U.S. patent application number 13/838570 was filed with the patent office on 2014-02-20 for identification and use of bacterial [2fe-2s] dihydroxy-acid dehydratases.
This patent application is currently assigned to Butamax(TM) Advanced Biofuels LLC. The applicant listed for this patent is Butamax(TM) Advanced Biofuels LLC. Invention is credited to Dennis FLINT, Steven Cary Rothman, Wonchul Suh, Jean-Francois Tomb, Rick W. Ye.
Application Number | 20140051137 13/838570 |
Document ID | / |
Family ID | 41402186 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140051137 |
Kind Code |
A1 |
FLINT; Dennis ; et
al. |
February 20, 2014 |
IDENTIFICATION AND USE OF BACTERIAL [2Fe-2S] DIHYDROXY-ACID
DEHYDRATASES
Abstract
A group of bacterial dihydroxy-acid dehydratases having a
[2Fe-2S] cluster was discovered. Bacterial [2Fe-2S] DHADs were
expressed as heterologous proteins in bacteria and yeast cells,
providing DHAD activity for conversion of 2,3-dihydroxyisovalerate
to .alpha.-ketoisovalerate or 2,3-dihydroxymethylvalerate to
.alpha.-ketomethylvalerate. Isobutanol and other compounds may be
synthesized in pathways that include bacterial [2Fe-2S] DHAD
activity.
Inventors: |
FLINT; Dennis; (Newark,
DE) ; Rothman; Steven Cary; (Wilmington, DE) ;
Suh; Wonchul; (Hockessin, DE) ; Tomb;
Jean-Francois; (Wilmington, DE) ; Ye; Rick W.;
(Hockessin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Butamax(TM) Advanced Biofuels LLC; |
|
|
US |
|
|
Assignee: |
Butamax(TM) Advanced Biofuels
LLC
Wilmington
DE
|
Family ID: |
41402186 |
Appl. No.: |
13/838570 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12569636 |
Sep 29, 2009 |
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13838570 |
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61100792 |
Sep 29, 2008 |
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Current U.S.
Class: |
435/136 ;
435/160; 435/252.3; 435/252.31; 435/252.32; 435/252.33; 435/252.34;
435/254.2; 435/254.21; 435/254.22; 435/254.23; 506/8 |
Current CPC
Class: |
Y02E 50/10 20130101;
G16B 30/00 20190201; C12N 9/88 20130101; C12P 13/08 20130101; C12P
13/06 20130101; C12P 7/40 20130101; C12Y 402/01009 20130101; C12P
7/16 20130101 |
Class at
Publication: |
435/136 ; 506/8;
435/252.3; 435/252.33; 435/252.34; 435/252.31; 435/252.32;
435/254.21; 435/254.22; 435/254.23; 435/254.2; 435/160 |
International
Class: |
C12N 9/88 20060101
C12N009/88; C12P 7/16 20060101 C12P007/16; G06F 19/22 20060101
G06F019/22 |
Claims
1. A method for identifying [2Fe-2S] DHAD enzymes comprising: (a)
querying one or more amino acid sequences with a Profile Hidden
Markov Model prepared using the proteins of SEQ ID NOs:164, 168,
230, 232, 298, 310, 344, and 346, wherein a match with an E-value
of less than 10-5 provides a first subset of sequences whereby said
first subset of sequences correspond to one or more DHAD related
proteins; (b) analyzing the first subset of sequences that
correspond to one or more DHAD related proteins of step (a) for the
presence of three conserved cysteines that correspond to positions
56, 129, and 201 in the Streptococcus mutans dihydroxy-acid
dehydratase amino acid sequence (SEQ ID NO: 168) whereby a second
subset of sequences encoding [2Fe-2S] DHAD enzymes are identified;
and (c) analyzing the second subset of sequences of step (b) for
the presence of signature conserved amino acids at positions
corresponding to positions in the Streptococcus mutans DHAD amino
acid sequence (SEQ ID NO: 168) that are aspartic acid at position
88, arginine or asparagine at position 142, asparagine at position
208, and leucine at position 454 whereby a third subset of
sequences encoding [2Fe-2S] DHAD enzymes are further
identified.
2. The method of claim 1 further comprising (d) expressing a
polypeptide having a sequence identifiable by any one or all of
steps a), b), and c) in a cell; and (e) confirming that said
polypeptide has DHAD activity in the cell.
3. The method of claim 1 further comprising (d) purifying a protein
encoded by a sequence identifiable by any one or all of steps a),
b), and c); and (e) confirming that said protein is a [2Fe-2S] DHAD
enzyme by UV-vis and EPR spectroscopy.
4. The method of claim 1 further comprising selecting one or more
sequences corresponding to bacterial [2Fe-2S] DHAD enzyme sequences
identified in any one or all of steps a), b), and c).
5. The method of claim 2 wherein the cell lacks endogenous DHAD
activity.
6. The method of claim 4 further comprising (d) expressing said
selected one or more sequences corresponding to bacterial [2Fe-2S]
DHAD enzyme sequences in a cell; and (e) confirming that said
enzyme sequence has DHAD activity in the cell.
7. The method of claim 4 further comprising (d) purifying a protein
encoded by said selected one or more sequences corresponding to
bacterial [2Fe-2S] DHAD enzyme sequences whereby a purified protein
is produced; and (e) confirming that the protein is a [2Fe-2S] DHAD
enzyme by UV-vis and EPR spectroscopy.
8. A microbial host cell comprising at least one heterologous
[2Fe-2S] DHAD enzyme identifiable by the method of claim 1.
9. The microbial host cell of claim 8 wherein the cell is bacterial
cell or a yeast cell.
10. The microbial host cell of claim 9 wherein the bacterial host
cell is a member of a genus of bacteria selected from the group
consisting of Clostridium, Zymomonas, Escherichia, Salmonella,
Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus,
Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter,
Corynebacterium, and Brevibacterium, Lactococcus, Leuconostoc,
Oenococcus, Pediococcus, and Streptococcus.
11. The microbial host cell of claim 9 wherein the yeast cell is a
member of a genus of yeast selected from the group consisting of
Saccharomyces, Schizosaccharomyces, Hansenula, Candida,
Kluyveromyces, Yarrowia and Pichia.
12. The microbial host cell of claim 8 wherein the cell produces
isobutanol.
13. A method for the production of isobutanol comprising: (a)
providing the microbial host cell of claim 8 wherein said host cell
comprises an isobutanol biosynthetic pathway; and (b) growing the
host cell of step (a) under conditions wherein isobutanol is
produced.
14. A method for the conversion of 2,3-dihydroxyisovalerate to
.alpha.-ketoisovalerate comprising: (a) providing the microbial
host of claim 8 and a source of 2,3-dihydroxyisovalerate; and (b)
growing the microbial host cell of (a) under conditions where the
2,3-dihydroxyisovalerate is converted to .alpha.-ketoisovalerate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to and claims the benefit of
priority of U.S. Provisional Application No. 61/100,792, filed Sep.
29, 2008, the entirety of which is herein incorporated by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to the field of industrial
microbiology and the expression of dihydroxy-acid dehydratase
activity. More specifically, bacterial dihydroxy-acid dehydratases
with a [2Fe-2S] cluster are identified and expressed as
heterologous proteins in bacterial and yeast hosts.
BACKGROUND OF THE INVENTION
[0003] Dihydroxy-acid dehydratase (DHAD), also called acetohydroxy
acid dehydratase, catalyzes the conversion of
2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate and of
2,3-dihydroxymethylvalerate to .alpha.-ketomethylvalerate. The DHAD
enzyme, classified as E.C. 4.2, 1.9, is part of naturally occurring
biosynthetic pathways producing valine, isoleucine, leucine and
pantothenic acid (vitamin B5). Increased expression of DHAD
activity is desired for enhanced microbial production of branched
chain amino acids or of pantothenic acid.
[0004] DHAD catalyzed conversion of 2,3-dihydroxyisovalerate to
.alpha.-ketoisovalerate is also a common step in the multiple
isobutanol biosynthetic pathways that are disclosed in commonly
owned and co-pending US Patent Application Publication US
20070092957 A1. Disclosed therein is engineering of recombinant
microorganisms for production of isobutanol. Isobutanol is useful
as a fuel additive, whose availability may reduce the demand for
petrochemical fuels.
[0005] For improved production of compounds synthesized in pathways
including dihydroxy-acid dehydratase, it is desirable to express a
heterologous enzyme that provides this enzymatic activity in the
production host of interest. Obtaining high functional expression
of dihydroxy-acid dehydratases in a heterologous host is
complicated by the enzyme's requirement for an Fe--S duster, which
involves availability and proper loading of the duster into the
DHAD apo-protein.
[0006] Fe--S duster requiring DHAD enzymes are known in the art and
are found either in the [4Fe-4S] or [2Fe-2S] form. Some bacterial
enzymes are known, the best characterized of which is from E. coli
(Flint, D H, et al. (1993) J. Biol. Chem. 268:14732-14742). However
these bacterial enzymes are all in the [4Fe-4S] form. The only
[2Fe-2S] form reported to date is a spinach enzyme (Flint and
Emptage (1988) J. Biol. Chem. 263:3558-3564).
[0007] It is desirable to use the [2Fe-2S] form of the enzyme in
host cells to enhance the production of introduced biosynthetic
pathways as the [2Fe-2S] form creates a lesser burden on Fe--S
cluster synthesis and/or assembly. Unfortunately, only one [2Fe-25]
form of this enzyme is known.
[0008] There exists a need therefore to identify new [2Fe-2S] forms
of DHAD for use in recombinant host cells where the conversion of
2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate is a metabolic
pathway step in a desired biosynthetic pathway.
SUMMARY OF THE INVENTION
[0009] Provided herein is a method for identifying [2Fe-2S] DHAD
enzymes, said method comprising:
[0010] a) querying one or more amino acid sequences with a Profile
Hidden Markov Model prepared using the proteins of SEQ ID NOs:164,
168, 230, 232, 298, 310, 344, and 346, wherein a match with an
E-value of less than 10.sup.-5 provides a first subset of sequences
whereby said first subset of sequences correspond to one or more
DHAD related proteins;
[0011] b) analyzing the first subset of sequences that correspond
to one or more DHAD related proteins of step (a) for the presence
of three conserved cysteines that correspond to positions 56, 129,
and 201 in the Streptococcus mutans dihydroxy-acid dehydratase
amino acid sequence (SEQ ID NO: 168) whereby a second subset of
sequences encoding [2Fe-2S] DHAD enzymes are identified; and
[0012] c) analyzing said second subset of sequences of step (b) for
the presence of signature conserved amino acids at positions
corresponding to positions in the Streptococcus mutans DHAD amino
acid sequence (SEQ ID NO: 168) that are aspartic acid at position
88, arginine or asparagine at position 142, asparagine at position
208, and leucine at position 454 whereby a third subset of
sequences encoding [2Fe-2S] DHAD enzymes are further
identified.
[0013] In another aspect of the invention, the above method further
comprises:
[0014] d) expressing a polypeptide having a sequence identifiable
by any one or all of steps a), b), and c) in a cell; and
[0015] e) confirming that said polypeptide has DHAD activity in the
cell.
[0016] In another aspect of the invention, the method above further
comprises:
[0017] d) purifying a protein encoded by a sequence identifiable by
any one or all of steps a), b), and c); and
[0018] e) confirming that said protein is a [2Fe-2S] DHAD enzyme by
UV-vis and EPR spectroscopy.
[0019] In another aspect of the invention, the method above further
comprises selecting one or more sequences corresponding to
bacterial [2Fe-2S] DHAD enzyme sequences identified in any one or
all of steps a), b), and c). Said selected sequences may be
expressed in a cell; and the DHAD activity in the cell may be
confirmed. Said selected sequences may be further purified such
that a purified protein is obtained and the [2Fe-2S] DHAD enzyme
activity of said purified protein may be confirmed by UV-vis and
EPR spectroscopy.
Another aspect of the invention is directed to a microbial host
cell comprising at least one heterologous [2Fe-2S] DHAD enzyme
identifiable by the methods described herein. Said cell maybe a
bacterial cell or a yeast cell. The cell may also be a recombinant
cell that produces isobutanol.
[0020] Another aspect of the invention is a method for the
production of isobutanol comprising:
[0021] a) providing the microbial host cell comprising at least one
heterologous [2Fe-2S] DHAD enzyme identifiable by the methods
described herein wherein said host cell further comprises an
isobutanol biosynthetic pathway; and
[0022] b) growing the host cell of step (a) under conditions
wherein isobutanol is produced.
[0023] Another aspect of the invention is a method for the
conversion of 2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate
comprising:
[0024] a) providing a microbial host cell comprising at least one
heterologous [2Fe-28] DHAD enzyme identifiable by the methods
described herein and a source of 2,3-dihydroxyisovalerate; and
[0025] b) growing the microbial host cell of (a) under conditions
where the 2,3-dihydroxyisovalerate is converted to
.alpha.-ketoisovalerate.
BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS
[0026] The invention can be more fully understood from the
following detailed description, figure, and the accompanying
sequence descriptions, which form a part of this application.
[0027] FIG. 1 shows the conserved cysteine regions, with C for
cysteine in bold, in representative bacterial [2Fe-2S] DHADs and a
[4Fe-4S] DHAD, Single letter amino acid abbreviations are used.
[0028] FIG. 2 shows a phylogenetic tree of DHAD related proteins.
Branches for [4Fe-4S] and [2Fe-2S] DHADs as well as EDDs, aldonic
acid dehydratases and an undefined group (Und) are marked, Select
individual DHADs are labeled.
[0029] FIG. 3 shows biosynthetic pathways for isobutanol
production.
[0030] FIG. 4 shows graphs of stability of activity in air of A) S.
mutans DHAD, and B) E. coli DHAD.
[0031] FIG. 5 shows a plot of the UV-visible spectrum of S. mutans
DHAD.
[0032] FIG. 6 shows a plot of the electron paramagnetic resonance
(EPR) spectrum of S. mutans DHAD at different temperatures between
20.degree. K and 90.degree. K.
[0033] FIG. 7 shows HPLC analysis of an extract of yeast cells that
express acetolactate synthase, KARI, and S. mutans DHAD genes, with
an isobutanol peak at 47.533 minutes.
[0034] FIG. 8 shows a graph of stability of L. lactis DHAD in
air.
[0035] FIG. 9 shows a plot of the UV-visible spectrum of the
purified L. lactis DHAD.
[0036] Table 1 is a table of the Profile HMM for dihydroxy-acid
dehydratases based on enzymes with assayed function prepared as
described in Example 1. Table 1 is submitted herewith
electronically and is incorporated herein by reference.
[0037] The following sequences conform with 37 C.F.R. 1.821-1.825
("Requirements for Patent Applications Containing Nucleotide
Sequences and/or Amino Acid Sequence Disclosures--the Sequence
Rules") and are consistent with World Intellectual Property
Organization (WIPO) Standard ST.25 (1998) and the sequence listing
requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and
Section 208 and Annex C of the Administrative Instructions). The
symbols and format used for nucleotide and amino acid sequence data
comply with the rules set forth in 37 C.F.R. .sctn.1.822.
