coli cells. Phosphatases remove
phosphate groups (e.g., converting NMPs to nucleosides, converting
NDPs to NMPs, or converting NTPs to NDPs), which reduce NTP
production due to futile cycles of nucleotide
phosphorylation/dephosphorylation. Nucleases cleave nucleic acids
into monomers or oligomers, which lead to RNA product degradation
(e.g., by RNase) and/or DNA template degradation (e.g., by DNase).
Proteases cleave proteins into amino acids or peptides, which
degrade pathway enzymes. Deaminases remove amino groups, which
reduced NTP concentrations by conversion of pathway intermediates
to non-useful substrates (e.g., xanthine and hypoxanthine) and can
lead to mutations in RNA products (e.g., C to U). Hydrolases (e.g.,
nucleoside hydrolase or nucleotide hydrolase) cleave nucleosides or
nucleotides into base and sugar moieties, which reduce NTP
concentrations due to irreversible degradation of nucleotides.
Oxidoreductases catalyze the transfer of electrons from one
molecule (the oxidant) to another molecule (the reductant).
Oxidation and/or reduction reactions can, for example, damage
nucleobases in DNA and/or RNA, leading to errors in transcription
and/or translation, or damage proteins or enzymes leading to loss
of function.
[0095] Thus, it is advantageous in many embodiments to eliminate
these native enzymatic activities or other undesired enzymatic
activities in an enzyme preparation, a cell lysate, and/or a
reaction mixture. Herein, "elimination" of enzymatic activities may
be partial (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of
the activity is eliminated) or complete (100% of the activity is
eliminated) of an undesired enzymatic activity. As discussed
herein, enzymatic activity may be eliminated by genetic
modification, conditional inactivation, and/or physical separation.
Other elimination methods may also be used. The undesired enzymatic
activity may stem from at least one (e.g., 1, 2, 3, 4 or 5) native
(endogenous) enzyme, including but not limited to, phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and/or
hydrolases.
[0096] In some embodiments, undesired phosphatase activity is
eliminated in an enzyme preparation, a cell lysate, and/or a
reaction mixture. In some embodiments, undesired nuclease activity
is eliminated in an enzyme preparation, a cell lysate, and/or a
reaction mixture. In some embodiments, undesired protease activity
is eliminated in an enzyme preparation, a cell lysate, and/or a
reaction mixture. In some embodiments, undesired deaminase activity
is eliminated in an enzyme preparation, a cell lysate, and/or a
reaction mixture. In some embodiments, undesired hydrolase activity
is eliminated in an enzyme preparation, a cell lysate, and/or a
reaction mixture.
[0097] Undesired (e.g., native) enzymatic activity(ies) may be
eliminated using genetic, conditional, or separation approaches. In
some embodiments, a genetic approach is used to remove undesired
enzymatic activity. Thus, in some embodiments, cells are modified
to reduce or eliminate undesired enzymatic activities. Examples of
genetic approaches that may be used to reduce or eliminate
undesired enzymatic activity include, but are not limited to,
secretion, gene knockouts, and protease targeting. In some
embodiments, a conditional approach is used to remove undesired
enzymatic activity. Thus, in some embodiments, undesired enzymes
exhibiting undesired activities remain in an enzyme preparation, a
cell lysate, and/or a reaction mixture and are selectively
inactivated. Examples of conditional approaches that may be used to
reduce or eliminate undesired enzymatic activity include, but are
not limited to, changes in temperature, pH, salt, detergent,
organic solvent (e.g., alcohol), and the use of chemical
inhibitors. In some embodiments, a separation/purification approach
is used to remove undesired enzymatic activity. Thus, in some
embodiments, undesired enzymes exhibiting undesired activities are
physically removed from an enzyme preparation, a cell lysate,
and/or a reaction mixture. Examples of separation approaches that
may be used to reduce or eliminate undesired enzymatic activity
include, but are not limited to, precipitation, immobilization,
filtration, and chromatography.
[0098] Genetic Approaches. In some embodiments, cells expressing an
enzyme and/or DNA template of a NTP and/or RNA production pathway
are modified to reduce or eliminate undesired enzymatic activities.
In some embodiments, a gene encoding an enzyme exhibiting an
undesired activity is deleted from the cells. In some embodiments,
a gene encoding an enzyme exhibiting an undesired activity is
mutated such that the resulting gene product is rendered
non-functional. In some embodiments, an enzyme exhibiting an
undesired activity is modified to include a site-specific
protease-recognition sequence in their protein sequence such that
the enzyme may be "targeted" and cleaved for inactivation (see,
e.g., U.S. Publication No. 2012/0052547 A1, published on Mar. 1,
2012; International Publication No. WO 2015/021058 A2, published
Feb. 12, 2015; and International Publication Number WO 2012/030980,
published Mar. 8, 2012, each of which is incorporated by reference
herein).
[0099] Cleavage of an enzyme containing a site-specific
protease-recognition sequence results from contact with a cognate
site-specific protease that is sequestered in the periplasm of the
cell (separate from the target enzyme) during the cell growth phase
(e.g., as engineered cells are cultured) and is brought into
contact with the enzyme during the ATP production phase (e.g.,
following cell lysis to produce a cell lysate). Thus, engineered
cells of the present disclosure comprise, in some embodiments, (i)
an engineered nucleic acid encoding an enzyme exhibiting an
undesired activity and includes a site-specific
protease-recognition sequence in the protein sequence of the
enzyme, and (ii) an engineered nucleic acid encoding a
site-specific protease that cleaves the site-specific
protease-recognition sequence of the enzyme and includes a
periplasmic-targeting sequence. This periplasmic-targeting sequence
is responsible for sequestering the site-specific protease to the
periplasmic space of the cell until the cell is lysed. Examples of
periplasmic-targeting sequences are known.
[0100] Examples of proteases that may be used in accordance with
the present disclosure include, without limitation, alanine
carboxypeptidase, astacin, bacterial leucyl aminopeptidase, cancer
procoagulant, cathepsin B, clostripain, cytosol alanyl
aminopeptidase, elastase, endoproteinase Brg-C, enterokinase,
gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl
endopeptidase, human rhinovirus 3C protease, hypodermin C,
Iga-specific serine endopeptidase, leucyl aminopeptidase, leucyl
endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl
aminopeptidase, methionyl aminopeptidase, myxobacter, nardilysin,
pancreatic endopeptidase E, picornain 2B, picornain 3C,
proendopeptidase, prolyl aminopeptidase, proprotein convertase I,
proprotein convertase II, russellysin, saccharopepsin,
semenogelase, T-plasminogen activator, thrombin, tissue kallikrein,
tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase,
U-plasminogen activator, V8, venombin B, venombin BB and Xaa-pro
aminopeptidase.
[0101] Conditional Approaches. In some embodiments, an enzyme
preparation, a cell lysate, and/or a reaction mixture includes an
enzyme exhibiting undesired activity that is selectively
inactivated. In some embodiments, an enzyme exhibiting undesired
activity is selectively inactivated by exposing the enzyme to
elimination conditions (e.g., high or low temperature, acidic or
basic pH value, high salt or low salt, detergent, and/or organic
solvent).
[0102] In some embodiments, an enzyme preparation, an enzyme
preparation, a cell lysate, and/or a reaction mixture is exposed to
a temperature that temporarily or irreversibly inactivates the
enzyme exhibiting undesired activity. "Temperature inactivation"
refers to the process of heating or cooling an enzyme preparation,
a cell lysate, and/or a reaction mixture to a temperature
sufficient to inactivate (or at least partially inactivate) native
target enzyme. Generally, the process of temperature inactivation
involves denaturation of (unfolding of) the deleterious enzyme. The
temperature at which an enzyme denature varies among organisms. In
E. coli, for example, enzymes generally denature at temperatures
above 41.degree. C. The denaturation temperature may be higher or
lower than 41.degree. C. for other organisms. Enzymes of a cell
lysate, as provide here, may be temperature inactivated at a
temperature of 0.degree. C.-95.degree. C., or higher. In some
embodiments, enzymes of a cell lysate are temperature inactivated
at a temperature of 0-90.degree. C., 0-80.degree. C., 0-70.degree.
C., 0-60.degree. C., 0-50.degree. C., 0-40.degree. C., 0-30.degree.
C., 0-20.degree. C., 0-10.degree. C., or 0-5.degree. C. In some
embodiments, enzymes of a cell lysate are temperature inactivated
at a temperature of 5-95.degree. C., 10-95.degree. C.,
20-95.degree. C., 30-95.degree. C., 40-95.degree. C., 50-95.degree.
C., 60-95.degree. C., 70-95.degree. C., 80-95.degree. C., or
90-95.degree. C. For example, enzymes of a cell lysate may be
temperature inactivated at a temperature of approximately
40.degree. C., 42.degree. C., 45.degree. C., 50.degree. C.,
55.degree. C., 60.degree. C., 65.degree. C., 70.degree. C.,
75.degree. C., 80.degree. C., 85.degree. C., 90.degree. C., or
95.degree. C. In some embodiments, enzymes of a cell lysate are
temperature inactivated at a temperature of 50-80.degree. C. In
some embodiments, enzymes of a cell lysate are temperature
inactivated at a temperature of approximately 70.degree. C. In some
embodiments, enzymes of a cell lysate are temperature inactivated
at a temperature of approximately 60.degree. C.
[0103] In some embodiments, an enzyme preparation, a cell lysate,
and/or a reaction mixture is exposed to an acid or base (change in
pH) that temporarily or irreversibly inactivates an enzyme
exhibiting undesired activity. "Acid or base inactivation" refers
to the process of adjusting an enzyme preparation, a cell lysate,
and/or a reaction mixture to a pH sufficient to inactivate (or at
least partially inactivate) an enzyme. Generally, the process of
acid or base inactivation involves denaturation of (unfolding of)
the enzyme. The pH at which enzymes denature varies among
organisms. In E. coli, for example, native enzymes generally
denature at pH above 7.5 or below 6.5. The denaturation pH may be
higher or lower than the denaturation pH for other organisms.
Enzymes of an enzyme preparation, a cell lysate, and/or a reaction
mixture, as provide herein, may be base inactivated at a pH of
7.5-14, or higher. In some embodiments, enzymes of a cell lysate is
base inactivated at a pH of 8-14, 8.5-14, 9-14, 9.5-14, 10-14,
10.5-14, 11-14, 11.5-14, 12-14, 12.5-14, 13-14, or 13.5-14. In some
embodiments, enzymes of an enzyme preparation, a cell lysate,
and/or a reaction mixture are base inactivated at a pH of 7.5-13.5,
7.5-13, 7.5-12.5, 7.5-12, 7.5-11.5, 7.5-11, 7.5-10.5, 7.5-10,
7.5-9.5, 7.5-9, 7.5-8.5, or 7.5-8. For example, enzymes of an
enzyme preparation, a cell lysate, and/or a reaction mixture may be
base inactivated at a pH of approximately 7.5, 8, 8.5, 9, 9.5, 10,
10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14. Enzymes of an enzyme
preparation, a cell lysate, and/or a reaction mixture, as provide
herein, may be acid inactivated at a pH of 6.5-0, or lower. In some
embodiments, enzymes of an enzyme preparation, a cell lysate,
and/or a reaction mixture are acid inactivated at a pH of 6.5-0.5,
6.5-1, 6.5-1.5, 6.5-2, 6.5-2.5, 6.5-3, 6.5-3.5, 6.5-4, 6.5-4.5,
6.5-5, or 6.5-6. In some embodiments, enzymes of an enzyme
preparation, a cell lysate, and/or a reaction mixture are acid
inactivated at a pH of 6-0, 5.5-0, 5-0, 4.5-0, 4-0, 3.5-0, 3-0,
2.5-0, 2-0, 1.5-0, 1-0, or 0.5-0. For example, enzymes of an enzyme
preparation, a cell lysate, and/or a reaction mixture may be acid
inactivated at a pH of approximately 6.5, 6, 5.5, 5, 4.5, 4, 3.5,
3, 2.5, 2, 1.5, 1, 0.5, or 0.
[0104] In some embodiments, an enzyme preparation, a cell lysate,
and/or a reaction mixture is exposed to a high salt or low salt
(change in salt concentration) that temporarily or irreversibly
inactivates an enzyme exhibiting undesired activity. "Salt
inactivation" refers to the process of adjusting an enzyme
preparation, a cell lysate, and/or a reaction mixture to a salt
concentration sufficient to inactivate (or partially inactivate) an
enzyme. Generally, the process of salt inactivation involves
denaturation of (unfolding of) the enzyme. The salt concentration
at which enzymes denature varies among organisms. In E. coli, for
example, native enzymes generally denature at a salt concentration
above 600 mM. The denaturation salt concentration may be higher or
lower than the denaturation salt concentration for other organisms.
Salts are combinations of anions and cations. Non-limiting examples
of cations include lithium, sodium, potassium, magnesium, calcium
and ammonium. Non-limiting examples of anions include acetate,
chloride, sulfate, and phosphate. Enzymes of an enzyme preparation,
a cell lysate, and/or a reaction mixture, as provided herein, may
be salt inactivated at a salt concentration of 600-1000 mM, or
higher. In some embodiments, enzymes of an enzyme preparation, a
cell lysate, and/or a reaction mixture are salt inactivated at a
salt concentration of 700-1000 mM, 750-1000 mM, 800-1000 mM,
850-1000 mM, 900-1000 mM, 950-1000 mM. In some embodiments, enzymes
of an enzyme preparation, a cell lysate, and/or a reaction mixture
are salt inactivated at a salt concentration of 600-950 mM, 600-900
mM, 600-850 mM, 600-800 mM, 600-750 mM, 600-700 mM, or 600-650 mM.
For example, enzymes of an enzyme preparation, a cell lysate,
and/or a reaction mixture may be salt inactivated at a salt
concentration of approximately 600 mM, 650 mM, 700 mM, 750 mM, 800
mM, 850 mM, 900 mM, 950 mM, or 1000 mM. Enzymes of an enzyme
preparation, a cell lysate, and/or a reaction mixture, as provided
herein, may be salt inactivated at a salt concentration of 400-0
mM, or lower. In some embodiments, enzymes of an enzyme
preparation, a cell lysate, and/or a reaction mixture are salt
inactivated at a salt concentration of 350-0 mM, 300-0 mM, 250-0
mM, 200-0 mM, 150-0 mM, 100-0 mM, or 50-0 mM. In some embodiments,
enzymes of an enzyme preparation, a cell lysate, and/or a reaction
mixture are salt inactivated at a salt concentration of 400-50 mM,
400-100 mM, 400-150 mM, 400-200 mM, 400-250 mM, 400-300 mM, or
400-350 mM. For example, enzymes of an enzyme preparation, a cell
lysate, and/or a reaction mixture may be salt inactivated at a salt
concentration of approximately 400 mM, 350 mM, 300 mM, 250 mM, 200
mM, 150 mM, 100 mM, 50 mM, or 0 mM.
[0105] In some embodiments, an organic solvent is added to an
enzyme preparation, a cell lysate, and/or a reaction mixture to
inactivate an enzyme exhibiting undesired activity. Non-limiting
examples of organic solvents include ethanol, methanol, ether,
dioxane, acetone, methyl ethyl ketone, acetonitrile, dimethyl
sulfoxide, and toluene.
[0106] In some embodiments, a detergent is added to an enzyme
preparation, a cell lysate, and/or a reaction mixture to inactivate
an enzyme exhibiting undesired activity. Non-limiting examples of
detergents include sodium dodecyl sulfate (SDS), ethyl
trimethylammonium bromide (ETMAB), lauryl trimethyl ammonium
bromide (LTAB), and lauryl trimethylammonium chloride (LTAC).
[0107] In some embodiments, a chemical inhibitor is added to an
enzyme preparation, a cell lysate, and/or a reaction mixture to
inactivate an enzyme exhibiting undesired activity. Non-limiting
examples of chemical inhibitors include sodium orthovanadate
(inhibitor of protein phosphotyrosyl phosphatases), sodium fluoride
(inhibitor of phosphoseryl and phosphothreonyl phosphatases),
sodium pyrophosphate (phosphatase inhibitor), sodium phosphate,
and/or potassium phosphate. In some embodiments, chemical
inhibitors are selected from a chemical inhibitor library.
[0108] For any of the conditional approaches used herein, it should
be understood that any of the pathway enzymes present in the cell
lysate or reaction mixture may also be exposed to the elimination
conditions (e.g., high or low temperature, acidic or basic pH
value, high salt or low salt, detergent and/or organic solvent).
Thus, in some embodiments, the pathway enzymes (e.g., polyphosphate
kinase, NMP kinase, NDP kinase, and/or polymerase) can withstand
elimination conditions. An enzyme is considered to withstand
elimination conditions if the enzyme, following exposure to the
elimination conditions, retains at least 10% (e.g., at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%, or at least 90%) of its enzymatic activity
(relative to enzymatic activity prior to exposure to the
inactivation condition).
[0109] For example, when native enzymes of an enzyme preparation, a
cell lysate, and/or a reaction mixture are heat-inactivated (e.g.,
exposed to a temperature of at least 40.degree. C., or
40-95.degree. C., for at least 2 min, or 2-60 min), the pathway
enzymes may be thermostable enzymes. Thus, in some embodiments, at
least one of a polyphosphate kinase, NMP kinase, NDP kinase,
nucleoside kinase, phosphoribosyltransferase, nucleoside
phosphorylase, ribokinase, phosphopentomutase, and polymerase is
thermostable. An enzyme (e.g., kinase or polymerase) is considered
thermostable if the enzyme (a) retains activity after temporary
exposure to high temperatures that denature native enzymes or (b)
functions at a high rate after temporary exposure to a medium to
high temperature where native enzymes function at low rates.
Thermostable enzymes are known, and non-limiting examples of
thermostable enzymes for use as provided herein. Other non-limiting
examples of pathway enzymes that can withstand elimination
conditions are also provided herein.
[0110] Separation Approaches. In some embodiments, a native enzyme
exhibiting undesired activity is physically removed from an enzyme
preparation, a cell lysate, and/or a reaction mixture. In some
embodiments, an enzyme exhibiting undesired activity is
precipitated from an enzyme preparation, a cell lysate, and/or a
reaction mixture. In some embodiments, an enzyme exhibiting
undesired activity is filtered (e.g., based on size) from an enzyme
preparation, a cell lysate, and/or a reaction mixture. In some
embodiments, an enzyme exhibiting undesired activity is removed
from an enzyme preparation, a cell lysate, and/or a reaction
mixture via capture and/or chromatography (e.g., by differential
affinity to a stationary phase).
[0111] In some embodiments, an enzyme exhibiting undesired activity
is removed from an enzyme preparation, a cell lysate, and/or a
reaction mixture via affinity chromatography. Examples of affinity
chromatography include, but are not limited to, Protein A
chromatography, Protein G chromatography, metal binding
chromatography (e.g., nickel chromatography), lectin
chromatography, and GST chromatography.
[0112] In some embodiments, an enzyme exhibiting undesired activity
is removed from an enzyme preparation, a cell lysate, and/or a
reaction mixture via ion exchange chromatography. Examples of anion
exchange chromatography (AEX) include, but are not limited to,
diethylaminoethyl (DEAE) chromatography, quaternary aminoethyl
(QAE) chromatography, and quaternary amine(Q) chromatography.
Examples of cation exchange chromatography include, but are not
limited to, carboxymethyl (CM) chromatography, sulfoethyl (SE)
chromatography, sulfopropyl (SP) chromatography, phosphate (P)
chromatography, and sulfonate (S) chromatography.
[0113] In some embodiments, an enzyme exhibiting undesired activity
is removed from an enzyme preparation, a cell lysate, and/or a
reaction mixture via hydrophobic interaction chromatography (HIC).
Examples of hydrophobic interaction chromatography include, but are
not limited to, Phenyl Sepharose chromatography, Butyl Sepharose
chromatography, Octyl Sepharose chromatography, Capto Phenyl
chromatography, Toyopearl Butyl chromatography, Toyopearl Phenyl
chromatography, Toyopearl Hexyl chromatography, Toyopearl Ether
chromatography, and Toyopearl PPG chromatography. Any of the
chemistries detailed above could be alternatively be used to
immobilize or capture pathway enzymes.
Thermostable Enzymes
[0114] Any of the pathway enzymes provided herein (e.g., nucleases,
kinases, polymerases, etc.) may be thermostable enzymes.
Thermostability refers to the quality of enzymes to resist
denaturation at relatively high or low temperature. For example, if
an enzyme is denatured (inactivated) at a temperature of 42.degree.
C., an enzyme having similar activity (e.g., kinase activity) is
considered "thermostable" if it does not denature at 42.degree.
C.
[0115] An enzyme (e.g., kinase or polymerase) is considered
thermostable if the enzyme (a) retains activity after temporary
exposure to high temperatures that denature other native enzymes or
(b) functions at a high rate after temporary exposure to a medium
to high temperature where native enzymes function at low rates.
[0116] An enzyme (e.g., kinase or polymerase) is also considered
thermostable if the enzyme (a) retains activity after temporary
exposure to low temperatures that denature other native enzymes or
(b) functions at a high rate after temporary exposure to a medium
to low temperature where native enzymes function at low rates.
[0117] In some embodiments, a thermostable enzyme retains greater
than 10% activity following temporary exposure to relatively high
temperature (e.g., higher than 41.degree. C. for kinases obtained
from E. coli, higher than 37.degree. C. for many RNA polymerases)
that would otherwise denature a similar (non-thermostable) native
enzyme. In some embodiments, a thermostable enzyme retains 10-100%,
25-100%, or 50-100% activity following temporary exposure to
relatively high temperature that would otherwise denature a similar
(non-thermostable) native enzyme. For example, a thermostable
enzyme may retain 10-90%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%,
10-60%, 10-55%, 25-90%, 25-85%, 25-80%, 25-75%, 25-70%, 25-65%,
25-60%, 25-55%, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%,
50-60%, or 50-55% temporary exposure to relatively high temperature
that would otherwise denature a similar (non-thermostable) native
enzyme. In some embodiments, a thermostable enzyme retains 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 98%, 99%, or 100% activity following temporary
exposure to relatively high temperature that would otherwise
denature a similar (non-thermostable) native enzyme.
[0118] In some embodiments, a thermostable enzyme retains greater
than 50% activity following temporary exposure to relatively low
temperature (e.g., lower than 32.degree. C. for kinases obtained
from E. coli, lower than 32.degree. C. for many RNA polymerases)
that would otherwise denature a similar (non-thermostable) native
enzyme. In some embodiments, a thermostable enzyme retains 50-100%
activity following temporary exposure to relatively low temperature
that would otherwise denature a similar (non-thermostable) native
enzyme. For example, a thermostable enzyme may retain 50-90%,
50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, or 50-55% activity
following temporary exposure to relatively low temperature that
would otherwise denature a similar (non-thermostable) native
enzyme. In some embodiments, a thermostable enzyme retains 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, 99%, or 100% activity following temporary exposure to
relatively low temperature that would otherwise denature a similar
(non-thermostable) native enzyme.
[0119] In some embodiments, the activity of a thermostable enzyme
after temporary exposure to medium to high temperature (e.g.,
42-80.degree. C.) is greater than (e.g., 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%
greater than) the activity of a similar (non-thermostable) native
enzyme.
[0120] In some embodiments, the activity of a thermostable enzyme
after temporary exposure to medium to low temperature (e.g.,
32-0.degree. C.) is greater than (e.g., 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%
greater than) the activity of a similar (non-thermostable) native
enzyme.
[0121] The activity of a thermostable kinase, for example, may be
measured by the amount of NMP or NDP the kinase is able to
phosphorylate. Thus, in some embodiments, a thermostable kinase, at
relatively high temperature (e.g., 42.degree. C.) converts greater
than 50% of NMP to NDP, or greater than 50% of NDP to NTP, in the
same amount of time required to complete a similar conversion at
37.degree. C. In some embodiments, a thermostable kinase, at
relatively high temperature (e.g., 42.degree. C.) converts greater
than 60% of NMP to NDP, or greater than 60% of NDP to NTP, in the
same amount of time required to complete a similar conversion at
37.degree. C. In some embodiments, a thermostable kinase, at
relatively high temperature (e.g., 42.degree. C.) converts greater
than 70% of NMP to NDP, or greater than 70% of NDP to NTP, in the
same amount of time required to complete a similar conversion at
37.degree. C. In some embodiments, a thermostable kinase, at
relatively high temperature (e.g., 42.degree. C.) converts greater
than 80% of NMP to NDP, or greater than 80% of NDP to NTP, in the
same amount of time required to complete a similar conversion at
37.degree. C. In some embodiments, a thermostable kinase, at
relatively high temperature (e.g., 42.degree. C.) converts greater
than 90% of NMP to NDP, or greater than 90% of NDP to NTP, in the
same amount of time required to complete a similar conversion at
37.degree. C.
[0122] In some embodiments, a thermostable kinase, at relatively
low temperature (e.g., 32.degree. C.) converts greater than 50% of
NMP to NDP, or greater than 50% of NDP to NTP, in the same amount
of time required to complete a similar conversion at 37.degree. C.
In some embodiments, a thermostable kinase, at relatively low
temperature (e.g., 32.degree. C.) converts greater than 60% of NMP
to NDP, or greater than 60% of NDP to NTP, in the same amount of
time required to complete a similar conversion at 37.degree. C. In
some embodiments, a thermostable kinase, at relatively low
temperature (e.g., 32.degree. C.) converts greater than 70% of NMP
to NDP, or greater than 70% of NDP to NTP, in the same amount of
time required to complete a similar conversion at 37.degree. C. In
some embodiments, a thermostable kinase, at relatively low
temperature (e.g., 32.degree. C.) converts greater than 80% of NMP
to NDP, or greater than 80% of NDP to NTP, in the same amount of
time required to complete a similar conversion at 37.degree. C. In
some embodiments, a thermostable kinase, at relatively low
temperature (e.g., 32.degree. C.) converts greater than 90% of NMP
to NDP, or greater than 90% of NDP to NTP, in the same amount of
time required to complete a similar conversion at 37.degree. C.
[0123] The activity of a thermostable polymerase, for example, is
assessed based on fidelity and polymerization kinetics (e.g., rate
of polymerization). Thus, one unit of a thermostable T7 polymerase,
for example, may incorporate 10 nmoles of NTP into acid insoluble
material in 30 minutes at temperatures above 37.degree. C. (e.g.,
at 50.degree. C.). In another example, one unit of a thermostable
T7 polymerase may incorporate 10 nmoles of NTP into acid insoluble
material in 30 minutes at temperatures below 32.degree. C. (e.g.,
at 25.degree. C.)
[0124] In some embodiments, thermostable enzymes (e.g., kinases or
polymerases) may remain active (able to catalyze a reaction) at a
temperature of 42.degree. C. to 80.degree. C., or higher. In some
embodiments, thermostable enzymes remain active at a temperature of
42-80.degree. C., 42-70.degree. C., 42-60.degree. C., 42-50.degree.