TABLE-US-00001 TABLE 2a Representative bacterial [2Fe--2S] DHAD
proteins and encoding sequences SEQ ID NO: SEQ ID NO: Organism of
derivation Nucleic acid Peptide Mycobacterium sp. MCS 1 2
Mycobacterium gilvum PYR-GCK 3 4 Mycobacterium smegmatis str. 5 6
MC2 155 Mycobacterium vanbaalenii PYR-1 7 8 Nocardia farcinica IFM
10152 9 10 Rhodococcus sp. RHA1 11 12 Mycobacterium ulcerans Agy99
13 14 Mycobacterium avium subsp. 15 16 paratuberculosis K-10
Mycobacterium tuberculosis H37Ra 17 18 Mycobacterium leprae TN * 19
20 Kineococcus radiotolerans SRS30216 21 22 Janibacter sp. HTCC2649
23 24 Nocardioides sp. JS614 25 26 Renibacterium salmoninarum ATCC
27 28 33209 Arthrobacter aurescens TC1 29 30 Leifsonia xyli subsp.
xyli str. CTCB07 31 32 Marine actinobacterium PHSC20C1 33 34
Clavibacter michiganensis subsp. 35 36 michiganensis NCPPB 382
Saccharopolyspora erythraea NRRL 37 38 2338 Acidothermus
cellulolyticus 11B 39 40 Corynebacterium efficiens YS-314 41 42
Brevibacterium linens BL2 43 44 Tropheryma whipplei TW08/27 45 46
Methylobacterium extorquens PA1 47 48 Methylobacterium
chloromethanicum 428 429 Methylobacterium nodulans ORS 2060 49 50
Rhodopseudomonas palustris BisB5 51 52 Rhodopseudomonas palustris
BisB18 53 54 Bradyrhizobium sp. ORS278 55 56 Bradyrhizobium
japonicum USDA 110 57 58 Fulvimarina pelagi HTCC2506 59 60
Aurantimonas sp. SI85-9A1 61 62 Hoeflea phototrophica DFL-43 63 64
Mesorhizobium loti MAFF303099 65 66 Mesorhizobium sp. BNC1 67 68
Parvibaculum lavamentivorans DS-1 69 70 Loktanella vestfoldensis
SKA53 71 72 Roseobacter sp. CCS2 73 74 Dinoroseobacter shibae DFL
12 75 76 Roseovarius nubinhibens ISM 77 78 Sagittula stellata E-37
79 80 Roseobacter sp. AzwK-3b 81 82 Roseovarius sp. TM1035 83 84
Oceanicola batsensis HTCC2597 85 86 Oceanicola granulosus HTCC2516
87 88 Rhodobacterales bacterium HTCC2150 89 90 Paracoccus
denitrificans PD1222 91 92 Oceanibulbus indolifex HEL-45 93 94
Sulfitobacter sp. EE-36 95 96 Roseobacter denitrificans OCh 114 97
98 Jannaschia sp. CCS1 99 100 Caulobacter sp. K31 101 102
Candidatus Pelagibacter ubique 103 104 HTCC1062 Erythrobacter
litoralis HTCC2594 105 106 Erythrobacter sp. NAP1 107 108 Comamonas
testosterone KF-1 109 110 Sphingomonas wittichii RW1 111 112
Burkholderia xenovorans LB400 113 114 Burkholderia phytofirmans
PsJN 115 116 Bordetella petrii DSM 12804 117 118 Bordetella
bronchiseptica RB50 119 120 Bradyrhizobium sp. ORS278 121 122
Bradyrhizobium sp. BTAi1 123 124 Bradhyrhizobium japonicum 125 126
Sphingomonas wittichii RW1 127 128 Rhodobacterales bacterium
HTCC2654 129 130 Solibacter usitatus Ellin6076 131 132 Roseiflexus
sp. RS-1 133 134 Rubrobacter xylanophilus DSM 9941 135 136
Salinispora tropica CNB-440 137 138 Acidobacteria bacterium
Ellin345 139 140 Thermus thermophilus HB27 141 142 Maricaulis maris
MCS10 143 144 Parvularcula bermudensis HTCC2503 145 146
Oceanicaulis alexandrii HTCC2633 147 148 Plesiocystis pacifica
SIR-1 149 150 Bacillus sp. NRRL B-14911 151 152 Oceanobacillus
iheyensis HTE831 153 154 Staphylococcus saprophyticus subsp. 155
156 saprophyticus ATCC 15305 Bacillus selenitireducens MLS10 157
158 Streptococcus pneumoniae SP6-BS73 159 160 Streptococcus
sanguinis SK36 161 162 Streptococcus thermophilus LMG 18311 163 164
Streptococcus suis 89/1591 165 166 Streptococcus mutans UA159 167
168 Leptospira borgpetersenii serovar 169 170 Hardjo-bovis L550
Candidatus Vesicomyosocius okutanii 171 172 HA Candidatus Ruthia
magnifica str. Cm 173 174 (Calyptogena magnifica) Methylococcus
capsulatusstr. Bath 175 176 uncultured marine bacterium 177 178
EB80_02D08 uncultured marine gamma 179 180 proteobacterium
EBAC31A08 uncultured marine gamma 181 182 proteobacterium EBAC20E09
uncultured gamma proteobacterium 183 184 eBACHOT4E07 Alcanivorax
borkumensis SK2 185 186 Chromohalobacter salexigens DSM 3043 187
188 Marinobacter algicola DG893 189 190 Marinobacter aquaeolei VT8
191 192 Marinobacter sp. ELB17 193 194 Pseudoalteromonas
haloplanktis 195 196 TAC125 Acinetobacter sp. ADP1 197 198
Opitutaceae bacterium TAV2 199 200 Flavobacterium sp. MED217 201
202 Cellulophaga sp. MED134 203 204 Kordia aigicida OT-1 205 206
Flavobacteriales bacterium ALC-1 207 208 Psychroflexus torquis ATCC
700755 209 210 Flavobacteriales bacterium HTCC2170 211 212
unidentified eubacterium SCB49 213 214 Gramella forsetii KT0803 215
216 Robiginitalea biformata HTCC2501 217 218 Tenacibaculum sp.
MED152 219 220 Polaribacter irgensii 23-P 221 222 Pedobacter sp.
BAL39 223 224 Flavobacteria bacterium BAL38 225 226 Flavobacterium
psychrophilum JIP02/86 227 228 Flavobacterium johnsoniae UW101 229
230 Lactococcus lactis subsp. cremoris SK11 231 232 Psychromonas
ingrahamii 37 233 234 Microscilla marina ATCC 23134 235 236
Cytophaga hutchinsonii ATCC 33406 237 238 Rhodopirellula baltica SH
1 239 240 Blastopirellula marina DSM 3645 241 242 Planctomyces
maris DSM 8797 243 244 Algoriphagus sp. PR1 245 246 Candidatus
Sulcia muelleri str. Hc 247 248 (Homalodisca coagulata) Candidatus
Carsonella ruddii PV 249 250 Synechococcus sp. RS9916 251 252
Synechococcus sp. WH 7803 253 254 Synechococcus sp. CC9311 255 256
Synechococcus sp. CC9605 257 258 Synechococcus sp. WH 8102 259 260
Synechococcus sp. BL107 261 262 Synechococcus sp. RCC307 263 264
Synechococcus sp. RS9917 265 266 Synechococcus sp. WH 5701 267 268
Prochlorococcus marinus str. MIT 9313 269 270 Prochlorococcus
marinus str. NATL2A 271 272 Prochlorococcus marinus str. MIT 9215
273 274 Prochlorococcus marinus str. AS9601 275 276 Prochlorococcus
marinus str. MIT 9515 277 278 Prochlorococcus marinus subsp.
pastoris 279 280 str. CCMP1986 Prochlorococcus marinus str. MIT
9211 281 282 Prochlorococcus marinus subsp. marinus 283 284 str.
CCMP1375 Nodulana spumigena CCY9414 285 286 Nostoc punctiforme PCC
73102 287 288 Nostoc sp. PCC 7120 289 290 Trichodesmium erythraeum
IMS101 291 292 Acaryochloris marina MBIC11017 293 294 Lyngbya sp.
PCC 8106 295 296 Synechocystis sp. PCC 6803 297 298 Microcystis
aeruginosa PCC 7806 426 427 Cyanothece sp. CCY0110 299 300
Thermosynechococcus elongatus BP-1 301 302 Synechococcus sp.
JA-2-3B'a(2-13) 303 304 Gloeobacter violaceus PCC 7421 305 306
Nitrosomonas eutropha C91 307 308 Nitrosomonas europaea ATCC 19718
309 310 Nitrosospira multiformis ATCC 25196 311 312 Chloroflexus
aggregans DSM 9485 313 314 Leptospirillum sp. Group II UBA 315 316
Leptospirillum sp. Group II UBA 317 318 Halorhodospira halophila
SL1 319 320 Nitrococcus mobilis Nb-231 321 322 Alkalilimnicola
ehrlichei MLHE-1 323 324 Deinococcus geothermalis DSM 11300 325 326
Polynucleobacter sp. QLW-P1DMWA-1 327 328 Polynucleobacter
necessarius STIR1 329 330 Azoarcus sp. EbN1 331 332 Burkholderia
phymatum STM815 333 334 Burkholderia xenovorans LB400 335 336
Burkholderia multivorans ATCC 17616 337 338 Burkholderia
cenocepacia PC184 339 340 Burkholderia mallei GB8 horse 4 341 342
Ralstonia eutropha JMP134 343 344 Ralstonia metallidurans CH34 345
346 Ralstonia solanacearum UW551 347 348 Ralstonia pickettii 12J
349 350 Limnobacter sp. MED105 351 352 Herminiimonas arsenicoxydans
353 354 Bordetella parapertussis 355 356 Bordetella petrii DSM
12804 357 358 Polaromonas sp. JS666 359 360 Polaromonas
naphthalenivorans CJ2 361 362 Rhodoferax ferrireducens T118 363 364
Verminephrobacter eiseniae EF01-2 365 366 Acidovorax sp. JS42 367
368 Delftia acidovorans SPH-1 369 370 Methylibium petroleiphilum
PM1 371 372 gamma proteobacterium KT 71 373 374 Tremblaya princeps
375 376 Blastopirellula marina DSM 3645 377 378 Planctomyces maris
DSM 8797 379 380 Salinibacter ruber DSM 13855 387 388
TABLE-US-00002 TABLE 2b Additional representative bacterial
[2Fe--2S] DHAD proteins and encoding sequences Nucleic Amino acid
acid SEQ ID SEQ ID Organism of derivation NO: NO: Burkholderia
ambifaria AMMD 443 444 Bradyrhizobium sp. BTAi1 445 446 Delftia
acidovorans SPH-1 447 448 Microcystis aeruginosa NIES-843 449 450
uncultured marine microorganism 451 452 HF4000_APKG8C21
Burkholderia ubonensis Bu 453 454 Gemmata obscuriglobus UQM 2246
455 456 Mycobacterium abscessus 457 458 Synechococcus sp. PCC 7002
459 460 Burkholderia graminis C4D1M 461 462 Methylobacterium
radiotolerans JCM 2831 463 464 Leptothrix cholodnii SP-6 465 466
Verrucomicrobium spinosum DSM 4136 467 468 Cyanothece sp. ATCC
51142 469 470 Opitutus terrae PB90-1 471 472 Leptospira bifiexa
serovar Patoc strain `Patoc 1 473 474 (Paris)` Methylacidiphilum
infernorum V4 475 476 Cupriavidus taiwanensis 477 478
Chthoniobacter flavus Ellin428 479 480 Cyanothece sp. PCC 7822 481
482 Phenylobacterium zucineum HLK1 483 484 Leptospirillum sp. Group
II `5-way CG` 485 486 Arthrospira maxima CS-328 487 488 Oligotropha
carboxidovorans OM5 489 490 Rhodospirillum centenum SW 491 492
Cyanothece sp. PCC 8801 493 494 Thermus aquaticus Y51MC23 495 496
Cyanothece sp. PCC 7424 497 498 Acidithiobacillus ferrooxidans ATCC
23270 499 500 Cyanothece sp. PCC 7425 501 502 Arthrobacter
chlorophenolicus A6 503 504 Burkholderia multivorans CGD2M 505 506
Thermomicrobium roseum DSM 5159 507 508 bacterium Ellin514 509 510
Desulfobacterium autotrophicum HRM2 511 512 Thioalkalivibrio sp.
K90mix 513 514 Flavobacteria bacterium MS024-3C 515 516
Flavobacteria bacterium MS024-2A 517 518 `Nostoc azollae` 0708 519
520 Acidobacterium capsulatum ATCC 51196 521 522 Gemmatimonas
aurantiaca T-27 523 524 Gemmatimonas aurantiaca T-27 525 526
Rhodococcus erythropolis PR4 527 528 Deinococcus deserti VCD115 529
530 Rhodococcus opacus B4 531 532 Chryseobacterium gleum ATCC 35910
533 534 Thermobaculum terrenum ATCC BAA-798 535 536 Kribbella
flavida DSM 17836 537 538 Gordonia bronchialis DSM 43247 539 540
Geodermatophilus obscurus DSM 43160 541 542 Xylanimonas
cellulosilytica DSM 15894 543 544 Sphingobacterium spiritivorum
ATCC 33300 545 546 Meiothermus silvanus DSM 9946 547 548
Meiothermus ruber DSM 1279 549 550 Nakamuralla multipartita DSM
44233 551 552 Cellulomonas flavigena DSM 20109 553 554 Rhodothermus
marinus DSM 4252 555 556 Planctomyces limnophilus DSM 3776 557 558
Beutenbergia cavernae DSM 12333 559 560 Spirosoma linguale DSM 74
561 562 Sphaerobacter thermophilus DSM 20745 563 564 Lactococcus
lactis 565 566 Thermus thermophilus HB8 567 568 Anabaena variabilis
ATCC 29413 569 570 Roseovarius sp. 217 571 572 uncultured
Prochlorococcus marinus clone 573 574 HF10-88D1 Burkholderia
xenovorans LB400 575 576 Saccharomonospora viridis DSM 43017 577
578 Pedobacter heparinus DSM 2366 579 580 Microcoleus
chthonoplastes PCC 7420 581 582 Acidimicrobium ferrooxidans DSM
10331 583 584 Rhodobacterales bacterium HTCC2083 585 586 Candidatus
Pelagibacter sp. HTCC7211 587 588 Chitinophaga pinensis DSM 2588
589 590 Alcanivorax sp. DG881 591 592 Micrococcus luteus NCTC 2665
593 594 Verrucomicrobiae bacterium DG1235 595 596 Synechococcus sp.
PCC 7335 597 598 Brevundimonas sp. BAL3 599 600 Dyadobacter
fermentans DSM 18053 601 602 gamma proteobacterium NOR5-3 603 604
gamma proteobacterium NOR51-B 605 606 Cyanobium sp. PCC 7001 607
608 Jonesia denitrificans DSM 20603 609 610 Brachybacterium faecium
DSM 4810 611 612 Paenibacillus sp. JDR-2 613 614 Octadecabacter
antarcticus 307 615 616 Variovorax paradoxus S110 617 618
TABLE-US-00003 TABLE 3 SEQ ID Numbers of Additional Proteins and
Encoding sequences SEQ ID NO: SEQ ID Encoding NO: Description seq
protein Vibrio cholerae KARI 389 390 Pseudomonas aeruginosa 422 423
PAO1 KARI Pseudomonas fluorescens 391 392 PF5 KARI Achromobacter
393 394 xylosoxidans butanol dehydrogenase sadB Escherichia coli
str. K-12 383 384 substr. MG1655 Phosphogluconate dehydratase
Azospirillum brasilense 385 386 arabonate dehydratase Escherichia
coli str. K-12 381 382 substr. MG1655 DHAD
[0038] SEQ ID NOs:395-409, 412-421, 424, and 431-436 are primers
for PCR, cloning or sequencing analysis used a described in the
Examples herein.
[0039] SEQ ID NO:410 is the nucleotide sequence of the pDM1
vector.
[0040] SEQ ID NO:411 is the nucleotide sequence of the pLH532
vector.
[0041] SEQ ID NO:425 is the S. cerevisiae FBA promoter.
[0042] SEQ ID NO:430 is the nucleotide sequence of the pRS423 FBA
ilvD(Strep) vector.
[0043] SEQ ID NO:437 is the nucleotide sequence of the pNY13
vector
[0044] SEQ ID NO:438 is the alsS coding region from B.
subtilis.
[0045] SEQ ID NO:439 is the S. cerevisiae GPD promoter.
[0046] SEQ ID NO:440 is the S. cerevisiae CYC1 terminator.
[0047] SEQ ID NO:442 is the S. cerevisiae ILV5 gene.
DETAILED DESCRIPTION OF THE INVENTION
[0048] As disclosed herein, applicants have solved the stated
problem through the discovery of methods of identifying [2Fe-2S]
DHADs. Through the discovery these enzymes and their use in
recombinant hosts, a heretofore unappreciated activity advantage in
pathway engineering with DHADs has been identified.
[0049] The present invention relates to recombinant yeast or
bacterial cells engineered to provide heterologous expression of
dihydroxy-acid dehydratase (DHAD) having a [2Fe-2S] cluster. The
expressed DHAD functions as a component of a biosynthetic pathway
for production of a compound such as valine, Isoleucine, leucine,
pantothenic acid, or isobutanol. These amino acids and pantothenic
acid may be used as nutritional supplements, and isobutanol may be
used as a fuel additive to reduce demand for petrochemicals.
[0050] The following abbreviations and definitions will be used for
the interpretation of the specification and the claims.
[0051] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having," "contains" or
"containing," or any other variation thereof, are intended to cover
a non-exclusive inclusion. For example, a composition, a mixture,
process, method, article, or apparatus that comprises a list of
elements is not necessarily limited to only those elements but may
include other elements not expressly listed or inherent to such
composition, mixture, process, method, article, or apparatus.
Further, unless expressly stated to the contrary, "or" refers to an
inclusive or and not to an exclusive or. For example, a condition A
or B is satisfied by any one of the following: A is true (or
present) and B is false (or not present), A is false (or not
present) and B is true (or present), and both A and B are true (or
present).
[0052] Also, the indefinite articles "a" and "an" preceding an
element or component of the invention are intended to be
nonrestrictive regarding the number of instances (i.e. occurrences)
of the element or component. Therefore "a" or "an" should be read
to include one or at least one, and the singular word form of the
element or component also includes the plural unless the number is
obviously meant to be singular.
[0053] The term "invention" or "present invention" as used herein
is a non-limiting term and is not intended to refer to any single
embodiment of the particular invention but encompasses all possible
embodiments as described in the specification and the claims.