C., 50-80.degree. C., 50-70.degree. C., 50-60.degree. C.,
60-80.degree. C., 60-70.degree. C., or 70-80.degree. C. For
example, thermostable enzymes may remain active at a temperature of
42.degree. C., 43.degree. C., 44.degree. C., 45.degree. C.,
46.degree. C., 47.degree. C., 48.degree. C., 49.degree. C.,
50.degree. C., 51.degree. C., 52.degree. C., 53.degree. C.,
54.degree. C., 55.degree. C., 55.degree. C., 56.degree. C.,
57.degree. C., 58.degree. C., 59.degree. C., 60.degree. C.,
61.degree. C., 62.degree. C., 63.degree. C., 64.degree. C.,
65.degree. C., 66.degree. C., 67.degree. C., 68.degree. C.,
69.degree. C., 70.degree. C., 71.degree. C., 72.degree. C.,
73.degree. C., 74.degree. C., 75.degree. C., 76.degree. C.,
77.degree. C., 78.degree. C., 79.degree. C., or 80.degree. C.
Thermostable enzymes may remain active at relatively high
temperatures for 15 minutes to 48 hours, or longer. For example,
thermostable enzymes may remain active at relatively high
temperatures for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 24, 36, 42, or 48 hours.
[0125] In some embodiments, thermostable enzymes (e.g., kinases or
polymerases) may remain active (able to catalyze a reaction) at a
temperature of 32.degree. C. to 0.degree. C., or lower. In some
embodiments, thermostable enzymes remain active at a temperature of
32-5.degree. C., 32-10.degree. C., 32-20.degree. C., 32-25.degree.
C., 32-30.degree. C., 30-0.degree. C., 25-0.degree. C.,
20-0.degree. C., 10-0.degree. C., or 5-0.degree. C. For example,
thermostable enzymes may remain active at a temperature of
32.degree. C., 31.degree. C., 30.degree. C., 29.degree. C.,
28.degree. C., 27.degree. C., 26.degree. C., 25.degree. C.,
24.degree. C., 23.degree. C., 22.degree. C., 21.degree. C.,
20.degree. C., 19.degree. C., 18.degree. C., 17.degree. C.,
16.degree. C., 15.degree. C., 14.degree. C., 13.degree. C.,
12.degree. C., 10.degree. C., 9.degree. C., 8.degree. C., 7.degree.
C., 6.degree. C., 5.degree. C., 4.degree. C., 3.degree. C.,
2.degree. C., 1.degree. C., or 0.degree. C. Thermostable enzymes
may remain active at relatively low temperatures for 15 minutes to
48 hours, or longer. For example, thermostable enzymes may remain
active at relatively low temperatures for 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 36, 42, or 48
hours.
[0126] Non-limiting examples of thermostable NMP kinases are listed
in Tables 4A-4D. Other thermostable kinases include thermostable
nucleoside diphosphate kinases (see, e.g., Table 5), thermostable
pyruvate kinases, and thermostable polyphosphate kinases (see,
e.g., Table 2). Other thermostable kinases are encompassed by the
present disclosure.
[0127] Non-limiting examples of RNA polymerases are listed in Table
6. Other RNA polymerases, including thermostable RNA polymerases,
are encompassed by the present disclosure.
[0128] Thermostable RNA polymerases may be prepared by modifying
wild-type enzymes. Such modifications (e.g., mutations) are known.
For example, variant thermostable T7 RNA polymerases may include
one or more of the following point mutations: V426 L, A702V, V795I,
S430P, F849I, S633P, F880Y, C510R, and S767G (EP2377928 and
EP1261696A1, each of which is incorporated herein by reference). In
some embodiments, a variant thermostable T7 RNA polymerase includes
V426 L, A702V, and V795I mutations. In some embodiments, a variant
thermostable T7 RNA polymerase includes S430P, F849I, S633P, and
F880Y mutations. In some embodiments, a variant thermostable T7 RNA
polymerase includes F880Y, S430P, F849I, S633P, C510R, and S767G
mutations. In some embodiments, a variant thermostable T7 RNA
polymerase includes Y639V, H784G, E593G, and V685A mutations. In
some embodiments, a variant thermostable T7 RNA polymerase includes
S430P, N433T, S633P, F849I, and F880Y mutations. Other variant and
recombinant thermostable polymerases are encompassed by the present
disclosure.
[0129] In some embodiments, a thermostable T7 polymerase is used to
produce a RNA of interest. For example, a thermostable T7
polymerase (e.g., incubated at a temperature of 37-60.degree. C.)
having a concentration of 0.1-5% total protein may be used to
synthesize RNA of interest at a rate of greater than 1 g/L/hr (or,
e.g., 1 g/L/hr-20 g/L/hr).
[0130] It should be understood that while many embodiments of the
present disclosure describe the use of thermostable
polymerases/enzymes, other enzymes/polymerases may be used. In some
embodiments, polymerase may be exogenously added to
heat-inactivated cell lysates, for example, to compensate for any
reduction or loss of activity of the thermostable enzyme(s).
Fusion Enzymes
[0131] Any of the pathway enzymes provided herein (e.g., nucleases,
kinases, polymerases, etc.) may be individual enzymes, enzymes with
multiple activities, or fusion enzymes. A fusion enzyme may be
created by joining two or more gene or gene segments that code for
separate proteins. Translation of this fusion gene results in a
single or multiple polypeptides with functional properties derived
from each of the original proteins, e.g., a fusion protein that
acts as a nuclease, acts as a kinase, and/or acts as a polymerase.
Other enzymes may also be expressed as a fusion protein.
[0132] Some enzymes that exist in nature are multifunctional (e.g.,
CMP-UMP kinases). Thus, the term "enzyme" encompasses "enzymatic
activities," regardless of how they are supplied.
[0133] A fusion enzyme is considered to "act as a nuclease" if the
enzyme exhibits nuclease activity (cleaves or depolymerizes a
nucleic acid; e.g., RNase R). A fusion enzyme is considered to "act
as a kinase" if the enzyme exhibits kinase activity (catalyzes the
transfer of a phosphate group from one molecule to another
molecule; e.g., polyphosphate kinase). A fusion enzyme is
considered to "act as a polymerase" if the enzyme exhibits
polymerase activity (assembles nucleotides to produce nucleic
acids; e.g., RNA polymerase).
Energy Sources
[0134] There are several energy and phosphate sources that may be
used, as provided herein, for the production of NTP and/or RNA.
Non-limiting examples of sources of phosphate include NTP (e.g.,
ATP, GTP, UTP, CTP), polyphosphate (e.g., hexametaphosphate), and
pyrophosphate (PPi). In some embodiments, NTP, whether chemically
synthesized, a product of fermentation, or extracted from a natural
source, is included in a reaction mixture for the production of
RNA. In some embodiments, polyphosphate and polyphosphate kinase
are included in a reaction mixture for the production of NTP and/or
RNA. In some embodiments, acetate, ADP, pyrophosphate, and at least
two acetate kinases (e.g., acetate kinase (diphosphate) EC 2.7.2.12
and acetate kinase (phosphorylating) EC.7.2.1) are included in a
reaction mixture for the production of NTP and/or RNA. In some
embodiments, citrate, AMP, pyrophosphate, citrate lyase (the
citrate lyase complex), a phosphoenolpyruvate carboxykinase (PEPCK)
or a phosphoenolpyruvate carboxylase (PEPC), and a pyruvate
phosphate dikinase (PPDK) are included in a reaction mixture for
the production of NTP and/or RNA. In some embodiments, sulfite,
AMP, pyrophosphate, adenylyl sulfate reductase, and sulfate
adenylyltransferase are included in a reaction mixture for the
production of NTP and/or RNA. Other energy sources are also
encompassed by the present disclosure.
[0135] In some embodiments, an energy source is ATP produced from
pyrophosphate through cyclical phosphorylation of acetate, from
pyrophosphate and citrate, or from pyrophosphate and sulfite.
Methods for ATP production from the above pathways are described
herein. A summary of the ATP production pathways and pathway
enzymes are provided in Table 7 below.
TABLE-US-00010 TABLE 7 Summary of Exemplary ATP Production Pathways
and Enzymes ATP Production Pathway Enzymes ATP production from
acetate kinase (diphosphate) pyrophosphate through (EC 2.7.2.12)
the acetate phosphorylation/ acetate kinase (phosphorylating)
dephosphorylation cycle (EC 2.7.2.1) ATP production from citrate
citrate lyase (EC 4.1.3.6) and pyrophosphate phosphoenolpyruvate
carboxykinase (PEPCK) (EC 4.1.1.38) pyruvate phosphate dikinase
(PPDK) (EC 2.7.9.1, 2.7.9.2) phosphenolpyruvate carboxylase (PEPC)
(EC 4.1.1.31) ATP production from sulfite sulfate
adenylytransferase and pyrophosphate (EC 2.7.7.4) adenylyl sulfate
reductase (EC 1.8.99.2)
ATP Production from Pyrophosphate and ADP Through an Acetate
Phosphorylation/Dephosphorylation Cycle
[0136] Some aspects of the present disclosure use methods for
producing ATP from pyrophosphate (high-energy phosphate donor) and
ADP (ultimate energy/phosphate acceptor) through an acetate
phosphorylation/dephosphorylation cycle (see, e.g., FIG. 14). The
first acetate kinase (AcK1; EC 2.7.2.12) phosphorylates acetate
using inorganic pyrophosphate (PP.sub.i), which produces
acetyl-phosphate and inorganic phosphate (P.sub.i). The
acetyl-phosphate is then dephosphorylated by a second acetate
kinase (AcK2; EC 2.7.2.1), which transfers the high-energy
phosphate group from the acetyl-phosphate to ADP and produces ATP
and acetate. The resulting acetate is then free to be
phosphorylated again by AcK1, thereby completing a reaction
cycle.
[0137] In some embodiments, the methods of producing ATP from
pyrophosphate and ADP include culturing cells engineered to express
a first acetate kinase, a second acetate kinase, or two different
acetate kinases. In some embodiments, the methods include culturing
cells engineered to express a first acetate kinase and a second
acetate kinase. In some embodiments, the first acetate kinase and
the second acetate kinase are expressed as a single fusion
(chimeric) protein.
[0138] In some embodiments, at least one of the enzymes is a
thermostable enzyme. In some embodiments, at least two of the
enzymes are thermostable enzymes. In some embodiments, all of the
enzymes are thermostable enzymes. Thus, in some embodiments, the
methods include culturing cells engineered to express a
thermostable acetate kinase. In other embodiments, the methods
include culturing cells engineered to express a first thermostable
acetate kinase and a second thermostable acetate kinase.
[0139] In some embodiments, the methods of producing ATP from
pyrophosphate through the cyclical phosphorylation of acetate
include lysing (e.g., thermal, osmotic, mechanical (e.g.,
sonication), chemical, or enzymatic lysis) the cultured cells to
produce at least one (e.g., at least two) cell lysate. It should be
understood that multiple cell lysates (and thus multiple cell
populations, e.g., from the same organism (e.g., bacteria) or from
different organisms (e.g., bacteria, yeast and/or plant) may be
used in an enzymatic reaction as provided herein. For example, one
cell population may be engineered to express a first acetate kinase
of the ATP production pathway, while another cell population may be
engineered to express a second acetate kinase of the ATP production
pathway. Thus, in some embodiments, the methods comprise culturing
a population of cells engineered to express an acetate kinase,
and/or culturing a cell population engineered to express at least
one additional acetate kinase. Following lysis of the cells, the
cell lysates are combined such that the enzymes are present in a
single cell lysate/reaction mixture.
[0140] In some embodiments, the methods of producing ATP from
pyrophosphate through the cyclical phosphorylation of acetate
further include heating the cell lysate(s) (or a cell lysate
mixture) to a temperature that inactivates native enzymatic
activity but does not inactivate any of the thermostable enzymes of
the ATP production pathway, to produce a heat-inactivated lysate.
The cell lysate(s), in some embodiments, is heated to a temperature
of at least 50.degree. C. For example, the cell lysate(s) may be
heated to a temperature of at least 55.degree. C., 60.degree. C.,
65.degree. C., 70.degree. C., 75.degree. C., 80.degree. C.,
85.degree. C., or 90.degree. C. A native enzyme (or other
non-thermostable enzyme) is considered inactive, in some
embodiments, when its level of activity is reduced by at least 50%.
In some embodiments, a native enzyme (or other non-thermostable
enzyme) is considered inactive when its level of activity is
reduced by at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100%.
[0141] The cell lysate(s) may be heated for a period of time
sufficient to inactive native enzymes (or other non-thermostable
enzymes) of the cell. For example, the cell lysate(s) may be heated
for at least 2, 3, 4, or at least 5 minutes. In some embodiments,
the cell lysate(s) are heated for longer than 5 minutes. In some
embodiments, the cell lysate(s) is heated for longer than 15
minutes. In some embodiments, the cell lysate(s) is heated for less
than 2 minutes. In some embodiments, the cell lysate(s) are heated
for a period of time sufficient to reduce activity of native
enzymes (or other non-thermostable enzymes) by at least 50% (e.g.,
at least 60%, 70%, 80%, or 90%).
[0142] Following heat inactivation, in some embodiments, at least
one (e.g., at least two or at least three) purified enzymes may be
added to the cell lysate/reaction mixture. Thus, a reaction
mixture, in some embodiments, may include a combination of enzymes
present in the cell lysate (expressed by the engineered host
cell(s)) and at least one purified enzyme. At least one purified
enzyme may be a first acetate kinase and/or a second acetate
kinase. In some embodiments, a cell lysate may be cooled (e.g., to
50.degree. C.) following a heat-inactivation step, prior to adding
the purified enzyme(s).
[0143] In some embodiments, the methods of producing ATP from
pyrophosphate through the cyclical phosphorylation of acetate also
include incubating the heat-inactivated lysate(s) in the presence
of acetate, adenosine diphosphate (ADP), and an inorganic phosphate
to produce ATP. The inorganic phosphate may be, for example,
pyrophosphate. Other inorganic phosphates and/or orthophosphate
polymers, including but not limited to tripolyphosphate,
tetrapolyphosphate, pentapolyphosphate, hexametaphosphate and
mixtures thereof, may be used.
[0144] Also encompassed herein are cells and cell lysates used for
the production of ATP from pyrophosphate through the cyclical
phosphorylation of acetate. Thus, an engineered cell (e.g.,
bacterial cell, yeast cell, and/or plant cell) or cell lysate(s) of
the present disclosure may include at least one (e.g., at least
two) acetate kinase. In some embodiments, an engineered cell (e.g.,
bacterial cell, yeast cell, and/or plant cell) or cell lysate(s) of
the present disclosure includes at least one (e.g., at least two)
thermostable acetate kinase.
TABLE-US-00011 TABLE 8 Exemplary Acetate Kinase Enzymes Enzyme Name
Reaction catalyzed EC No. Native Organism NCBI No. Acetate
Phosphorylates acetate 2.7.2.12 Entamoeba histolytica XP_655990.1
kinase to acetyl-phosphate Acetate Phosphorylates ADP to 2.7.2.1
Cryptococcus neoformans XP_012053491.1 kinase ATP
ATP Production from Pyrophosphate, AMP, and Citrate
[0145] Some aspects of the present disclosure use methods for
producing ATP from pyrophosphate, AMP, and citrate (see, e.g.,
FIGS. 15A-15B). A three-step enzymatic pathway is shown in FIG.
15A. In the first step, citrate lyase converts citrate to acetate
and oxaloacetate. In the second step, phosphoenolpyruvate
carboxykinase (PEPCK) converts pyrophosphate and oxaloacetate
generated in the first step to phosphoenolpyruvate (PEP), carbon
dioxide (CO.sub.2), and inorganic phosphate (P.sub.i). In the third
step, pyruvate phosphate dikinase (PPDK) converts inorganic
pyrophosphate (PP.sub.i), AMP, and PEP generated in the second step
to pyruvate, P.sub.i, and ATP. The combined chemical reaction uses
one mole of citrate, one mole of AMP, and two moles of PP.sub.i to
yield one mole of acetate, one mole of pyruvate, one mole of
CO.sub.2, two moles of P.sub.i, and one mole of ATP (FIG. 15B).
Alternatively, phosphenolpyruvate carboxylase (PEPC) may be used to
catalyze the carboxylation of PEP to oxaloacetate, which may be
reversible under certain conditions.
[0146] These methods, in some embodiments, include culturing cells
engineered to express a citrate lyase, a PEPCK (or at least one
PEPC), a PPDK, or a combination of at least two or at least three
of the foregoing enzymes. In some embodiments, citrate lyase and
PEPCK (or PEPC), PEPCK (or PEPC) and PPDK, or citrate lyase and
PPDK are expressed as a single fusion (chimeric) protein.
[0147] In some embodiments, at least one of the enzymes is a
thermostable enzyme. In some embodiments, at least two or at least
three of the enzymes are thermostable enzymes. In some embodiments,
all of the enzymes are thermostable enzymes. Thus, in some
embodiments, the methods include culturing cells engineered to
express a thermostable citrate lyase, a thermostable PEPCK, a PPDK,
or a combination of at least two or at least three of the foregoing
thermostable enzymes.
[0148] In some embodiments, the methods of producing ATP from
citrate include lysing (e.g., thermal, osmotic, mechanical (e.g.,
sonication), chemical, or enzymatic lysis) the cultured cells to
produce at least one (e.g., at least two, or three) cell lysate. It
should be understood that multiple cell lysates (and thus multiple
cell populations, e.g., from the same organism (e.g., bacteria) or
from different organisms (e.g., bacteria, yeast and/or plant cells)
may be used in an enzymatic reaction as provided herein. For
example, one cell population may be engineered to express one or
more enzymes of the ATP production pathway, while another cell
population (or several other cell populations) may be engineered to
express another (at least one other) enzyme of the ATP production
pathway. Thus, in some embodiments, the methods comprise culturing
a population of cells engineered to express a citrate lyase,
culturing a cell population engineered to express a PEPCK (a
thermostable PEPCK), and/or culturing a cell population engineered
to express a PPDK. Following lysis of the cells, the cell lysates
are combined such that the enzymes are present in a single cell
lysate/reaction mixture.
[0149] In some embodiments, the methods of producing ATP from
citrate further include heating the cell lysate(s) (or a cell
lysate mixture) to a temperature that inactivates native enzymatic
activity but does not inactivate any of the thermostable enzymes of
the ATP production pathway, to produce a heat-inactivated lysate.
The cell lysate(s), in some embodiments, is heated to a temperature
of at least 50.degree. C. For example, the cell lysate(s) may be
heated to a temperature of at least 55.degree. C., 60.degree. C.,
65.degree. C., 70.degree. C., 75.degree. C., 80.degree. C.,
85.degree. C., or 90.degree. C. A native enzyme (or other
non-thermostable enzyme) is considered inactive, in some
embodiments, when its level of activity is reduced by at least 50%.
In some embodiments, a native enzyme (or other non-thermostable
enzyme) is considered inactive when its level of activity is
reduced by at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100%.
[0150] The cell lysate(s) may be heated for a period of time
sufficient to inactive native enzymes (or other non-thermostable
enzymes) of the cell. For example, the cell lysate(s) may be heated
for at least 2, 3, 4, or at least 5 minutes. In some embodiments,
the cell lysate(s) are heated for longer than 5 minutes. In some
embodiments, the cell lysate(s) are heated for a period of time
sufficient to reduce activity of native enzymes (or other
non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70%,
80%, or 90%).
[0151] Following heat inactivation, in some embodiments, at least
one (e.g., at least two or at least three) purified enzymes may be
added to the cell lysate/reaction mixture. Thus, a reaction
mixture, in some embodiments, may include a combination of enzymes
present in the cell lysate (expressed by the engineered host
cell(s)) and at least one purified enzyme. At least one purified
enzyme may be selected from the group consisting of citrate lyase,
PEPCK (or PEPC), and PPDK. In some embodiments, a cell lysate may
be cooled (e.g., to 50.degree. C.) following a heat-inactivation
step, prior to adding the purified enzyme(s).
[0152] In some embodiments, the methods of producing ATP from
citrate also include incubating the heat-inactivated lysate(s) in
the presence of citrate, adenosine monophosphate (AMP), and
inorganic phosphate to produce ATP. The inorganic phosphate may be,
for example, pyrophosphate. Other inorganic phosphates and/or
orthophosphate polymers, including but not limited to
tripolyphosphate, tetrapolyphosphate, pentapolyphosphate,
hexametaphosphate and mixtures thereof, may be used.
[0153] Also encompassed herein are cells and cell lysates used for
the production of ATP from citrate. Thus, an engineered cell (e.g.,
bacterial cell, yeast cell, and/or plant cell) or cell lysate(s) of
the present disclosure may include at least one (e.g., at least two
or at least three) enzyme selected from the group consisting of
citrate lyase, PEPCK (or PEPC), and PPDK. In some embodiments, an
engineered cell (e.g., bacterial cell, yeast cell, and/or plant
cell) or cell lysate(s) of the present disclosure includes at least
one (e.g., at least two or at least three) enzyme selected from the
group consisting of thermostable citrate lyase, thermostable PEPCK
(or thermostable PEPC), and thermostable PPDK.
TABLE-US-00012 TABLE 9 Exemplary ATP Production from Pyrophosphate
and Citrate Pathway Enzymes Pathway Step Enzyme Name EC No. Native
Organism NCBI No. 1 Citrate lyase 4.1.3.6 Escherichia coli
AAC28949.1; AAC73717.2; AAC73716.1 Caloramator australicus
CCJ33900.1; CCJ33901.1; CCJ33902.1 2 phosphoenolpyruvate 4.1.1.38
Propionibacterium freudenreichii AJQ89945.1 carboxykinase (PEPCK);
LC062511.1 also known as Entamoeba histolytica XP_654765.1
phosphoenolpyruvate XP_650862.1 carboxytransphosphorylase
phosphenolpyruvate 4.1.1.31 Pseudomonas fluorescens ABA72812.1
carboxylase (PEPC) 3 pyruvate phosphate 2.7.9.1 Clostridium
symbiosum AAA22917.1 dikinase (PPDK)
ATP Production from Pyrophosphate, AMP, and Sulfite
[0154] Some aspects of the present disclosure use methods for
producing ATP from pyrophosphate, AMP, and sulfite (see, e.g., FIG.
16). In the first step, adenylyl sulfate reductase converts
adenosine monophosphate (AMP) to adenosine 5'-phosphosulfate (APS)
with consumption of sulfite. In the second step, sulfate
adenylyltransferase catalyzes the conversion of APS to sulfate with
generation of ATP and consumption of pyrophosphate.
[0155] In some embodiments, the methods of producing ATP from
pyrophosphate, AMP, and sulfite include culturing cells engineered
to express a adenylyl sulfate reductase, a sulfate
adenylyltransferase, or a combination of a adenylyl sulfate
reductase and a sulfate adenylyltransferase. In some embodiments,
the adenylyl sulfate reductase and the sulfate adenylyltransferase
are expressed as a single fusion (chimeric) protein or a
bifunctional protein.
[0156] In some embodiments, reducing agents may be added that serve
as electron sinks. Examples of such reducing agents include but are
not limited to the following: dithiothreitol (DTT) or glutathione
or ferricyanide or dithioerythritol or Tris-2-carboxyethylphosphine
hydrochloride (TCEP). While individual enzymes will differ in their
cofactor preferences, there may be instances where biological
cofactors such as NAD.sup.+, NADP.sup.+, NADH, or NADPH may be used
by an enzyme to absorb these electrons. In these instances,
cofactors such as these may also be included.
[0157] In some embodiments, at least one of the enzymes is a
thermostable enzyme. In some embodiments, at least two (of the
enzymes are thermostable enzymes. In some embodiments, all of the
enzymes are thermostable enzymes. Thus, in some embodiments, the
methods include culturing cells engineered to express a
thermostable adenylyl sulfate reductase and a thermostable sulfate
adenylyltransferase.
[0158] In some embodiments, the methods of producing ATP from
sulfite include lysing (e.g., thermal, osmotic, mechanical (e.g.,
sonication), chemical, or enzymatic lysis) the cultured cells to
produce at least one (e.g., 2, 3, 4 or 5) cell lysate. It should be
understood that multiple cell lysates (and thus multiple cell
populations, e.g., from the same organism (e.g., bacteria) or from
different organisms (e.g., bacteria, yeast, and/or plant) may be
used in an enzymatic reaction as provided herein. For example, one
cell population may be engineered to express an adenylyl sulfate
reductase, while another cell population (or several other cell
populations) may be engineered to express a sulfate
adenylyltransferase. Thus, in some embodiments, the methods
comprise culturing a population of cells engineered to express an
adenylyl sulfate reductase, and/or culturing a cell population
engineered to express a sulfate adenylyltransferase. Following
lysis of the cells, the cell lysates are combined such that the
enzymes are present in a single cell lysate/reaction mixture.
[0159] In some embodiments, the methods of producing ATP from
sulfite further include heating the cell lysate(s) (or a cell
lysate mixture) to a temperature that inactivates native enzymatic
activity but does not inactivate any of the thermostable enzymes of
the ATP production pathway, to produce a heat-inactivated lysate.
The cell lysate(s), in some embodiments, is heated to a temperature
of at least 50.degree. C. For example, the cell lysate(s) may be
heated to a temperature of at least 55.degree. C., 60.degree. C.,
65.degree. C., 70.degree. C., 75.degree. C., 80.degree. C.,
85.degree. C., or 90.degree. C. A native enzyme (or other
non-thermostable enzyme) is considered inactive, in some
embodiments, when its level of activity is reduced by at least 50%.
In some embodiments, a native enzyme (or other non-thermostable
enzyme) is considered inactive when its level of activity is
reduced by at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100%.
[0160] The cell lysate(s) may be heated for a period of time
sufficient to inactive native enzymes (or other non-thermostable
enzymes) of the cell. For example, the cell lysate(s) may be heated
for at least 2, 3, 4, or at least 5 minutes. In some embodiments,
the cell lysate(s) are heated for longer than 5 minutes. In some
embodiments, the cell lysate(s) are heated for a period of time
sufficient to reduce activity of native enzymes (or other
non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70%,
80%, or 90%).
[0161] Following heat inactivation, in some embodiments, at least
one (e.g., at least two or at least three) purified enzymes may be
added to the cell lysate/reaction mixture. Thus, a reaction
mixture, in some embodiments, may include a combination of cell
lysate, enzymes present in the cell lysate (expressed by the
engineered host cell(s)), and at least one purified enzyme. At
least one purified enzyme may be a first acetate kinase and/or a
second acetate kinase. In some embodiments, a cell lysate may be
cooled (e.g., to 50.degree. C.) following a heat-inactivation step,
prior to adding the purified enzyme(s).
[0162] In some embodiments, the methods of producing ATP from
sulfite also include incubating the heat-inactivated lysate(s) in
the presence of sulfite, adenosine monophosphate (AMP), and
inorganic phosphate to produce ATP. The inorganic phosphate may be,
for example, pyrophosphate. Other inorganic phosphates and/or
orthophosphate polymers, including but not limited to
tripolyphosphate, tetrapolyphosphate, pentapolyphosphate,
hexametaphosphate and mixtures thereof, may be used.
[0163] Also encompassed herein are cells and cell lysates used for
the production of ATP. Thus, an engineered cell (e.g., bacterial
cell, yeast cell, and/or plant cell) or cell lysate(s) of the
present disclosure may include at least one (e.g., at least two)
adenylyl sulfate reductase and/or at least one sulfate
adenylyltransferase. In some embodiments, an engineered cell (e.g.,
bacterial cell, yeast cell, and/or plant cell) or cell lysate(s) of
the present disclosure includes at least one (e.g., at least two,
at least three, or at least four) thermostable adenylyl sulfate
reductase and/or at least one thermostable sulfate
adenylyltransferase.