[0054] As used herein, the term "about" modifying the quantity of
an ingredient or reactant of the invention employed refers to
variation in the numerical quantity that can occur, for example,
through typical measuring and liquid handling procedures used for
making concentrates or use solutions in the real world; through
inadvertent error in these procedures; through differences in the
manufacture, source, or purity of the ingredients employed to make
the compositions or carry out the methods; and the like. The term
"about" also encompasses amounts that differ due to different
equilibrium conditions for a composition resulting from a
particular initial mixture. Whether or not modified by the term
"about", the claims include equivalents to the quantities. In one
embodiment, the term "about" means within 10% of the reported
numerical value, preferably within 5% of the reported numerical
value The term "[2Fe-2S] DHAD" refers to DHAD enzymes having a
bound [2Fe-2S] duster.
[0055] The term "[4Fe-4S] DHAD" refers to DHAD enzymes having a
bound [4Fe-4S] duster.
[0056] There term "Dihydroxy-acid dehydratase" will be abbreviated
DHAD and will refer to an enzyme that converts
2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate.
[0057] The term "isobutanol biosynthetic pathway" refers to an
enzyme pathway to produce isobutanol from pyruvate.
[0058] The term "a facultative anaerobe" refers to a microorganism
that can grow in both aerobic and anaerobic environments.
[0059] The term "carbon substrate" or "fermentable carbon
substrate" refers to a carbon source capable of being metabolized
by host organisms of the present invention and particularly carbon
sources selected from the group consisting of monosaccharides,
oligosaccharides, polysaccharides, and one-carbon substrates or
mixtures thereof. Carbon substrates may include C6 and C5 sugars
and mixtures thereof.
[0060] The term "gene" refers to a nucleic acid fragment that is
capable of being expressed as a specific protein, optionally
including regulatory sequences preceding (5' non-coding sequences)
and following (3' non-coding sequences) the coding sequence.
"Native gene" refers to a gene as found in nature with its own
regulatory sequences. "Chimeric gene" refers to any gene that is
not a native gene, comprising regulatory and coding sequences that
are not found together in nature. Accordingly, a chimeric gene may
comprise regulatory sequences and coding sequences that are derived
from different sources, or regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different than that found in nature. "Endogenous gene" refers to a
native gene in its natural location in the genome of an organism. A
"foreign gene" or "heterologous gene" refers to a gene not normally
found in the host organism, but that is introduced into the host
organism by gene transfer. Foreign genes can comprise native genes
inserted into a non-native organism, or chimeric genes. A
"transgene" is a gene that has been introduced into the genome by a
transformation procedure.
[0061] As used herein the term "coding region" refers to a DNA
sequence that codes for a specific amino acid sequence. "Suitable
regulatory sequences" refer to nucleotide sequences located
upstream (5.degree. non-coding sequences), within, or downstream
(3' non-coding sequences) of a coding sequence, and which influence
the transcription, RNA processing or stability, or translation of
the associated coding sequence. Regulatory sequences may include
promoters, translation leader sequences, introns, polyadenylation
recognition sequences, RNA processing site, effector binding site
and stem-loop structure.
[0062] The term "promoter" refers to a DNA sequence capable of
controlling the expression of a coding sequence or functional RNA.
In general, a coding sequence is located 3' to a promoter sequence.
Promoters may be derived in their entirety from a native gene, or
be composed of different elements derived from different promoters
found in nature, or even comprise synthetic DNA segments. It is
understood by those skilled in the art that different promoters may
direct the expression of a gene in different tissues or cell types,
or at different stages of development, or in response to different
environmental or physiological conditions. Promoters which cause a
gene to be expressed in most cell types at most times are commonly
referred to as "constitutive promoters". It is further recognized
that since in most cases the exact boundaries of regulatory
sequences have not been completely defined, DNA fragments of
different lengths may have identical promoter activity.
[0063] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of effecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0064] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide.
[0065] As used herein the term "transformation" refers to the
transfer of a nucleic acid fragment into a host organism, resulting
in genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
or "recombinant" or "transformed" organisms.
[0066] The terms "plasmid" and "vector" as used herein, refer to an
extra chromosomal element often carrying genes which are not part
of the central metabolism of the cell, and usually in the form of
circular double-stranded DNA molecules. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell.
[0067] As used herein the term "codon degeneracy" refers to the
nature in the genetic code permitting variation of the nucleotide
sequence without effecting the amino acid sequence of an encoded
polypeptide. The skilled artisan is well aware of the "codon-bias"
exhibited by a specific host cell in usage of nucleotide codons to
specify a given amino acid. Therefore, when synthesizing a gene for
improved expression in a host cell, i is desirable to design the
gene such that its frequency of codon usage approaches the
frequency of preferred codon usage of the host cell.
[0068] The term "codon-optimized" as it refers to genes or coding
regions of nucleic acid molecules for transformation of various
hosts, refers to the alteration of codons in the gene or coding
regions of the nucleic acid molecules to reflect the typical codon
usage of the host organism without altering the polypeptide encoded
by the DNA.
[0069] As used herein, an "isolated nucleic acid fragment" or
"isolated nucleic acid molecule" will be used interchangeably and
will mean a polymer of RNA or DNA that is single- or
double-stranded, optionally containing synthetic, non-natural or
altered nucleotide bases. An isolated nucleic acid fragment in the
form of a polymer of DNA may be comprised of one or more segments
of cDNA, genomic DNA or synthetic DNA.
[0070] A nucleic acid fragment is "hybridizable" to another nucleic
acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a
single-stranded form of the nucleic acid fragment can anneal to the
other nucleic acid fragment under the appropriate conditions of
temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook, J., Fritsch,
E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,
2.sup.nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor,
N.Y. (1989), particularly Chapter 11 and Table 11.1 therein
(entirely incorporated herein by reference). The conditions of
temperature and ionic strength determine the "stringency" of the
hybridization. Stringency conditions can be adjusted to screen for
moderately similar fragments (such as homologous sequences from
distantly related organisms), to highly similar fragments (such as
genes that duplicate functional enzymes from closely related
organisms). Post-hybridization washes determine stringency
conditions. One set of preferred conditions uses a series of washes
starting with 6.times.SSC, 0.5% SDS at room temperature for 15 min,
then repeated with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30
min, and then repeated twice with 0.2.times.SSC, 0.5% SDS at
50.degree. C. for 30 min. A more preferred set of stringent
conditions uses higher temperatures in which the washes are
identical to those above except for the temperature of the final
two 30 min washes in 0.2.times.SSC, 0.5% SDS was increased to
60.degree. C. Another preferred set of highly stringent conditions
uses two final washes in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
An additional set of stringent conditions include hybridization at
0.1.times.SSC, 0.1% SDS, 65.degree. C. and washes with 2.times.SSC,
0.1% SDS followed by 0.1.times.SSC, 0.1% SDS, for example.
[0071] Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the
length of the nucleic acids and the degree of complementation,
variables well known in the art. The greater the degree of
similarity or homology between two nucleotide sequences, the
greater the value of Tm for hybrids of nucleic acids having those
sequences. The relative stability (corresponding to higher Tm) of
nucleic acid hybridizations decreases in the following order:
RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating Tm have been
derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8). In one embodiment the length for a hybridizable
nucleic acid is at least about 10 nucleotides. Preferably a minimum
length for a hybridizable nucleic acid is at least about 15
nucleotides; more preferably at least about 20 nucleotides; and
most preferably the length is at least about 30 nucleotides.
Furthermore, the skilled artisan will recognize that the
temperature and wash solution salt concentration may be adjusted as
necessary according to factors such as length of the probe.
[0072] A "substantial portion" of an amino acid or nucleotide
sequence is that portion comprising enough of the amino acid
sequence of a polypeptide or the nucleotide sequence of a gene to
putatively identify that polypeptide or gene, either by manual
evaluation of the sequence by one skilled in the art, or by
computer-automated sequence comparison and identification using
algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol.,
215:403-410 (1993)). In general, a sequence of ten or more
contiguous amino acids or thirty or more nucleotides is necessary
in order to putatively identify a polypeptide or nucleic acid
sequence as homologous to a known protein or gene. Moreover, with
respect to nucleotide sequences, gene specific oligonucleotide
probes comprising 20-30 contiguous nucleotides may be used in
sequence-dependent methods of gene identification (e.g., Southern
hybridization) and isolation (e.g., in situ hybridization of
bacterial colonies or bacteriophage plaques). In addition, short
oligonucleotides of 12-15 bases may be used as amplification
primers in PCR in order to obtain a particular nucleic acid
fragment comprising the primers. Accordingly, a "substantial
portion" of a nucleotide sequence comprises enough of the sequence
to specifically identify and/or isolate a nucleic acid fragment
comprising the sequence. The instant specification teaches the
complete amino acid and nucleotide sequence encoding particular
proteins. The skilled artisan, having the benefit of the sequences
as reported herein, may now use all or a substantial portion of the
disclosed sequences for purposes known to those skilled in this
art. Accordingly, the instant invention comprises the complete
sequences as reported in the accompanying Sequence Listing, as well
as substantial portions of those sequences as defined above.
[0073] The term "complementary" is used to describe the
relationship between nucleotide bases that are capable of
hybridizing to one another. For example, with respect to DNA,
adenosine is complementary to thymine and cytosine is complementary
to guanine.
[0074] The term "percent identity", as known in the art, is a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" or "sequence identity" also means
the degree of sequence relatedness between polypeptide or
polynucleotide sequences, as the case may be, as determined by the
match between strings of such sequences. "Identity" and
"similarity" can be readily calculated by known methods, including
but not limited to those described in:
1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford
University: NY (1988); 2.) Biocomputing: Informatics and Genome
Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer
Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular
Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence
Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY
(1991).
[0075] Preferred methods to determine identity are designed to give
the best match between the sequences tested. Methods to determine
identity and similarity are codified in publicly available computer
programs. Sequence alignments and percent identity calculations may
be performed using the MegAlign.TM. program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences is performed using the "Clustal
method of alignment" which encompasses several varieties of the
algorithm including the "Clustal V method of alignment"
corresponding to the alignment method labeled Clustal V (described
by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et
al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the
MegAlign.TM. program of the LASERGENE bioinformatics computing
suite (DNASTAR Inc.). For multiple alignments, the default values
correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default
parameters for pairwise alignments and calculation of percent
identity of protein sequences using the Clustal method are
KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For
nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5,
WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences
using the Clustal V program, it is possible to obtain a "percent
identity" by viewing the "sequence distances" table in the same
program. Additionally the "Clustal W method of alignment" is
available and corresponds to the alignment method labeled Clustal W
(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins,
D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992) Thompson, J.
D., Higgins, D. G., and Gibson T. J. (1994) Nuc. Acid Res. 22: 4673
4680) and found in the MegAlign.TM. v6.1 program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc.). Default parameters
for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2,
Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein
Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After
alignment of the sequences using the Clustal W program, it is
possible to obtain a "percent identity" by viewing the "sequence
distances" table in the same program.
[0076] It is well understood by one skilled in the art that many
levels of sequence identity are useful in identifying polypeptides,
from other species, wherein such polypeptides have the same or
similar function or activity. Useful examples of percent identities
include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, or 95%, or any integer percentage from 55% to 100% may be
useful in describing the present invention, such as 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99%. Suitable nucleic acid fragments not only have the
above homologies but typically encode a polypeptide having at least
50 amino acids, preferably at least 100 amino acids, more
preferably at least 150 amino acids, still more preferably at least
200 amino acids, and most preferably at least 250 amino acids.
[0077] The term "sequence analysis software" refers to any computer
algorithm or software program that is useful for the analysis of
nucleotide or amino acid sequences. "Sequence analysis software"
may be commercially available or independently developed. Typical
sequence analysis software will include, but is not limited to: 1.)
the GCG suite of programs (Wisconsin Package Version 9.0, Genetics
Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX
(Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR
(DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes
Corporation, Ann Arbor, Mich.); and 5.) the FASTA program
incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput.
Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992,
111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within
the context of this application it will be understood that where
sequence analysis software is used for analysis, that the results
of the analysis will be based on the "default values" of the
program referenced, unless otherwise specified. As used herein
"default values" will mean any set of values or parameters that
originally load with the software when first initialized.
[0078] Standard recombinant DNA and molecular cloning techniques
used here are well known in the art and are described by Sambrook,
J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis");
and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W.,
Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, published by Greene
Publishing Assoc. and Wiley-Interscience (1987). Additional methods
used here are in Methods in Enzymology, Volume 194, Guide to Yeast
Genetics and Molecular and Cell Biology (Part A, 2004, Christine
Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San
Diego, Calif.).
Discovery of [2Fe-2S] DHADs
[0079] DHAD proteins are known to contain a bound iron-sulfur
(Fe--S) duster that is required for enzyme activity. The only DHAD
with a [2Fe-2S] duster reported to date is a spinach enzyme (Hint
and Emptage (1988) J. Biol. Chem. 263:3558-3564). Some bacterial
enzymes are also known, the best characterized of which is from E.
coli (Hint, D H, et al. (1993) J. Biol. Chem. 268:14732-14742),
which has a [4Fe-4S] duster.
[0080] Applicants have now determined that there is a class of
bacterial DHADs that contain a [2Fe-2S] duster ([2Fe-2S] DHADs).
Applicants have found that the group of [2Fe-2S] DHADs may be
distinguished from a group of [4Fe-4S] DHADs by the presence of
three conserved cysteine residues in the protein. The three
conserved cysteines are similar to three essential cysteines
reported in the Azospirillum brasiliense arabonate (an aldonic
acid) dehydratase (Watanabe, S et al. J. Biol. Chem. (2006)
281:33521-33536) that was reported to contain a [4Fe-4S] cluster.
In the Azospirillum brasiliense arabonate dehydratase protein,
cysteines located at amino acid positions 56, 124, and 197 were
determined to be essential for enzyme activity and likely involved
in coordination with the Fe--S duster. Surprisingly, applicants
have found that three conserved cysteines, which are in
corresponding positions to the three essential cysteines of the
Azospirillum brasiliense arabonate dehydratase, are characteristic
of DHADs containing a [2Fe-2S] duster. Applicants have found that
the E. coli DHAD, that contains a [4Fe-4S] duster, has two of the
conserved cysteines, but not the third conserved cysteine. Shown in
FIG. 1 is a comparison of the sequence regions of the conserved
cysteines for the E. coli [4Fe-4S] cluster-containing DHADs and for
representatives of a phylogenetic group of [2Fe-2S] duster DHADs
that was identified herein in Example 1 and is described below.
[0081] Applicants have developed a method for identifying [2Fe-2S]
DHADs. In the present invention, bacterial [2Fe-2S] DHADs, which
may be identified by this method, may be used for heterologous
expression in bacteria.
[0082] To structurally characterize DHAD enzymes a Profile Hidden
Markov Model (HMM) was prepared as described in Example 1 using
amino acid sequences of DHAD proteins with experimentally verified
function, as determined in Example 2 herein, and is given in Table
1. These DHADs are from Nitrosomonas europaea (DNA SEQ ID NO:309;
Protein SEQ ID NO:310), Synechocystis sp. PCC6803 (DNA SEQ ID:297;
Protein SEQ ID NO:298), Streptococcus mutans (DNA SEQ ID NO:167;
Protein SEQ ID NO:168), Streptococcus thermophilus (DNA SEQ ID
NO:163; SEQ ID No:164), Raistonia metallidurans (DNA SEQ ID NO:345;
Protein SEQ ID NO:346), Raistonia eutropha (DNA SEQ ID NO:343;
Protein SEQ ID NO:344), and Lactococcus lactis (DNA SEQ ID NO:231;
Protein SEQ ID NO:232). In addition the DHAD from Flavobacterium
johnsoniae (DNA SEQ ID NO:229; Protein SEQ ID NO:230) was found to
have dihydroxy-acid dehydratase activity when expressed in E. coli
and was used in making the Profile. This Profile HMM for DHADs may
be used to identify DHAD related proteins. Any protein that matches
the Profile HMM with an E value of <10.sup.-5 is a DHAD related
protein, which includes [4Fe-4S] DHADs, [2Fe-2S] DHADs, aldonic
acid dehydratases, and phosphogluconate dehydratases. A
phylogenetic tree of sequences matching this Profile HMM is shown
in FIG. 2.
[0083] Sequences matching the Profile HMM given herein are then
analyzed for the presence of the three conserved cysteines
described above. The exact positions of the three conserved
cysteines may vary, and these may be identified in the context of
the surrounding sequence using multiple sequence alignments
performed with the Clustal W algorithm (Thompson, J. D., Higgins,
D. G., and Gibson T. J. (1994) Nuc. Acid Res. 22: 4673 4680)
employing the following parameters: 1) for pairwise alignment
parameters, a Gap opening=10; Gap extend=0.1; matrix is Gonnet 250;
and mode-Slow-accurate, 2) for multiple alignment parameters, Gap
opening=10; Gap extension=0.2; and matrix is Gonnet series. For
example, the three conserved cysteines are located at amino acid
positions 56, 129, and 201 in the Streptococcus mutans DHAD (SEQ ID
NO: 168), and at amino acid positions 61, 135, and 207 in the
Lactococcus lactis DHAD (SEQ ID NO: 232). The exact positions of
the three conserved cysteines in other protein sequences correspond
to these positions in the S. mutans or the L. lactis amino acid
sequence. One skilled in the art will readily be able to identify
the presence or absence of each of the three conserved cysteines in
the amino acid sequence of a DHAD protein using pairwise or
multiple sequence alignments. In addition, other methods may be
used to determine the presence of the three conserved cysteines,
such as analysis by eye.