TABLE-US-00013 TABLE 10 Exemplary ATP Production from
Pyrophosphate, AMP, and Sulfite Pathway Enzymes Pathway Uniprot
Step Enzyme Name EC No. Native Organism NCBI No. ID 1 Adenylyl
sulfate 1.8.99.2 Archaeoglobus fulgidus CAA45030.1, Q59115,
reductase CAA45029.1 Q59116 Archaeoglobus profundus WP_012940649.1,
WP_012940650 Thermodesulforhabdus SFM96889.1 norvegica Thiobacillus
denitrificans AAQ18138.1, Q5VLA6, AAQ18139.1 Q5VLA7 Desulfovibrio
vulgaris YP_010068.1, Q59339, YP_010067.1 Q59338 2 Sulfate 2.7.7.4
Archaeoglobus fulgidus KUJ93479.1 A0A124 adenylyltransferase FBI0
Archaeoglobus profundus WP_012940652.1 Thermodesulforhabdus
WP_093395234.1, norvegica SFM89448.1 Thiobacillus denitrificans
AAQ18137.1 Desulfovibrio vulgaris WP_012611243.1, WP_010938590.1
Escherichia coli ANO79304.1
Depolymerization of Cellular RNA
[0164] In some embodiments, cellular RNA serves as the substrate
for the production of NTP and/or RNA. Depolymerization
(degradation) of cellular RNA results in a pool comprising
nucleoside diphosphates (NDPs) or 5'-nucleoside monophosphates
(5'-NMPs), depending on the enzymes used for depolymerization.
[0165] Cellular RNA, in some embodiments, is depolymerized into
NDPs using, for example, a polynucleotide phosphorylase (PNPase)
(see, e.g., Table 1). In some embodiments, the concentration of
PNPase used in a reaction mixture is 0.001-10 mg/mL (e.g., 0.001,
0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 5, or 10
mg/mL). In some embodiments, the concentration of PNPase is a
reaction mixture is 0.5-5 mg/mL. In some embodiments, the
concentration of PNPase is a reaction mixture is 5 mg/mL. In some
embodiments, the concentration of PNPase is a reaction mixture is
greater than 10 mg/mL.
[0166] Cellular RNA, in other embodiments, is depolymerized into
NMPs using, for example, a nuclease (e.g., RNase R or P1 nuclease)
(see, e.g., Table 1). Depending on the enzyme, enzymatic
depolymerization of RNA may yield 3'-NMPs, 5'-NMPs or a combination
of 3'-NMPs and 5'-NMPs. Because it is not possible to polymerize
3'-NTPs (converted from 3'-NDPs, which are converted from 3'-NMPs),
enzymes (e.g., RNase R and/or P1 nuclease) that yield 5'-NMPs
(which are then converted to 5'-NDPs, and then 5'-NTPs) are
preferred. In some embodiments, the concentration of nuclease
(e.g., RNase R and/or P1 nuclease) used in a reaction mixture is
0.001-10 mg/mL (e.g., 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 5, or 10 mg/mL). In some embodiments, the
concentration of nuclease in a reaction mixture is 0.0.5-5 mg/mL.
In some embodiments, the concentration of nuclease in a reaction
mixture is 5 mg/mL. In some embodiments, the concentration of
nuclease in a reaction mixture is greater than 10 mg/mL.
[0167] The PNPase and/or the RNase, in some embodiments, is
obtained from or is a component of a cell lysate of cells that
express the PNPase and/or the RNase.
[0168] The amount of cellular RNA required to synthesize a RNA
product of interest may vary, depending on, for example, the
desired length and yield of the RNA product as well as the
nucleotide composition of the RNA product relative to the
nucleotide composition of the cellular RNA starting material.
Typically, for a bacterial cell or a yeast cell, for example,
cellular RNA content ranges from 5-50% of the total cell mass. The
percent of total cell mass can be calculated, for example, using
the following equation: (kilogram (kg) of RNA/kilogram of dry cell
weight).times.100%.
[0169] Conditions suitable for the production of NMPs and
conditions suitable for the production of NDPs are known in the art
or may be determined by one of ordinary skill in the art, taking
into consideration, for example, optimal conditions for nuclease
(e.g., RNase) activity, including pH (e.g., pH 3-8), temperature
(e.g., 15.degree. C. to 70.degree. C.), length of time (e.g., 5
min-72 hrs), and salt concentration (e.g., sodium chloride,
potassium chloride, sodium acetate, potassium acetate at a
concentration of 5 mM to 1 M) of the reaction mixture as well as
any exogenous cofactors. In some embodiments, buffer is added to a
cell lysate, for example, to achieve a particular pH value and/or
salt concentration. Examples of buffers include, without
limitation, phosphate buffer, Tris buffer, MOPS buffer, HEPES
buffer, citrate buffer, acetate buffer, malate buffer, MES buffer,
histidine buffer, PIPES buffer, bis-tris buffer, and ethanolamine
buffer.
[0170] In some embodiments, a reaction mixture during a RNA
depolymerization reaction is incubated for 24 hours at a
temperature of 37.degree. C. In some embodiments, a reaction
mixture during a RNA depolymerization reaction is incubated for
5-30 min at a temperature of 37.degree. C. In some embodiments, a
reaction mixture during a RNA depolymerization reaction has a pH of
7.0 and is incubated for 15 minutes at a temperature of 37.degree.
C. In some embodiments, a reaction mixture during a RNA
depolymerization reaction may be incubated under conditions that
result in greater than 65% conversion of RNA to NDP or RNA to
5'-NMPs. In some embodiments, RNA is converted to NDP or 5'-NMPs at
a rate of (or at least) 50 mM/hr, 100 mM/hr or 200 mM/hr. In other
embodiments, a reaction mixture during an RNA depolymerization
reaction is incubated at a higher temperature (for example,
50.degree. C.-70.degree. C.), as in Example 5.
Polymerization of RNA Product
[0171] In some embodiments, NTPs, either produced by a method
provided herein or supplied from commercial sources, are used in a
biosynthetic pathway for the production of a RNA product of
interest. A DNA designed to encode the RNA product serves as the
template for the synthesis of the RNA. The DNA template may be
engineered, in some instances, to have a transcriptional promoter
that selectively drives transcription of the RNA of interest.
Polymerization of RNA requires NTPs, a DNA template comprising a
transcriptional promoter, and a polymerase (e.g., RNA polymerase)
specific to the transcriptional promoter. Typically, a polymerase
for use as provided herein is a single subunit polymerase, is
highly selective for its cognate transcriptional promoters, has
high-fidelity, and is highly efficient.
[0172] In some embodiments, the concentration of the DNA template
in a reaction mixture is 0.001-10 .mu.g/.mu.l. In some embodiments,
the concentration of the DNA template in a reaction mixture is
0.001 .mu.g/.mu.l, 0.05 .mu.g/.mu.l, 0.1 .mu.g/.mu.l, 0.5
.mu.g/.mu.l, 1.0 .mu.g/.mu.l, 5 .mu.g/.mu.l, or 10 .mu.g/.mu.l.
[0173] Conditions suitable for the production of RNA are known in
the art or may be determined by one of ordinary skill in the art,
taking into consideration, for example, optimal conditions for
polymerase (e.g., T7 RNA polymerase) activity, including pH (e.g.,
pH 3-8), temperature (e.g., 15.degree. C. to 70.degree. C.), length
of time (e.g., 5 min-72 hrs), and salt concentration (e.g., sodium
chloride, potassium chloride, sodium acetate, potassium acetate at
a concentration of 5 mM to 1 M) of the reaction mixture as well as
any exogenous cofactors. In some embodiments, buffer is added to a
cell lysate, for example, to achieve a particular pH value and/or
salt concentration. Examples of buffers include, without
limitation, phosphate buffer, Tris buffer, MOPS buffer, HEPES
buffer, citrate buffer, acetate buffer, malate buffer, MES buffer,
histidine buffer, PIPES buffer, bis-tris buffer, and ethanolamine
buffer.
[0174] In some embodiments, a reaction mixture during a RNA
polymerization reaction is incubated for 0.5-24 hours at a
temperature of 37.degree. C. In some embodiments, a reaction
mixture during a RNA polymerization reaction is incubated for
0.5-24 hours at a temperature of 50.degree. C.
Cells and Cell Lysates
[0175] Cells of the present disclosure, in some embodiments,
express cellular RNA, enzymes that depolymerizes RNA (e.g.,
RNases), pathway enzymes (e.g., recombinant enzymes such as
polyphosphate kinase), and/or polymerases (e.g., RNA polymerases).
In some embodiments, the engineered cells include a DNA template
containing a promoter, and optionally a transcriptional terminator,
operably linked to a nucleotide sequence encoding a RNA product of
interest.
[0176] In some embodiments, the cells are engineered cells.
Engineered cells are cells that comprise a engineered (e.g.,
recombinant or synthetic) nucleic acid, or are otherwise modified
such that they are structurally and/or functionally distinct from
their naturally-occurring counterparts. Thus, a cell that contains
an engineered nucleic acid is considered an "engineered cell."
[0177] A cell "expresses" a product if the product, encoded by a
nucleic acid (e.g., an engineered nucleic acid), is produced in the
cell. It is known in the art that gene expression refers to the
process by which genetic instructions in the form of a nucleic acid
are used to synthesize a product, such as a protein (e.g., an
enzyme).
[0178] Cells may be prokaryotic cells or eukaryotic cells. In some
embodiments, cells are bacterial cells, yeast cells, insect cells,
mammalian cells, plant cells, or other types of cells.
[0179] Bacterial cells of the present disclosure include, without
limitation, Escherichia spp., Streptomyces spp., Zymomonas spp.,
Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium
spp., Clostridium spp., Corynebacterium spp., Streptococcus spp.,
Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus
spp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp.,
Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus
spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp.,
Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter
spp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp.,
Thermus spp., Stenotrophomonas spp., Chromobacterium spp.,
Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp.,
Pantoea spp, and Vibrio natriegens.
[0180] Yeast cells of the present disclosure include, without
limitation, engineered Saccharomyces spp., Schizosaccharomyces,
Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.
[0181] In some embodiments, cells of the present disclosure are
Escherichia coli cells, Bacillus subtilis cells, Pseudomonas putida
cells, Saccharomyces cerevisiae cells, or Lactobacillus brevis
cells. In some embodiments, cells of the present disclosure are
Escherichia coli cells.
[0182] Typically, cells are cultured. Culturing is the process by
which cells are grown under controlled conditions, typically
outside of their natural environment. For example, cells, such as
bacterial cells, may be grown as a cell suspension in liquid
nutrient broth, also referred to as liquid culture medium.
[0183] Examples of commonly used bacterial Escherichia coli growth
media include, without limitation, LB (Lysogeny Broth) Miller broth
(1% NaCl): 1% peptone, 0.5% yeast extract, and 1% NaCl; LB
(Lysogeny Broth) Lennox Broth (0.5% NaCl): 1% peptone, 0.5% yeast
extract, and 0.5% NaCl; SOB medium (Super Optimal Broth): 2%
peptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl.sub.2, 10 mM MgSO.sub.4; SOC medium (Super Optimal broth with
Catabolic repressor): SOB+20 mM glucose; 2.times.YT broth (2.times.
Yeast extract and Tryptone): 1.6% peptone, 1% yeast extract, and
0.5% NaCl; TB (Terrific Broth) medium: 1.2% peptone, 2.4% yeast
extract, 72 mM K.sub.2HPO.sub.4, 17 mM KH.sub.2PO.sub.4 and 0.4%
glycerol; and SB (Super Broth) medium: 3.2% peptone, 2% yeast
extract, and 0.5% NaCl and or Korz medium (Korz, D J et al.
1995).
[0184] Examples of high density bacterial Escherichia coli growth
media include, but are not limited to, DNAGro.TM. medium,
ProGro.TM. medium, AutoX.TM. medium, DetoX.TM. medium, InduX.TM.
medium, and SecPro.TM. medium.
[0185] In some embodiments, cells are cultured under conditions
that result in expression of enzymes or nucleic acids. Such culture
conditions may depend on the particular product being expressed and
the desired amount of the product.
[0186] In some embodiments, cells are cultured at a temperature of
30.degree. C. to 40.degree. C. For example, engineered cells may be
cultured at a temperature of 30.degree. C., 31.degree. C.,
32.degree. C., 33.degree. C., 34.degree. C., 35.degree. C.,
36.degree. C., 37.degree. C., 38.degree. C., 39.degree. C. or
40.degree. C. Typically, cells, such as engineered E. coli cells,
are cultured at a temperature of 37.degree. C.
[0187] In some embodiments, cells are cultured for a period of time
of 12 hours to 72 hours, or more. For example, engineered cells may
be cultured for a period of time of 12, 18, 24, 30, 36, 42, 48, 54,
60, 66, or 72 hours. Typically, cells, such as engineered bacterial
cells, are cultured for a period of time of 12 to 24 hours. In some
embodiments, cells are cultured for 12 to 24 hours at a temperature
of 37.degree. C.
[0188] In some embodiments, cells are cultured (e.g., in liquid
cell culture medium) to an optical density, measured at a
wavelength of 600 nm (OD.sub.600), of 5 to 200. In some
embodiments, cells are cultured to an OD.sub.600 of 5, 10, 15, 20,
25, 50, 75, 100, 150, or 200.
[0189] In some embodiments, cells are cultured to a density of
1.times.10.sup.8 (OD.sub.600<1) to 2.times.10.sup.11
(OD.about.200) viable cells/ml cell culture medium. In some
embodiments, cells are cultured to a density of 1.times.10.sup.8,
2.times.10.sup.8, 3.times.10.sup.8, 4.times.10.sup.8,
5.times.10.sup.8, 6.times.10.sup.8, 7.times.10.sup.8,
8.times.10.sup.8, 9.times.10.sup.8, 1.times.10.sup.9,
2.times.10.sup.9, 3.times.10.sup.9, 4.times.10.sup.9,
5.times.10.sup.9, 6.times.10.sup.9, 7.times.10.sup.9,
8.times.10.sup.9, 9.times.10.sup.9, 1.times.10.sup.10,
2.times.10.sup.10, 3.times.10.sup.10, 4.times.10.sup.10,
5.times.10.sup.10, 6.times.10.sup.10, 7.times.10.sup.10,
8.times.10.sup.10, 9.times.10.sup.10, 1.times.10.sup.11, or
2.times.10.sup.11 viable cells/ml. (Conversion factor: OD
1=8.times.10.sup.8 cells/ml).
[0190] In some embodiments, cells are cultured in a bioreactor. A
bioreactor refers simply to a container in which cells are
cultured, such as a culture flask, a dish, or a bag that may be
single-use (disposable), autoclavable, or sterilizable. The
bioreactor may be made of glass, or it may be polymer-based, or it
may be made of other materials.
[0191] Examples of bioreactors include, without limitation, stirred
tank (e.g., well mixed) bioreactors and tubular (e.g., plug flow)
bioreactors, airlift bioreactors, membrane stirred tanks, spin
filter stirred tanks, vibromixers, fluidized bed reactors, and
membrane bioreactors. The mode of operating the bioreactor may be a
batch or continuous processes and will depend on the engineered
cells being cultured. A bioreactor is continuous when the feed and
product streams are continuously being fed and withdrawn from the
system. A batch bioreactor may have a continuous recirculating
flow, but no continuous feeding of nutrient or product harvest. For
intermittent-harvest and fed-batch (or batch fed) cultures, cells
are inoculated at a lower viable cell density in a medium that is
similar in composition to a batch medium. Cells are allowed to grow
exponentially with essentially no external manipulation until
nutrients are somewhat depleted and cells are approaching
stationary growth phase. At this point, for an intermittent harvest
batch-fed process, a portion of the cells and product may be
harvested, and the removed culture medium is replenished with fresh
medium. This process may be repeated several times. For production
of recombinant proteins and antibodies, a fed-batch process may be
used. While cells are growing exponentially, but nutrients are
becoming depleted, concentrated feed medium (e.g., 10-15 times
concentrated basal medium) is added either continuously or
intermittently to supply additional nutrients, allowing for further
increase in cell concentration and the length of the production
phase. Fresh medium may be added proportionally to cell
concentration without removal of culture medium (broth). To
accommodate the addition of medium, a fedbatch culture is started
in a volume much lower that the full capacity of the bioreactor
(e.g., approximately 40% to 50% of the maximum volume).
[0192] Some methods of the present disclosure are directed to
large-scale (commercial-scale) production of RNA (e.g., mRNA). For
large-scale production methods, cells may be grown in liquid
culture medium in a volume of 5 liters (L) to 250,000 L, or more.
In some embodiments, cells may be grown in liquid culture medium in
a volume of greater than (or equal to) 10 L, 100 L, 1000 L, 10000
L, or 100000 L. In some embodiments, cells are grown in liquid
culture medium in a volume of 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35
L, 40 L, 45 L, 50 L, 100 L, 500 L, 1000 L, 5000 L, 10000 L, 100000
L, 150000 L, 200000 L, 250000 L, or more. In some embodiments,
cells may be grown in liquid culture medium in a volume of 5 L to
10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35
L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25
L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10L to 45 L, 10 L to
50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L
to 40 L, 15 L to 45 L, or 15 to 50 L. In some embodiments, cells
may be grown in liquid culture medium in a volume of 100 L to
300000 L, 100 L to 200000 L, or 100 L to 100000 L.
[0193] Typically, culturing of cells is followed by lysing the
cells. Lysing is the process by which cells are broken down, for
example, by viral, heat, chemical, enzymatic, mechanical, or
osmotic mechanisms. A cell lysate is a fluid containing the
contents of lysed cells (e.g., lysed engineered cells), including,
for example, organelles, membrane lipids, proteins, nucleic acids
and inverted membrane vesicles. Cell lysates of the present
disclosure may be produced by lysing any population of engineered
cells, as provided herein.
[0194] Cell lysis can disturb carefully controlled cellular
environments, resulting in protein degradation and modification by
unregulated endogenous proteases and phosphatases. Thus, in some
embodiments, protease inhibitors and/or phosphatase inhibitors
and/or nuclease inhibitors and/or hydrolase inhibitors and/or
deaminase inhibitors may be added to the cell lysate or cells
before lysis, or these activities may be removed by heat
inactivation, gene inactivation, or protease targeting.
[0195] Cell lysates, in some embodiments, may be combined with a
nutrient. For example, cell lysates may be combined with
Na.sub.2HPO.sub.4, KH.sub.2PO.sub.4, NH.sub.4Cl, NaCl, MgSO.sub.4,
CaCl.sub.2. Examples of other nutrients include, without
limitation, magnesium sulfate, magnesium chloride, magnesium
orotate, magnesium citrate, potassium phosphate monobasic,
potassium phosphate dibasic, potassium phosphate tribasic, sodium
phosphate monobasic, sodium phosphate dibasic, sodium phosphate
tribasic, ammonium phosphate monobasic, ammonium phosphate dibasic,
ammonium sulfate, ammonium chloride, and ammonium hydroxide.
[0196] Cell lysates, in some embodiments, may be combined with a
cofactor. For example, cell lysates may be combined with adenosine
diphosphate (ADP), adenosine triphosphate (ATP), nicotinamide
adenine dinucleotide (NAD+), or other non-protein chemical
compounds required for activity of an enzyme (e.g., inorganic ions
and coenzymes).
[0197] The volume of cell lysate used for a single reaction may
vary. In some embodiments, the volume of a cell lysate is 0.001 to
250 m.sup.3.
Nucleic Acids
[0198] A "nucleic acid" is at least two nucleotides covalently
linked together, and in some instances, may contain phosphodiester
bonds (e.g., a phosphodiester "backbone"). Nucleic acids (e.g.,
components, or portions, of nucleic acids) may be naturally
occurring or engineered. "Naturally occurring" nucleic acids are
present in a cell that exists in nature in the absence of human
intervention. "Engineered nucleic acids" include recombinant
nucleic acids and synthetic nucleic acids. A "recombinant nucleic
acid" refers to a molecule that is constructed by joining nucleic
acid molecules (e.g., from the same species or from different
species) and, typically, can replicate in a living cell. A
"synthetic nucleic acid" refers to a molecule that is biologically
synthesized, chemically synthesized, or by other means synthesized
or amplified. A synthetic nucleic acid includes nucleic acids that
are chemically modified or otherwise modified but can base pair
with naturally-occurring nucleic acid molecules. Recombinant and
synthetic nucleic acids also include those molecules that result
from the replication of either of the foregoing. Engineered nucleic
acids may contain portions of nucleic acids that are naturally
occurring, but as a whole, engineered nucleic acids do not occur
naturally and require human intervention. In some embodiments, a
nucleic acid encoding a product of the present disclosure is a
recombinant nucleic acid or a synthetic nucleic acid. In other
embodiments, a nucleic acid encoding a product is naturally
occurring.
[0199] An engineered DNA template encoding RNA, as provided herein,
may be operably linked to a promoter, which is a control region of
a nucleic acid at which initiation and rate of transcription of the
remainder of a nucleic acid are controlled. A promoter drives
expression or drives transcription of the nucleic acid that it
regulates.
[0200] 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 of a given gene or
sequence. Such a promoter may be endogenous.
[0201] In some embodiments, a coding nucleic acid sequence may be
positioned under the control of a recombinant or heterologous
promoter, which refers to a promoter that is not normally
associated with the encoded sequence in its natural environment.
Such promoters may include promoters of other genes; promoters
isolated from any other cell; and synthetic promoters or enhancers
that are not "naturally occurring" such as, for example, those that
contain different elements of different transcriptional regulatory
regions and/or mutations that alter expression through methods of
genetic engineering that are known in the art. 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 polymerase
chain reaction (PCR).
[0202] A promoter is considered to be operably linked to a
nucleotide sequence when it is in a correct functional location and
orientation in relation to the nucleotide sequence it regulates to
control ("drive") transcriptional initiation and/or expression of
that nucleotide sequence.
[0203] Engineered nucleic acids of the present disclosure may
contain a constitutive promoter or an inducible promoter. In some
embodiments, the constitutive promotor or the inducible promoter is
operably linked to a coding sequence, and optionally one or more
transcriptional terminators. In some embodiments, the coding
sequence encodes a protein or a RNA product. A "constitutive
promoter" refers to a promoter that is constantly active in a cell.
An "inducible promoter" refers to a promoter that initiates or
enhances transcriptional activity when in the presence of,
influenced by, or contacted by an inducer or inducing agent, or
activated in the absence of a factor that causes repression.
Inducible promoters for use in accordance with the present
disclosure include any inducible promoter described herein or known
to one of ordinary skill in the art. Examples of inducible
promoters include, without limitation,
chemically/biochemically-regulated and physically-regulated
promoters such as organic solvent-regulated promoters,
tetracycline-regulated promoters, steroid-regulated promoters,
metal-regulated promoters, pathogenesis-regulated promoters,
temperature/heat-inducible, phosphate-regulated (e.g., PhoA), and
light-regulated promoters.
[0204] An engineered DNA template encoding RNA, as provided herein,
may also be operably linked to one or more transcriptional
terminators, which are control regions of a nucleic acid which
cause a polymerase to stop transcribing and dissociate from the DNA
template.
[0205] A terminator may be one or more sequences naturally
associated with a gene or sequence, as may be obtained by isolating
the 3'-non-coding sequences located downstream of the coding
segment of a given gene or sequence. Such a terminator may be
endogenous or engineered for improved termination efficiency.
Endogenous and/or engineered terminator sequences from one or more
sources can be added in series for improved termination
efficiency.
[0206] Circular DNA templates encoding RNA may include one or more
transcriptional terminators to minimize or prevent transcription of
non-template DNA sequence, for example, a sequence that is part of
a plasmid backbone.
[0207] Engineered nucleic acids may be introduced into host cells
using any means known in the art, including, without limitation,
transformation, transfection (e.g., chemical (e.g., calcium
phosphate, cationic polymers, or liposomes) or non-chemical (e.g.,
electroporation, sonoporation, impalefection, optical transfection,
hydrodynamic transfection)), and transduction (e.g., viral
transduction).
[0208] Enzymes or other proteins encoded by a naturally-occurring,
intracellular nucleic acid may be referred to as "endogenous
enzymes" or "endogenous proteins."
Compositions
[0209] In some embodiments, a reaction mixture for the production
of nucleoside triphosphates (NTPs) comprises nucleoside
diphosphates (NDPs), a polyphosphate kinase, and a polyphosphate.
In some embodiments, the reaction mixture further comprises a
nucleoside kinase and/or a NDP kinase.
[0210] In some embodiments, a reaction mixture for the production
of NTPs comprises 5' nucleoside monophosphates, a polyphosphate
kinase, and polyphosphate. In some embodiments, the reaction
mixture further comprises a nucleoside kinase, a NMP kinase, and/or
a NDP kinase.
[0211] In some embodiments, a reaction mixture for the production
of NTPs comprises nucleosides, a polyphosphate kinase, and a
polyphosphate. In some embodiments, the reaction mixture further
comprises a nucleoside kinase, a NMP kinase, and/or a NDP
kinase.
[0212] In some embodiments, a reaction mixture for the production
of NTPs comprises nucleobases, a phosphoribosyltransferase, a
phosphoribosylpyrophosphate, a polyphosphate kinase, and a
polyphosphate. In some embodiments, the reaction mixture further
comprises a nucleoside kinase, a NMP kinase, and/or a NDP
kinase.
[0213] In some embodiments, a reaction mixture for the production
of NTPs comprises nucleobases, D-ribose, a ribokinase, a
phosphopentomutase, a nucleoside phosphorylase, a polyphosphate
kinase, and a polyphosphate. In some embodiments, the reaction
mixture further comprises a nucleoside kinase, a NMP kinase, and/or
a NDP kinase.
[0214] In some embodiments, a reaction mixture for the production
of ribonucleic acid (RNA) comprises nucleoside diphosphates (NDPs),
a polyphosphate kinase, a polyphosphate, a deoxyribonucleic acid
(DNA) template, and a polymerase. In some embodiments, the reaction
mixture further comprises a nucleoside kinase, a NMO kinase, and/or
a NDP kinase.
[0215] In some embodiments, a reaction mixture for the production
of RNA comprises 5' nucleoside monophosphates, a polyphosphate
kinase, a polyphosphate, a deoxyribonucleic acid (DNA) template,
and a polymerase. In some embodiments, the reaction mixture further
comprises a nucleoside kinase, a NMP kinase, and/or a NDP
kinase.
[0216] In some embodiments, a reaction mixture for the production
of RNA comprises nucleosides, a nucleoside kinase, a polyphosphate
kinase, a polyphosphate, a deoxyribonucleic acid (DNA) template,
and a polymerase. In some embodiments, the reaction mixture further
comprises a nucleoside kinase, a NMP kinase, and/or a NDP
kinase.
[0217] In some embodiments, a reaction mixture for the production
of RNA comprises nucleobases, a phosphoribosyltransferase, a
phosphoribosylpyrophosphate, a polyphosphate kinase, a
polyphosphate, a deoxyribonucleic acid (DNA) template, and a
polymerase. In some embodiments, the reaction mixture further
comprises a nucleoside kinase, a NMP kinase, and/or a NDP
kinase.
[0218] In some embodiments, a reaction mixture for the production
of RNA comprises nucleobases, D-ribose, a ribokinase, a
phosphopentomutase, a nucleoside phosphorylase, a polyphosphate
kinase, a polyphosphate, a deoxyribonucleic acid (DNA) template,
and a polymerase. In some embodiments, the reaction mixture further
comprises a nucleoside kinase, a NMP kinase, and/or a NDP
kinase.
Additional Embodiments
[0219] Additional embodiments of the present disclosure are
encompassed by the following numbered paragraphs.
[0220] 1. A method for producing nucleoside triphosphates (NTPs),
comprising: [0221] incubating in a reaction mixture nucleoside
diphosphates (NDPs), a polyphosphate kinase, and a polyphosphate
under conditions appropriate for the production of NTPs, optionally
wherein the reaction mixture further comprises a nucleoside kinase
and/or a NDP kinase.
[0222] 2. The method of paragraph 1, wherein the NDPs comprise ADP,
GDP, CDP, and/or UDP.
[0223] 3. The method of paragraph 1 or 2, wherein the NDPs are
chemically synthesized, a product of fermentation, or extracted
from a natural source.
[0224] 4. The method of any one of paragraphs 1-3, wherein the a
polyphosphate kinase is selected from PPK1 family enzymes and PPK2
family enzymes.
[0225] 5. The method of paragraph 4, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0226] 6. The method of any one of paragraphs 1-5, wherein the
polyphosphate comprises hexametaphosphate.
[0227] 7. The method of any one of paragraphs 1-6, wherein the
polyphosphate kinase, the nucleoside kinase, and/or the NDP kinase
is prepared from cells that express the polyphosphate kinase, the
nucleoside kinase, and/or the NDP kinase.
[0228] 8. The method of any one of paragraphs 1-7, wherein the
reaction mixture comprises a cell lysate or an enzyme preparation
from cells that express the polyphosphate kinase, the nucleoside
kinase, and/or the NDP kinase.
[0229] 9. The method of paragraph 8, wherein native enzymatic
activity of enzymes in the cell lysate or enzyme preparation have
been eliminated.
[0230] 10. The method of paragraph 9, wherein native enzymatic
activity of enzymes in the cell lysate or enzyme preparation have
been eliminated via genetic modification, enzyme secretion from a
cell, and/or protease targeting.
[0231] 11. The method of paragraph 9 or 10, wherein native
enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via temperature, pH, salt,
detergent, alcohol, and/or chemical inhibitors.
[0232] 12. The method of any one of paragraphs 9-11, wherein native
enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0233] 13. The method of any one of paragraphs 9-12, wherein the
native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0234] 14. The method of any one of paragraphs 1-13, wherein the
polyphosphate kinase, the nucleoside kinase, and/or the NDP kinase
can withstand elimination conditions.
[0235] 15. A method for producing nucleoside triphosphates (NTPs),
comprising: [0236] incubating in a reaction mixture 5' nucleoside
monophosphates (5' NMPs), a polyphosphate kinase, and a
polyphosphate under conditions appropriate for the production of
NTPs, optionally wherein the reaction mixture further comprises a
nucleoside kinase, a NMP kinase, and/or a NDP kinase.
[0237] 16. The method of paragraph 15, wherein the 5' NMPs comprise
5' AMP, 5' GMP, 5' CMP and/or 5' UMP.
[0238] 17. The method of paragraph 15 or 16, wherein the 5' NMPs
are chemically synthesized, a product of fermentation, or extracted
from a natural source.
[0239] 18. The method of any one of paragraphs 15-17, wherein the
polyphosphate kinase is selected from PPK1 family enzymes and PPK2
family enzymes.
[0240] 19. The method of paragraph 18, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0241] 20. The method of any one of paragraphs 15-19, wherein the
polyphosphate comprises hexametaphosphate.
[0242] 21. The method of any one of paragraphs 15-20, wherein the
polyphosphate kinase, the nucleoside kinase, the NMP kinase, and/or
the NDP kinase is prepared from cells that express the
polyphosphate kinase, the nucleoside kinase, the NMP kinase, and/or
the NDP kinase.
[0243] 22. The method of any one of paragraphs 15-21, wherein the
reaction mixture comprises a cell lysate or an enzyme preparation
from cells that express the polyphosphate kinase, the nucleoside
kinase, the NMP kinase, and/or the NDP kinase.
[0244] 23. The method of paragraph 22, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0245] 24. The method of paragraph 23, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0246] 25. The method of paragraph 23 or 24, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0247] 26. The method of any one of paragraphs 23-25, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0248] 27. The method of any one of paragraphs 23-26, wherein the
native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0249] 28. The method of any one of paragraphs 15-27, wherein the
polyphosphate kinase, the nucleoside kinase, the NMP kinase, and/or
the NDP kinase can withstand elimination conditions.
[0250] 29. A method for producing nucleoside triphosphates (NTPs),
comprising: [0251] incubating in a reaction mixture nucleosides, a
polyphosphate kinase, and a polyphosphate under conditions
appropriate for the production of NTPs, optionally wherein the
reaction mixture further comprises a nucleoside kinase, a NMP
kinase, and/or a NDP kinase.
[0252] 30. The method of paragraph 29, wherein the nucleosides
comprise adenosine, guanosine, cytidine, and/or uridine.
[0253] 31. The method of paragraph 29 or 30, wherein the
nucleosides are chemically synthesized, a product of fermentation,
or extracted from a natural source.
[0254] 32. The method of any one of paragraphs 29-31, wherein the
polyphosphate kinase is selected from PPK1 family enzymes and PPK2
family enzymes.
[0255] 33. The method of paragraph 32, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0256] 34. The method of any one of paragraphs 29-33, wherein the
polyphosphate comprises hexametaphosphate.
[0257] 35. The method of any one of paragraphs 29-34, wherein the
polyphosphate kinase, the nucleoside kinase, the NMP kinase and/or
the NDP kinase is prepared from cells that express the
polyphosphate kinase, the nucleoside kinase, the NMP kinase, and/or
the NDP kinase.
[0258] 36. The method of any one of paragraphs 29-35, wherein the
reaction mixture comprises a cell lysate or an enzyme preparation
from cells that express the polyphosphate kinase, the nucleoside
kinase, the NMP kinase, and/or the NDP kinase.
[0259] 37. The method of paragraph 36, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0260] 38. The method of paragraph 37, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0261] 39. The method of paragraph 37 or 38, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0262] 40. The method of any one of paragraphs 37-39, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0263] 41. The method of any one of paragraphs 37-40, wherein the
native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0264] 42. The method of any one of paragraphs 29-41, wherein the
polyphosphate kinase, the nucleoside kinase, the NMP kinase, and/or
the NDP kinase can withstand elimination conditions.
[0265] 43. A method for producing nucleoside triphosphates (NTPs),
comprising: [0266] incubating in a reaction mixture nucleobases, a
phosphoribosyltransferase, a phosphoribosylpyrophosphate, a
polyphosphate kinase, and a polyphosphate under conditions
appropriate for the production of NTPs, optionally wherein the
reaction mixture further comprises a nucleoside kinase, a NMP
kinase, and/or a NDP kinase.
[0267] 44. The method of paragraph 43, wherein the nucleobases
comprise adenine, guanidine, cytosine, and/or uracil.
[0268] 45. The method of paragraph 43 or 44, wherein the
nucleobases are chemically synthesized, a product of fermentation,
or extracted from a natural source.
[0269] 46. The method of any one of paragraphs 43-45, wherein the
polyphosphate kinase is selected from PPK1 family enzymes and PPK2
family enzymes.
[0270] 47. The method of paragraph 46, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0271] 48. The method of any one of paragraphs 43-47, wherein the
polyphosphate comprises hexametaphosphate.
[0272] 49. The method of any one of paragraphs 43-48, wherein the
polyphosphate kinase, the nucleoside kinase, the NMP kinase and/or
the NDP kinase is prepared from cells that express the
polyphosphate kinase, the nucleoside kinase, the NMP kinase, and/or
the NDP kinase.
[0273] 50. The method of any one of paragraphs 43-49, wherein the
reaction mixture comprises a cell lysate or an enzyme preparation
from cells that express the polyphosphate kinase, the nucleoside
kinase, the NMP kinase, and/or the NDP kinase.
[0274] 51. The method of paragraph 50, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0275] 52. The method of paragraph 51, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0276] 53. The method of paragraph 51 or 52, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0277] 54. The method of any one of paragraphs 51-53, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0278] 55. The method of any one of paragraphs 51-54, wherein the
native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0279] 56. The method of any one of paragraphs 43-55, wherein the
polyphosphate kinase, the nucleoside kinase, the NMP kinase, and/or
the NDP kinase can withstand elimination conditions.
[0280] 57. A method for producing nucleoside triphosphates (NTPs),
comprising: [0281] incubating in a reaction mixture nucleobases,
ribose, ribokinase, phosphopentomutase, a nucleoside phosphorylase,
a polyphosphate kinase, and a polyphosphate under conditions
appropriate for the production of NTPs, optionally wherein the
reaction mixture further comprises a nucleoside kinase, a NMP
kinase, and/or a NDP kinase.
[0282] 58. The method of paragraph 57, wherein the nucleobases
comprise adenine, guanidine, cytosine, and/or uracil.
[0283] 59. The method of paragraph 57 or 58, wherein the
nucleobases are chemically synthesized, a product of fermentation,
or extracted from a natural source.
[0284] 60. The method of any one of paragraphs 57-59, wherein the
polyphosphate kinase is selected from PPK1 family enzymes and PPK2
family enzymes.
[0285] 61. The method of paragraph 60, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0286] 62. The method of any one of paragraphs 57-61, wherein the
polyphosphate comprises hexametaphosphate.
[0287] 63. The method of any one of paragraphs 57-62, wherein the
ribokinase, the phosphopentomutase, the nucleoside phosphorylase,
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
and/or the NDP kinase is prepared from cells that express the
ribokinase, the phosphopentomutase, the nucleoside phosphorylase,
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
and/or the NDP kinase.
[0288] 64. The method of any one of paragraphs 57-63, wherein the
reaction mixture comprises a lysate prepared from cells that
express the ribokinase, the phosphopentomutase, the nucleoside
phosphorylase, the polyphosphate kinase, the nucleoside kinase, the
NMP kinase, and/or the NDP kinase.
[0289] 65. The method of paragraph 64, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0290] 66. The method of paragraph 65, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0291] 67. The method of paragraph 65 or 66, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, organic solvent,
and/or chemical inhibitors.
[0292] 68. The method of any one of paragraphs 65-67, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0293] 69. The method of any one of paragraphs 65-68, wherein the
native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0294] 70. The method of any one of paragraphs 57-69, wherein the
ribokinase, the phosphopentomutase, the nucleoside phosphorylase,
the polyphosphate kinase, the NMP kinase, the NDP kinase, and/or
the nucleoside kinase is modified to withstand elimination
conditions.
[0295] 71. A method for producing nucleoside triphosphates (NTPs),
comprising: [0296] incubating in a reaction mixture cellular
ribonucleic acid (RNA), a polynucleotide phosphorylase (PNPase),
inorganic phosphate, a polyphosphate kinase, and a polyphosphate
under conditions appropriate for the production of NDPs and NTPs,
optionally wherein the reaction mixture further comprises a
nucleoside kinase and/or a NDP kinase.
[0297] 72. The method of paragraph 71, wherein the cellular RNA
comprises ribosomal RNA, messenger RNA, and/or transfer RNA.
[0298] 73. The method of paragraph 61 or 72, wherein the cellular
RNA is from a unicellular organism or a multicellular organism.
[0299] 74. The method of any one of paragraphs 71-73, wherein the
polyphosphate kinase is selected from PPK1 family enzymes and PPK2
family enzymes.
[0300] 75. The method of paragraph 74, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0301] 76. The method of any one of paragraphs 71-75, wherein the
polyphosphate comprises hexametaphosphate.
[0302] 77. The method of any one of paragraphs 71-76, wherein the
PNPase, the polyphosphate kinase, the nucleoside kinase, and/or the
NDP kinase is prepared from cells that express the PNPase, the
polyphosphate kinase, the nucleoside kinase, and/or the NDP
kinase.
[0303] 78. The method of any one of paragraphs 71-77, wherein the
reaction mixture comprises a cell lysate or an enzyme preparation
from cells that express the PNPase, the polyphosphate kinase, the
nucleoside kinase, and/or the NDP kinase.
[0304] 79. The method of paragraph 78, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0305] 80. The method of paragraph 79, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0306] 81. The method of paragraph 79 or 80, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0307] 82. The method of any one of paragraphs 79-81, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0308] 83. The method of any one of paragraphs 79-82, wherein the
native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0309] 84. The method of any one of paragraphs 79-83, wherein the
PNPase, the polyphosphate kinase, the nucleoside kinase, and/or the
NDP kinase can withstand elimination conditions.
[0310] 85. A method for producing nucleoside triphosphates (NTPs),
comprising: [0311] (a) incubating in a reaction mixture cellular
ribonucleic acid (RNA), a ribonuclease, a polyphosphate kinase, and
a polyphosphate under conditions appropriate for the production of
5' NMPs and NTPs, optionally wherein the reaction mixture further
comprises a nucleoside kinase, a NMP kinase, and/or a NDP
kinase.
[0312] 86. The method of paragraph 85, wherein the cellular RNA
comprises ribosomal RNA, messenger RNA, and/or transfer RNA.
[0313] 87. The method of paragraph 85 or 86, wherein the cellular
RNA is from a unicellular organism or a multicellular organism.
[0314] 88. The method of any one of paragraphs 85-87, wherein the
polyphosphate kinase is selected from PPK1 family enzymes and PPK2
family enzymes.
[0315] 89. The method of paragraph 88, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0316] 90. The method of any one of paragraphs 85-89, wherein the
polyphosphate comprises hexametaphosphate.
[0317] 91. The method of any one of paragraphs 85-90, wherein the
ribonuclease, the polyphosphate kinase, the nucleoside kinase, the
NMP kinase, and/or the NDP kinase is prepared from cells that
express the ribonuclease, the polyphosphate kinase, the nucleoside
kinase, the NMP kinase, and/or the NDP kinase.
[0318] 92. The method of any one of paragraphs 85-91, wherein the
reaction mixture comprises a cell lysate or an enzyme preparation
from cells that express the ribonuclease, the polyphosphate kinase,
the nucleoside kinase, the NMP kinase, and/or the NDP kinase.
[0319] 93. The method of paragraph 92, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0320] 94. The method of paragraph 93, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0321] 95. The method of paragraph 93 or 94, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0322] 96. The method of any one of paragraphs 93-95, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0323] 97. The method of any one of paragraphs 93-96, wherein the
native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0324] 98. The method of any one of paragraphs 93-97, wherein the
ribonuclease, the polyphosphate kinase, the nucleoside kinase, the
NMP kinase, and/or the NDP kinase can withstand elimination
conditions.
[0325] 99. A method for producing ribonucleic acid (RNA),
comprising: [0326] incubating in a reaction mixture nucleoside
diphosphates (NDPs), a polyphosphate kinase, a polyphosphate, a DNA
template encoding a RNA of interest, and a RNA polymerase under
conditions appropriate for the production of the RNA of interest,
optionally wherein the reaction mixture further comprises a
nucleoside kinase and/or a NDP kinase.
[0327] 100. The method of paragraph 99, wherein the NDPs comprise
ADP, GDP, CDP, and/or UDP.
[0328] 101. The method of paragraph 99 or 100, wherein the NDPs are
chemically synthesized, a product of fermentation, or extracted
from a natural source.
[0329] 102. The method of any one of paragraphs 99-101, wherein the
polyphosphate kinase is selected from PPK1 family enzymes and PPK2
family enzymes.
[0330] 103. The method of paragraph 102, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0331] 104. The method of any one of paragraphs 99-103, wherein the
polyphosphate comprises hexametaphosphate.
[0332] 105. The method of any one of paragraphs 99-104, wherein the
polyphosphate kinase, the DNA template, the polymerase, the
nucleoside kinase, and/or the NDP kinase is prepared from cells
that express the polyphosphate kinase, the DNA template, the
polymerase, the nucleoside kinase, and/or the NDP kinase.
[0333] 106. The method of any one of paragraphs 99-105, wherein the
reaction mixture comprises a cell lysate or an enzyme preparation
from cells that express the polyphosphate kinase, the DNA template,
the polymerase, the nucleoside kinase, and/or the NDP kinase.
[0334] 107. The method of paragraph 106, wherein native enzymatic
activity of enzymes in the cell lysate or enzyme preparation have
been eliminated.
[0335] 108. The method of paragraph 107, wherein native enzymatic
activity of enzymes in the cell lysate or enzyme preparation have
been eliminated via genetic modification, enzyme secretion from a
cell, and/or protease targeting.
[0336] 109. The method of paragraph 107 or 108, wherein native
enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via temperature, pH, salt,
detergent, alcohol, and/or chemical inhibitors.
[0337] 110. The method of any one of paragraphs 107-109, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0338] 111. The method of any one of paragraphs 107-110, wherein
the native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0339] 112. The method of any one of paragraphs 99-111, wherein the
polyphosphate kinase, the polymerase, the nucleoside kinase, and/or
the NDP kinase can withstand elimination conditions.
[0340] 113. The method of any one of paragraphs 99-112, wherein the
polymerase comprises a RNA polymerase.
[0341] 114. A method for producing ribonucleic acid (RNA),
comprising: [0342] incubating in a reaction mixture 5' nucleoside
monophosphates (5' NMPs), a polyphosphate kinase, a polyphosphate,
a DNA template encoding a RNA of interest, and a polymerase under
conditions appropriate for the production of the RNA of interest,
optionally wherein the reaction mixture further comprises a
nucleoside kinase, a NMP kinase, and/or a NDP kinase.
[0343] 115. The method of paragraph 114, wherein the 5' NMPs
comprise 5' AMP, 5' GMP, 5' CMP and/or 5' UMP.
[0344] 116. The method of paragraph 114 or 115, wherein the 5' NMPs
are chemically synthesized, a product of fermentation, or extracted
from a natural source.
[0345] 117. The method of any one of paragraphs 114-116, wherein
the polyphosphate kinase is selected from PPK1 family enzymes and
PPK2 family enzymes.
[0346] 118. The method of paragraph 117, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0347] 119. The method of any one of paragraphs 114-118, wherein
the polyphosphate comprises hexametaphosphate.
[0348] 120. The method of any one of paragraphs 114-119, wherein
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
the NDP kinase, the DNA template, and/or the polymerase is prepared
from cells that express the polyphosphate kinase, the nucleoside
kinase, the NMP kinase, the NDP kinase, the DNA template, and/or
the polymerase.
[0349] 121. The method of any one of paragraphs 114-120, wherein
the reaction mixture comprises a cell lysate or an enzyme
preparation from cells that express the polyphosphate kinase, the
nucleoside kinase, the NMP kinase, the NDP kinase, the DNA
template, and/or the polymerase.
[0350] 122. The method of paragraph 121, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0351] 123. The method of paragraph 122, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0352] 124. The method of paragraph 122 or 123, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0353] 125. The method of any one of paragraphs 122-124, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0354] 126. The method of any one of paragraphs 122-125, wherein
the native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0355] 127. The method of any one of paragraphs 114-126, wherein
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
the NDP kinase, and/or the polymerase can withstand elimination
conditions.
[0356] 128. The method of any one of paragraphs 114-127, wherein
the polymerase comprises a RNA polymerase.
[0357] 129. A method for producing ribonucleic acid (RNA),
comprising: [0358] incubating in a reaction mixture nucleosides, a
polyphosphate kinase, a polyphosphate, a DNA template encoding a
RNA of interest, and/or a polymerase under conditions appropriate
for the production of the RNA of interest, optionally wherein the
reaction mixture further comprises a nucleoside kinase, a NMP
kinase, and/or a NDP kinase.
[0359] 130. The method of paragraph 129, wherein the nucleosides
comprise adenosine, guanosine, cytidine, and/or uridine.
[0360] 131. The method of paragraph 129 or 130, wherein the
nucleosides are chemically synthesized, a product of fermentation,
or extracted from a natural source.
[0361] 132. The method of any one of paragraphs 129-131, wherein
the polyphosphate kinase is selected from PPK1 family enzymes and
PPK2 family enzymes.
[0362] 133. The method of paragraph 132, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0363] 134. The method of any one of paragraphs 129-133, wherein
the polyphosphate comprises hexametaphosphate.
[0364] 135. The method of any one of paragraphs 129-134, wherein
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
the NDP kinase, the DNA template, and/or the polymerase is prepared
from cells that express the least one polyphosphate kinase, the
nucleoside kinase, the polyphosphate, the nucleoside kinase, the
NMP kinase, the NDP kinase, the DNA template, and/or the
polymerase.
[0365] 136. The method of any one of paragraphs 129-135, wherein
the reaction mixture comprises a cell lysate or an enzyme
preparation from cells that express the least one polyphosphate
kinase, the nucleoside kinase, the NMP kinase, the NDP kinase, the
DNA template, and/or the polymerase.
[0366] 137. The method of paragraph 136, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0367] 138. The method of paragraph 137, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0368] 139. The method of paragraph 137 or 138, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0369] 140. The method of any one of paragraphs 137-139, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0370] 141. The method of any one of paragraphs 137-140, wherein
the native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0371] 142. The method of any one of paragraphs 129-141, wherein
the least one polyphosphate kinase, the polyphosphate, the
nucleoside kinase, the NMP kinase, the NDP kinase, and/or the
polymerase can withstand elimination conditions.
[0372] 143. The method of any one of paragraphs 129-142, wherein
the polymerase comprises a RNA polymerase.
[0373] 144. A method for producing ribonucleic acid (RNA),
comprising: [0374] incubating in a reaction mixture nucleobases, a
phosphoribosyltransferase, a phosphoribosylpyrophosphate, a
polyphosphate kinase, and a polyphosphate, a DNA template encoding
a RNA of interest, and/or a polymerase under conditions appropriate
for the production of the RNA of interest, optionally wherein the
reaction mixture further comprises a nucleoside kinase, a NMP
kinase, and/or a NDP kinase.
[0375] 145. The method of paragraph 144, wherein the nucleobases
comprise adenine, guanidine, cytosine, and/or uracil.
[0376] 146. The method of paragraph 144 or 145, wherein the
nucleobases are chemically synthesized, a product of fermentation,
or extracted from a natural source.
[0377] 147. The method of any one of paragraphs 144-146, wherein
the polyphosphate kinase is selected from PPK1 family enzymes and
PPK2 family enzymes.
[0378] 148. The method of paragraph 148, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0379] 149. The method of any one of paragraphs 144-148, wherein
the polyphosphate comprises hexametaphosphate.
[0380] 150. The method of any one of paragraphs 144-149, wherein
the phosphoribosyltransferase, the polyphosphate kinase, the
nucleoside kinase, a NMP kinase, the NDP kinase, the DNA template,
and/or the polymerase is prepared from cells that express the
phosphoribosyltransferase, the polyphosphate kinase, the nucleoside
kinase, the NMP kinase, the NDP kinase, the DNA template, and/or
the polymerase.
[0381] 151. The method of any one of paragraphs 144-150, wherein
the reaction mixture comprises a cell lysate or an enzyme
preparation from cells that express the phosphoribosyltransferase,
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
the NDP kinase, the DNA template, and/or the polymerase.
[0382] 152. The method of paragraph 151, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0383] 153. The method of paragraph 152, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0384] 154. The method of paragraph 152 or 153, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0385] 155. The method of any one of paragraphs 152-154, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0386] 156. The method of any one of paragraphs 152-155, wherein
the native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0387] 157. The method of any one of paragraphs 144-156, wherein
the phosphoribosyltransferase, the polyphosphate kinase, the
nucleoside kinase, the NMP kinase, the NDP kinase, and/or the
polymerase can withstand elimination conditions.
[0388] 158. The method of any one of paragraphs 144-157, wherein
the polymerase comprises a RNA polymerase.
[0389] 159. A method for producing ribonucleic acid (RNA),
comprising: [0390] incubating in a reaction mixture nucleobases, a
ribose, a ribokinase, a phosphopentomutase, a nucleoside
phosphorylase, a polyphosphate kinase, a polyphosphate, a DNA
template encoding a RNA of interest, and a polymerase under
conditions appropriate for the production of the RNA of interest,
optionally wherein the reaction mixture further comprises a
nucleoside kinase, a NMP kinase, and/or at least one NDP
kinase.
[0391] 160. The method of paragraph 159, wherein the nucleobases
comprise adenine, guanidine, cytosine, and/or uracil.
[0392] 161. The method of paragraph 159 or 160, wherein the
nucleobases are chemically synthesized, a product of fermentation,
or extracted from a natural source.
[0393] 162. The method of any one of paragraphs 159-161, wherein
the polyphosphate kinase is selected from PPK1 family enzymes and
PPK2 family enzymes.
[0394] 163. The method of paragraph 162, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0395] 164. The method of any one of paragraphs 159-163, wherein
the polyphosphate comprises hexametaphosphate.
[0396] 165. The method of any one of paragraphs 159-164, wherein
the ribokinase, the phosphopentomutase, the nucleoside
phosphorylase, the polyphosphate kinase, the nucleoside kinase, the
NMP kinase, the NDP kinase, the DNA template, and/or the polymerase
is from at least one lysate prepared from engineered cells modified
to express the ribokinase, the phosphopentomutase, the nucleoside
phosphorylase, the polyphosphate kinase, the nucleoside kinase, the
NMP kinase, the NDP kinase, the DNA template, and/or the
polymerase.
[0397] 166. The method of any one of paragraphs 159-165, wherein
the reaction mixture a lysate prepared from cells that express the
ribokinase, the phosphopentomutase, the nucleoside phosphorylase,
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
the NDP kinase, the DNA template, and/or the polymerase.
[0398] 167. The method of paragraph 166, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated.
[0399] 168. The method of paragraph 167, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0400] 169. The method of paragraph 167 or 168, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, organic solvent,
and/or chemical inhibitors.
[0401] 170. The method of any one of paragraphs 167-169, wherein
native enzymatic activity of enzymes in the cell lysate or enzyme
preparation have been eliminated via separation, precipitation,
filtration, capture, and/or chromatography.
[0402] 171. The method of any one of paragraphs 167-170, wherein
the native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0403] 172. The method of any one of paragraphs 159-171, wherein
the ribokinase, the phosphopentomutase, the nucleoside
phosphorylase, the polyphosphate kinase, the nucleoside kinase, the
NMP kinase, the NDP kinase, and/or the polymerase is modified to
withstand elimination conditions.
[0404] 173. The method of any one of paragraphs 159-172, wherein
the at least one polymerase comprises at least one RNA
polymerase.
[0405] 174. A method for producing ribonucleic acid (RNA),
comprising: [0406] (a) incubating in a reaction mixture cellular
ribonucleic acid (RNA), a polynucleotide phosphorylase (PNPase),
and inorganic phosphate under conditions appropriate for the
production of nucleoside diphosphates (NDPs); [0407] (b)
eliminating the PNPase; and [0408] (c) incubating in the reaction
mixture, or in a second reaction mixture, the NDPs, a polyphosphate
kinase, a polyphosphate, a DNA template encoding a RNA of interest,
and a polymerase under conditions appropriate for the production of
the RNA of interest, optionally wherein the reaction mixture of
step (c) further comprises a nucleoside kinase and/or a NDP
kinase.