[0084] The DHAD Profile HMM matching proteins that have two but not
the third (position 56) conserved cysteine include [4Fe-4S] DHADs
and phosphogluconate dehydratases (EDDs). Proteins having the three
conserved cysteines include arabonate dehydratases and [2Fe-2S]
DHADs, and are members of a [2Fe-2S] DHAD/aldonic acid dehydratase
group. The [2Fe-2S] DHADs may be distinguished from the aldonic
acid dehydratases by analyzing for signature conserved amino acids
found to be present in the [2Fe-2S] DHADs or in the aldonic acid
dehydratases at positions corresponding to the following positions
in the Streptococcus mutans DHAD amino acid sequence. These
signature amino acids are in [2Fe-2S] DHADs or in aldonic acid
dehydratases, respectively, at the following positions (with
greater than 90% occurance): 88 asparagine vs glutamic acid; 113
not conserved vs glutamic add; 142 arginine or asparagine vs not
conserved; 165: not conserved vs glycine; 208 asparagine vs not
conserved; 454 leucine vs not conserved; 477 phenylalanine or
tyrosine vs not conserved; and 487 glycine vs not conserved.
[0085] The disclosed methods for identification of [2Fe-2S] DHAD
enzymes can be carried out on a single sequence or on a group of
sequences. In a preferred embodiment, one or more sequence
databases are queried with a Profile HMM as described herein.
Suitable sequence databases are known to those skilled in the art
and include but are not limited to the Genbank non-redundant
protein database, the SwissProt database, or UniProt database or
other available databases such as GQPat (GenomeQuest, Westboro,
Mass.) and BRENDA (Biobase, Beverly, Mass.).
[0086] Among the [2Fe-2S] DHADs identifiable by this method,
bacterial [2Fe-2S] DHADs may readily be identifiable by the natural
source organism being a type of bacteria. Any bacterial [2Fe-2S]
DHAD identifiable by this method may be suitable for heterologous
expression in a microbial host cell. It will also be appreciated
that any bacterial [2Fe-2S] DHAD expressly disclosed herein by
sequence may be suitable for heterologous expression in bacterial
cells, Preferred bacterial [2Fe-2S] DHAD enzymes can be expressed
in a host cell and provide DHAD activity.
[0087] Initially, 193 different bacterial [2Fe-2S] DHADs with
sequence identities of less than 95% (greater than 95% identity
proteins removed to simplify the analysis) were identified as
described in Example 1 and the amino acid and coding sequences of
these proteins are provided in the sequence listing, with SEQ ID
NOs listed in Table 2a.
[0088] A subsequent analysis described in Example 11 returned 268
different bacterial [2Fe-25] DHADs, Amino acid and coding sequences
that were not identical to any of the 193 bacterial [2Fe-2S] DHADs
provided by the initial identification are included in the sequence
listing, with SEQ ID NOs listed in Table 2b.
[0089] Any [2Fe-2S] DHAD protein matching a sequence identifiable
through the methods disclosed herein or a sequence expressly
disclosed herein with an identity of at least about 95%, 96%, 97%,
98%, or 99% is a [2Fe-2S] DHAD that may be used for heterologous
expression in bacterial cells as disclosed herein. Among the
bacterial [2Fe-2S] DHADs expressly disclosed herein, there is 100%
conservation of the signature amino acids at positions: 88 aspartic
acid, 142 arginine or asparagine, 208 asparagine, and 454
leucine.
[0090] In addition, bacterial [2Fe-2S] DHADs that may be used in
the present invention are identifiable by their position in the
[2Fe-2S] DHAD branch of a phylogenetic tree of DHAD related
proteins such as that shown in FIG. 2 and described in Example 1,
In addition, bacterial [2Fe-2S] DHADs that may be used are
identifiable using sequence comparisons with any of the 281
bacterial [2Fe-25] DHADs whose sequences are provided herein, where
sequence identity may be at least about 80%-85%, 85%-90%, 90%-95%
or 95%-99%.
[0091] Additionally, the sequences of [2Fe-2S] DHADs provided
herein may be used to identify other homologs in nature. For
example each of the DHAD encoding nucleic acid fragments described
herein may be used to isolate genes encoding homologous proteins.
Isolation of homologous genes using sequence-dependent protocols is
well known in the art. Examples of sequence-dependent protocols
include, but are not limited to: 1.) methods of nucleic acid
hybridization; 2.) methods of DNA and RNA amplification, as
exemplified by various uses of nucleic acid amplification
technologies [e.g., polymerase chain reaction (PCR), Mullis et al.,
U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et
al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement
amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A.,
89; 392 (1992)]; and 3.) methods of library construction and
screening by complementation.
[0092] For example, genes encoding similar proteins or polypeptides
to the [2Fe-2S] DHAD encoding genes provided herein could be
isolated directly by using all or a portion of the instant nucleic
acid fragments as DNA hybridization probes to screen libraries from
any desired organism using methodology well known to those skilled
in the art. Specific oligonucleotide probes based upon the
disclosed nucleic acid sequences can be designed and synthesized by
methods known in the art (Maniatis, supra). Moreover, the entire
sequences can be used directly to synthesize DNA probes by methods
known to the skilled artisan (e.g., random primers DNA labeling,
nick translation or end-labeling techniques), or RNA probes using
available in vitro transcription systems. In addition, specific
primers can be designed and used to amplify a part of (or
full-length of) the instant sequences. The resulting amplification
products can be labeled directly during amplification reactions or
labeled after amplification reactions, and used as probes to
isolate full-length DNA fragments by hybridization under conditions
of appropriate stringency.
[0093] Typically, in PCR-type amplification techniques, the primers
have different sequences and are not complementary to each other.
Depending on the desired test conditions, the sequences of the
primers should be designed to provide for both efficient and
faithful replication of the target nucleic acid. Methods of PCR
primer design are common and well known in the art (Thein and
Wallace, "The use of oligonucleotides as specific hybridization
probes in the Diagnosis of Genetic Disorders", in Human Genetic
Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50,
IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular
Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:
Current Methods and Applications. Humania: Totowa, N.J.).
[0094] Generally two short segments of the described sequences may
be used in polymerase chain reaction protocols to amplify longer
nucleic acid fragments encoding homologous genes from DNA or RNA.
The polymerase chain reaction may also be performed on a library of
cloned nucleic acid fragments wherein the sequence of one primer is
derived from the described nucleic acid fragments, and the sequence
of the other primer takes advantage of the presence of the
polyadenylic acid tracts to the 3.degree. end of the mRNA precursor
encoding microbial genes.
[0095] Alternatively, the second primer sequence may be based upon
sequences derived from the cloning vector. For example, the skilled
artisan can follow the RACE protocol (Frohman et al., PNAS USA
85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of
the region between a single point in the transcript and the 3' or
5' end. Primers oriented in the 3' and 5.degree. directions can be
designed from the instant sequences. Using commercially available
3' RACE or 5' RACE systems (e.g., BRL, Gaithersburg, Md.), specific
3' or 5' cDNA fragments can be isolated (Ohara et al., PNAS USA
86:5673 (1989); Loh et al., Science 243:217 (1989)).
[0096] Alternatively, the provided [2Fe-2S] DHAD encoding sequences
may be employed as hybridization reagents for the identification of
homologs. The basic components of a nucleic acid hybridization test
include a probe, a sample suspected of containing the gene or gene
fragment of interest, and a specific hybridization method. Probes
are typically single-stranded nucleic acid sequences that are
complementary to the nucleic acid sequences to be detected. Probes
are "hybridizable" to the nucleic acid sequence to be detected. The
probe length can vary from 5 bases to tens of thousands of bases,
and will depend upon the specific test to be done. Typically a
probe length of about 15 bases to about 30 bases is suitable. Only
part of the probe molecule need be complementary to the nucleic
acid sequence to be detected. In addition, the complementarity
between the probe and the target sequence need not be perfect.
Hybridization does occur between imperfectly complementary
molecules with the result that a certain fraction of the bases in
the hybridized region are not paired with the proper complementary
base.
[0097] Hybridization methods are well defined. Typically the probe
and sample must be mixed under conditions that will permit nucleic
acid hybridization. This involves contacting the probe and sample
in the presence of an inorganic or organic salt under the proper
concentration and temperature conditions. The probe and sample
nucleic acids must be in contact for a long enough time that any
possible hybridization between the probe and sample nucleic acid
may occur. The concentration of probe or target in the mixture will
determine the time necessary for hybridization to occur. The higher
the probe or target concentration, the shorter the hybridization
incubation time needed. Optionally, a chaotropic agent may be
added. The chaotropic agent stabilizes nucleic acids by inhibiting
nuclease activity. Furthermore, the chaotropic agent allows
sensitive and stringent hybridization of short oligonucleotide
probes at room temperature (Van Ness and Chen, Nucl. Acids Res.
19:5143-5151 (1991)). Suitable chaotropic agents include
guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate,
lithium tetrachloroacetate, sodium perchlorate, rubidium
tetrachloroacetate, potassium iodide and cesium trifluoroacetate,
among others. Typically, the chaotropic agent will be present at a
final concentration of about 3 M. If desired, one can add formamide
to the hybridization mixture, typically 30-50% (v/v).
[0098] Various hybridization solutions can be employed. Typically,
these comprise from about 20 to 60% volume, preferably 30%, of a
polar organic solvent. A common hybridization solution employs
about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride,
about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES
or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g.,
sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL
(Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about
250-500 kdal) and serum albumin. Also included in the typical
hybridization solution will be unlabeled carrier nucleic acids from
about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or
salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to
2% wt/vol glycine. Other additives may also be included, such as
volume exclusion agents that include a variety of polar
water-soluble or swellable agents (e.g., polyethylene glycol),
anionic polymers (e.g., polyacrylate or polymethylacrylate) and
anionic saccharidic polymers (e.g., dextran sulfate).
[0099] Nucleic acid hybridization is adaptable to a variety of
assay formats. One of the most suitable is the sandwich assay
format. The sandwich assay is particularly adaptable to
hybridization under non-denaturing conditions. A primary component
of a sandwich-type assay is a solid support. The solid support has
adsorbed to it or covalently coupled to it immobilized nucleic acid
probe that is unlabeled and complementary to one portion of the
sequence.
Expression of Heterologous Bacterial 2Fe-2S DHADs in Bacterial and
Yeast Hosts
[0100] Applicants have found that a heterologous [2Fe-2S] DHAD
provides DHAD activity when expressed in a microbial cell. Any
[2Fe-2S] DHAD which may be identified as described herein, may be
expressed in a heterologous microbial cell. Expression of any of
these proteins provides DHAD activity for a biosynthetic pathway
that includes conversion of 2,3-dihydroxyisovalerate to
.alpha.-ketoisovalerate or 2,3-dihydroxymethylvalerate to
.alpha.-ketomethylvalerate. Expression of a [2Fe-2S] DHAD, as
opposed to a 4Fe-4D DHAD, lowers the requirement for Fe and S in
dusters to obtain enzyme activity. In addition, the S. mutans
[2Fe-2S] DHAD was shown herein to have higher stability in air as
compared to the sensitivity in air of the E. coli [4Fe-4S] DHAD,
which is desirable for obtaining better activity in a heterologous
host cell.
[0101] Bacterial cells that may be hosts for expression of a
heterologous bacterial [2Fe-2S] DHAD include, but are not limited
to, Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus,
Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus,
Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter,
Corynebacterium, and Brevibacterium, Lactococcus, Leuconostoc,
Oenococcus, Pediococcus, and Streptococcus. Engineering expression
of a heterologous bacterial [2Fe-2S] DHAD may increase DHAD
activity in a host bacterial cell that naturally expresses a
[2Fe-2S] DHAD or a [4Fe-4S] DHAD. Such host cells may include, for
example, E. coli and Bacillus subtilis. Furthermore, engineering
expression of a heterologous bacterial [2Fe-2S] DHAD provides DHAD
activity in a host bacterial cell that has no endogenous DHAD
activity. Such host cells may include, for example, Lactobacillus,
Enterococcus, Pediococcus and Leuconostoc.
[0102] Specific hosts include: Escherichia coli, Alcaligenes
eutrophus, Bacillus licheniformis, Paenibacillus macerans,
Rhodococcus erythropolis, Pseudomonas putida. Lactobacillus
plantarum, Enterococcus faecium. Enterococcus gallinarium,
Enterococcus faecalis, and Bacillus subtilis.
[0103] A host bacterial cell may be engineered to express a
heterologous bacterial [2Fe-2S] DHAD by methods well known to one
skilled in the art. The coding region for the DHAD to be expressed
may be codon optimized for the target host cell, as well known to
one skilled in the art. Vectors useful for the transformation of a
variety of host cells are common and commercially available from
companies such as EPICENTRE.RTM. (Madison, Wis.), Invitrogen Corp.
(Carlsbad, Calif.), Stratagene (La Jolla, Calif.), and New England
Biolabs, Inc. (Beverly, Mass.). Typically the vector contains a
selectable marker and sequences allowing autonomous replication or
chromosomal integration in the desired host. In addition, suitable
vectors comprise a promoter region which harbors transcriptional
initiation controls and a transcriptional termination control
region, between which a coding region DNA fragment may be inserted,
to provide expression of the inserted coding region. Both control
regions may be derived from genes homologous to the transformed
host cell, although it is to be understood that such control
regions may also be derived from genes that are not native to the
specific species chosen as a production host.
[0104] Initiation control regions or promoters, which are useful to
drive expression of bacterial [2Fe-2S] DHAD coding regions in the
desired bacterial host cell are numerous and familiar to those
skilled in the art. Virtually any promoter capable of driving these
genetic elements is suitable for the present invention including,
but not limited to, lac, am, tot, trp, IP.sub.L, IP.sub.R, T7, tac,
and trc promoters (useful for expression in Escherichia coli,
Alcaligenes, and Pseudomonas); the amy, apr, and npr promoters, and
various phage promoters useful for expression in Bacillus subtilis,
Bacillus licheniformis, and Paenibacillus macerans; nisA (useful
for expression Gram-positive bacteria, Eichenbaum et al. Appl.
Environ. Microbiol. 64(8):2763-2769 (1998)); and the synthetic P11
promoter (useful for expression in Lactobacillus plantarum, Rud et
al., Microbiology 152:1011-1019 (2006)).
[0105] Termination control regions may also be derived from various
genes native to the preferred hosts. Optionally, a termination site
may be unnecessary; however, it is most preferred if included.
[0106] Certain vectors are capable of replicating in a broad range
of host bacteria and can be transferred by conjugation. The
complete and annotated sequence of pRK404 and three related
vectors: pRK437, pRK442, and pRK442(H), are available. These
derivatives have proven to be valuable tools for genetic
manipulation in Gram-negative bacteria (Scott et al., Plasmid
50(1):74-79 (2003)). Several plasmid derivatives of
broad-host-range Inc P4 plasmid RSF1010 are also available with
promoters that can function in a range of Gram-negative bacteria.
Plasmid pAYC36 and pAYC37, have active promoters along with
multiple cloning sites to allow for heterologous gene expression in
Gram-negative bacteria, Some vectors that are useful for
transformation of Bacillus subtilis and Lactobacillus include
pAM.beta.1 and derivatives thereof (Renault et al., Gene
183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231
(1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al.
Appl. Environ. Microbiol. 62:1481-1486 (1996)); pMG1, a conjugative
plasmid (Tanimoto et al., J. Bacteria. 184:5800-5804 (2002));
pNZ9520 (Kleerebezem et al., Appl. Environ, Microbiol. 63:4581-4584
(1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol.
67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. Agents
Chemaher. 38:1899-1903 (1994)), Several plasmids from Lactobacillus
plantarum have also been reported (van Kranenburg et al., Appl.
Environ. Microbiol. 71(3):1223-1230 (2005)).
[0107] Chromosomal gene replacement tools are also widely
available. For example, a thermosensitive variant of the
broad-host-range replicon pWV101 has been modified to construct a
plasmid pVE6002 which can be used to effect gene replacement in a
range of Gram-positive bacteria (Maguin et al., J. Bacteriol.
174(17):5633-5638 (1992)). Additionally, in vitro transposomes are
available from commercial sources such as EPICENTRE.RTM. to create
random mutations in a variety of genomes.
[0108] Yeast cells that may be hosts for expression of a
heterologous bacterial [2Fe-2S] DHAD are any yeast cells that are
amenable to genetic manipulation and include, but are not limited
to, Saccharomyces, Schizosaccharomyces, Hansenula, Candida,
Kluyveromyces, Yarrowia and Pichia. Suitable strains include, but
are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Kluyveromyces lactic, Kluyveromyces thermotolerans, Candida
glabrata, Candida albicans, Pichia stipitis and Yarrowia
lipolytica. Most suitable is Saccharomyces cerevisiae.
[0109] Expression is achieved by transforming with a gene
comprising a sequence encoding any of these [2Fe-2S] DHADs. The
coding region for the DHAD to be expressed may be codon optimized
for the target host cell, as well known to one skilled in the art.
Methods for gene expression in yeast are known in the art (see for
example Methods in Enzymology, Volume 194, Guide to Yeast Genetics
and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and
Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).
Expression of genes in yeast typically requires a promoter,
operably linked to a coding region of interest, and a
transcriptional terminator. A number of yeast promoters can be used
in constructing expression cassettes for genes in yeast, including,
but not limited to promoters derived from the following genes:
CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5. GAPDH, ADC1, TRP1, URA3,
LEU2, ENO, TPI, CUP1, FBA, GPD, GPM, and AOX1. Suitable
transcriptional terminators include, but are not limited to FBAt,
GPDt, GPMt, ERG10t, GAL1t, CYC1, and ADH1.
[0110] Suitable promoters, transcriptional terminators, and
[2Fe-2S] DHAD coding regions may be cloned into E. coli-yeast
shuttle vectors, and transformed into yeast cells. These vectors
allow strain propagation in both E. coli and yeast strains.
Typically the vector used contains a selectable marker and
sequences allowing autonomous replication or chromosomal
integration in the desired host. Typically used plasmids in yeast
are shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American
Type Culture Collection, Rockville, Md.), which contain an E. coli
replication origin (e.g., pMB1), a yeast 2.mu. origin of
replication, and a marker for nutritional selection. The selection
markers for these four vectors are His3 (vector pRS423), Trp1
(vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426).
Construction of expression vectors with a chimeric gene encoding
the described DHADs may be performed by either standard molecular
cloning techniques in E. coli or by the gap repair recombination
method in yeast.
[0111] The gap repair cloning approach takes advantage of the
highly efficient homologous recombination in yeast. Typically, a
yeast vector DNA is digested (e.g., in its multiple cloning site)
to create a "gap" in its sequence. A number of insert DNAs of
interest are generated that contain a .gtoreq.21 bp sequence at
both the 5' and the 3' ends that sequentially overlap with each
other, and with the 5' and 3' terminus of the vector DNA. For
example, to construct a yeast expression vector for "Gene X', a
yeast promoter and a yeast terminator are selected for the
expression cassette. The promoter and terminator are amplified from
the yeast genomic DNA, and Gene X is either PCR amplified from its
source organism or obtained from a cloning vector comprising Gene X
sequence. There is at least a 21 bp overlapping sequence between
the 5' end of the linearized vector and the promoter sequence,
between the promoter and Gene X, between Gene X and the terminator
sequence, and between the terminator and the 3' end of the
linearized vector. The "gapped" vector and the insert DNAs are then
co-transformed into a yeast strain and plated on the medium
containing the appropriate compound mixtures that allow
complementation of the nutritional selection markers on the
plasmids. The presence of correct insert combinations can be
confirmed by PCR mapping using plasmid DNA prepared from the
selected cells. The plasmid DNA isolated from yeast (usually low in
concentration) can then be transformed into an E. coli strain, e.g.
TOP10, followed by mini preps and restriction mapping to further
verify the plasmid construct. Finally the construct can be verified
by sequence analysis.
[0112] Like the gap repair technique, integration into the yeast
genome also Lakes advantage of the homologous recombination system
in yeast. Typically, a cassette containing a coding region plus
control elements (promoter and terminator) and auxotrophic marker
is PCR-amplified with a high-fidelity DNA polymerase using primers
that hybridize to the cassette and contain 40-70 base pairs of
sequence homology to the regions 5' and 3' of the genomic area
where insertion is desired. The PCR product is then transformed
into yeast and plated on medium containing the appropriate compound
mixtures that allow selection for the integrated auxotrophic
marker. For example, to integrate "Gene X" into chromosomal
location "Y", the promoter-coding regionX-terminator construct is
PCR amplified from a plasmid DNA construct and joined to an
autotrophic marker (such as URA3) by either SOE PCR or by common
restriction digests and cloning. The full cassette, containing the
promoter-coding regionX-terminator-URA3 region, is PCR amplified
with primer sequences that contain 40-70 bp of homology to the
regions 5' and 3' of location "Y" on the yeast chromosome. The PCR
product is transformed into yeast and selected on growth media
lacking uracil. Transformants can be verified either by colony PCR
or by direct sequencing of chromosomal DNA.
Confirming DHAD Activity
[0113] The presence of DHAD activity in a cell engineered to
express a bacterial [2Fe-2S] DHAD can be confirmed using methods
known in the art. As one example, and as demonstrated in the
Examples herein, crude extracts from cells engineered to express a
bacterial [2Fe-2S] DHAD may be used in a DHAD assay as described by
Hint and Emptage (J. Biol. Chem. (1988) 263(8): 3558-64) using
dinitrophenylhydrazine. In another example, and as demonstrated in
the Examples herein, DHAD activity may be assayed by expressing a
bacterial DHAD identifiable by the methods disclosed herein in a
yeast strain that lacks endogenous DHAD activity. If DHAD activity
is present, the yeast strain will grow in the absence of
branched-chain amino acids. DHAD activity may also be confirmed by
more indirect methods, such as by assaying for a downstream product
in a pathway requiring DHAD activity. Any product that has
.alpha.-ketoisovalerate or .alpha.-ketomethylvalerate as a pathway
intermediate may be measured as an assay for DHAD activity. A list
of such products includes, but is not limited to, valine,
isoleucine, leucine, pantothenic add, 2-methyl-1-butanol,
3-methyl-1-butanol and isobutanol.
Isobutanol Production
[0114] Expression of a bacterial [2Fe-2S] DHAD in bacteria or
yeast, as described herein, provides the transformed, recombinant
host cell with dihydroxy-acid dehydratase activity for conversion
of 2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate or
2,3-dihydroxymethylvalerate to .alpha.-ketomethylvalerate. Any
product that has .alpha.-ketoisovalerate or
.alpha.-ketomethylvalerate as a pathway intermediate may be
produced with greater effectiveness in a bacterial or yeast strain
disclosed herein having the described heterologous [2Fe-2S] DHAD. A
list of such products includes, but is not limited to, valine,
isoleucine, leucine, pantothenic acid, 2-methyl-1-butanol,
3-methyl-1-butanol and isobutanol.
[0115] For example, in yeast biosynthesis of valine includes steps
of acetolactate conversion to 2,3-dihydroxy-isovalerate by
acetohydroxy acid reductoisomerase (ILV5), conversion of
2,3-dihydroxy-isovalerate to .alpha.-ketoisovalerate (also called
2-keto-isovalerate) by dihydroxy-acid dehydratase, and conversion
of .alpha.-ketoisovalerate to valine by branched-chain amino acid
transaminase (BAT2) and branched-chain animo acid aminotransferase
(BAT1). Biosynthesis of leucine includes the same steps to
.alpha.-ketoisovalerate, followed by conversion of
.alpha.-ketoisovalerate to alpha-isopropylmalate by
alpha-isopropylmalate synthase (LEU9, LEU4), conversion of
alpha-isopropylmalate to beta-isopropylmalate by isopropylmalate
isomerase (LEU1), conversion of beta-isopropylmalate to
alpha-ketoisocaproate by beta-IPM dehydrogenase (LEU2), and finally
conversion of alpha-ketoisocaproate to leucine by branched-chain
amino acid transaminase (BAT2) and branched-chain amino acid
aminotransferase (BAT1). The bacterial pathway is similar,
involving differently named proteins and genes. Increased
conversion of 2,3-dihydroxy-isovalerate to .alpha.-ketoisovalerate
will increase flow in these pathways, particularly if one or more
additional enzymes of a pathway is overexpressed. Thus it is
desired for production of valine or leucine to use a strain
disclosed herein.
[0116] Biosynthesis of pantothenic acid includes a step performed
by DHAD, as well as steps performed by ketopantoate
hydroxymethyltransferase and pantothenate synthase. Engineering of
expression of these enzymes for enhanced production of pantothenic
acid biosynthesis in microorganisms is described in U.S. Pat. No.
6,177,264.
[0117] The .alpha.-ketoisovalerate product of DHAD is an
intermediate in isobutanol biosynthetic pathways disclosed in
commonly owned and co-pending US Patent Publication 20070092957 A1,
which is herein incorporated by reference. A diagram of the
disclosed isobutanol biosynthetic pathways is provided in FIG. 3.
Production of isobutanol in a strain disclosed herein benefits from
increased DHAD activity. As disclosed herein, DHAD activity is
provided by expression of a bacterial [2Fe-2S] DHAD in a bacterial
or yeast cell. As described in US 20070092957 A1, steps in an
example isobutanol biosynthetic pathway include conversion of:
[0118] pyruvate to acetolactate (see FIG. 1, pathway step a
therein), as catalyzed for example by acetolactate synthase, [0119]
acetolactate to 2,3-dihydroxyisovalerate (see FIG. 1, pathway step
b therein) as catalyzed for example by acetohydroxy acid
isomeroreductase; [0120] 2,3-dihydroxyisovalerate to
.alpha.-ketoisovalerate (see FIG. 1, pathway step c therein) as
catalyzed for example by acetohydroxy acid dehydratase, also called
dihydroxy-acid dehydratase (DHAD); [0121] .alpha.-ketoisovalerate
to isobutyraldehyde (see FIG. 1, pathway step d therein) as
catalyzed for example by branched-chain .alpha.-keto acid
decarboxylase; and [0122] isobutyraldehyde to isobutanol (see FIG.
1, pathway step e therein) as catalyzed for example by
branched-chain alcohol dehydrogenase.
[0123] The substrate to product conversions, and enzymes involved
in these reactions, for steps f, g, h, l, j, and k of alternative
pathways are described in US 20070092957 A1.
[0124] Genes that may be used for expression of the pathway step
enzymes named above other than the bacterial [2Fe-2S] DHADs
disclosed herein, as well as those for two additional isobutanol
pathways, are described in US 20070092957 A1, and additional genes
that may be used can be identified by one skilled in the art
through bioinformatics or experimentally as described above. The
preferred use in all three pathways of ketol-acid reductoisomerase
(KARI) enzymes with particularly high activities is disclosed in
commonly owned and co-pending US Patent Application Publication No.
US20080261230A1. Examples of high activity KARIs disclosed therein
are those from Vibrio cholerae (DNA: SEQ ID NO:389; protein SEQ ID
NO:390), Pseudomonas aeruginosa PAO1, (DNA: SEQ ID NO: 422; protein
SEQ ID NO:423), and Pseudomonas fluorescens PF5 (DNA: SEQ ID
NO:391; protein SEQ ID NO:392).
[0125] Additionally described in US 20070092957 A1 are construction
of chimeric genes and genetic engineering of bacteria and yeast for
isobutanol production using the disclosed biosynthetic
pathways.
Growth for Production
[0126] Recombinant bacteria or yeast hosts disclosed herein are
grown in fermentation media which contains suitable carbon
substrates. Additional carbon substrates may include but are not
limited to monosaccharides such as fructose, oligosaccharides such
as lactose maltose, galactose, or sucrose, polysaccharides such as
starch or cellulose or mixtures thereof and unpurified mixtures
from renewable feedstocks such as cheese whey permeate, cornsteep
liquor, sugar beet molasses, and barley malt. Other carbon
substrates may include ethanol, lactate, succinate, or
glycerol.
[0127] Additionally the carbon substrate may also be one-carbon
substrates such as carbon dioxide, or methanol for which metabolic
conversion into key biochemical intermediates has been
demonstrated. In addition to one and two carbon substrates,
methylotrophic organisms are also known to utilize a number of
other carbon containing compounds such as methylamine, glucosamine
and a variety of amino acids for metabolic activity. For example,
methylotrophic yeasts are known to utilize the carbon from
methylamine to form trehalose or glycerol (Bellion et al., Microb.
Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s):
Murrell, J. Collin; Kelly, Don P. Publisher: intercept, Andover,
UK). Similarly, various species of Candida will metabolize alanine
or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)).
Hence it is contemplated that the source of carbon utilized in the
present invention may encompass a wide variety of carbon containing
substrates and will only be limited by the choice of organism.
[0128] Although it is contemplated that all of the above mentioned
carbon substrates and mixtures thereof are suitable in the present
invention, preferred carbon substrates are glucose, fructose, and
sucrose, or mixtures of these with C5 sugars such as xylose and/or
arabinose for yeasts cells modified to use C5 sugars. Sucrose may
be derived from renewable sugar sources such as sugar cane, sugar
beets, cassava, sweet sorghum, and mixtures thereof. Glucose and
dextrose may be derived from renewable grain sources through
saccharification of starch based feedstocks including grains such
as corn, wheat, rye, barley, oats, and mixtures thereof. In
addition, fermentable sugars may be derived from renewable
cellulosic or lignocellulosic biomass through processes of
pretreatment and saccharification, as described, for example, in
co-owned and co-pending U.S. Patent Application Publication No.
2007/0031918A1, which is herein incorporated by reference. Biomass
refers to any cellulosic or lignocellulosic material and includes
materials comprising cellulose, and optionally further comprising
hemicellulose, lignin, starch, oligosaccharides and/or
monosaccharides. Biomass may also comprise additional components,
such as protein and/or lipid. Biomass may be derived from a single
source, or biomass can comprise a mixture derived from more than
one source; for example, biomass may comprise a mixture of corn
cobs and corn stover, or a mixture of grass and leaves. Biomass
includes, but is not limited to, bioenergy crops, agricultural
residues, municipal solid waste, industrial solid waste, sludge
from paper manufacture, yard waste, wood and forestry waste.
Examples of biomass include, but are not limited to, corn grain,
corn cobs, crop residues such as corn husks, corn stover, grasses,
wheat, wheat straw, barley, barley straw, hay, rice straw,
switchgrass, waste paper, sugar cane bagasse, sorghum, soy,
components obtained from milling of grains, trees, branches, roots,
leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits,
flowers, animal manure, and mixtures thereof.
[0129] In addition to an appropriate carbon source, fermentation
media must contain suitable minerals, salts, cofactors, buffers and
other components, known to those skilled in the art, suitable for
the growth of the cultures and promotion of an enzymatic pathway
comprising a bacterial [2Fe-2S] DHAD.
Culture Conditions
[0130] Typically cells are grown at a temperature in the range of
about 20.degree. C. to about 40.degree. C. in an appropriate
medium. Suitable growth media in the present invention are common
commercially prepared media such as Luria Bertani (LB) broth,
Sabouraud Dextrose (SD) broth, Yeast Medium (YM) broth, or broth
that includes yeast nitrogen base, ammonium sulfate, and dextrose
(as the carbon/energy source) or YPD Medium, a blend of peptone,
yeast extract, and dextrose in optimal proportions for growing most
Saccharomyces cerevisiae strains. Other defined or synthetic growth
media may also be used, and the appropriate medium for growth of
the particular microorganism will be known by one skilled in the
art of microbiology or fermentation science. The use of agents
known to modulate catabolite repression directly or indirectly,
e.g., cyclic adenosine 2':3'-monophosphate, may also be
incorporated into the fermentation medium.
[0131] Suitable pH ranges for the fermentation of yeast are
typically between pH 3.0 to pH 9.0. where pH 5.0 to pH 8.0 is
preferred as the initial condition. Suitable pH ranges for the
fermentation of other microorganisms are between pH 3.0 to pH 7.5,
where pH 4.5.0 to pH 6.5 is preferred as the initial condition.
[0132] Fermentations may be performed under aerobic or anaerobic
conditions, where anaerobic or microaerobic conditions are
preferred.
Industrial Batch and Continuous Fermentations
[0133] Fermentation may be a batch method of fermentation. A
classical batch fermentation is a dosed system where the
composition of the medium is set at the beginning of the
fermentation and not subject to artificial alterations during the
fermentation. Thus, at the beginning of the fermentation the medium
is inoculated with the desired organism or organisms, and
fermentation is permitted to occur without adding anything to the
system. Typically, however, "batch" fermentation is batch with
respect to the addition of carbon source and attempts are often
made at controlling factors such as pH and oxygen concentration. In
batch systems the metabolite and biomass compositions of the system
change constantly up to the time the fermentation is stopped.
Within batch cultures cells moderate through a static lag phase to
a high growth log phase and finally to a stationary phase where
growth rare is diminished or halted. If untreated, cells in the
stationary phase will eventually die. Cells in log phase generally
are responsible for the bulk of production of end product or
intermediate.
[0134] A variation on the standard batch system is the fed-batch
system. Fed-batch fermentation processes are also suitable in the
present invention and comprise a typical batch system with the
exception that the substrate is added in increments as the
fermentation progresses. Fed-batch systems are useful when
catabolite repression is apt to inhibit the metabolism of the cells
and where it is desirable to have limited amounts of substrate in
the media. Measurement of the actual substrate concentration in
fed-batch systems is difficult and is therefore estimated on the
basis of the changes of measurable factors such as pH, dissolved
oxygen and the partial pressure of waste gases such as CO2. Batch
and fed-batch fermentations are common and well known in the art
and examples may be found in Thomas D. Brock in Biotechnology: A
Textbook of Industrial Microbiology, Second Edition (1989) Sinauer
Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl.