[0409] 175. The method of paragraph 174, wherein the cellular RNA
comprises ribosomal RNA, messenger RNA, and/or transfer RNA.
[0410] 176. The method of paragraph 174 or 175, wherein the
cellular RNA is from a unicellular organism or a multicellular
organism.
[0411] 177. The method of any one of paragraphs 174-176, wherein
the polyphosphate kinase is selected from PPK1 family enzymes and
PPK2 family enzymes.
[0412] 178. The method of paragraph 177, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0413] 179. The method of any one of paragraphs 174-178, wherein
the polyphosphate comprises hexametaphosphate.
[0414] 180. The method of any one of paragraphs 174-179, wherein
the PNPase is prepared from cells that express the PNPase.
[0415] 181. The method of any one of paragraphs 174-180, wherein
the reaction mixture of (a) comprises a cell lysate prepared from
cells that express the PNPase.
[0416] 182. The method of paragraph 181, wherein step (b) comprises
eliminating the PNPase via temperature, pH, salt, detergent,
alcohol, and/or chemical inhibitors.
[0417] 183. The method of paragraph 181 or 182, wherein step (b)
comprises eliminating the PNPase via wherein native enzymatic
activity of enzymes in the cell lysate or enzyme preparation have
been eliminated via separation, precipitation, filtration, capture,
and/or chromatography.
[0418] 184. The method of any one of paragraphs 174-183, wherein
the polyphosphate kinase, the nucleoside kinase, the NDP kinase,
the DNA template, and/or the polymerase is prepared from cells that
express the polyphosphate kinase, the nucleoside kinase, the NDP
kinase, the DNA template, and/or the polymerase.
[0419] 185. The method of any one of paragraphs 174-183, wherein
the reaction mixture of step (c) comprises a cell lysate prepared
from cells that express the polyphosphate kinase, the nucleoside
kinase, the NDP kinase, the DNA template, and/or the
polymerase.
[0420] 186. The method of paragraph 185, wherein native enzymatic
activity of enzymes in the cell lysate of step (c) have been
eliminated.
[0421] 187. The method of paragraph 186, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0422] 188. The method of paragraph 186 or 187, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0423] 189. The method of any one of paragraphs 186-188, wherein
native enzymatic activity of enzymes in the cell lysate have been
eliminated via separation, precipitation, filtration, capture,
and/or chromatography.
[0424] 190. The method of any one of paragraphs 186-189, wherein
the native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0425] 191. The method of any one of paragraphs 186-190, wherein
the polyphosphate kinase, the nucleoside kinase, the NDP kinase,
and/or the polymerase can withstand elimination conditions.
[0426] 192. The method of any one of paragraphs 174-191, wherein
the polymerase comprises a RNA polymerase.
[0427] 193. A method for producing ribonucleic acid (RNA),
comprising: [0428] (a) incubating in a reaction mixture cellular
ribonucleic acid (RNA), a polynucleotide phosphorylase (PNPase),
inorganic phosphate, a polyphosphate kinase, a polyphosphate, a DNA
template encoding a RNA of interest, and a polymerase under
conditions appropriate for the production of nucleoside
diphosphates, optionally wherein the reaction mixture further
comprises a nucleoside kinase and/or a NDP kinase; [0429] (b)
eliminating the PNPase; and [0430] (c) incubating the reaction
mixture under conditions appropriate for the production of the RNA
of interest.
[0431] 194. The method of paragraph 193, wherein the cellular RNA
comprises ribosomal RNA, messenger RNA, and/or transfer RNA.
[0432] 195. The method of paragraph 193 or 194, wherein the
cellular RNA is from a unicellular organism or a multicellular
organism.
[0433] 196. The method of any one of paragraphs 193-195, wherein
the polyphosphate kinase is selected from PPK1 family enzymes and
PPK2 family enzymes.
[0434] 197. The method of paragraph 196, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0435] 198. The method of any one of paragraphs 193-197, wherein
the polyphosphate comprises hexametaphosphate.
[0436] 199. The method of any one of paragraphs 193-198, wherein
the PNPase, the polyphosphate kinase, the nucleoside kinase, the
NDP kinase, the DNA template, and/or the polymerase is prepared
from cells that express the PNPase, the polyphosphate kinase, the
nucleoside kinase, the NDP kinase, the DNA template, and/or the
polymerase.
[0437] 200. The method of any one of paragraphs 193-199, wherein
the reaction mixture of (a) comprises a cell lysate prepared from
cells that express the PNPase, the polyphosphate kinase, the
nucleoside kinase, the NDP kinase, the DNA template, and/or the
polymerase.
[0438] 201. The method of paragraph 200, wherein step (b) comprises
eliminating the PNPase and native enzymatic activities in the cell
lysate via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0439] 202. The method of paragraph 200 or 201, wherein step (b)
comprises eliminating the PNPase and native enzymatic activities in
the cell lysate via separation, precipitation, filtration, capture,
and/or chromatography.
[0440] 203. The method of any one of paragraphs 200-202, wherein
step (b) comprises eliminating native enzymatic activities in the
cell lysate via genetic modification, enzyme secretion from a cell,
and/or protease targeting.
[0441] 204. The method of any one of paragraphs 201-203, wherein
the native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0442] 205. The method of any one of paragraphs 201-204, wherein
the polyphosphate kinase, the nucleoside kinase, the NDP kinase,
and/or the polymerase can withstand elimination conditions.
[0443] 206. The method of any one of paragraphs 193-205, wherein
the polymerase comprises a RNA polymerase.
[0444] 207. A method for producing ribonucleic acid (RNA),
comprising: [0445] (a) incubating in a reaction mixture cellular
ribonucleic acid (RNA) and a ribonuclease under conditions
appropriate for the production of 5' nucleoside monophosphates (5'
NMPs); [0446] (b) eliminating the ribonuclease; and [0447] (c)
incubating in the reaction mixture, or in a second reaction
mixture, the 5' NMPs, a polyphosphate kinase, a polyphosphate, a
DNA template encoding a RNA of interest, and a polymerase under
conditions appropriate for the production of the RNA of interest,
optionally wherein the reaction mixture of step (c) further
comprises a nucleoside kinase, a NMP kinase, and/or a NDP
kinase.
[0448] 208. The method of paragraph 207, wherein the cellular RNA
comprises ribosomal RNA, messenger RNA, and/or transfer RNA.
[0449] 209. The method of paragraph 207 or 208, wherein the
cellular RNA is from a unicellular organism or a multicellular
organism.
[0450] 210. The method of any one of paragraphs 207-209, wherein
the polyphosphate kinase is selected from PPK1 family enzymes and
PPK2 family enzymes.
[0451] 211. The method of paragraph 210, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0452] 212. The method of any one of paragraphs 207-211, wherein
the polyphosphate comprises hexametaphosphate.
[0453] 213. The method of any one of paragraphs 207-212, wherein
the ribonuclease is prepared from cells that express the
ribonuclease.
[0454] 214. The method of any one of paragraphs 207-213, wherein
the reaction mixture of (a) comprises a cell lysate prepared from
cells that express the ribonuclease.
[0455] 215. The method of paragraph 214, wherein step (b) comprises
eliminating the ribonuclease via temperature, pH, salt, detergent,
alcohol, and/or chemical inhibitors.
[0456] 216. The method of paragraph 214 or 215, wherein step (b)
comprises eliminating the ribonuclease via separation,
precipitation, filtration, capture, and/or chromatography.
[0457] 217. The method of any one of paragraphs 207-216, wherein
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
the NDP kinase, the DNA template, and/or the polymerase is prepared
from cells that express the polyphosphate kinase, the nucleoside
kinase, the NMP kinase, the NDP kinase, the DNA template, and/or
the polymerase.
[0458] 218. The method of any one of paragraphs 207-216, wherein
the reaction mixture of step (c) comprises a cell lysate prepared
from cells that express the polyphosphate kinase, the nucleoside
kinase, the NMP kinase, the NDP kinase, the DNA template, and/or
the polymerase.
[0459] 219. The method of paragraph 218, wherein native enzymatic
activity of enzymes in the cell lysate of step (c) have been
eliminated.
[0460] 220. The method of paragraph 219, wherein native enzymatic
activity of enzymes in the cell lysate have been eliminated via
genetic modification, enzyme secretion from a cell, and/or protease
targeting.
[0461] 221. The method of paragraph 219 or 220, wherein native
enzymatic activity of enzymes in the cell lysate have been
eliminated via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0462] 222. The method of any one of paragraphs 219-221, wherein
native enzymatic activity of enzymes in the cell lysate have been
eliminated via separation, precipitation, filtration, capture,
and/or chromatography.
[0463] 223. The method of any one of paragraphs 219-222, wherein
the native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0464] 224. The method of any one of paragraphs 219-223, wherein
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
the NDP kinase, and/or the polymerase can withstand elimination
conditions.
[0465] 225. The method of any one of paragraphs 207-224, wherein
the polymerase comprises at least on RNA polymerase.
[0466] 226. A method for producing ribonucleic acid (RNA),
comprising: [0467] (a) incubating in a reaction mixture cellular
ribonucleic acid (RNA), a ribonuclease, a polyphosphate kinase, a
polyphosphate, a DNA template encoding a RNA of interest, and a
polymerase under conditions appropriate for the production of 5'
nucleoside monophosphates (5' NMPs); [0468] (b) eliminating the
ribonuclease; and [0469] (c) incubating the reaction mixture under
conditions appropriate for the production of the RNA of
interest.
[0470] 227. The method of paragraph 226, wherein the cellular RNA
comprises ribosomal RNA, messenger RNA, and/or transfer RNA.
[0471] 228. The method of paragraph 226 or 214, wherein the
cellular RNA is from a unicellular organism or a multicellular
organism.
[0472] 229. The method of any one of paragraphs 226-228, wherein
the polyphosphate kinase is selected from PPK1 family enzymes and
PPK2 family enzymes.
[0473] 230. The method of paragraph 229, wherein the polyphosphate
kinase comprises a Class III polyphosphate kinase 2 from
Deinococcus geothermalis.
[0474] 231. The method of any one of paragraphs 226-230, wherein
the polyphosphate comprises hexametaphosphate.
[0475] 232. The method of any one of paragraphs 226-231, wherein
the ribonuclease, the polyphosphate kinase, the nucleoside kinase,
the NMP kinase, the NDP kinase, the DNA template, and/or the
polymerase is prepared from cells that express the ribonuclease,
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
the NDP kinase, the DNA template, and/or the polymerase.
[0476] 233. The method of any one of paragraphs 227-232, wherein
the reaction mixture of (a) comprises a cell lysate prepared from
cells that express the ribonuclease, the polyphosphate kinase, the
nucleoside kinase, the NMP kinase, the NDP kinase, the DNA
template, and/or the polymerase.
[0477] 234. The method of paragraph 233, wherein step (b) comprises
eliminating the ribonuclease and native enzymatic activities in the
cell lysate via temperature, pH, salt, detergent, alcohol, and/or
chemical inhibitors.
[0478] 235. The method of paragraph 233 or 234, wherein step (b)
comprises eliminating the ribonuclease and native enzymatic
activities in the cell lysate via separation, precipitation,
filtration, capture, and/or chromatography.
[0479] 236. The method of any one of paragraphs 233-235, wherein
step (b) comprises eliminating native enzymatic activities in the
cell lysate via genetic modification, enzyme secretion from a cell,
and/or protease targeting.
[0480] 237. The method of any one of paragraphs 234-236, wherein
the native enzymatic activities are selected from phosphatases,
nucleases, proteases, deaminases, oxidoreductases, and
hydrolases.
[0481] 238. The method of any one of paragraphs 234-237, wherein
the polyphosphate kinase, the nucleoside kinase, the NMP kinase,
the NDP kinase, and/or the polymerase can withstand elimination
conditions.
[0482] 239. The method of any one of paragraphs 226-238, wherein
the polymerase comprises a RNA polymerase.
Examples
Example 1--Cell-Free Synthesis of RNA Starting from Either Lysate
RNA or Purified E. coli RNA
[0483] Materials and Methods
[0484] Strains and Lysates
[0485] E. coli strain BL21(DE3) was transformed with pETDuet-1
vectors encoding codon-optimized versions of the following enzymes:
RNase R (K544R) from E. coli (EcRNR), UMP kinase from Pyrococcus
furiosus (PfPyrH), CMP kinase from Thermus thermophilus (TthCmk),
GMP kinase from Thermotoga maritima (TmGmk), NDP kinase from
Aquifex aeolicus (AaNdk), and Class III polyphosphate kinase 2 from
Deinococcus geothermalis (DgPPK2). All enzymes, except for DgPPK2,
contained N-terminal hexahistidine tags. The resulting strains were
grown in a 37.degree. C. batch fermentation process in Korz media
supplemented with 40 g/L glucose and 50 mg/L carbenicillin until
OD.sub.600=20, induced with 0.8 mM isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG), and grown for an
additional 1 hour before harvest via centrifugation. After harvest,
biomass pellets were stored at -80.degree. C.
[0486] Biomass pellets were then used to prepare cell lysates.
Lysates were prepared by thawing biomass pellets on ice, then
resuspending in 1.5 volumes resuspension buffer. For the strain
expressing EcRNR, biomass was resuspended in 58.8 mM potassium
phosphate dibasic. For strains expressing PfPyrH, TthCmk, TmGmk,
and AaNdk, biomass was resuspended in 50 mM Tris-HCl (pH 8.5) with
50 mM NaCl. For the strain expressing DgPPK2, biomass was
resuspended in 100 mM MOPS-NaOH (pH 7.0). For all strains, biomass
was lysed by 2-3 passes of mechanical homogenization at 15,000 psi
at 4.degree. C. Lysates were then clarified by centrifugation at
16,000.times.g for 1 hour at 4.degree. C.
[0487] Extraction and Purification of E. coli RNA
[0488] RNA was extracted and purified from high-density E. coli
lysates (protein concentration: 40-50 mg/mL) according to
established protocols (Mohanty, B. K., Giladi, H., Maples, V. F.,
& Kushner, S. R. (2008). Analysis of RNA decay, processing, and
polyadenylation in Escherichia coli and other prokaryotes. Methods
in Enzymology, 447, 3-29).
[0489] Protein Expression and Purification
[0490] E. coli strain BL21(DE3) was transformed with a pBAD24
vector encoding a thermostable mutant T7 RNA polymerase with an
N-terminal hexahistidine tag. The resulting strain was cultivated
in baffled shake flasks using lysogeny broth (LB) media
supplemented with 50 mg/L carbenicillin. Cultures were grown at
37.degree. C. with shaking until OD600=0.6, then induced with 0.2%
(w/v) L-arabinose and grown for an additional 4 hours. Biomass was
then harvested by centrifugation and stored at -80.degree. C. prior
to lysis. Lysates were prepared by thawing biomass pellets on ice,
resuspending in 1.5 volumes equilibration/wash buffer (20 mM sodium
phosphate pH 7.4, 500 mM sodium chloride, 30 mM imidazole), and 2-3
passes of mechanical homogenization at 15,000 psi and 4.degree. C.
Lysates were then clarified by centrifugation. Recombinant protein
was purified by fast protein liquid chromatography (FPLC) using an
AKTAPrime Plus equipped with a HisTrap HP column (GE Healthcare
Life Sciences) following standard protocols. Fractions containing
recombinant protein were then combined and buffer exchanged by
dialysis into 2.times. phosphate buffered saline (PBS) supplemented
with 5 mM DTT and 0.01% Triton X-100. Post-dialysis, protein stocks
were diluted with an equal volume of glycerol and stored at
-20.degree. C.
[0491] For E. coli RNase R, cultures were grown, expression was
induced, and lysates were prepared following the protocols
described herein. The enzyme was then purified following the
protocols described herein, except that the purified enzyme was
buffer-exchanged by dialysis into 2.times.PBS supplemented with 500
mM NaCl prior to mixing with glycerol.
[0492] DNA Template Preparation
[0493] Linear DNA templates were amplified from synthetic DNA by
PCR and purified using solid-phase reversible immobilization (SPRI)
on paramagnetic beads. Plasmid DNA templates were prepared by
cloning the sequence of interest into a suitable plasmid vector,
transforming the resulting plasmid into E. coli strain DH10b,
culturing the transformants in LB media, and purifying the plasmids
using Plasmid Maxi or Giga kits (Qiagen).
[0494] Cell-Free RNA Synthesis from Lysate RNA
[0495] Lysates expressing EcRNR, TthCmk, PfPyrH, TmGmk, AaNdk, and
DgPPK2 were each diluted to 42 mg/mL in 50 mM Tris, 50 mM NaCl (pH
7.0), then combined in equal proportion. Depolymerization of RNA in
the lysates was initiated by adding an equal volume of 3 mM
ethylenediaminetetraacetic acid (EDTA) in 50 mM Tris, 50 mM NaCl
(pH 7.0) and incubating at 37.degree. C. for 15 minutes.
Depolymerization was monitored by quantifying acid-soluble
nucleotides by absorbance at 260 nm. Magnesium sulfate and sodium
hexametaphosphate were added to the lysates to final concentrations
of 30 mM and 1 mM, respectively, then the lysates were incubated at
70.degree. C. for 15 minutes to inactivate EcRNR and endogenous E.
coli enzymes. After 15 minutes, the temperature was reduced to
50.degree. C. and the reaction was assembled with the following
composition:
TABLE-US-00014 TABLE 11 Reaction Conditions Magnesium sulfate 30 mM
Sodium hexametaphosphate 4 mM Kinase lysates 21 g/L total protein
Spermidine 2 mM Template DNA 25 mg/L RNA polymerase 0.3 g/L
[0496] Reactions were incubated at 50.degree. C. for 2 hours,
treated with TURBO DNase (Thermo Fisher), and analyzed by agarose
gel electrophoresis.
[0497] Cell-Free RNA Synthesis from Purified E. coli RNA
[0498] Purified E. coli RNA (approximately 8 g/L) was depolymerized
by incubating with 1 g/L purified E. coli RNase R in a buffer
containing 50 mM Tris-HCl (pH 7.0), 50 mM sodium chloride, and 2 mM
magnesium sulfate. The depolymerization reaction was incubated at
37.degree. C. for 30 minutes and monitored by quantifying
acid-soluble nucleotides by absorbance at 260 nm. After 30 minutes,
the depolymerization reaction was combined with the TthCmk, TmGmk,
PfPyrH, AaNdk, and DgPPK2 lysates each diluted in 50 mM Tris-HCl
(pH 7.0), 50 mM NaCl, and mixed in equal proportion. Magnesium
sulfate and sodium hexametaphosphate were then added to final
concentrations of 30 mM and 1 mM, respectively, and the reaction
heated to 70.degree. C. for 15 minutes. After heat inactivation,
reactions were assembled as described herein with the following
composition:
TABLE-US-00015 TABLE 12 Reaction Conditions Magnesium sulfate 30 mM
Sodium hexametaphosphate 4 mM Kinase lysates 13 g/L total protein
Spermidine 2 mM Template DNA 25 mg/L RNA polymerase 0.3 g/L
[0499] Results
[0500] Cell-free synthesis of RNA was performed using lysate RNA as
a substrate. RNA from pooled lysates overexpressing pathway enzymes
was first depolymerized by overexpressed E. coli RNase R (EcRNR),
an exonuclease that produces 5' NMPs. Depolymerization was rapid,
producing approximately 14 mM acid-soluble nucleotides after 15
minutes (FIG. 8A). The lysate mix containing pathway enzymes and 5'
NMPs was then heat treated to inactivate EcRNR and endogenous E.
coli enzymes, while the thermostable pathway enzymes remained
active. After heat inactivation, the RNA synthesis reaction was
assembled, incubated at 50.degree. C., and visualized on an agarose
gel (FIG. 8B). Reactions with two different templates, including a
linear PCR product (Template 1) and a hairpin template encoded on a
plasmid (Template 2), yielded distinct bands on an agarose gel
(FIG. 8B). No bands were observed in the conditions without RNA
polymerase. As a positive control, reactions were performed where
purified 5' NMPs (4 mM each AMP, CMP, GMP, and UMP) were added.
These reactions produced products that migrated at the same size as
reactions using NMPs produced by depolymerizing RNA.
[0501] Cell-free synthesis of RNA was also performed using purified
E. coli RNA as substrate. RNA was first incubated with purified E.
coli RNase R (K544R) to release 5' NMPs (FIG. 9A). After 30
minutes, the depolymerization reaction was combined with a lysate
mix containing pathway enzymes and heat treated to inactivate EcRNR
and endogenous E. coli enzymes, while the thermostable pathway
enzymes remained active. After heat inactivation, the RNA synthesis
reaction was assembled, incubated at 50.degree. C., and visualized
on an agarose gel (FIG. 9B). Reactions with two different
templates, including a linear PCR product (Template 1) and a
hairpin template encoded on a plasmid (Template 2), yielded defined
bands on an agarose gel, though yields appeared lower for Template
2 (FIG. 9B). Again, no bands were observed in the conditions
without RNA polymerase.
[0502] Taken together, these results demonstrate that cell-free RNA
synthesis can be used to produce an RNA of interest from a variety
of NMP source material, including high-purity purchased NMPs, NMPs
produced by enzymatic depolymerization of lysate RNA, and NMPs
produced by enzymatic depolymerization of purified RNA.
Example 2--Cell-Free Synthesis of RNA Using a Non-Thermostable
Wild-Type Polymerase or a Thermostable Polymerase Mutant, and
Purified NMPs as a Substrate
[0503] Materials and Methods
[0504] Biomass, lysates, purified proteins, and DNA template were
prepared as described herein. Cell-free synthesis of RNA was
performed essentially as described in Example 1, except that EcRNR
was omitted and 4 mM each AMP, CMP, GMP, and UMP were added to the
reaction.
[0505] Results
[0506] Cell-free synthesis of RNA was performed using purified NMPs
as a substrate at a 37.degree. C. reaction temperature. Lysates
containing pathway enzymes were assembled and heat treated to
inactivate endogenous E. coli enzymes, while the thermostable
pathway enzymes remained active. After heat inactivation, the
reaction was assembled using either wild-type T7 RNA polymerase or
a thermostable mutant, incubated at 37.degree. C. for 2 hours, and
visualized on an agarose gel (FIG. 10). Reactions with two
different templates, including a linear PCR product (Template 1)
and a hairpin template encoded on a plasmid (Template 2), yielded
distinct bands with both polymerases. At 37.degree. C., RNA yield
(based on gel band intensity) appeared greater with the wild-type
polymerase than the thermostable mutant, especially with Template
2. No bands were observed in the conditions without RNA
polymerase.
[0507] These results demonstrate that the RNA polymerase used for
cell-free RNA synthesis does not need to be thermostable, and that
RNA can be produced using a wild-type T7 RNA polymerase in a
37.degree. C. reaction.
Example 3--Cell-Free Synthesis of RNA Using a Class III
Polyphosphate Kinase 2 from Deinococcus geothermalis (DgPPK2) and
NMPs as a Substrate
[0508] Materials and Methods
[0509] Cell-free RNA synthesis reactions were assembled essentially
as described in Example 2, with the following composition:
TABLE-US-00016 TABLE 13 Reaction Conditions Magnesium sulfate 45 mM
Sodium hexametaphosphate 13 mM DgPPK2 lysate 7 g/L total protein
Template DNA 50 mg/L Spermidine 2 mM RNA polymerase 0.3 g/L
As a positive control, a 5-enzyme lysate system containing
uridylate kinase, cytidylate kinase, guanylate kinase, nucleotide
diphosphate kinase and a polyphosphate kinase was used in the
reaction according to Examples 1-2. The dsRNA synthesized in the
reactions was purified via an adapted RNASwift extraction protocol
and quantitated using a reverse phase ion pair chromatography as
described herein (Nwokeji, A. O., Kilby, P. M., Portwood, D. E.,
& Dickman, M. J. (2016). RNASwift: A rapid, versatile RNA
extraction method free from phenol and chloroform. Analytical
Biochemistry, 512, 36-46).
[0510] Results
[0511] Cell-free synthesis of RNA was performed in reactions
comprising DgPPK2 as the sole kinase in the reaction, and NMPs as a
substrate. As a positive control, RNA was synthesized in the
presence of the 5-enzyme lysate system containing uridylate kinase,
cytidylate kinase, guanylate kinase, nucleotide diphosphate kinase,
and polyphosphate kinase. Control reaction were performed in the
absence of polymerase.
[0512] The response factor for each reaction was determined. The
response factor was calculated as ratio of area of the dsRNA of
interest to that of a commercially-available dsRNA internal
standard. Comparison of the response factors demonstrated that
reactions containing DgPPK2 as the sole kinase synthesized
.about.48% of the dsRNA synthesized that was synthesized in
reactions containing the 5-enzyme lysate system (FIG. 11A). The
dsRNA product synthesized in the DgPPK2 reaction and the 5-enzyme
lysate system reaction were analyzed by HPLC. The HPLC
chromatograms of the dsRNA product from the reactions were similar
demonstrating that the dsRNA product produced by the DgPPK2-only
system was similar to the product produced by the 5-enzyme system
(FIG. 11B).
[0513] Taken together, these results demonstrate that the DgPPK2
could be used as a sole kinase to synthesize cell-free dsRNA from
NMPs or NDPs.
Example 4--Depolymerization of RNA from Various Sources Using
Purified RNase R or Purified Nuclease P1
[0514] Materials and Methods
[0515] Extraction and Purification of RNA and Nuclease
[0516] RNA was extracted and purified from high-density E. coli
lysates (protein concentration: 40-50 mg/mL) according to
established protocols (Mohanty, B. K., Giladi, H., Maples, V. F.,
& Kushner, S. R. (2008). Analysis of RNA decay, processing, and
polyadenylation in Escherichia coli and other prokaryotes. Methods
in Enzymology, 447, 3-29).
[0517] RNA from Vibrio was purified from V. natriegens cell broth
using RNASwift Protocol (Nwokeji, A. O., Kilby, P. M., Portwood, D.
E., & Dickman, M. J. (2016). RNASwift: A rapid, versatile RNA
extraction method free from phenol and chloroform. Analytical
Biochemistry, 512, 36-46).
[0518] Yeast derived RNA extract was purchased from commercial
sources. The RNA powder was .about.85% to .about.90% pure and
required no further purification. RNase R was purified from an E.
coli strain overexpressing RNase R and grown to high cell density.
Proteins were purified by immobilized metal affinity chromatography
using HisTrap HP columns connected to an AKTAPrime Plus FPLC system
(GE Healthcare). Purified Nuclease P1, a 5' phosphodiesterase, was
obtained from commercial sources.
[0519] Depolymerization of RNA with Exogenous Nuclease
[0520] E. coli RNA, V. natriegens RNA, Yeast derived RNA powder
resuspended in nuclease-free water (RNA content of 11 mg/mL), RNase
R solution (1 mg/mL in 300 mM potassium phosphate buffer pH 7.4,
200 mM KCl, 2 mM MgCl.sub.2) and Purified Nuclease P1 (also
referred 5' phosphodiesterase) (1-2 mg/mL in 100 mM potassium
phosphate buffer pH 7.4, 1 mM ZnCl.sub.2, 10 mM MgCl.sub.2) were
pre-equilibrated at 2.degree. C. before initiating the reaction. At
time t=0, 50 .mu.L, of RNA and 50 .mu.L nuclease solution were
mixed and the reaction initiated by transferring to a preheated
37.degree. C. block. After initiation, reactions were incubated at
37.degree. C. and periodically sampled by transferring 10 .mu.L to
acid quench solution (90 .mu.L of 0.2M sulfuric acid) on ice. After
completion of the time course, quenched samples were clarified by
centrifugation at 3,200.times.g for 20 minutes at 2.degree. C.