Biochem. Biolechnol., 36:227, (1992), herein incorporated by
reference.
[0135] The fermentation culture may be adapted to continuous
fermentation methods. Continuous fermentation is an open system
where a defined fermentation medium is added continuously to a
bioreactor and an equal amount of conditioned media is removed
simultaneously for processing. Continuous fermentation generally
maintains the cultures at a constant high density.
[0136] Continuous fermentation allows for the modulation of one
factor or any number of factors that affect cell growth or end
product concentration. For example, one method will maintain a
limiting nutrient such as the carbon source or nitrogen level at a
fixed rate and allow all other parameters to moderate. In other
systems a number of factors affecting growth can be altered
continuously while the cell concentration, measured by the
turbidity of the culture medium, is kept constant. Continuous
systems strive to maintain steady state growth conditions and thus
the cell loss due to the medium being drawn off must be balanced
against the cell growth rate in the fermentation. Methods of
modulating nutrients and growth factors for continuous fermentation
processes as well as techniques for maximizing the rate of product
formation are well known in the art of industrial microbiology and
a variety of methods are detailed by Brock, supra.
[0137] It is contemplated that batch, fed-batch, continuous
processes, or any known mode of fermentation is suitable for growth
of the described recombinant microbial host cell. Additionally, it
is contemplated that cells may be immobilized on a substrate as
whole cell catalysts and subjected to fermentation conditions for
isobutanol production.
Methods for Product Isolation from the Fermentation Medium
[0138] Bioproduced isobutanol may be isolated from the fermentation
medium using methods known in the art such as for ABE fermentations
(see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648
(1998), Groot et al., Process Biochem. 27:61-75 (1992), and
references therein). For example, solids may be removed from the
fermentation medium by centrifugation, filtration, decantation, or
the like. Then, the isobutanol may be isolated from the
fermentation medium using methods such as distillation, azeotropic
distillation, liquid-liquid extraction, adsorption, gas stripping,
membrane evaporation, or pervaporation.
[0139] Because isobutanol forms a low boiling point, azeotropic
mixture with water, distillation can be used to separate the
mixture up to its azeotropic composition. Distillation may be used
in combination with another separation method to obtain separation
around the azeotrope. Methods that may be used in combination with
distillation to isolate and purify butanol include, but are no
limited to, decantation, liquid-liquid extraction, adsorption, and
membrane-based techniques. Additionally, butanol may be isolated
using azeotropic distillation using an entrainer (see for example
Doherty and Malone, Conceptual Design of Distillation Systems,
McGraw Hill, New York, 2001).
[0140] The butanol-water mixture forms a heterogeneous azeotrope so
that distillation may be used in combination with decantation to
isolate and purify the isobutanol. In this method, the isobutanol
containing fermentation broth is distilled to near the azeotropic
composition. Then, the azeotropic mixture is condensed, and the
isobutanol is separated from the fermentation medium by
decantation. The decanted aqueous phase may be returned to the
first distillation column as reflux. The isobutanol rich decanted
organic phase may be further purified by distillation in a second
distillation column.
[0141] The isobutanol may also be isolated from the fermentation
medium using liquid-liquid extraction in combination with
distillation. In this method, the isobutanol is extracted from the
fermentation broth using liquid-liquid extraction with a suitable
solvent. The isobutanol-containing organic phase is then distilled
to separate the butanol from the solvent.
[0142] Distillation in combination with adsorption may also be used
to isolate isobutanol from the fermentation medium. In this method,
the fermentation broth containing the isobutanol is distilled to
near the azeotropic composition and then the remaining water is
removed by use of an adsorbent, such as molecular sieves (Aden et
al. Lignocellulosic Biomass to Ethanol Process Design and Economics
Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic
Hydrolysis for Corn Stover, Report NREUTP-510-32438, National
Renewable Energy Laboratory, June 2002).
[0143] Additionally, distillation in combination with pervaporation
may be used to isolate and purify the isobutanol from the
fermentation medium. In this method, the fermentation broth
containing the isobutanol is distilled to near the azeotropic
composition, and then the remaining water is removed by
pervaporation through a hydrophilic membrane (Guo et al., J. Membr.
Sci. 245, 199-210 (2004)).
EXAMPLES
[0144] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating a preferred embodiment of the invention, are given by
way of illustration only. From the above discussion and these
Examples, one skilled in the art can ascertain the essential
characteristics of this invention and, without departing from the
spirit and scope thereof, can make various changes and
modifications of the invention to adapt it to various uses and
conditions.
General Methods
[0145] Standard recombinant DNA and molecular cloning techniques
described in the Examples are well known in the art and are
described by Sambrook, J., Fritsch, E. F. and Maniatis, T.
Molecular Cloning: A Laboratory Manual; Cold Spring Harbor
Laboratory Press: Cold Spring Harbor, N.Y., (1989) (Maniatis) and
by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with
Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols
in Molecular Biology, pub. by Greene Publishing Assoc. and
Wiley-Interscience (1987) amd by Methods in Yeast Genetics, 2005,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0146] Materials and methods suitable for the maintenance and
growth of bacterial cultures are well known in the art. Techniques
suitable for use in the following Examples may be found as set out
in Manual of Methods for General Bacteriology (Phillipp Gerhardt,
R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.
Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society
for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, Second
Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All
reagents, restriction enzymes and materials used for the growth and
maintenance of bacterial cells were obtained from Aldrich Chemicals
(Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life
Technologies (Rockville, Md.), or Sigma Chemical Company (St.
Louis, Mo.) unless otherwise specified.
[0147] Microbial strains were obtained from The American Type
Culture Collection (ATCC), Manassas, Va., unless otherwise noted.
The oligonucleotide primers used in the following Examples were
synthesized by Sigma-Genosys (Woodlands, Tex.) or Integrated DNA
Technologies (Coralsville, Iowa).
[0148] Synthetic complete medium is described in Amberg, Burke and
Strathern, 2005, Methods in Yeast Genetics, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
HPLC
[0149] Analysis for fermentation by-product composition is well
known to those skilled in the art. For example, one high
performance liquid chromatography (HPLC) method utilizes a Shodex
SH-1011 column with a Shodex SH-G guard column (both available from
Waters Corporation, Milford, Mass.), with refractive index (RI)
detection. Chromatographic separation is achieved using 0.01 M
H.sub.2SO.sub.4 as the mobile phase with a flow rate of 0.5 mL/min
and a column temperature of 50.degree. C. Isobutanol retention time
is about 47.6 minutes.
[0150] The meaning of abbreviations is as follows: "s" means
second(s), "min" means minute(s), "h" means hour(s), "psi" means
pounds per square inch, "nm" means nanometers, "d" means day(s),
".mu.L" means microliter(s), "mL" means milliliter(s), "L" means
liter(s), "mm" means millimeter(s), "nn" means nanometers, "mM"
means millimolar, ".mu.M" means micromolar, "M" means molar, "mmol"
means millimole(s), ".mu.mol" means micromole(s)", "g" means
gram(s), ".mu.g" means microgram(s) and "ng" means nanogram(s),
"PCR" means polymerase chain reaction, "OD" means optical density,
"OD600" means the optical density measured at a wavelength of 600
nm, "kDa" means kilodaltons, "g" means the gravitation constant,
"bp" means base pair(s), "kbp" means kilobase pair(s), ".about."
means about, "% w/v" means weight/volume percent, % v/v" means
volume/volume percent, "HPLC" means high performance liquid
chromatography, and "GC" means gas chromatography.
Example 1
Identification of Bacterial Dihydroxy-Acid Dehydratases with
[2Fe-2S] Cluster
Phylogenetic Analysis
[0151] Phylogenetic relationships were determined for
dihydroxy-acid dehydratases (DHADs) and related proteins. Related
proteins were identified through BlastP searches of publicly
available databases using amino acid sequences of E. coli DHAD (SEQ
ID NO:382), E. coli phosphogluconate dehydratase (EDD; SEQ ID
NO:384; coding region SEQ ID NO:383), and Azospirillum brasiliense
arabonate dehydratase (SEQ ID NO:386; coding region SEQ ID NO:385),
with the following search parameters: E value=10, word size=3,
Matrix=Blosum62, and Gap opening=11 and gap extension=1. Blast
searches employing the three different protein sequences generated
overlapping sets of sequence matches. Sequences were selected from
the search results based on E value cutoff of 10.sup.-5 with
removal of 95% identity sequences. Sequences that were shorter than
350 amino acids and sequences that were longer than 650 amino acids
were also removed. The resultant set of 976 amino acid sequences
included dihydroxy-acid dehydratases, phosphogluconate
dehydratrases, and aldonic acid dehydratases.
[0152] A profile HMM was generated from the experimentally verified
DHADs described in Example 2. See details below on building,
calibrating, and searching with this profile HMM. An hmmer search,
using this profile HMM as a query, against the 976 sequences
matched all sequences with an E value of <10.sup.-5. Multiple
sequence alignments of the amino acid sequences were performed with
the Clustal W algorithm (Thompson, J. D., Higgins, D. G., and
Gibson T. J. (1994) Nuc. Acid Res. 22: 4673 4680) employing the
following parameters: 1) for pairwise alignment parameters, a Gap
opening=10; Gap extend=0.1; matrix is Gonnet 250; and
mode-Slow-accurate, 2) for multiple alignment parameters, Gap
opening=10; Gap extension=0.2; and matrix is Gonnet series.
Phylogenetic trees were generated from sequence alignments based on
the Neighbor Joining method. A tree representing phylogenetic
relationships among the 976 sequences is shown in FIG. 2. Four main
main branches emerged from this analysis. They are labeled "4Fe-4S
DHAD", "2Fe-2S DHAD", "aldonic acid dehydratase", and "EDD", based
on the criteria detailed below. A fifth small branch of 17
sequences is marked as "Und" for undefined.
[0153] The aligned sequences were initially analyzed for the
presence of three cysteines determined to be essential for enzyme
activity and likely involved in Fe--S coordination in the
Azospirillum brasiliense arabonate dehydratase, which was reported
as a [4Fe-4S] duster protein (Watanabe, S et al. J. Biol. Chem.
(2006) 281:33521-33536). Each of the 976 sequences has the
cysteines corresponding to two of the Azospirillum brasiliense
arabonate dehydratase essential cysteines (at positions 124 and
197). Within the phylogenetic tree, there is a branch of 168
sequences that includes the [4Fe-4S] phosphogluconate dehydratase
of Zymomonas mobilis (Rodriguez, M. et. al. (1996) Biochem Mol Biol
Int. 38:783-789), Only the four amino adds alanine, valine or
serine or glycine, but not cysteine, were found at the position of
the third essential cysteine of the A. brasiliense arabonate
dehydratase (position 56). This 168 sequence branch is labeled in
FIG. 2 as "EDD".
[0154] A different branch of the tree contains 322 sequences, among
which is the known [4Fe-4S] cluster DHAD of E. coli. This 322
sequence branch is labeled in FIG. 2 as "4Fe-4S DHAD". All
sequences within this branch, and in the branch of 17 sequences
("Und"), contain glycine at the position corresponding to the third
cysteine. The remaining 469 sequences, which are clustered in two
branches and comprise both the aldonic acid recognizing A.
brasiliense arabonate dehydratase and a set of DHADs, possess all
three cysteines. These cysteines are at positions 56, 129, and 201
in the S. mutans DHAD (SEQ ID NO: 168) and at positions 61, 135,
and 207 in the L. lactis DHAD (SEQ ID NO: 232). Shown in FIG. 1 are
examples of regions from multiple sequence alignments that include
the conserved cysteines.
[0155] Further analysis of multiple sequence alignments and of
phylogenetic trees was performed to identify DHAD-specific residues
(signatures) In distinguish DHADs from the arabonate dehydratases
and other aldonic acid dehydratases. Among sequences containing the
three specified conserved cysteines, a Glade of 274 sequences was
found to contain the DHADs from S. mutans and L. lactis. The A.
brasiliense arabonate dehydratase was found in a separate Glade of
195 sequences. Multiple sequence alignments containing the
sequences from the DHAD group of 274, the aldonic acid dehydratase
group, and the "[4Fe-4S] DHAD" branch were analyzed for conserved
residues at each position. A set of residues that are conserved in
a majority of both DHAD groups, but not in the aldonic acid
dehydratases group, was detected and is shown in Table 4.
Additionally, residues that are conserved in the aldonic acid
dehydratases, but not in either of the two DHAD groups were also
found. Such differentially conserved residues may act as substrate
specificity determinants in their respective enzymes.
TABLE-US-00004 TABLE 4 Conserved residues* discriminating DHADs
from aldonic acid dehydratases [4Fe--4S] DHAD-274 Aldonic acid
Position* DHAD group Dehydratase 88 Asp Asp Glu 113 NC** NC Glu 142
Arg Arg or Asn NC 165 NC NC Gly 208 Asn Asn NC 454 Leu Leu NC 477
Phe Phe or Tyr NC 487 Gly Gly NC Residue(s) conserved in a >90%
majority of representatives. *Position numbering is a based on the
position in the S. mutans DHAD **Not Conserved
[0156] The group of DHADs forming the 274-sequence clade that does
not include [4Fe-4S] duster E. coli DHAD, [4Fe-4S] duster Z.
mobilis phosphogluconate dehydratase, or the reportedly [4Fe-4S]
duster A. brasillense arabonate dehydratase, was differentially
identified from the other groups by phylogeny and conserved
residues found in multiple sequence alignments as described above.
Consistent with the proposal that the group includes [2Fe-2S]
duster DHADs, the Arabidopsis thaliana DHAD and the S. solfataricus
DHAD are part of the group. Because Arabidopsis thaliana is a plant
as is spinach, and the spinach DHAD has been identified as a
[2Fe-2S] duster DHAD (Flint and Emptage (1988) J. Biol. Chem.
263:3558-3564), the Arabidopsis thaliana DHAD may be a [2Fe-2S]
duster DHAD. The S. solfataricus DHAD is reported to be oxygen
resistant like the spinach [2Fe-2S] duster DHAD, (Kim and Lee
(2006) J. Biochem. 139, 591-596) which is an indication that the S.
solfataricus DHAD may be a [2Fe-2S] duster DHAD.
[0157] The 274 sequence Glade is labeled on FIG. 4 as "2Fe-2S
DHAD". 193 of these sequences are from bacterial sources. The three
conserved cysteines and the conserved residues specified in Table 4
can be identified in multiple sequence alignments of the 193
bacterial DHADs, employing the alignment procedure described above.
The sequences of the 193 bacterial [2Fe-2S] DHADs are provided in
the sequence listing and the SEC) ID NOs are listed in Table
2a.
[0158] Within the [2Fe-2S] DHAD group, the identities of several
bacterial proteins have been confirmed by functional analysis as
DHADs, which is described in other examples herein. Other examples
herein show that proteins in this group, such as the S. mutans DHAD
and the L. lactic DHAD, contain a [2Fe-2S] duster.
Preparation of Profile HMM
[0159] Seven bacterial DHADs that were identified as members of the
[2Fe-2S] phylogenetic group were expressed in E. coli and
dihydroxy-acid dehydratase activity was found as described in
Example 2 below. These DHADs are from Nitrosomonas europaea (SEQ ID
NO:310), Synechocystis sp. PCC6803 (SEQ ID NO: 298), Streptococcus
mutans (SEQ ID NO:168), Streptococcus thermophilus (SEQ ID NO:164),
Raistonia metailidurans (SEQ ID NO:346), Raistonia eutrophy (SEQ ID
NO:344), and Lactococcus lactis (SEQ ID NO:232). In addition the
DHAD from Flavobacterium johnsoniae (SEQ ID NO:230) was found to
have dihydroxy-acid dehydratase activity when expressed in E. coli.
The amino acid sequences of these experimentally determined
functional bacterial DHADs were analyzed using the HMMER software
package (The theory behind profile HMMs is described in R. Durbin,
S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis:
probabilistic models of proteins and nucleic acids, Cambridge
University Press, 1998; Krogh et al., 1994; J. Mol. Biol.
235:1501-1531), following the user guide which is available from
HMMER (Janelia Farm Research Campus, Ashburn, Va.). The output of
the HMMER software program is a Profile Hidden Markov Model (HMM)
that characterizes the input sequences. As stated in the user
guide, Profile HMMs are statistical models of multiple sequence
alignments. They capture position-specific information about how
conserved each column of the alignment is, and which amino add
residues are most likely to occur at each position. Thus HMMs have
a formal probabilistic basis. Profile HMMs for a large number of
protein families are publicly available in the PFAM database
(Janelia Farm Research Campus, Ashburn, Va.).
The Profile HMM was built as follows:
Step 1. Build a Sequence Alignment
[0160] The eight sequences for the functionally verified DHADs
listed above were aligned using Clustal W with default
parameters.
Step 2. Build a Profile HMM
[0161] The hmmbuild program was run on the set of aligned sequences
using default parameters. hmmbuild reads the multiple sequence
alignment file, builds a new Profile HMM, and saves the Profile HMM
to file. Using this program an un-calibrated profile was generated
from the multiple alignment for each set of subunit sequences
described above.
[0162] The following information based on the HMMER software user
guide gives some description of the way that the hmmbuild program
prepares a Profile HMM. A Profile HMM is capable of modeling gapped
alignments, e.g. including insertions and deletions, which lets the
software describe a complete conserved domain (rather than just a
small ungapped motif). Insertions and deletions are modeled using
insertion (I) states and deletion (D) states, All columns that
contain more than a certain fraction x of gap characters will be
assigned as an insert column. By default, x is set to 0.5. Each
match state has an I and a D state associated with ft. HMMER calls
a group of three states (M/D/I) at the same consensus position in
the alignment a "node". These states are interconnected with arrows
called state transition probabilities. M and I states are emitters,
while D states are silent. The transitions are arranged so that at
each node, either the M state is used (and a residue is aligned and
scored) or the D state is used (and no residue is aligned,
resulting in a deletion-gap character, Insertions occur between
nodes, and I states have a self-transition, allowing one or more
inserted residues to occur between consensus columns.
[0163] The scores of residues in a match state (i.e. match state
emission scores), or in an insert state (i.e. insert state emission
scores) are proportional to Log.sub.--2 (p_x)/(null_x). Where p_x
is the probability of an amino acid residue, at a particular
position in the alignment, according to the Profile HMM and null_x
is the probability according to the Null model. The Null model is a
simple one state probabilistic model with pre-calculated set of
emission probabilities for each of the 20 amino acids derived from
the distribution of amino acids in the SWISSPROT release 24.
[0164] State transition scores are also calculated as log odds
parameters and are proportional to Log.sub.--2 (t_x). Where t_x is
the probability of transiting to an emitter or non-emitter
state.
Step 3. Calibrate the Profile HMM
[0165] The Profile HMM was read using hmmcalibrate which scores a
large number of synthesized random sequences with the Profile (the
default number of synthetic sequences used is 5,000), fits an
extreme value distribution (EVD) to the histogram of those scores,
and re-saves the HMM file now including the EVD parameters. These
EVD parameters (.mu. and .lamda.) are used to calculate the
E-values of bit scores when the profile is searched against a
protein sequence database. hmmcalibrate writes two parameters into
the HMM file on a line labeled "EVD": these parameters are the .mu.
(location) and .lamda. (scale) parameters of an extreme value
distribution (EVD) that best fits a histogram of scores calculated
on randomly generated sequences of about the same length and
residue composition as SWISS-PROT. This calibration was done once
for the Profile HMM.
[0166] The calibrated Profile HMM for the DHAD set of sequences is
provided in Table 1. The Profile HMM is provided in a chart that
gives the probability of each amino acid occurring at each position
in the amino acid sequence. The highest probability is highlighted
for each position. The first line for each position reports the
match emission scores: probability for each amino acid to be in
that state (highest score is highlighted). The second line reports
the insert emission scores, and the third line reports on state
transition scores: M.fwdarw.M, M.fwdarw.I, M.fwdarw.D; I.fwdarw.M,
I.fwdarw.I; D.fwdarw.M, D.fwdarw.D; B.fwdarw.M; M.fwdarw.E.
[0167] For example, the DHAD Profile HMM shows that methionine has
a 1757 probability of being in the first position, the highest
probability which is highlighted. In the second position glutamic
acid has the highest probability, which is 1356. In the third
position lysine has the highest probability, which is 1569.
Step 4. Test the Specificity and Sensitivity of the Built Profile
HMMs
[0168] The Profile HMM was evaluated using hmmsearch, which reads a
Profile HMM from hmmfile and searches a sequence file for
significantly similar sequence matches. The sequence file searched
contained 976 sequences (see above). During the search, the size of
the database (Z parameter) was set to 1 billion. This size setting
ensures that significant E-values against the current database will
remain significant in the foreseeable future. The E-value cutoff
was set at 10.
[0169] A hmmer search with the Profile HMM generated from the
alignment of the eight DHADs with experimentally verified function,
matched all 976 sequences with an E value <10.sup.-5. This
result indicates that members of the dehydratase superfamily share
significant sequence similarity. A hmmer search with a cutoff of E
value 10.sup.-5 was used to separate DHAD related dehydratases from
other more remote but related proteins, as described above.
Example 2
Expression and Characterization of Bacterial [2Fe-2S]
Dihydroxy-Acid Dehydratases in E. coli
[0170] The ilvD coding regions from different bacteria, which from
the phylogenetic analysis described in Example 1 are in the
[2Fe-2S] group, were expressed under the control of the T7 promoter
in vector pET28a (Novagen) in E. coli. Each ilvD coding region was
amplified with a specific forward primer with an NheI restriction
site and a specific reverse primer with a NotI restriction site
(listed in Table 5).
TABLE-US-00005 TABLE 5 SEQ ID NOs of primers used for PCR of DHAD
coding regions from the listed organisms. Reverse Forward primer
Primer Organism SEQ ID NO SEQ ID NO Nitrosomonas europaea ATCC
19718 424 401 Synechocystis sp. PCC 6803 399 400 Streptococcus
mutans UA159 (ATCC 435 436 700610) Streptococcus thermophilus LMG
18311 395 396 Ralstonia metallidurans CH34 404 405 Ralstonia
eutropha H16 (ATCC 17699) 406 407 Lactococcus lactis 420 421
[0171] The genomic DNA of each bacterial strain was used as a
template. Genomic DNA was prepared from each strain listed in Table
1 using a MasterPure DNA Purification Kit (Epicentre, Madison,
Wis.). The plasmid vector was amplified with primers pET28a-F(NotI)
(SEQ ID NO:397) and pET28a-R(NheI) (SEQ ID NO:398) to remove the
his tag region. Both gene and plasmid fragments were digested with
NheI and NotI before ligation. The ligation mixture was transformed
into E. coli (Top 10) competent cells (Invitrogen). Transformants
were grown in LB agar plates supplemented with 50 .mu.g/ml of
kanamycin. Positive clones that were confirmed by sequencing were
transformed into the E. coli Tuner (DE3) strain (Novagen) for
expression. Selected colonies were grown in LB liquid medium
supplemented with kanamycin at 30.degree. C. Induction was carried
out by adding 0.5 mM of IPTG when the E. coli culture reached an
O.D. of 0.3 to 0.4 at 600 nm. The culture was harvested after 5
hours of induction. Cell pellets were washed with Tris buffer (pH
8.0).
[0172] Enzymatic activity of the crude extract was assayed at
37.degree. C. as follows. Cells to be assayed for DHAD were
suspended in 2-5 volumes of 50 mM Tris, 10 mM MgSO.sub.4, pH 8.0
(TM8) buffer, then broken by sonication at 0.degree. C. The crude
extract from the broken cells was centrifuged to pellet the cell
debris. The supernatants were removed and stored on ice until
assayed (initial assay was within 2 hrs of breaking the cells). It
was found that the DHADs assayed herein were stable in crude
extracts kept on ice for a few hours. The activity was also
preserved when small samples were frozen in liquid N.sub.2 and
stored at -80.degree. C.
[0173] The supernatants were assayed using the reagent
2,4-dinitrophenyl hydrazine as described in Flint and Emptage (J.
Biol. Chem. (1988) 263: 3558-64). When the activity was so high
that it became necessary to dilute the crude extract to obtain an
accurate assay, the dilution was done in 5 mg/ml BSA in TM8.
[0174] Protein assays were performed using the Pierce Better
Bradford reagent (cat 23238) using BSA as a standard. Dilutions for
protein assays were made in TM8 buffer when necessary.
[0175] All of the DHADs were active when expressed in E. coli, and
the specific activities are given in Table 6. The DHAD from
Streptococcus mutans had the highest specific activity.
TABLE-US-00006 TABLE 6 Activities of bacterial [2Fe--2S] DHADs in
E. coli Specific SEQ ID NO of Activity DHAD coding (.mu.mol
Organism Source of DHAD sequence min.sup.-1 mg.sup.-1) Nitrosomonas
europaea ATCC 19718 309 3.3 Synechocystis sp. PCC 6803 297 3.5
Streptococcus mutans UA159 (ATCC 167 7.9 700610) Streptococcus
thermophilus LMG 18311 163 6.6 Ralstonia metallidurans CH34 404 2.4
Ralstonia eutropha H16 (ATCC 17699) 406 4.1 Lactococcus lactis 231
2.1 Vector control N/A 0.07
Example 3
Purification and Characterization of DHAD from Streptococcus mutans
Expressed in E. coli
[0176] The DHAD from S. mutans was further purified and
characterized. For purification of the S. mutans DHAD, six liters
of culture of the E. coli Tuner strain harboring the pET28a plasmid
with the S. mutans ilvD were grown and induced with IPTG. The
enzyme was purified by breaking the cells, as described in Example
2, in a 50 mM Tris buffer pH 8.0 containing 10 mM MgCl.sub.2 (TM8
buffer), centrifuging to remove cell debris, then loading the
supernatant of the crude extract on a Q Sepharose (GE Healthcare)
column and eluting the CHAD with an increasing concentration of
NaCl in TM8 buffer. The fractions containing the DHAD based on the
color appearance (brownish color is due to the presence of the
Fe--S cluster) were pooled and loaded onto a Sephacryl S-100 (GE
Healthcare) column and eluted with TM8 buffer. As judged by SOS
gels, the purity of the protein eluted from the Sephacryl column
was estimated to be 60-80%. The activity of the partially purified
enzyme was assayed at 37.degree. C. as described by Flint et al.
(J. Biol. Chem. (1988) 263(8): 3558-64). The specific activity of
the purified protein was 40 .mu.mol min.sup.-1 mg.sup.-1. The
k.sub.cat for the purified enzyme was estimated to be 50-70
sec.sup.-1.
[0177] Stability of the purified DHAD in air was studied by
incubating the purified enzyme at 23.degree. C. for various time
intervals in the presence of ambient air, followed by an activity
assay as described above. The activity of the DHAD from
Streptococcus mutans, in contrast to DHAD purified similarly from
E. coli, was stable even after 72 hours of incubation as shown in
FIG. 4, where (A) shows results from the S. mutans DHAD and (B)
shows results from the E. coli DHAD.
[0178] The UV-visible spectrum of the purified S. mutans is shown
in FIG. 5. The number of peaks above 300 nm is typical of proteins
with [2Fe-2S] dusters. The S. mutans DHAD was reduced with sodium
dithionite and EPR spectra were obtained at varying temperatures.
FIG. 6 show spectra measured at temperatures between 20.degree. K
and 70.degree. K. That the EPR spectrum of S. mutans is measureable
up to 70.degree. K is indicative that it contains a [2Fe-2S]
duster. It is well known for example that the EPR spectra of
proteins containing [4Fe-4S] dusters are not observable at
temperatures much above 10.degree. K. (See, for example, Rupp, et
al. Biochimica et Biophysica Acta (1978) 537:255-269.)
Example 4
Construction of Dihydroxy-Acid Dehydratase (DHAD) Expression
Cassettes for Lactobacillus plantarum
[0179] The purpose of this example is to describe how to done and
express a dihydroxy-acid dehydratase gene (ilvD) from different
bacterial sources in Lactobacillus plantarum PN0512 (ATCC
PTA-7727). A shuttle vector pDM1 (SEQ D NO:410) was used for
cloning and expression of ilvD genes from Lactococcus lactis subsp
lactis NCDO2118 (NCIMB 702118) [Codon et al., J. Bacterial. (1992)
174:6580-6589] and Streptococcus mutans UA159 (ATCC 700610) in L.
plantarum PN0512. Plasmid pDM1 contains a minimal pLF1 replicon
(-0.7 Kbp) and pemK-pemI toxin-antitoxin(TA) from Lactobacillus
plantarum ATCC14917 plasmid pLF1, a P15A replicon from pACYC184,
chloramphenicol marker for selection in both E. coli and L.
plantarum, and P30 synthetic promoter [Rud et al, Microbiology
(2006) 152:1011-1019]. Plasmid pLF1 (C.-F. Lin et al., GenBank
accession no. AF508808) is closely related to plasmid p256 [Sorvig
et al., Microbiology (2005) 151:421-431], whose copy number was
estimated to be .about.5-10 copies per chromosome for L. plantarum
NC7. A P30 synthetic promoter was derived from L. plantarum rRNA
promoters that are known to be among the strongest promoters in
lactic acid bacteria (LAB) [Rud et al. Microbiology (2005)
152:1011-1019].
[0180] The Lactococcus lactis ilvD coding region (SEQ D NO:231) was
PCR-amplified from Lactococcus lactis subsp lactis NCD02118 genomic
DNA with primers 3T-ilvDLI(BamHI) (SEQ ID NO:408) and
5B-ilvDLI(NotI) (SEQ ID NO:409). L. lactis subsp lactis NCDO2118
genomic DNA was prepared with a Puregene Gentra Kit (QIAGEN, CA).
The 1.7 Kbp L. lactis ilvD PCR product (ilvDLI) was digested with
NotI and treated with the Klenow fragment of DNA polymerase to make
blunt ends. The resulting L. lactis ilvD coding region fragment was
digested with BamHI and gel-purified using a QIAGEN gel extraction
kit (QIAGEN, CA). Plasmid pDM1 was digested with ApaLI, treated
with the Klenow fragment of DNA polymerase to make blunt ends, and
then digested with BamHI. The gel purified L. lactis ilvD coding
region fragment was ligated into the BamHI and ApaLI(blunt) sites
of the plasmid pDM1. The ligation mixture was transformed into E.
coli Top10 cells (Invitrogen, CA). Transformants were plated for
selection on LB chloramphenicol plates. Positive clones were
screened by SalI digestion, giving one fragment with an expected
size of 5.3 Kbp. The positive clones were further confirmed by DNA
sequencing. The correct done was named pDM1-ilvD(L. lactis).
[0181] The S. mutans UA159 (ATCC 700610) ilvD coding region from
the plasmid pET28a was cloned on the plasmid pDM1. The construction
of pET28a containing the S. mutans ilvD was described in Example 2.
The plasmid pET28a containing the S. mutans ilvD was digested with
XbaI and NotI, treated with the Klenow fragment of DNA polymerase
to make blunt ends, and a 1,759 bp fragment containing the S.
mutans ilvD coding region was gel-purified. Plasmid pDM1 was
digested with BamHI, treated with the Klenow fragment of DNA
polymerase to make blunt ends, and then digested with PvuII. The
gel purified fragment containing S. mutans ilvD coding region was
ligated into the BamHI(blunt) and PvuII sites of the plasmid pDM1.
The ligation mixture was transformed into E. coli Top10 cells
(Invitrogen, CA). Transformants were plated for selection on LB
chloramphenicol plates. Positive clones were screened by ClaI
digestion, giving one fragment with an expected size of 5.5 Kbp.
The correct clone was named pDM1-ilvD(S. mutans).
Example 5
Measurement of Expressed DHAD Activity in L. plantarum PN0512
[0182] L. plantarum PN0512 was transformed with plasmid
pDM1-ilvD(L. lactis) or pDM1-ilvD(S. mutans) by electroporation.
Electro-competent cells were prepared by the following procedure. 5
ml of Lactobacilli MRS medium containing 1% glycine was inoculated
with PN0512 cells and grown overnight at 30.degree. C. 100 ml MRS
medium with 1% glycine was inoculated with the overnight culture to
an OD600=0.1 and grown to an OD600=0.7 at 30.degree. C. Cells were
harvested at 3700.times.g for 8 min at 4.degree. C., washed with
100 ml cold 1 mM MgCl.sub.2, centrifuged at 3700.times.g for 8 min
at 4.degree. C., washed with 100 ml cold 30% PEG-1000 (81188,
Sigma-Aldrich, St. Louis, Mo.), recentrifuged at 3700.times.g for
20 min at 4.degree. C., then resuspended in 1 ml cold 30% PEG-1000.
60 .mu.l of electro-competent cells were mixed with .about.100 ng
plasmid DNA in a cold 1 mm gap electroporation cuvette and
electroporated in a BioRad Gene Pulser (Hercules. CA) at 1.7 kV, 25
.mu.F, and 400.OMEGA.. Cells were resuspended in 1 ml MRS medium
containing 500 mM sucrose and 100 mM MgCl.sub.2, incubated at
30.degree. C. for 2 hrs, and then plated on MRS medium plates
containing 10 .mu.g/ml of chloramphenicol.
[0183] L. plantarum PN0512 transformants carrying pDM1-ilvD(L.
lactis) or pDM1-ilvD(S. mutans) as well as control transformants
with the pDM1 vector alone, were grown overnight in Lactobacilli
MRS medium at 30.degree. C. 120 ml of MRS medium supplemented with
100 mM MOPS (pH7.5), 40 .mu.M ferric citrate, 0.5 mM L-cysteine,
and 10 .mu.g/ml chloramphenicol was inoculated with overnight
culture to an OD600=0.1 in a 125 ml screw cap flask, for each
overnight sample. The cultures were anaerobically incubated at
37.degree. C. until reaching an OD600 of 1-2. Cultures were
centrifuged at 3700.times.g for 10 min at 4.degree. C. Pellets were
washed with 50 mM potassium phosphate buffer pH 6.2 (6.2 g/L
KH.sub.2PO.sub.4 and 1.2 g/L K.sub.2HPO.sub.4) and re-centrifuged.