Depolymerization was first quantified by absorbance of acid-soluble
nucleotides at 260 nm. The total nucleotide pool (e.g., 100%
depolymerization) was determined by alkaline hydrolysis of RNA: 50
.mu.L RNA was combined with 150 .mu.L of 0.2M potassium hydroxide,
then heated to 99.degree. C. for 20 minutes. Alkaline-hydrolyzed
samples were then quenched and analyzed as described above.
Depolymerization was also quantified by LC-MS analysis of 5', 2',
and 3' NMPs: 10 .mu.L of sample was quenched in 30 .mu.L 100%
acetonitrile and diluted into 500 .mu.L of deionized water
containing 10 .mu.M adipic acid used as internal standard. The
sample was then centrifuged and passed through a 0.2 .mu.m filter
before LC-MS analysis.
[0521] Nucleotide Analysis
[0522] Analysis of 2', 3', and 5' NMPs was performed by mass
spectrometry and liquid chromatography using an ABSCIEX API 5000
Mass spectrometer and a standard Agilent 1200 HPLC equipped with
Sequant Zinc-hilic column (2.1.times.50 mm, 3 .mu.m i.d.). The
mobile phases consisted of 20 mM ammonium acetate in 90%
acetonitrile (A) and 20 mM ammonium acetate in 10% acetonitrile
(B). The separation method consisted of the following: starting
gradient of 6% B followed by a gradient to 8.5% B over 600 seconds,
a gradient to 13% B over 400 seconds, followed by a gradient from
to 20% B over 60 seconds, a wash at 50% B for 60 seconds, and
finally, re-equilibration at 6% B for 220 seconds. Quantitation was
performed in negative mode electro-spray ionization (ESI) using the
following mass spec transitions: 2'3'5' AMP: 346.1-134.1, 2'3'5'
UMP: 323.0--97, 2'3'5' CMP: 322--97, 2'3'5' GMP: 362.1--211. Peak
areas were compared to standard curves consisting of purified
compounds (purchased from Sigma-Aldrich except for 2' and 3' CMP,
UMP, and GMP which were purchased from Biolog Life Science
Institute) and normalized to an internal standard (adipic acid)
that was spiked into the samples prior to analysis. For analysis of
samples, standard curves were prepared in deionized water, diluted
with internal standard and filtered as in the sample preparation
steps described herein.
[0523] Results
[0524] V. natriegens RNA was digested using purified E. coli RNase
R, and E. coli RNA and yeast-derived RNA extract were digested
using both E. coli RNase R and Nuclease P1. Depolymerization was
monitored by release of acid-soluble nucleotides. Treatment of E.
coli and V. natriegens RNA with RNase R showed a time-dependent
conversion of RNA to acid-soluble nucleotides reaching .about.98 to
.about.100% depolymerization after 30 minutes of incubation (FIG.
12A). Treatment of yeast-derived RNA extract reached .about.94%
depolymerization with Nuclease P1 (FIG. 12A). Also, treatment of E.
coli RNA with Nuclease P1 resulted in .about.90% depolymerization
(FIG. 12A). Subsequent analyses by LC-MS revealed release of 5'
NMPs in E. coli RNA and yeast-derived RNA extract treated with
Nuclease P1 (FIG. 12B)
[0525] These results demonstrate that varying sources of RNA (e.g.,
Vibrio RNA, E. coli RNA, and yeast RNA) can be digested to 5' NMP
using different nucleases (e.g., RNase R and Nuclease P1).
Example 5--Effects of Temperature and Lysate Inactivation on RNA
Depolymerization
[0526] Materials and Methods
[0527] Strains and Lysates
[0528] Strain GL17-086
(BL21(DE3)..DELTA.tolC..DELTA.ph[DE3]1+2*..DELTA.ph[285p]*..DELTA.fhuA*..-
DELTA.lamB*rna::tolC) and strain GL17-109 (BL21(DE3).
.DELTA.tolC..DELTA.ph[DE3]1+2*..DELTA.ph[285p]*..DELTA.fhuA*..DELTA.lamB*-
.DELTA.rna*.
.DELTA.phoA*..DELTA.appA*..DELTA.amn*..DELTA.nagD*..DELTA.ushA::tolC)
were used in the study described herein. For both strains, 1L
cultures were grown under batch-growth conditions in KORZ media (5
g/L (NH.sub.4).sub.2SO.sub.4, 15.7 g/L K.sub.2HPO.sub.4, 4.2 g/L
KH.sub.2PO.sub.4, 1.7 g/L citric acid, 0.6 g/L MgSO.sub.4, 0.1%
Thiamine-HCl, 0.01% Pluronic, trace metals, and 40 g/L glucose) for
approximately seven hours. After growth the cells were centrifuged
at 6000 g for 20 minutes and then stored at -80.degree. C. Frozen
biomass was thawed in 1.5.times. volumes of a 58.8 mM dibasic
potassium phosphate solution and lysed by passing through an
EmulsiFlex C3 homogenizer (Avestin) for 3 passes, recirculating,
with pulses of 15000-20000 psi. Lysate was clarified by spinning at
15000 g for 1 hour at 4.degree. C. The lysate was then stored at
-80.degree. C. in single-use aliquots.
[0529] Analysis of RNA Polymerization Products at Various
Temperatures
[0530] Frozen lysates for both strains (086 and 109) were incubated
at varying temperatures to profile the RNA depolymerization
products and their potential degradation. Lysate aliquots were
thawed on ice and then dispensed into PCR strip-tubes. Tubes were
then incubated at 40.degree. C., 50.degree. C., 60.degree. C., and
70.degree. C. up to an hour with intermittent sampling. The initial
to timepoint was taken by quenching while the lysates were still on
ice. For all other times, samples were quenched in 5.times. volumes
of acetonitrile with 60 .mu.l diluted into 500 .mu.l of a 50 .mu.M
adipic acid solution in 50 mM ammonium acetate, and then run on an
LC-QQQ. Using MRM, all four main nucleotides and their associated
derivates (ATP, ADP, AMP, adenine, adenosine, GTP, GDP, GMP,
guanine, guanosine, CTP, CDP, CMP, cytidine, cytosine, UTP, UDP,
UMP, uridine, and uracil) were quantified at each timepoint. Base
hydrolysis of lysates, where RNA is completely hydrolyzed down to
NMPs, was performed by incubating the lysate in 3.times. volumes of
0.2M NaOH at 99.degree. C. for 20 minutes. It was then neutralized
with an equal volume of 150 mM HCl and 20 .mu.l of this solution
was diluted into 200 .mu.l of a 50 mM ammonium acetate solution
with 50 .mu.M adipic acid. Base hydrolyzed lysate values represent
100% depolymerization. Under some conditions these lysates were
incubated as-is, and in others conditions the lysates were mixed
with either 0.5 mg/ml Nuclease P1 (Sigma N8630) or RNase R. RNase R
was prepared as follows: cells were resuspended in 1.5.times.
volumes of lysis buffer (50 mM potassium phosphate, pH=7.4, 500 mM
NaCl, 20 mM imidazole), lysed in an EmulsiFlex C3 Homogenizer
(Avestin) for three passes at 15000-25000 psi, clarified for one
hour at 16000 g, 4.degree. C. The supernatant was then purified via
FPLC, then dialyzed overnight in 2.times.PBS. Precipitated protein
post-dialysis was recovered by adding 500 mM NaCl, mixed with
glycerol to yield a 50% glycerol solution for aliquoting and
storing at -20.degree. C.
[0531] Analysis of Dilution Effects and Pre-Heat-Kill Effects on
RNA Depolymerization
[0532] Experiments were performed with the lysate of GL17-109,
prepared as described herein. Lysates were prepared under a wide
range of conditions. Common across all conditions, reactions
received an added 150 mM potassium phosphate (pH=7.4), 100 mM KCl,
and 0.1 mM ZnCl.sub.2, and all of the following conditions were
tested with RNase R, Nuclease P1, or no exogenous nuclease added.
Both nuclease stock solutions were made as described previously.
Depolymerization reactions were carried out under 80% and 50%
lysate dilutions into water. These were screened with 0.5 or 0.31
mg/ml, respectively, of exogenous nuclease spiked in. Lysate
mixtures were also prepared by making a 1 mg/ml nuclease lysate and
mixing it into a non-spiked lysate yielding 0.064 and 0.04 mg/ml
nuclease in 80% dilution conditions, or 0.04 and 0.025 mg/ml
nuclease in 50% dilution conditions. Lastly, conditions were tested
where lysates that did not contain added nuclease were heat killed
at 70.degree. C. for 15 minutes and then mixed with either purified
nuclease, or the still-active 1 mg/ml nuclease lysate. Samples were
incubated at 37.degree. C. for 30 minutes and sampled by quenching
in 5.times. volumes of acetonitrile, and diluting into 1 ml of a 50
.mu.M adipic acid solution in 50 mM ammonium acetate. The quench
solution was then spun down, filtered through a 0.2 .mu.m filter
plate, and the filtrate was run on an LC-QQQ to measure NMPs,
nucleosides, and nucleobases.
[0533] Results
[0534] RNA nucleases (RNases) are known to have activity profiles
that span temperature profiles more broadly than many other enzymes
from mesophilic sources, occasionally showing activity up to
60.degree. C. despite originating from an organism evolved to grow
at 37.degree. C. To evaluate the impact of elevated temperatures on
RNA depolymerization in an E. coli lysate, two separate studies
were conducted. First, lysates from two separate strains were
incubated at temperatures greater than 37.degree. C. over the
course of an hour, with or without the addition of one of two
RNases--RNase R or RNase P1. The second study involved eliminating
a lysate to remove deleterious enzymatic activities and mixing this
inactive lysate with an active lysate to study potential impacts on
RNA depolymerization, or mixing exogenous nucleases into the
inactivated lysate.
[0535] Improved depolymerization was observed when lysates were
incubated at 60.degree. C. and 70.degree. C. (FIG. 13).
Depolymerization improved in several ways. When RNA depolymerizes,
it predominantly produces NMPs. In an active lysate, however,
theses NMPs continue to be degraded by other enzymes, with a very
large accumulation of two main products--guanosine and uracil.
Incubating lysates at 60.degree. C. and 70.degree. C. significantly
decreased the accumulation of these nucleobases, even in lysates
that did not receive exogenous RNase R or Nuclease P1 (FIG. 13). As
each mole of nucleobase correlates to an irreversible loss of NMPs
from RNA, this demonstrated improved NMP stability in lysates.
Additionally, when exogenous nuclease is added under these
temperature conditions, dramatic improvement in net NMP
accumulation is seen, as shown in FIG. 13 at 70.degree. C. with
RNase R added. Base hydrolysis of the GL17-109 lysate showed a
maximum concentration of 32.6 mM NMPs. Correcting for dilution of
the added nuclease, the accumulation of .about.22 mM NMPs seen at
70.degree. C. with RNase R is a 75% yield. Assuming that tRNA
represents about 15% of the total RNA pool and that it is
inaccessible to these nucleases, this would represent a 94% yield
of all accessible RNA. Depolymerization in GL17-086 was poorer
across the board than GL17-109, likely due to the greater
phosphorylytic activity (data not shown).
[0536] The impacts of pre-heat kill and lysate dilutions showed a
small benefit, but only under a more heavily diluted lysate. In
this study, two types of lysates were made--a "reagent" lysate,
which contains the RNA to be depolymerized, and the "catalyst"
lysate, containing exogenous nuclease (RNase R or Nuclease P1).
Many different conditions were evaluated, as shown in Table 14
below, to determine their impact on RNA depolymerization. Lysates
that were diluted to only 80% showed similar depolymerization
performance, regardless of whether portions of the lysate were
heat-killed or not. However, lysates that were diluted to 50%
performed slightly better when a large portion of the RNA was in an
inactivated lysate. Across all of the conditions tested at this
dilution, an average depolymerization yield improvement of 2.7% was
observed. The performance of the depolymerization reaction under
these conditions does not show a dramatic improvement, but by
performing equivalently or only slightly better, this does leave
the possibility open for implementing a pre-depolymerization heat
kill should other parts of the process require it, for example
halting growth of any unlysed cells that may remain in the culture
or reducing the protein content of the lysate if a pelleting step
is included after heat-treatment.
TABLE-US-00017 TABLE 14 Summary of RNA depolymerization yields
across varying mixtures of lysates, nucleases, and inactivated
lysates. Percent yields are based on a basis of 32.6 mM NMPs, as
quantified through lysate base hydrolysis of RNA. No Pre-Heat Kill
Pre-Heat Kill % Depol. % Depol. % Depol. Yield % Depol. Yield Yield
(Nuclease Yield (Nuclease Reaction (RNase R) P.sub.1) (RNase R)
P.sub.1) 80% background lysate + 0.5 26 40 27 40 mg/mL Nuclease 72%
background lysate + 8% of 1 26 33 22 30 mg/mL nuclease lysate
(0.064 mg/mL nuclease final) 75% background lysate + 5% of 1 29 31
24 29 mg/mL nuclease lysate (0.04 mg/mL nuclease final) 80%
background lysate - no 18 23 10 10 nuclease control 50% background
lysate + 0.31 41 49 44 56 mg/mL Nuclease 46% background lysate + 4%
of 1 33 41 40 36 mg/mL nuclease lysate (0.04 mg/mL nuclease final)
47.5% background lysate + 2.5% of 27 37 27 41 1 mg/mL nuclease
lysate (0.025 mg/mL nuclease final) 50% background lysate - no 20
34 10 12 nuclease control
Example 6: Cell-Free Production of RNA Using Various Nucleotide
Sources
[0537] Materials and Methods
[0538] Yeast RNA powder obtained from commercial sources was
dissolved in water at 45-60 g/L and depolymerized using 1.2 g/L P1
nuclease at 70.degree. C., pH 5.5-5.8 for 1 hour in the presence of
0.05 mM zinc chloride. The resulting depolymerized material was
clarified by centrifugation and filtered using a 10 kDa MWCO
filter. The resulting stream contained 5' nucleotide monophosphates
(NMPs) at a total concentration of .about.90-100 mM (.about.20-25
mM each AMP, CMP, GMP, and UMP).
[0539] E. coli BL21(DE3) derivatives carrying pBAD24-derived
vectors encoding individual kinase enzymes (TthCmk, PfPyrH, TmGmk,
AaNdk, and DgPPK2) were cultivated in fermentations with Korz media
supplemented with 50 mg/L carbenicillin using standard techniques
[Korz, D. J., Rinas, U., Hellmuth, K., Sanders, E. A., &
Deckwer, W. D. (1995). Simple fed-batch technique for high cell
density cultivation of Escherichia coli. Journal of biotechnology,
39(1), 59-65.]. Protein expression was induced by adding
L-arabinose. After harvest, lysates were prepared in 60 mM
phosphate buffer using high-pressure homogenization, resulting in
mixtures of approximately 40 g/L total protein.
[0540] E. coli BL21(DE3) derivatives carrying pUC19-derived vectors
containing one or more transcriptional templates (each consisting
of a T7 promoter, target sequence, and one or more transcriptional
terminators) encoding a 524 bp double-stranded RNA sequence. Cells
were cultivated in fermentations in Korz media using standard
techniques [Phue, J. N., Lee, S. J., Trinh, L., & Shiloach, J.
(2008). Modified Escherichia coli B (BL21), a superior producer of
plasmid DNA compared with Escherichia coli K (DH5alpha).
Biotechnology and bioengineering, 101(4), 831.]. After harvest,
lysates were created by high pressure homogenization, diluted, and
heat-treated following similar procedures.
[0541] E. coli BL21(DE3) derivatives carrying pBAD24-derived
vectors encoding thermostable T7 RNA polymerase enzymes were
cultivated, enzyme expression was induced, and lysates were
prepared using similar procedures. Polymerase enzymes were
partially purified using two steps of ammonium sulfate
fractionation.
[0542] Reactions were assembled in 30 mM phosphate buffer, pH 7
according to Table 15.
TABLE-US-00018 TABLE 15 Reaction Conditions Depolymerized
Nucleotide source cellular RNA NMPs NDPs Nucleotides 15% v/v 4 mM
each 4 mM each Magnesium sulfate 45 mM Sodium hexametaphosphate 13
mM Kinase lysates 2 g/L total protein Template DNA lysate 5.4% v/v
RNA polymerase 0.1 g/L
[0543] In assembling the cell-free reactions, lysates containing
kinase enzymes and template DNA were diluted, combined in equal
proportion, and mixed with reaction additives such as magnesium
sulfate and sodium hexametaphosphate. Lysates were incubated at
70.degree. C. for 15 minutes to inactivate other enzymatic
activities while preserving the activities of the overexpressed
kinases. Cell-free reactions were initiated by the addition of RNA
polymerase, incubated at 48.degree. C. for 1 hour, and analyzed
according to established protocols [Nwokeoji, A. O., Kilby, P. M.,
Portwood, D. E., & Dickman, M. J. (2016). RNASwift: A rapid,
versatile RNA extraction method free from phenol and chloroform.
Analytical biochemistry, 512, 36].
[0544] Results
[0545] Cell-free RNA synthesis reactions were performed using
cellular RNA, an equimolar mix of nucleoside 5'-monophosphates
(AMP, CMP, GMP, UMP), or an equimolar mix of 5' nucleoside
diphosphates (ADP, CDP, GDP, UDP). Similar titers of dsRNA product
were produced for each nucleotide source (FIG. 17).
[0546] These results demonstrate that the cell-free reactions
described herein can be used to synthesize RNA from multiple
sources of nucleotides, including cellular RNA, nucleoside
5'-monophosphates, and nucleoside diphosphates.
Example 7: Cell-Free Production of RNA Using Wild-Type RNA
Polymerase
[0547] Materials and Methods
[0548] E. coli BL21(DE3) derivatives carrying pBAD24-derived
vectors encoding hexahistidine-tagged thermostable or wild-type T7
RNA polymerase enzymes were cultivated, enzyme expression was
induced, and lysates were prepared using procedures described
herein. Polymerase enzymes were purified by fast protein liquid
chromatography (FPLC) as described herein. Cell-free reactions were
performed as described herein except that reactions were performed
at a range of temperatures (37-48.degree. C.) for 2 hours. Titers
of dsRNA product were quantified as described herein.
[0549] Results
[0550] Cell-free RNA synthesis reactions were performed using
wild-type T7 RNA polymerase or a thermostable mutant (FIG. 18).
Reactions performed with the thermostable mutant produced dsRNA
product at 37.degree. C. and 48.degree. C. In contrast, reactions
performed with the wild-type polymerase produced product at
37.degree. C. but not 48.degree. C.
[0551] These results demonstrate that the cell-free reactions
described herein do not require thermostable RNA polymerases
provided they are incubated at appropriate temperatures.
Example 8: Cell-Free Production of NTPs Using Various Nucleotide
Sources
[0552] Materials and Methods
[0553] Cell-free reactions were performed as described in Example
6, except the template DNA lysate and RNA polymerase were omitted.
Nucleotides were analyzed by HPLC using an adaptation of published
methods [de Korte, D., Haverkort, W. A., Roos, D., & van
Gennip, A. H. (1985). Anion-exchange high performance liquid
chromatography method for the quantitation of nucleotides in human
blood cells. Clinica chimica acta; international journal of
clinical chemistry, 148(3), 185.]
[0554] Results
Cell-free reactions to produce nucleotide 5'-triphosphates (NTPs:
ATP, CTP, GTP and UTP) were performed using cellular RNA, an
equimolar mix of nucleoside 5'-monophosphates (AMP, CMP, GMP, UMP),
or an equimolar mix of 5' nucleoside diphosphates (ADP, CDP, GDP,
UDP). Similar titers of each NTP were produced for each nucleotide
source (FIG. 19).
[0555] These results demonstrate that the cell-free reactions
described herein can also be used to produce NTPs simply by
omitting the RNA polymerase and DNA template. Similarly to the
cell-free reactions producing RNA described in Example 6, cell-free
reactions producing NTPs can utilize multiple sources of
nucleotides, including cellular RNA, nucleoside 5'-monophosphates,
and nucleoside diphosphates.
REFERENCES
[0556] 1. Maekewa K., Tsunasawa S., Dibo G., Sakiyama F. 1991.
Primary structure of nuclease P1 from Penicillium citrinum. Eur. J.
Biochem. 200:651-661. [0557] 2. Volbeda A., Lahm A., Sakiyama F.,
Suck D. 1991. Crystal structure of Penicillium citrinum P1 nuclease
at 2.8-A resolution. EMBO J. 10:1607-1618(1991) [0558] 3. Romier
C., Dominguez R., Lahm A., Dahl O., Suck D. 1998. Recognition of
single-stranded DNA by nuclease P1: high resolution crystal
structures of complexes with substrate analogs. Proteins 32:414-424
[0559] 4. Cheng Z. F., Deutscher M. P. 2002. Purification and
characterization of the Escherichia coli exoribonuclease RNase R.
Comparison with RNase II. J. Biol. Chem. 277:21624-21629. [0560] 5.
Zilhao R., Camelo L., Arraiano C. M. 1993. DNA sequencing and
expression of the gene rnb encoding Escherichia coli ribonuclease
II. Mol. Microbiol. 8:43-51 [0561] 6. March P. E., Ahnn J., Inouye
M. 1985. The DNA sequence of the gene (mc) encoding ribonuclease
III of Escherichia coli. Nucleic Acids Res. 13:4677-4685 [0562] 7.
Chen S. M., Takiff H. E., Barber A. M., Dubois G. C., Bardwell J.
C., Court D. L. 1990. Expression and characterization of RNase III
and Era proteins. Products of the mc operon of Escherichia coli. J.
Biol. Chem. 265:2888-2895 [0563] 8. Robertson H. D., Webster R. E.,
Zinder N. D. 1968. Purification and properties of ribonuclease III
from Escherichia coli. J. Biol. Chem. 243:82-91. [0564] 9. Molina
L., Bernal P., Udaondo Z., Segura A., Ramos J. L. 2013. Complete
Genome Sequence of a Pseudomonas putida Clinical Isolate, Strain
H8234. Genome Announc. 1:E00496-13; and Cheng, Z. F. and M. P.
Deutscher. 2002. Purification and characterization of the
Escherichia coli exoribonuclease RNAse R. Comparison with RNAse II.
J Biol Chem. 277(24). [0565] 10. Even S., Pellegrini O., Zig L.,
Labas V., Vinh J., Brechemmier-Baey D., Putzer H. 2005.
Ribonucleases J1 and J2: two novel endoribonucleases in B. subtilis
with functional homology to E. coli RNase E. Nucleic Acids Res.
33:2141-2152. [0566] 11. Li de la Sierra-Gallay I., Zig L., Jamalli
A., Putzer H. 2008. Structural insights into the dual activity of
RNase J. Nat. Struct. Mol. Biol. 15:206-212. [0567] 12. Ball T. K.,
Saurugger P. N., Benedick M. J. 1987. The extracellular nuclease
gene of Serratia marcescens and its secretion from Escherichia
coli. Gene 57:183-192. [0568] 13. Biedermann K., Jepsen P. K.,
Riise E., Svendsen I. 1989. Purification and characterization of a
Serratia marcescens nuclease produced by Escherichia coli.
Carlsberg Res. Commun. 54:17-27. [0569] 14. Shlyapnikov S. V.,
Lunin V. V., Perbandt M., Polyakov K. M., Lunin V. Y., Levdikov V.
M., Betzel C., Mikhailov A. M. 2000. Atomic structure of the
Serratia marcescens endonuclease at 1.1 A resolution and the enzyme
reaction mechanism. Acta Crystallogr. D 56:567-572. [0570] 15. Zuo
Y., Deutscher M. P. 2002. Mechanism of action of RNase T. I.
Identification of residues required for catalysis, substrate
binding, and dimerization. J. Biol. Chem. 277:50155-50159. [0571]
16. Zuo Y., Zheng H., Wang Y., Chruszcz M., Cymborowski M., Skarina
T., Savchenko A., Malhotra A., Minor W. 2007. Crystal structure of
RNase T, an exoribonuclease involved in tRNA maturation and end
turnover. Structure 15:417-428. [0572] 17. Huang S., Deutscher M.
P. 1992. Sequence and transcriptional analysis of the Escherichia
coli rnt gene encoding RNase T. J. Biol. Chem. 267:25609-25613.
[0573] 18. Chauhan A. K., Miczak A., Taraseviciene L., Apirion D.
1991. Sequencing and expression of the me gene of Escherichia coli.
Nucleic Acids Res. 19:125-129. [0574] 19. Cormack R. S., Genereaux
J. L., Mackie G. A. 1993. RNase E activity is conferred by a single
polypeptide: overexpression, purification, and properties of the
ams/rne/hmp1 gene product. Proc. Natl. Acad. Sci. U.S.A.
90:9006-9010. [0575] 20. Motomura., K., Hirota, R., Okada, M.,
Ikeda, T., Ishida, T., & Kuroda, A, (2014), A New Subfamily of
Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside
Monophosphate Phosphorylation and Nucleoside Diphosphate
Phosphorylation, Applied and Environmental Microbiology, 80(8),
2602-2608. http://doi.org/10.1128/AEM 0.03971-13 [0576] 21. Elkin,
S. R., Kumar, A., Price, C. W., & Columbus, L. (2013). A Broad
Specificity Nucleoside Kinase from Thermoplasma acidophilum.
Proteins, 81(4), 568-582. doi.org/10.1002/prot.24212 [0577] 22.
Hansen, T., Arnfors, L., Ladenstein, R., & Schonheit, P.
(2007). The phosphofructokinase-B (MJ0406) from Methanocaldococcus
jannaschii represents a nucleoside kinase with a broad substrate
specificity. Extremophiles, 11(1), 105. [0578] 23. Ota, H.,
Sakasegawa, S., Yasuda, Y., Imamura, S., & Tamura, T. (2008). A
novel nucleoside kinase from Burkholderia thailandensis: a member
of the phosphofructokinase B-type family of enzymes. The FEBS
journal, 275(23), 5865. [0579] 24. Tomoike, F., Nakagawa, N.,
Kuramitsu, S., & Masui, R. (2011). A single amino acid limits
the substrate specificity of Thermus thermophilus uridine-cytidine
kinase to cytidine. Biochemistry, 50(21), 4597. [0580] 25. Henne
A., Brueggemann H., Raasch C., Wiezer A., Hartsch T., Liesegang H.,
Johann A., Lienard T., Gohl O., Martinez-Arias R., Jacobi C.,
Starkuviene V., Schlenczeck S., Dencker S., Huber R., Klenk H.-P.,
Kramer W., Merkl R., Gottschalk G., Fritz H.-J. 2004. The genome
sequence of the extreme thermophile Thermus thermophilus. Nat.