Pellets were frozen and stored at -80.degree. C. until assayed for
DHAD activity. Cell extract samples were assayed for DHAD activity
using a dinitrophenylhydrazine based method as described in Example
2. The DHAD activity results are given in Table 7. Specific
activity of L. lactic DHAD and S. mutans DHAD in L. plantarum
PN0512 showed 0.02 and 0.06 .mu.mol min.sup.-1 mg.sup.-1,
respectively, while the vector control sample exhibited no
detectable activity.
TABLE-US-00007 TABLE 7 DHAD activity in L. plantarum PN0512.
Specific Activity Source of DHAD Plasmid (.mu.mol min.sup.-1
mg.sup.-1) Vector control pDM1 0.00 Lactococcus lactis subsp lactis
pDM1-ilvD 0.02 NCDO2118 (L. lactis) Streptococcus mutans UA159
pDM1-ilvD 0.06 (S. mutans)
Example 6
Expression of Dihydroxy-Acid Dehydratase from S. mutans in
Yeast
[0184] The shuttle vector pRS423 FBA ilvD(Strep) (SEQ ID NO:430)
was used for the expression of DHAD from Streptococcus mutans. This
shuttle vector contained an F1 origin of replication (1423 to 1879)
for maintenance in E. coli and a 2 micron origin (nt 8082 to 9426)
for replication in yeast. The vector has an FBA promoter (nt 2111
to 3108; SEQ ID NO:425) and FBA terminator (nt 4861 to 5860). In
addition, it carries the His marker (nt 504 to 1163) for selection
in yeast and ampicillin resistance marker (nt 7092 to 7949) for
selection in E. coli. The ilvD coding region (nt 3116 to 4828) from
Streptococcus mutans UA159 (ATCC 700610) is between the FBA
promoter and FBA terminator forming a chimeric gene for
expression.
[0185] To test the expression of the DHAD from Streptococcus mutans
in yeast strain BY4741 (known in the art and obtainable from ATCC
#201388), the expression vector pRS423 FBA IlvD(Strep) was
transformed in combination with empty vector pRS426 into BY4741
cells (obtainable from ATCC, #201388). The transformants were grown
on synthetic medium lacking histidine and uracil (Teknova). Growth
on liquid medium for assay was carried out by adding 5 ml of an
overnight culture into 100 ml medium in a 250 ml flask. The
cultures were harvested when they reached 1 to 2 O.D. at 600 nm.
The samples were washed with 10 ml of 20 mM Tris (pH 7.5) and then
resuspended in 1 ml of the same Tris buffer. The samples were
transferred into 2.0 ml tubes containing 0.1 mm silica (Lysing
Matrix B, MP biomedicals). The cells were then broken in a
bead-beater (BIO101). The supernatant was obtained by
centrifugation in a microfuge at 13,000 rpm at 4.degree. C. for 30
minutes. Typically, 0.06 to 0.1 mg of crude extract protein was
used in DHAD assay at 37.degree. C. as described by Flint and
Emptage (J. Biol. Chem. (1988) 263(8): 3558-64) using
dinitrophenylhydrazine. The dehydratase from Streptococcus mutans
had a specific activity of 0.24 .mu.mol min.sup.-1 mg.sup.-1 when
expressed in yeast. A control strain containing empty vectors
pRS423 and pRS426 had a background of activity in the range of 0.03
to 0.06 .mu.mol min.sup.-1 mg.sup.-1.
Example 7
Expression of the IlvD Gene from L. lactis in Yeast
[0186] The ilvD coding region from L. lactis was amplified with the
forward primer IlvD(LI)-F (SEQ ID NO:420) and reverse primer
IlvD(LI)-R (SEQ ID NO:421). The amplified fragment was cloned into
the shuttle vector pNY13by gap repair. pNY13 (SEQ ID NO:437) was
derived from pRS423. This shuttle vector contained an F1 origin of
replication (1423 to 1879) for maintenance in E. coli and a 2
micron origin (nt 7537 to 8881) for replication in yeast. The
vector has an FBA promoter (nt 2111 to 3110) and FBA terminator (nt
4316 to 5315). In addition, it carries the His marker (nt 504 to
1163) for selection in yeast and Ampicillin resistance marker (nt
6547 to 7404) for selection in E. coli.
[0187] Positive clones were selected based on amplification with
the forward and reverse primers for the ilvD coding region and
further confirmed by sequencing. The new construct was designated
as pRS423 FBA ilvD(L. lactis). This construct was transformed into
yeast strain BY4743 (.DELTA.LEU1) (Open Biosystems, Huntsville,
Ala.; Catalog #YSC1021-666629) along with the empty vector pRS426
as described in Example 6. The growth and assay of yeast strains
containing the expression vector were also carried out according to
the procedures as described in Example 6. The dihydroxy-acid
dehydratase activity in yeast strains with the L. lactis IlvD gene
was determined to be in the range of 0.05 to 0.17 .mu.mol
min.sup.-1 mg.sup.-1. This activity was slightly above the control.
Complementation experiments were carried out to investigate the
expression of this DHAD and DHADs from other bacteria (example
8).
Example 8
Complementation of Yeast ILV3 Deletion Strain with Bacterial
DHADs
[0188] The endogenous DHAD enzyme of S. cerevisiae is encoded by
the ILV3 gene and the protein is targeted to the mitochondrion.
Deletion of this gene results in loss of endogenous DHAD activity
and provides a test strain where expression of heterologous
cytosolic DHAD activity can be readily assessed. Deletion of ILV3
results in an inability of the strain to grow in the absence of
branched-chained amino acids. Expression of different bacterial
DHADs was assayed by determining their ability to complement the
yeast ILV3 deletion strain such that it grows in the absence of
branched-chained amino acids.
[0189] Expression shuttle vectors containing ilvD gene sequences
encoding DHADs from the bacteria listed in Table 8 were
constructed. The basic elements of these expression constructs were
the same as the pRS423 FBA ilvD(strep) vector described in Example
6. Each of the ilvD coding regions was prepared by PCR as described
in Example 2 and cloned to replace the Streptococcus mutans ilvD
coding region in pRS423 FBA ilvD(Strep) creating the plasmids
listed in Table 8. These expression constructs were transformed
into the ILV3 deletion strain, BY4741 ilv3::URA3 and was prepared
as follows. An ilv3::URA3 disruption cassette was constructed by
PCR amplification of the URA3 marker from pRS426 (ATCC No. 77107)
with primers "ILV3::URA3 F" and "ILV3::URA3 R", given as SEQ ID
NOs: 431 and 432. These primers produced a 1.4 kb URA3 PCR product
that contained 70 bp 5' and 3' extensions identical to sequences
upstream and downstream of the ILV3 chromosomal locus for
homologous recombination. The PCR product was transformed into
BY4741 cells (ATCC 201388) using standard genetic techniques
(Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., pp. 201-202) and resulting
transformants were maintained on synthetic complete media lacking
uracil and supplemented with 2% glucose at 30.degree. C.
Transformants were screened by PCR using primers "ILV3 F Check" and
"URA3 REV Check", given as SEQ ID NOs:433 and 434, to verify
integration at the correct site and disruption of the
endogenous/LV3 locus.
[0190] The transformants with bacterial DHADs in Table 8 were
selected on plates with yeast synthetic medium lacking histidine.
Colonies selected were then patched onto plates lacking valine,
leucine and isoleucine. Strains containing the expression vectors
listed in Table 8 were able to grow on these plates lacking the
branched chain amino acids, while the control strain with the
control plasmid did not. This result indicated that DHADs from
these bacteria were actively expressed in S. cerevisiae.
TABLE-US-00008 TABLE 8 Bacterial DHADs tested and Expression
vectors SEQ ID NO. OF DHAD nucleic acid Organism Source of DHAD
sequence Vector designation Nitrosomonas europaea 309 pRS423 FBA
ilvD (europ) ATCC 19718 Synechocystis sp. PCC 297 pRS423 FBA ilvD
(Synech) 6803 Streptococcus thermophilus 163 pRS423 FBA ilvD
(thermo) LMC 18311 Ralstonia eutropha H16 406 pRS423 FBA ilvD (H16)
(ATCC 17699) Lactococcus lactis 231 pRS423 FBA ilvD (L. lactis)
Example 9
Purification and Characterization of DHAD from Lactococcus lactis
expressed in E. coli
[0191] The DHAD from L. lactis was purified and characterized. For
purification of L. lactis DHAD, 14 liters of culture of the E. coli
Tuner (DE3) strain (Novagen) harboring the pET28a plasmid
containing the L. lactis ilvD were grown and induced with IPTG. The
enzyme was purified by breaking the cells, as described in Example
2, in 120 mls of 50 mM Tris buffer pH 8.0 containing 10 mM
MgCl.sub.2 (TM8 buffer), centrifuging to remove cell debris, then
loading the supernatant of the crude extract on a 5.times.15 cm Q
Sepharose (GE Healthcare) column and eluting the DHAD with an
increasing concentration of NaCl in TM8 buffer. The fractions
containing the DHAD were pooled, made 1 M in
(NH.sub.4).sub.2SO.sub.4 and loaded onto a 2.6.times.15 cm
phenyl-Sepharose column (GE Healthcare) column equilibrated with 1
M (NH.sub.4).sub.2SO.sub.4 in TM8 buffer and eluted with a
decreasing gradient of (NH.sub.4).sub.2SO.sub.4. The fractions
containing DHAD off the phenyl-Sepharose column were pooled and
concentrated to 10 ml. This was loaded onto a 3.5.times.60 cm
Superdex-200 column (GE Healthcare) and eluted with TM8. The
fractions containing DHAD activity were pooled, concentrated, and
frozen as beads in N.sub.2(I). As judged by SDS gels, the purity of
the protein eluted from the Superdex-200 column was estimated to be
>80%. The activity of the enzyme was assayed at 37.degree. C. as
described by Flint et al. (J. Biol. Chem. (1988) 263(8): 3558-64).
The specific activity of the purified protein was 64 .mu.mol
min.sup.-1 mg.sup.-1 at pH 8 and 37.degree. C. The k.sub.cat for
the purified enzyme was 71 sec.sup.-1.
[0192] Stability of the purified DHAD in air was studied by
incubating the purified enzyme at 23.degree. C. for various time
intervals in the presence of ambient air, followed by an activity
assay as described above. The DHAD from L. lactic was almost fully
active even after 20 hours of incubation in air as shown in FIG.
8.
[0193] The UV-visible spectrum of the purified L. lactis is shown
in FIG. 9. It is characteristic of proteins with [2Fe-2S]
clusters.
Example 10
Use of Dihydroxy-Acid Dehydratase to Construct a Pathway for the
Production of Isobutanol in Yeast
[0194] The first three steps of an isobutanol biosynthetic pathway
are performed by the enzymes acetolactate synthase, ketol-acid
reductoisomerase (KARI), and dihydroxy-acid dehydratase.
Acetolactate synthase is encoded by alsS. KARI genes are known as
ILV5 in yeast or ilvC in bacteria. Once .alpha.-ketoisovalerate
(KIV) is formed from pyruvate by the reaction of these three
enzymes, it can be further converted to isobutanol in yeast by
alcohol dehydrogenases.
[0195] Vector pLH532 (SEQ ID NO:411) was constructed to express
KARI and alsS genes. This vector is derived from the 2 MICRON based
vector pHR81. In pLH532 the alsS coding region from B. subtilis (nt
14216 to 15931) was under the control of the CUP1 promoter (nt.
15939 to 16386). There were two KARI genes in pLH532: the ilvC
coding region from P. fluorescens Pf5 (nt. 10192 to 11208) was
under the control of the yeast ILV5 promoter (nt. 11200 to 12390),
and the yeast ILV5 coding region (nt. 8118-9167) was placed under
the control of the FBA promoter (nt. 7454-8110). The selection
marker was URA3 (nt. 3390 to 4190).
[0196] The yeast host for isobutanol production was BY4741
pdc1::FBAp-alsS-LEU2. This strain was constructed as follows.
First, the expression plasmid pRS426-FBAp-alsS was constructed. The
1.7 kb alsS coding region fragment of pRS426::GPD::alsS::CYC was
isolated by gel purification following BbvCI and PacI digestion.
This plasmid has a chimeric gene containing the GPD promoter (SEQ
ID NO:439), the alsS coding region from Bacillus subtilis (SEQ ID
NO:438), and the CYC1 terminator (SEQ ID NO:440) and was described
in Example 17 of US Patent Publication #US20070092957A1 which is
herein incorporated by reference. The ILV5 fragment from plasmid
pRS426::FBA::ILV5::CYC, also described in US20070092957 Example 17,
was removed by restriction digestion with BbvCI and Pad and the
remaining 6.6 kb vector fragment was gel purified. This vector has
a chimeric gene containing the FBA promoter (SEQ ID NO:425) and
CYC1 terminator bounding the coding region of the ILV5 gene of S.
cerevisiae (SEQ ID NO:442). These two purified fragments were
ligated overnight at 16.degree. C. and transformed into E. coli
TOP10 chemically competent cells (Invitrogen). Transformants were
obtained by plating cells on LB Amp100 medium. Insertion of alsS
into the vector was confirmed by restriction digestion pattern and
PCR (primers N98SeqF1 and N99SeqR2, SEQ ID NOs:412 and 413).
[0197] A pdc1::FBAp-alsS-LEU2 disruption cassette was created by
joining the FBAp-alsS segment from pRS426-FBAp-alsS to the LEU2
gene from pRS425 (ATCC No. 77106) by SOE PCR (as described by
Horton et al. (1989) Gene 77:61-68) using as template
pRS426-FBAp-alsS and pRS425 plasmid DNAs, with Phusion DNA
polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no.
F-540S) and primers 112590-48A and 112590-30B through D, given as
SEQ ID NOs:414, SEQ ID NOs:415, 416, and 417. The outer primers for
the SOE PCR (112590-48A and 112590-300) contained 5' and 3' 50 bp
regions homologous to regions upstream and downstream of the PDC1
promoter and terminator. The completed cassette PCR fragment was
transformed into BY4741 (ATCC No. 201388) and transformants were
maintained on synthetic complete media lacking leucine and
supplemented with 2% glucose at 30.degree. C. using standard
genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202).
Transformants were screened by PCR using primers 112590-30E and
112590-30F, given as SEQ ID NOs:419 and 418, to verify integration
at the PDC1 locus with deletion of the PDC1 coding region. The
correct transformants have the genotype: BY4741
pdc1::FBAp-alsS-LEU2.
[0198] To test whether the ilvD encoded DHAD from Streptococcus
mutans could be used for the biosynthesis of isobutanol, the
expression vector containing this ilvD, pRS423 FBA ilvD(strep)
prepared in Example 6, was co-transformed with vector pLH532 into
yeast strain BY4741 pdc1::FBAp-alsS-LEU2. Competent cell
preparation, transformation, and growth medium for selection of the
transformants were the same as described in Example 6. Selected
colonies were grown under oxygen-limiting conditions in 15 ml of
medium in 20 ml serum bottles with stoppers. The bottles were
incubated at 30.degree. C. in a shaker with a constant speed of 225
rotations per minute. After 48 hours of incubation, the samples
were analyzed with HPLC for the presence of isobutanol. Result from
the HPLC analysis is shown in FIG. 7. The presence of isobutanol
was indicated by a peak with a retention time of 47.533 min. This
result showed that expression of the ilvD gene from Streptococcus
mutans, along with expression of alsS and KARI genes, led to the
production of isobutanol in yeast.
Example 11
Query of Updated Databases to Identify Additional
[2Fe-2S]dihydroxy-Acid Dehydratases
[0199] A later second query of the updated public database was
performed to discover newly sequenced [2Fe-2S] dihydroxy-acid
dehydratases. At the 95% identity cutoff, an initial set of 1425
sequences was generated from a database query as described in
Example 1, "Phylogenetic analysis". Multiple sequence alignments
were then executed with ClustalW as described in Example 1.
Sequences were subsequently analyzed for the following conserved
residues at the corresponding positions in the S. mutans DHAD:
cysteines at positions 56, 129, and 201, aspartic acid at position
88, arginine or asparagine at position 142, asparagine at position
208, and leucine at position 454, In a addition to the original set
of 193, 88 novel [2Fe-2S]dihydroxy-acid dehydratases from bacteria
were identified and are listed in Table 2b.
TABLE-US-00009 TABLE 1 ##STR00001## ##STR00002## ##STR00003##
##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008##
##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018##
##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023##
##STR00024## ##STR00025## ##STR00026## ##STR00027## ##STR00028##
##STR00029## ##STR00030## ##STR00031## ##STR00032## ##STR00033##
##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038##
##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043##
##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048##
##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053##
##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140051137A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140051137A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References