Biotechnol. 22:547-553. [0581] 26. Tan Z W, Liu J, Zhang X F, Meng
F G, Zhang Y Z. Nan Fang Yi Ke Da Xue Xue Bao. 2010. Expression,
purification and enzymatic characterization of adenylate kinase of
Thermus thermophilus HB27 in Escherichia coli. January; 30(1):1-6
[0582] 27. Maeder D. L., Weiss R. B., Dunn D. M., Cherry J. L.,
Gonzalez J. M., DiRuggiero J., Robb F. T. 1999. Divergence of the
hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii
inferred from complete genomic sequences. Genetics 152:1299-1305.
[0583] 28. Methe, B. A., Nelson, K. E., Deming, J. W., Momen, B.,
Melamud, F., Zhang, X., Fraser, C. M. (2005). The psychrophilic
lifestyle as revealed by the genome sequence of Colwellia
psychrerythraea 34H through genomic and proteomic analyses.
Proceedings of the National Academy of Sciences of the United
States of America, 102(31), 10913-10918.
doi.org/10.1073/pnas.0504766102 [0584] 29. Medigue, C., Krin, E.,
Pascal, G., Barbe, V., Bernsel, A., Bertin, P. N., . . . Danchin,
A. (2005). Coping with cold: The genome of the versatile marine
Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. Genome
Research, 15(10), 1325-1335. doi.org/10.1101/gr.4126905 [0585] 30.
Ayala-del-Rio, H. L., Chain, P. S., Grzymski, J. J., Ponder, M. A.,
Ivanova, N., Bergholz, P. W., . . . Tiedje, J. M. (2010). The
Genome Sequence of Psychrobacter arcticus 273-4, a Psychroactive
Siberian Permafrost Bacterium, Reveals Mechanisms for Adaptation to
Low-Temperature Growth. Applied and Environmental Microbiology,
76(7), 2304-2312. doi.org/10.1128/AEM.02101-09 [0586] 31. Feil, H.,
Feil, W. S., Chain, P., Larimer, F., DiBartolo, G., Copeland, A., .
. . Lindow, S. E. (2005). Comparison of the complete genome
sequences of Pseudomonas syringae pv. syringae B728a and pv. tomato
DC3000. Proceedings of the National Academy of Sciences of the
United States of America, 102(31), 11064-11069.
doi.org/10.1073/pnas.0504930102 [0587] 32. Song, S., Inouye, S.,
Kawai, M., Fukami-Kobayashi, K., G , M., & Nakazawa, A. (1996).
Cloning and characterization of the gene encoding Halobacterium
halobium adenylate kinase. Gene, 175(1), 65-70. [0588] 33. Masui
R., Kurokawa K., Nakagawa N., Tokunaga F., Koyama Y., Shibata T.,
Oshima T., Yokoyama S., Yasunaga T., Kuramitsu S. Complete genome
sequence of Thermus thermophilus HB8. Submitted (November 2004) to
the EMBL/GenBank/DDBJ databases. [0589] 34. Ng, W. V., Kennedy, S.
P., Mahairas, G. G., Berquist, B., Pan, M., Shukla, H. D., . . .
DasSarma, S. (2000). Genome sequence of Halobacterium species
NRC-1. Proceedings of the National Academy of Sciences of the
United States of America, 97(22), 12176-42181. [0590] 35.
Marco-Marin C., Escamilla-Honrubia J. M., Rubio V. 2005. First-time
crystallization and preliminary X-ray crystallographic analysis of
a bacterial-archaeal type UMP kinase, a key enzyme in microbial
pyrimidine biosynthesis. Biochim. Biophys. Acta 1747:271-275.
[0591] 36. Marco-Marin C., Escamilla-Honrubia J. M., Rubio V. 2005.
First-time crystallization and preliminary X-ray crystallographic
analysis of a bacterial-archaeal type UMP kinase, a key enzyme in
microbial pyrimidine biosynthesis. Biochim. Biophys. Acta
1747:271-275. [0592] 37. Jensen, K. S., Johansson, E., &.
Jensen, K. F. (2007). Structural and enzymatic investigation of the
Sulfolobus solfataricus uridylate kinase shows competitive UTP
inhibition and the lack of GTP stimulation. Biochemistry, 46(10),
2745-2757. [0593] 38. Nelson K. E., Clayton R. A., Gill S. R.,
Gwinn M. L., Dodson R. J., Haft D. H., Hickey E. K., Peterson J.
D., Nelson W. C., Ketchum K. A., McDonald L. A., Utterback T. R.,
Malek J. A., Linher K. D., Garrett M. M., Stewart A. M., Cotton M.
D., Pratt M. S. Fraser C. M. 1999. Evidence for lateral gene
transfer between Archaea and Bacteria from genome sequence of
Thermotoga maritima. Nature 399:323-329. [0594] 39. Riley, M.,
Staley, J. T., Danchin, A., Wang, T. Z., Brettin, T. S., Hauser, L.
J., . . . Thompson, L. S. (2008). Genomics of an extreme
psychrophile, Psychromonas ingrahamii. BMC Genomics, 9, 210.
doi.org/10.1186/1471-2164-9-210 [0595] 40. Ishibashi, M., Tokunaga,
H., Hiratsuka, K., Yonezawa, Y., Tsurumaru, H., Arakawa, T., &
Tokunaga, M. (2001). NaCl-activated nucleoside diphosphate kinase
from extremely halophilic archaeon, Halobacterium salinarum,
maintains native conformation without salt. FEBS letters, 493(2-3),
134. [0596] 41. Polosina, Y. Y., Zamyatkin, D. F., Kostyukova, A.
S., Filimonov, V. V., & Fedorov, O. V. (2002). Stability of
Natrialba magadii NDP kinase: comparisons with other halophilic
proteins. Extremophiles: life under extreme conditions, 6(2), 135.
[0597] 42. Polosina, Y. Y., Zamyatkin, D. F., Kostyukova, A. S.,
Filimonov, V. V., & Fedorov, O. V. (2002), Stability of
Natrialba magadii NDP kinase: comparisons with other halophilic
proteins. Extremophiles: life under extreme conditions, 6(2), 135.
[0598] 43. Udaondo, Z., Molina, L, Daniels, C., Gomez, M. J.,
Molina-Henares, M. A., Matilla, M. A., . . . Ramos, J. L. (2013).
Metabolic potential of the organic-solvent tolerant Pseudomonas
putida DOT-T1E deduced from its annotated genome. Microbial
Biotechnology, 6(5), 598-611,
http://doi.org/10.1111/1751-7915.12061 [0599] 44. Nolling, J.,
Breton, G., Omelchenko, Ni. V., Makarova, K. S., Zeng, Q., Gibson,
R., Smith, D. R. (2001). Genome Sequence and Comparative Analysis
of the Solvent-Producing Bacterium Clostridium acetobutylicum.
Journal of Bacteriology, 183(16), 4823-4838.
http://doi.org/10.1128/JB.183.16.4823-4838.2001 [0600] 45. Brune,
M., Schumann, R., & Wittinghofer, F. (1985). Cloning and
sequencing of the adenylate kinase gene (adk) of Escherichia coli.
Nucleic Acids Research, 13(19), 7139-7151. [0601] 46. Pel, H. J.,
de Winde., J. H., Archer, D. B., Dyer, P. S., Hofmann, G., Schaap,
P. J., . . . & Andersen, M. R. (2007). Genome sequencing and
analysis of the versatile cell factory Aspergillus niger CBS 513,
88. Nature biotechnology, 25(2), 221. [0602] 47. Magdolen, V.,
Ochsner, U., & Bandlow, W. (1987). The complete nucleotide
sequence of the gene coding for yeast adenylate kinase. Current
genetics, 12(6), 405. [0603] 48. Pedersen, S., Skouv, J., Kajitani,
M., & Ishihama. (1984). Transcriptional organization of the
rpsA operon of Escherichia coli. Molecular & general genetics:
MGG, 196(1), 135. [0604] 49. Smallshaw, J., & Kelln, R. A.
(1992). Cloning, nucleotide sequence and expression of the
Escherichia coli K-12 pyrH gene encoding UMP kinase. Genetics (Life
Sci. Adv.), 11, 59-65, [0605] 50. Liljelund, P., Sanni, A.,
Friesen, J. D., & Lacroute, F. (1989). Primary structure of the
S. cerevisiae gene encoding uridine monophosphokinase. Biochemical
and biophysical research communications, 165(1), 464. [0606] 51.
Gentry, D., Bengra, C., Ikehara, K., & Cashel, M. (1993).
Guanylate kinase of Escherichia coli K-12. The Journal of
biological chemistry, 268(19), 14316. [0607] 52. Konrad, M. (1992).
Cloning and expression of the essential gene for guanylate kinase
from yeast. The Journal of biological chemistry, 267(36), 25652.
[0608] 53. Hama, H., Almaula, N., Lerner, C. G., Inouye, S., &
Inouye, M. (1991). Nucleoside diphosphate kinase from Escherichia
coli; its overproduction and sequence comparison with eukaryotic
enzymes. Gene, 105(1), 31. [0609] 54. Besir, H., Zeth, K., Bracher,
A., Heider, U., Iishibashi, M., Tokunaga, M., & Oesterhelt, D.
(2005). Structure of a halophilic nucleoside diphosphate kinase
from Halobacterium salinarum. FEBS letters, 579(29), 6595. [0610]
55. Deutscher, M. & Reuven N. (1991). Enzymatic basis for
hydrolytic versus phosphorolytic mRNA degradation in Escherichia
coli and Bacillus subtilis. PNAS, 88, 3277-3280, [0611] 56.
Nwokeji, A. O., Kilby, P. M., Portwood, D. E., & Dickman, M. J.
(2016). RNASwift: A rapid, versatile RNA extraction method free
from phenol and chloroform. Analytical Biochemistry, 512, 36-46.
[0612] 57. Mohanty, B. K., Giladi, H., Maples, V. F., &
Kushner, S. R. (2008). Analysis of RNA decay, processing, and
polyadenylation in Escherichia coli and other prokaryotes. Methods
in Enzymology, 447, 3-29. [0613] 58. Korz, D. J., Rinas, U.,
Hellmuth, K., Sanders, E. A., & Deckwer, W. D. (1995). Simple
fed-batch technique for high cell density cultivation of
Escherichia coli. Journal of biotechnology, 39(1), 59-654 [0614]
59. Phue, J. N., Lee, S. J., Trinh, L., & Shiloach, J. (2008).
Modified Escherichia coli B (BL21), a superior producer of plasmid
DNA compared with Escherichia coli K (DH5alpha). Biotechnology and
bioengineering, 101(4), 831. [0615] 60. de Korte, D., Haverkort, W.
A., Roos, D., & van Gennip, A. H. (1985). Anion-exchange high
performance liquid chromatography method for the quantitation of
nucleotides in human blood cells. Clinica chimica acta;
international journal of clinical chemistry, 148(3), 185.]
TABLE-US-00019 [0615] Sequences Deinococcus geothermalis DSM 11300
PPK2 (SEQ ID NO: 1)
MQLDRYRVPPGQRVRLSNWPTDDDGGLSKAEGEALLPDLQQRLANLQERL
YAESQQALLIVLQARDAGGKDGTVKHVIGAFNPSGVQVSNFKVPTEEERA
HDFLWRIHRQTPRLGMIGVFNRSQYEDVLVTRVHHLIDDQTAQRRLKHIC
AFESLLTDSGTRIVKFYLHISPEEQKKRLEARLADPSKHWKFNPGDLQER
AHWDAYTAVYEDVLTTSTPAAPWYVVPADRKWFRNLLVSQILVQTLEEMN PQFPAPAFNAADLRIV
Meiothermus ruber DM 1279 PPK2 (SEQ ID NO: 2)
MGFCSIEFLMGAQMKKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALA
VLNRRLEKLQELLYAEGQHKVLVVLQAMDAGGKDGTIRVVFDGVNPSGVR
VASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYEDVLVVRVKNLV
PQQVWQKRYRHIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPE
KRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEYAPWYVIPANKNWYRNW
LVSHILVETLEGLAMQYPQPETASEKIVIE Meiothermus silvanus DSM 9946 PPK2
(SEQ ID NO: 3) MAKTIGATLNLQDIDPRSTPGFNGDKEKALALLEKLTARLDELQEQLYAE
HQHRVLVILQGMDTSGKDGTIRHVFKNVDPLGVRVVAFKAPTPPELERDY
LWRVHQHVPANGELVIFNRSHYEDVLVARVHNLVPPAIWSRRYDHINAFE
KMLVDEGTTVLKFFLHISKEEQKKRLLERLVEADKHWKFDPQDLVERGYW
EDYMEAYQDVLDKTHTQYAPWHVIPADRKWYRNLQVSRLLVEALEGLRMK
YPRPKLNIPRLKSELEKM Thermosynechococcus elongatus BP-1 PPK2 (SEQ ID
NO: 4) MIPQDFLDEINPDRYIVPAGGNFHWKDYDPGDTAGLKSKVEAQELLAAGI
KKLAAYQDVLYAQNIYGLLIIFQAMDAAGKDSTIKHVMSGLNPQACRVYS
FKAPSAEELDHDFLWRANRALPERGCIGIFNRSYYEEVLVVRVHPDLLNR
QQLPPETKTKHIWKERFEDINHYERYLTRNGILILKFFLHISKAEQKKRF
LERISRPEKNWKFSIEDVRDRAHWDDYQQAYADVFRHTSTKWAPWHIIPA
NHKWFARLMVAHFIYQKLASLNLHYPMLSEAHREQLLEAKALLENEPDED Anaerolinea
thermophila UNI-1 PPK2 (SEQ ID NO: 5)
MGEAMERYFIKPGEKVRLKDWSPDPPKDFEGDKESTRAAVAELNRKLEVL
QERLYAERKHKVLVILQGMDTSGKDGVIRSVFEGVNPQGVKVANFKVPTQ
EELDHDYLWRVHKVVPGKGEIVIFNRSHYEDVLVVRVHNLVPPEVWKKRY
EQINQFERLLHETGTTILKFFLFISREEQKQRLLERLADPAKHWKFNPGD
LKERALWEEYEKAYEDVLSRTSTEYAPWILVPADKKWYRDWVISRVLVET
LEGLEIQLPPPLADAETYRRQLLEEDAPESR Caldilinea aerophila DSM 14535 PPK2
(SEQ ID NO: 6) MDVDRYRVPPGSTIHLSQWPPDDRSLYEGDKKQGKQDLSALNRRLETLQE
LLYAEGKHKVLIILQGMDTSGKDGVIRHVFNGVNPQGVKVASFKVPTAVE
LAHDFLWRIHRQTPGSGEIVIFNRSHYEDVLVVRVHGLVPPEVWARRYEH
INAFEKLLVDEGTTILKFFLHISKEEQRQRLLERLEMPEKRWKFSVGDLA
ERKRWDEYMAAYEAVLSKTSTEYAPWYIVPSDRKWYRNLVISHVIINALE
GLNMRYPQPEDIAFDTIVIE Chlorobaculum tepidum TLS PPK2 (SEQ ID NO: 7)
MKLDLDAFRIQPGKKPNLAKRPTRIDPVYRSKGEYHELLANHVAELSKLQ
NVLYADNRYAILLIFQAMDAAGKDSAIKHVMSGVNPQGCQVYSFKHPSAT
ELEHDFLWRTNCVLPERGRIGIFNRSYYEEVLVVRVHPEILEMQNIPHNL
AHNGKVWDHRYRSIVSHEQHLHCNGTRIVKFYLHLSKEEQRKRFLERIDD
PNKNWKFSTADLEERKFWDQYMEAYESCLQETSTKDSPWFAVPADDKKNA
RLIVSRIVLDTLESLNLKYPEPSPERRKELLDIRKRLENPENGK Oceanithermus
profundus DSM 14977 PPK2 (SEQ ID NO: 8)
MDVSRYRVPPGSGFDPEAWPTREDDDFAGGKKEAKKELARLAVRLGELQA
RLYAEGRQALLIVLQGMDTAGKDGTIRHVFRAVNPQGVRVTSFKKPTALE
LAHDYLWRVHRHAPARGEIGIFNRSHYEDVLVVRVHELVPPEVWGRRYDH
INAFERLLADEGTRIVKFFLHISKDEQKRRLEARLENPRKHWKFNPADLS
ERARWGDYAAAYAEALSRTSSDRAPWYAVPADRKWQRNRIVAQVLVDALE
AMDPRFPRVDFDPASVRVE Roseiflexus castenholzii DSM 13941 PPK2 (SEQ ID
NO: 9) MYAQRVVPGMRVRLHDIDPDANGGLNKDEGRARFAELNAELDVMQEELYA
AGIHALLLILQGMDTAGKDGAIRNVMLNLNPQGCRVESFKVPTEEELAHD
FLWRVHRVVPRKGMVGVFNRSHYEDVLVVRVHSLVPESVWRARYDQINAF
ERLLADTGTIIVKCFLHISKEEQEQRLLARERDVSKAWKLSAGDWRERAF
WDDYMAAYEEALTRCSTDYAPWYIIPANRKWYRDLAISEALVETLRPYRD
DWRRALDAMSRARRAELEAFRAEQHAMEGRPQGAGGVSRR Roseiflexus sp. RS-1 PPK2
(SEQ ID NO: 10) MHYAHTVIPGTQVRLRDIDPDASGGLTKDEGRERFASFNATLDAMQEELY
AAGVHALLLILQGMDTAGKDGAIRNVMHNLNPQGCRVESFKVPTEEELAH
DFLWRVHKVVPRKGMVGVFNRSHYEDVLVVRVHSLVPEHVWRARYDQINA
FERLLTDTGTIIVKCFLHISKDEQEKRLLAREQDVTKAWKLSAGDWRERE
RWDEYMAAYEEALTRCSTEYAPWYIIPANRKWYRDLAISEVLVETLRPYR
DDWQRALDAMSQARLAELKAFRHQQTAGATRL Truepera radiovictrix DSM 17093
PPK2 (SEQ ID NO: 11)
MSQGSAKGLGKLDKKVYARELALLQLELVKLQGWIKAQGLKVVVLFEGRD
AAGKGSTITRITQPLNPRVCRVVALGAPTERERTQWYFQRYVHHLPAAGE
MVLFDRSWYNRAGVERVMGFCTEAEYREFLHACPTFERLLLDAGIILIKY
WFSVSAAEQERRMRRRNENPAKRWKLSPMDLEARARWVAYSKAKDAMFYH
TDTKASPWYVVNAEDKRRAHLSCIAHLLSLIPYEDLTPPPLEMPPRDLAG
ADEGYERPDKAHQTWVPDYVPPTR Thermus thermophilus Adk (SEQ ID NO: 12)
MDVGQAVIFLGPPGAGKGTQASRLAQELGFKKLSTGDILRDHVARGTPLG
ERVRPIMERGDLVPDDLILELIREELAERVIFDGFPRTLAQAEALDRLLS
ETGTRLLGVVLVEVPEEELVRRILRRAELEGRSDDNEETVRRRLEVYREK
TEPLVGYYEARGVLKRVDGLGTPDEVYARIRAALGI Thermus thermophilus Cmk (SEQ
ID NO: 13) MRGIVTIDGPSASGKSSVARRVAAALGVPYLSSGLLYRAAAFLALRAGVD
PGDEEGLLALLEGLGVRLLAQAEGNRVLADGEDLTSFLHTPEVDRVVSAV
ARLPGVRAWVNRRLKEVPPPFVAEGRDMGTAVFPEAAHKFYLTASPEVRA
WRRARERPQAYEEVLRDLLRRDERDKAQSAPAPDALVLDTGGMTLDEVVA WVLAHIRR
Pyrococcus furiosus PyrH (SEQ ID NO: 14)
MRIVFDIGGSVLVPENPDIDFIKEIAYQLTKVSEDHEVAVVVGGGKLARK
YIEVAEKFNSSETFKDFIGIQITRANAMLLIAALREKAYPVVVEDFWEAW
KAVQLKKIPVMGGTHPGHTTDAVAALLAEFLKADLLVVITNVDGVYTADP
KKDPTAKKIKKMKPEELLEIVGKGIEKAGSSSVIDPLAAKIIARSGIKTI
VIGKEDAKDLFRVIKGDHNGTTIEP Thermotoga maritima Gmk (SEQ ID NO: 15)
MKGQLFVICGPSGAGKTSIIKEVLKRLDNVVFSVSCTTRPKRPHEEDGKD
YFFITEEEFLKRVERGEFLEWARVHGHLYGTLRSFVESHINEGKDVVLDI
DVQGALSVKKKYSNTVFIYVAPPSYADLRERILKRGTEKEADVLVRLENA
KWELMFMDEFDYIVVNENLEDAVEMVVSIVRSERAKVTRNQDKIERFKME VKGWKKL Aquifex
aeolicus Ndk (SEQ ID NO: 16)
MAVERTLIIVKPDAMEKGALGKILDRFIQEGFQIKALKMFRFTPEKAGEF
YYVHRERPFFQELVEFMSSGPVVAAVLEGEDAIKRVREIIGPTDSEEARK
VAPNSIRAQFGTDKGKNAIHASDSPESAQYEICFIFSGLEIV
[0616] All references, patents and patent applications disclosed
herein are incorporated by reference with respect to the subject
matter for which each is cited, which in some cases may encompass
the entirety of the document.
[0617] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0618] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0619] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
[0620] The terms "about" and "substantially" preceding a numerical
value mean.+-.10% of the recited numerical value.
[0621] Where a range of values is provided, each value between the
upper and lower ends of the range are specifically contemplated and
described herein.
Sequence CWU 1
1
161266PRTDeinococcus geothermalis 1Met Gln Leu Asp Arg Tyr Arg Val
Pro Pro Gly Gln Arg Val Arg Leu1 5 10 15Ser Asn Trp Pro Thr Asp Asp
Asp Gly Gly Leu Ser Lys Ala Glu Gly 20 25 30Glu Ala Leu Leu Pro Asp
Leu Gln Gln Arg Leu Ala Asn Leu Gln Glu 35 40 45Arg Leu Tyr Ala Glu
Ser Gln Gln Ala Leu Leu Ile Val Leu Gln Ala 50 55 60Arg Asp Ala Gly
Gly Lys Asp Gly Thr Val Lys His Val Ile Gly Ala65 70 75 80Phe Asn
Pro Ser Gly Val Gln Val Ser Asn Phe Lys Val Pro Thr Glu 85 90 95Glu
Glu Arg Ala His Asp Phe Leu Trp Arg Ile His Arg Gln Thr Pro 100 105
110Arg Leu Gly Met Ile Gly Val Phe Asn Arg Ser Gln Tyr Glu Asp Val
115 120 125Leu Val Thr Arg Val His His Leu Ile Asp Asp Gln Thr Ala
Gln Arg 130 135 140Arg Leu Lys His Ile Cys Ala Phe Glu Ser Leu Leu
Thr Asp Ser Gly145 150 155 160Thr Arg Ile Val Lys Phe Tyr Leu His
Ile Ser Pro Glu Glu Gln Lys 165 170 175Lys Arg Leu Glu Ala Arg Leu
Ala Asp Pro Ser Lys His Trp Lys Phe 180 185 190Asn Pro Gly Asp Leu
Gln Glu Arg Ala His Trp Asp Ala Tyr Thr Ala 195 200 205Val Tyr Glu
Asp Val Leu Thr Thr Ser Thr Pro Ala Ala Pro Trp Tyr 210 215 220Val
Val Pro Ala Asp Arg Lys Trp Phe Arg Asn Leu Leu Val Ser Gln225 230
235 240Ile Leu Val Gln Thr Leu Glu Glu Met Asn Pro Gln Phe Pro Ala
Pro 245 250 255Ala Phe Asn Ala Ala Asp Leu Arg Ile Val 260
2652280PRTMeiothermus ruber 2Met Gly Phe Cys Ser Ile Glu Phe Leu
Met Gly Ala Gln Met Lys Lys1 5 10 15Tyr Arg Val Gln Pro Asp Gly Arg
Phe Glu Leu Lys Arg Phe Asp Pro 20 25 30Asp Asp Thr Ser Ala Phe Glu
Gly Gly Lys Gln Ala Ala Leu Glu Ala 35 40 45Leu Ala Val Leu Asn Arg
Arg Leu Glu Lys Leu Gln Glu Leu Leu Tyr 50 55 60Ala Glu Gly Gln His
Lys Val Leu Val Val Leu Gln Ala Met Asp Ala65 70 75 80Gly Gly Lys
Asp Gly Thr Ile Arg Val Val Phe Asp Gly Val Asn Pro 85 90 95Ser Gly
Val Arg Val Ala Ser Phe Gly Val Pro Thr Glu Gln Glu Leu 100 105
110Ala Arg Asp Tyr Leu Trp Arg Val His Gln Gln Val Pro Arg Lys Gly
115 120 125Glu Leu Val Ile Phe Asn Arg Ser His Tyr Glu Asp Val Leu
Val Val 130 135 140Arg Val Lys Asn Leu Val Pro Gln Gln Val Trp Gln
Lys Arg Tyr Arg145 150 155 160His Ile Arg Glu Phe Glu Arg Met Leu
Ala Asp Glu Gly Thr Thr Ile 165 170 175Leu Lys Phe Phe Leu His Ile
Ser Lys Asp Glu Gln Arg Gln Arg Leu 180 185 190Gln Glu Arg Leu Asp
Asn Pro Glu Lys Arg Trp Lys Phe Arg Met Gly 195 200 205Asp Leu Glu
Asp Arg Arg Leu Trp Asp Arg Tyr Gln Glu Ala Tyr Glu 210 215 220Ala
Ala Ile Arg Glu Thr Ser Thr Glu Tyr Ala Pro Trp Tyr Val Ile225 230
235 240Pro Ala Asn Lys Asn Trp Tyr Arg Asn Trp Leu Val Ser His Ile
Leu 245 250 255Val Glu Thr Leu Glu Gly Leu Ala Met Gln Tyr Pro Gln
Pro Glu Thr 260 265 270Ala Ser Glu Lys Ile Val Ile Glu 275
2803268PRTMeiothermus silvanus 3Met Ala Lys Thr Ile Gly Ala Thr Leu
Asn Leu Gln Asp Ile Asp Pro1 5 10 15Arg Ser Thr Pro Gly Phe Asn Gly
Asp Lys Glu Lys Ala Leu Ala Leu 20 25 30Leu Glu Lys Leu Thr Ala Arg
Leu Asp Glu Leu Gln Glu Gln Leu Tyr 35 40 45Ala Glu His Gln His Arg
Val Leu Val Ile Leu Gln Gly Met Asp Thr 50 55 60Ser Gly Lys Asp Gly
Thr Ile Arg His Val Phe Lys Asn Val Asp Pro65 70 75 80Leu Gly Val
Arg Val Val Ala Phe Lys Ala Pro Thr Pro Pro Glu Leu 85 90 95Glu Arg
Asp Tyr Leu Trp Arg Val His Gln His Val Pro Ala Asn Gly 100 105
110Glu Leu Val Ile Phe Asn Arg Ser His Tyr Glu Asp Val Leu Val Ala
115 120 125Arg Val His Asn Leu Val Pro Pro Ala Ile Trp Ser Arg Arg
Tyr Asp 130 135 140His Ile Asn Ala Phe Glu Lys Met Leu Val Asp Glu
Gly Thr Thr Val145 150 155 160Leu Lys Phe Phe Leu His Ile Ser Lys
Glu Glu Gln Lys Lys Arg Leu 165 170 175Leu Glu Arg Leu Val Glu Ala
Asp Lys His Trp Lys Phe Asp Pro Gln 180 185 190Asp Leu Val Glu Arg
Gly Tyr Trp Glu Asp Tyr Met Glu Ala Tyr Gln 195 200 205Asp Val Leu
Asp Lys Thr His Thr Gln Tyr Ala Pro Trp His Val Ile 210 215 220Pro
Ala Asp Arg Lys Trp Tyr Arg Asn Leu Gln Val Ser Arg Leu Leu225 230
235 240Val Glu Ala Leu Glu Gly Leu Arg Met Lys Tyr Pro Arg Pro Lys
Leu 245 250 255Asn Ile Pro Arg Leu Lys Ser Glu Leu Glu Lys Met 260
2654300PRTThermosynechococcus elongatus 4Met Ile Pro Gln Asp Phe
Leu Asp Glu Ile Asn Pro Asp Arg Tyr Ile1 5 10 15Val Pro Ala Gly Gly
Asn Phe His Trp Lys Asp Tyr Asp Pro Gly Asp 20 25 30Thr Ala Gly Leu
Lys Ser Lys Val Glu Ala Gln Glu Leu Leu Ala Ala 35 40 45Gly Ile Lys
Lys Leu Ala Ala Tyr Gln Asp Val Leu Tyr Ala Gln Asn 50 55 60Ile Tyr
Gly Leu Leu Ile Ile Phe Gln Ala Met Asp Ala Ala Gly Lys65 70 75
80Asp Ser Thr Ile Lys His Val Met Ser Gly Leu Asn Pro Gln Ala Cys
85 90 95Arg Val Tyr Ser Phe Lys Ala Pro Ser Ala Glu Glu Leu Asp His
Asp 100 105 110Phe Leu Trp Arg Ala Asn Arg Ala Leu Pro Glu Arg Gly
Cys Ile Gly 115 120 125Ile Phe Asn Arg Ser Tyr Tyr Glu Glu Val Leu
Val Val Arg Val His 130 135 140Pro Asp Leu Leu Asn Arg Gln Gln Leu
Pro Pro Glu Thr Lys Thr Lys145 150 155 160His Ile Trp Lys Glu Arg
Phe Glu Asp Ile Asn His Tyr Glu Arg Tyr 165 170 175Leu Thr Arg Asn
Gly Ile Leu Ile Leu Lys Phe Phe Leu His Ile Ser 180 185 190Lys Ala
Glu Gln Lys Lys Arg Phe Leu Glu Arg Ile Ser Arg Pro Glu 195 200
205Lys Asn Trp Lys Phe Ser Ile Glu Asp Val Arg Asp Arg Ala His Trp
210 215 220Asp Asp Tyr Gln Gln Ala Tyr Ala Asp Val Phe Arg His Thr
Ser Thr225 230 235 240Lys Trp Ala Pro Trp His Ile Ile Pro Ala Asn
His Lys Trp Phe Ala 245 250 255Arg Leu Met Val Ala His Phe Ile Tyr
Gln Lys Leu Ala Ser Leu Asn 260 265 270Leu His Tyr Pro Met Leu Ser
Glu Ala His Arg Glu Gln Leu Leu Glu 275 280 285Ala Lys Ala Leu Leu
Glu Asn Glu Pro Asp Glu Asp 290 295 3005281PRTAnaerolinea
thermophila 5Met Gly Glu Ala Met Glu Arg Tyr Phe Ile Lys Pro Gly
Glu Lys Val1 5 10 15Arg Leu Lys Asp Trp Ser Pro Asp Pro Pro Lys Asp
Phe Glu Gly Asp 20 25 30Lys Glu Ser Thr Arg Ala Ala Val Ala Glu Leu
Asn Arg Lys Leu Glu 35 40 45Val Leu Gln Glu Arg Leu Tyr Ala Glu Arg
Lys His Lys Val Leu Val 50 55 60Ile Leu Gln Gly Met Asp Thr Ser Gly
Lys Asp Gly Val Ile Arg Ser65 70 75 80Val Phe Glu Gly Val Asn Pro
Gln Gly Val Lys Val Ala Asn Phe Lys 85 90 95Val Pro Thr Gln Glu Glu
Leu Asp His Asp Tyr Leu Trp Arg Val His 100 105 110Lys Val Val Pro
Gly Lys Gly Glu Ile Val Ile Phe Asn Arg Ser His 115 120 125Tyr Glu
Asp Val Leu Val Val Arg Val His Asn Leu Val Pro Pro Glu 130 135
140Val Trp Lys Lys Arg Tyr Glu Gln Ile Asn Gln Phe Glu Arg Leu
Leu145 150 155 160His Glu Thr Gly Thr Thr Ile Leu Lys Phe Phe Leu
Phe Ile Ser Arg 165 170 175Glu Glu Gln Lys Gln Arg Leu Leu Glu Arg
Leu Ala Asp Pro Ala Lys 180 185 190His Trp Lys Phe Asn Pro Gly Asp
Leu Lys Glu Arg Ala Leu Trp Glu 195 200 205Glu Tyr Glu Lys Ala Tyr
Glu Asp Val Leu Ser Arg Thr Ser Thr Glu 210 215 220Tyr Ala Pro Trp
Ile Leu Val Pro Ala Asp Lys Lys Trp Tyr Arg Asp225 230 235 240Trp
Val Ile Ser Arg Val Leu Val Glu Thr Leu Glu Gly Leu Glu Ile 245 250
255Gln Leu Pro Pro Pro Leu Ala Asp Ala Glu Thr Tyr Arg Arg Gln Leu
260 265 270Leu Glu Glu Asp Ala Pro Glu Ser Arg 275
2806270PRTCaldilinea aerophila 6Met Asp Val Asp Arg Tyr Arg Val Pro
Pro Gly Ser Thr Ile His Leu1 5 10 15Ser Gln Trp Pro Pro Asp Asp Arg
Ser Leu Tyr Glu Gly Asp Lys Lys 20 25 30Gln Gly Lys Gln Asp Leu Ser
Ala Leu Asn Arg Arg Leu Glu Thr Leu 35 40 45Gln Glu Leu Leu Tyr Ala
Glu Gly Lys His Lys Val Leu Ile Ile Leu 50 55 60Gln Gly Met Asp Thr
Ser Gly Lys Asp Gly Val Ile Arg His Val Phe65 70 75 80Asn Gly Val
Asn Pro Gln Gly Val Lys Val Ala Ser Phe Lys Val Pro 85 90 95Thr Ala
Val Glu Leu Ala His Asp Phe Leu Trp Arg Ile His Arg Gln 100 105
110Thr Pro Gly Ser Gly Glu Ile Val Ile Phe Asn Arg Ser His Tyr Glu
115 120 125Asp Val Leu Val Val Arg Val His Gly Leu Val Pro Pro Glu
Val Trp 130 135 140Ala Arg Arg Tyr Glu His Ile Asn Ala Phe Glu Lys
Leu Leu Val Asp145 150 155 160Glu Gly Thr Thr Ile Leu Lys Phe Phe
Leu His Ile Ser Lys Glu Glu 165 170 175Gln Arg Gln Arg Leu Leu Glu
Arg Leu Glu Met Pro Glu Lys Arg Trp 180 185 190Lys Phe Ser Val Gly
Asp Leu Ala Glu Arg Lys Arg Trp Asp Glu Tyr 195 200 205Met Ala Ala
Tyr Glu Ala Val Leu Ser Lys Thr Ser Thr Glu Tyr Ala 210 215 220Pro
Trp Tyr Ile Val Pro Ser Asp Arg Lys Trp Tyr Arg Asn Leu Val225 230
235 240Ile Ser His Val Ile Ile Asn Ala Leu Glu Gly Leu Asn Met Arg
Tyr 245 250 255Pro Gln Pro Glu Asp Ile Ala Phe Asp Thr Ile Val Ile
Glu 260 265 2707294PRTChlorobaculum tepidum 7Met Lys Leu Asp Leu
Asp Ala Phe Arg Ile Gln Pro Gly Lys Lys Pro1 5 10 15Asn Leu Ala Lys
Arg Pro Thr Arg Ile Asp Pro Val Tyr Arg Ser Lys 20 25 30Gly Glu Tyr
His Glu Leu Leu Ala Asn His Val Ala Glu Leu Ser Lys 35 40 45Leu Gln
Asn Val Leu Tyr Ala Asp Asn Arg Tyr Ala Ile Leu Leu Ile 50 55 60Phe
Gln Ala Met Asp Ala Ala Gly Lys Asp Ser Ala Ile Lys His Val65 70 75
80Met Ser Gly Val Asn Pro Gln Gly Cys Gln Val Tyr Ser Phe Lys His
85 90 95Pro Ser Ala Thr Glu Leu Glu His Asp Phe Leu Trp Arg Thr Asn
Cys 100 105 110Val Leu Pro Glu Arg Gly Arg Ile Gly Ile Phe Asn Arg
Ser Tyr Tyr 115 120 125Glu Glu Val Leu Val Val Arg Val His Pro Glu
Ile Leu Glu Met Gln 130 135 140Asn Ile Pro His Asn Leu Ala His Asn
Gly Lys Val Trp Asp His Arg145 150 155 160Tyr Arg Ser Ile Val Ser
His Glu Gln His Leu His Cys Asn Gly Thr 165 170 175Arg Ile Val Lys
Phe Tyr Leu His Leu Ser Lys Glu Glu Gln Arg Lys 180 185 190Arg Phe
Leu Glu Arg Ile Asp Asp Pro Asn Lys Asn Trp Lys Phe Ser 195 200
205Thr Ala Asp Leu Glu Glu Arg Lys Phe Trp Asp Gln Tyr Met Glu Ala
210 215 220Tyr Glu Ser Cys Leu Gln Glu Thr Ser Thr Lys Asp Ser Pro
Trp Phe225 230 235 240Ala Val Pro Ala Asp Asp Lys Lys Asn Ala Arg
Leu Ile Val Ser Arg 245 250 255Ile Val Leu Asp Thr Leu Glu Ser Leu
Asn Leu Lys Tyr Pro Glu Pro 260 265 270Ser Pro Glu Arg Arg Lys Glu
Leu Leu Asp Ile Arg Lys Arg Leu Glu 275 280 285Asn Pro Glu Asn Gly
Lys 2908269PRTOceanithermus profundus 8Met Asp Val Ser Arg Tyr Arg
Val Pro Pro Gly Ser Gly Phe Asp Pro1 5 10 15Glu Ala Trp Pro Thr Arg
Glu Asp Asp Asp Phe Ala Gly Gly Lys Lys 20 25 30Glu Ala Lys Lys Glu
Leu Ala Arg Leu Ala Val Arg Leu Gly Glu Leu 35 40 45Gln Ala Arg Leu
Tyr Ala Glu Gly Arg Gln Ala Leu Leu Ile Val Leu 50 55 60Gln Gly Met
Asp Thr Ala Gly Lys Asp Gly Thr Ile Arg His Val Phe65 70 75 80Arg
Ala Val Asn Pro Gln Gly Val Arg Val Thr Ser Phe Lys Lys Pro 85 90
95Thr Ala Leu Glu Leu Ala His Asp Tyr Leu Trp Arg Val His Arg His
100 105 110Ala Pro Ala Arg Gly Glu Ile Gly Ile Phe Asn Arg Ser His
Tyr Glu 115 120 125Asp Val Leu Val Val Arg Val His Glu Leu Val Pro
Pro Glu Val Trp 130 135 140Gly Arg Arg Tyr Asp His Ile Asn Ala Phe
Glu Arg Leu Leu Ala Asp145 150 155 160Glu Gly Thr Arg Ile Val Lys
Phe Phe Leu His Ile Ser Lys Asp Glu 165 170 175Gln Lys Arg Arg Leu
Glu Ala Arg Leu Glu Asn Pro Arg Lys His Trp 180 185 190Lys Phe Asn
Pro Ala Asp Leu Ser Glu Arg Ala Arg Trp Gly Asp Tyr 195 200 205Ala
Ala Ala Tyr Ala Glu Ala Leu Ser Arg Thr Ser Ser Asp Arg Ala 210 215
220Pro Trp Tyr Ala Val Pro Ala Asp Arg Lys Trp Gln Arg Asn Arg
Ile225 230 235 240Val Ala Gln Val Leu Val Asp Ala Leu Glu Ala Met
Asp Pro Arg Phe 245 250 255Pro Arg Val Asp Phe Asp Pro Ala Ser Val
Arg Val Glu 260 2659290PRTRoseiflexus castenholzii 9Met Tyr Ala Gln
Arg Val Val Pro Gly Met Arg Val Arg Leu His Asp1 5 10 15Ile Asp Pro
Asp Ala Asn Gly Gly Leu Asn Lys Asp Glu Gly Arg Ala 20 25 30Arg Phe
Ala Glu Leu Asn Ala Glu Leu Asp Val Met Gln Glu Glu Leu 35 40 45Tyr
Ala Ala Gly Ile His Ala Leu Leu Leu Ile Leu Gln Gly Met Asp 50 55
60Thr Ala Gly Lys Asp Gly Ala Ile Arg Asn Val Met Leu Asn Leu Asn65
70 75 80Pro Gln Gly Cys Arg Val Glu Ser Phe Lys Val Pro Thr Glu Glu
Glu 85 90 95Leu Ala His Asp Phe Leu Trp Arg Val His Arg Val Val Pro
Arg Lys 100 105 110Gly Met Val Gly Val Phe Asn Arg Ser His Tyr Glu
Asp Val Leu Val 115 120 125Val Arg Val His Ser Leu Val Pro Glu Ser
Val Trp Arg Ala Arg Tyr 130 135 140Asp Gln Ile Asn Ala Phe Glu Arg
Leu Leu Ala Asp Thr Gly Thr Ile145 150 155 160Ile Val Lys Cys Phe
Leu His Ile Ser Lys Glu Glu Gln Glu Gln Arg 165 170 175Leu Leu Ala
Arg Glu Arg Asp Val Ser Lys Ala Trp Lys Leu Ser Ala 180 185 190Gly
Asp Trp Arg Glu Arg Ala Phe Trp Asp Asp Tyr Met Ala Ala Tyr 195 200
205Glu Glu Ala Leu Thr Arg Cys Ser Thr
Asp Tyr Ala Pro Trp Tyr Ile 210 215 220Ile Pro Ala Asn Arg Lys Trp
Tyr Arg Asp Leu Ala Ile Ser Glu Ala225 230 235 240Leu Val Glu Thr
Leu Arg Pro Tyr Arg Asp Asp Trp Arg Arg Ala Leu 245 250 255Asp Ala
Met Ser Arg Ala Arg Arg Ala Glu Leu Glu Ala Phe Arg Ala 260 265
270Glu Gln His Ala Met Glu Gly Arg Pro Gln Gly Ala Gly Gly Val Ser
275 280 285Arg Arg 29010282PRTRoseiflexus castenholzii 10Met His
Tyr Ala His Thr Val Ile Pro Gly Thr Gln Val Arg Leu Arg1 5 10 15Asp
Ile Asp Pro Asp Ala Ser Gly Gly Leu Thr Lys Asp Glu Gly Arg 20 25
30Glu Arg Phe Ala Ser Phe Asn Ala Thr Leu Asp Ala Met Gln Glu Glu
35 40 45Leu Tyr Ala Ala Gly Val His Ala Leu Leu Leu Ile Leu Gln Gly
Met 50 55 60Asp Thr Ala Gly Lys Asp Gly Ala Ile Arg Asn Val Met His
Asn Leu65 70 75 80Asn Pro Gln Gly Cys Arg Val Glu Ser Phe Lys Val
Pro Thr Glu Glu 85 90 95Glu Leu Ala His Asp Phe Leu Trp Arg Val His
Lys Val Val Pro Arg 100 105 110Lys Gly Met Val Gly Val Phe Asn Arg
Ser His Tyr Glu Asp Val Leu 115 120 125Val Val Arg Val His Ser Leu
Val Pro Glu His Val Trp Arg Ala Arg 130 135 140Tyr Asp Gln Ile Asn
Ala Phe Glu Arg Leu Leu Thr Asp Thr Gly Thr145 150 155 160Ile Ile
Val Lys Cys Phe Leu His Ile Ser Lys Asp Glu Gln Glu Lys 165 170
175Arg Leu Leu Ala Arg Glu Gln Asp Val Thr Lys Ala Trp Lys Leu Ser
180 185 190Ala Gly Asp Trp Arg Glu Arg Glu Arg Trp Asp Glu Tyr Met
Ala Ala 195 200 205Tyr Glu Glu Ala Leu Thr Arg Cys Ser Thr Glu Tyr
Ala Pro Trp Tyr 210 215 220Ile Ile Pro Ala Asn Arg Lys Trp Tyr Arg
Asp Leu Ala Ile Ser Glu225 230 235 240Val Leu Val Glu Thr Leu Arg
Pro Tyr Arg Asp Asp Trp Gln Arg Ala 245 250 255Leu Asp Ala Met Ser
Gln Ala Arg Leu Ala Glu Leu Lys Ala Phe Arg 260 265 270His Gln Gln
Thr Ala Gly Ala Thr Arg Leu 275 28011274PRTTruepera radiovictrix
11Met Ser Gln Gly Ser Ala Lys Gly Leu Gly Lys Leu Asp Lys Lys Val1
5 10 15Tyr Ala Arg Glu Leu Ala Leu Leu Gln Leu Glu Leu Val Lys Leu
Gln 20 25 30Gly Trp Ile Lys Ala Gln Gly Leu Lys Val Val Val Leu Phe
Glu Gly 35 40 45Arg Asp Ala Ala Gly Lys Gly Ser Thr Ile Thr Arg Ile
Thr Gln Pro 50 55 60Leu Asn Pro Arg Val Cys Arg Val Val Ala Leu Gly
Ala Pro Thr Glu65 70 75 80Arg Glu Arg Thr Gln Trp Tyr Phe Gln Arg
Tyr Val His His Leu Pro 85 90 95Ala Ala Gly Glu Met Val Leu Phe Asp
Arg Ser Trp Tyr Asn Arg Ala 100 105 110Gly Val Glu Arg Val Met Gly
Phe Cys Thr Glu Ala Glu Tyr Arg Glu 115 120 125Phe Leu His Ala Cys
Pro Thr Phe Glu Arg Leu Leu Leu Asp Ala Gly 130 135 140Ile Ile Leu
Ile Lys Tyr Trp Phe Ser Val Ser Ala Ala Glu Gln Glu145 150 155
160Arg Arg Met Arg Arg Arg Asn Glu Asn Pro Ala Lys Arg Trp Lys Leu
165 170 175Ser Pro Met Asp Leu Glu Ala Arg Ala Arg Trp Val Ala Tyr
Ser Lys 180 185 190Ala Lys Asp Ala Met Phe Tyr His Thr Asp Thr Lys
Ala Ser Pro Trp 195 200 205Tyr Val Val Asn Ala Glu Asp Lys Arg Arg
Ala His Leu Ser Cys Ile 210 215 220Ala His Leu Leu Ser Leu Ile Pro
Tyr Glu Asp Leu Thr Pro Pro Pro225 230 235 240Leu Glu Met Pro Pro
Arg Asp Leu Ala Gly Ala Asp Glu Gly Tyr Glu 245 250 255Arg Pro Asp
Lys Ala His Gln Thr Trp Val Pro Asp Tyr Val Pro Pro 260 265 270Thr
Arg12186PRTThermus thermophilus 12Met Asp Val Gly Gln Ala Val Ile
Phe Leu Gly Pro Pro Gly Ala Gly1 5 10 15Lys Gly Thr Gln Ala Ser Arg
Leu Ala Gln Glu Leu Gly Phe Lys Lys 20 25 30Leu Ser Thr Gly Asp Ile
Leu Arg Asp His Val Ala Arg Gly Thr Pro 35 40 45Leu Gly Glu Arg Val
Arg Pro Ile Met Glu Arg Gly Asp Leu Val Pro 50 55 60Asp Asp Leu Ile
Leu Glu Leu Ile Arg Glu Glu Leu Ala Glu Arg Val65 70 75 80Ile Phe
Asp Gly Phe Pro Arg Thr Leu Ala Gln Ala Glu Ala Leu Asp 85 90 95Arg
Leu Leu Ser Glu Thr Gly Thr Arg Leu Leu Gly Val Val Leu Val 100 105
110Glu Val Pro Glu Glu Glu Leu Val Arg Arg Ile Leu Arg Arg Ala Glu
115 120 125Leu Glu Gly Arg Ser Asp Asp Asn Glu Glu Thr Val Arg Arg
Arg Leu 130 135 140Glu Val Tyr Arg Glu Lys Thr Glu Pro Leu Val Gly
Tyr Tyr Glu Ala145 150 155 160Arg Gly Val Leu Lys Arg Val Asp Gly
Leu Gly Thr Pro Asp Glu Val 165 170 175Tyr Ala Arg Ile Arg Ala Ala
Leu Gly Ile 180 18513208PRTThermus thermophilus 13Met Arg Gly Ile
Val Thr Ile Asp Gly Pro Ser Ala Ser Gly Lys Ser1 5 10 15Ser Val Ala
Arg Arg Val Ala Ala Ala Leu Gly Val Pro Tyr Leu Ser 20 25 30Ser Gly
Leu Leu Tyr Arg Ala Ala Ala Phe Leu Ala Leu Arg Ala Gly 35 40 45Val
Asp Pro Gly Asp Glu Glu Gly Leu Leu Ala Leu Leu Glu Gly Leu 50 55
60Gly Val Arg Leu Leu Ala Gln Ala Glu Gly Asn Arg Val Leu Ala Asp65
70 75 80Gly Glu Asp Leu Thr Ser Phe Leu His Thr Pro Glu Val Asp Arg
Val 85 90 95Val Ser Ala Val Ala Arg Leu Pro Gly Val Arg Ala Trp Val
Asn Arg 100 105 110Arg Leu Lys Glu Val Pro Pro Pro Phe Val Ala Glu
Gly Arg Asp Met 115 120 125Gly Thr Ala Val Phe Pro Glu Ala Ala His
Lys Phe Tyr Leu Thr Ala 130 135 140Ser Pro Glu Val Arg Ala Trp Arg
Arg Ala Arg Glu Arg Pro Gln Ala145 150 155 160Tyr Glu Glu Val Leu
Arg Asp Leu Leu Arg Arg Asp Glu Arg Asp Lys 165 170 175Ala Gln Ser
Ala Pro Ala Pro Asp Ala Leu Val Leu Asp Thr Gly Gly 180 185 190Met
Thr Leu Asp Glu Val Val Ala Trp Val Leu Ala His Ile Arg Arg 195 200
20514225PRTPyrococcus furiosus 14Met Arg Ile Val Phe Asp Ile Gly
Gly Ser Val Leu Val Pro Glu Asn1 5 10 15Pro Asp Ile Asp Phe Ile Lys
Glu Ile Ala Tyr Gln Leu Thr Lys Val 20 25 30Ser Glu Asp His Glu Val
Ala Val Val Val Gly Gly Gly Lys Leu Ala 35 40 45Arg Lys Tyr Ile Glu
Val Ala Glu Lys Phe Asn Ser Ser Glu Thr Phe 50 55 60Lys Asp Phe Ile
Gly Ile Gln Ile Thr Arg Ala Asn Ala Met Leu Leu65 70 75 80Ile Ala
Ala Leu Arg Glu Lys Ala Tyr Pro Val Val Val Glu Asp Phe 85 90 95Trp
Glu Ala Trp Lys Ala Val Gln Leu Lys Lys Ile Pro Val Met Gly 100 105
110Gly Thr His Pro Gly His Thr Thr Asp Ala Val Ala Ala Leu Leu Ala
115 120 125Glu Phe Leu Lys Ala Asp Leu Leu Val Val Ile Thr Asn Val
Asp Gly 130 135 140Val Tyr Thr Ala Asp Pro Lys Lys Asp Pro Thr Ala
Lys Lys Ile Lys145 150 155 160Lys Met Lys Pro Glu Glu Leu Leu Glu
Ile Val Gly Lys Gly Ile Glu 165 170 175Lys Ala Gly Ser Ser Ser Val
Ile Asp Pro Leu Ala Ala Lys Ile Ile 180 185 190Ala Arg Ser Gly Ile
Lys Thr Ile Val Ile Gly Lys Glu Asp Ala Lys 195 200 205Asp Leu Phe
Arg Val Ile Lys Gly Asp His Asn Gly Thr Thr Ile Glu 210 215
220Pro22515207PRTThermotoga maritima 15Met Lys Gly Gln Leu Phe Val
Ile Cys Gly Pro Ser Gly Ala Gly Lys1 5 10 15Thr Ser Ile Ile Lys Glu
Val Leu Lys Arg Leu Asp Asn Val Val Phe 20 25 30Ser Val Ser Cys Thr
Thr Arg Pro Lys Arg Pro His Glu Glu Asp Gly 35 40 45Lys Asp Tyr Phe
Phe Ile Thr Glu Glu Glu Phe Leu Lys Arg Val Glu 50 55 60Arg Gly Glu
Phe Leu Glu Trp Ala Arg Val His Gly His Leu Tyr Gly65 70 75 80Thr
Leu Arg Ser Phe Val Glu Ser His Ile Asn Glu Gly Lys Asp Val 85 90
95Val Leu Asp Ile Asp Val Gln Gly Ala Leu Ser Val Lys Lys Lys Tyr
100 105 110Ser Asn Thr Val Phe Ile Tyr Val Ala Pro Pro Ser Tyr Ala
Asp Leu 115 120 125Arg Glu Arg Ile Leu Lys Arg Gly Thr Glu Lys Glu
Ala Asp Val Leu 130 135 140Val Arg Leu Glu Asn Ala Lys Trp Glu Leu
Met Phe Met Asp Glu Phe145 150 155 160Asp Tyr Ile Val Val Asn Glu
Asn Leu Glu Asp Ala Val Glu Met Val 165 170 175Val Ser Ile Val Arg
Ser Glu Arg Ala Lys Val Thr Arg Asn Gln Asp 180 185 190Lys Ile Glu
Arg Phe Lys Met Glu Val Lys Gly Trp Lys Lys Leu 195 200
20516142PRTAquifex aeolicus 16Met Ala Val Glu Arg Thr Leu Ile Ile
Val Lys Pro Asp Ala Met Glu1 5 10 15Lys Gly Ala Leu Gly Lys Ile Leu
Asp Arg Phe Ile Gln Glu Gly Phe 20 25 30Gln Ile Lys Ala Leu Lys Met
Phe Arg Phe Thr Pro Glu Lys Ala Gly 35 40 45Glu Phe Tyr Tyr Val His
Arg Glu Arg Pro Phe Phe Gln Glu Leu Val 50 55 60Glu Phe Met Ser Ser
Gly Pro Val Val Ala Ala Val Leu Glu Gly Glu65 70 75 80Asp Ala Ile
Lys Arg Val Arg Glu Ile Ile Gly Pro Thr Asp Ser Glu 85 90 95Glu Ala
Arg Lys Val Ala Pro Asn Ser Ile Arg Ala Gln Phe Gly Thr 100 105
110Asp Lys Gly Lys Asn Ala Ile His Ala Ser Asp Ser Pro Glu Ser Ala
115 120 125Gln Tyr Glu Ile Cys Phe Ile Phe Ser Gly Leu Glu Ile Val
130 135 140
* * * * *
References