Beruflich Dokumente
Kultur Dokumente
NOV. 6, 2008
12/110,514
(22) Filed:
2-BUTANONE
(76) Inventors:
_ _
'
.
Wilmington, DE (Us)
(52)
Correspondence Address:
Publication Classi?cation
(57)
(2006.01)
ABSTRACT
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Figure 1
OH
VIII
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[0003]
[0006]
[0015]
time;
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[0016]
TABLE l-continued
SEQUENCE DESCRIPTIONS
[0019]
107
108
109
110
Ill
112
113
114
115
116
117
118
119
120
121
106
pneumoniae
SEQ ID
Protein
pneumoniae
cation.
SEQ ID
Nucleic
acid
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 37C.F.R. l.822.
l3
l4
90
91
ruber 219
furiosus
chnA, cyclohexanol dehydrogenase from
71
72
74
144
75
122
Acinleobacler sp.
TABLE 1
Description
SEQ ID
Nucleic
acid
SEQ ID
Protein
codon
opt.
123
124
125
126
subsp. alroseprica
amino alcohol O-phosphate lyase from Erwinia
carolovora subsp. alrosepli ca
133
134
80
81
145
146
82
83
pneumoniae
147
148
pneumoniae
76
78
77
79
lerrigena
budB, acetolactate synthase from Klebsiella
pneumoniae ATCC 25955
alsS, acetolactate synthase from Bacillus subrilis
budB, acetolactate synthase from Klebsiella
lerrigena
budC butanediol dehydrogenase from Klebsiella
butanediol dehydrogenase from Bacillus cereus
butanediol dehydrogenase from Bacillus cereus
84
86
85
87
88
89
l0
ll
12
92
93
94
95
96
97
98
99
100
101
102
103
104
105
lacris
pneumoniae
150
151
152
153
154
[0022]
149
Klebsiella pneumoniae
pneumoniae IAMlO63
Klebsiella pneumoniae
glycerol dehydratase reactivase large subunit from
Klebsiella pneumoniae
oxyloca.
[0028] SEQ ID NO:73 is the nucleotide sequence of
pDCQ2, Which is described in Example 13.
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[0029]
alroseplica.
tion.
[0032]
TABLE 2
Additional glycerol and diol dehydratase large, medium and small subunits
protein
Description
Corresponding subunits from same organism6
Glycerol dehydratase alpha subunit from Closlridium
bsubunit SEQ ID
136
137
138
139
140
141
pasleuriarium
Glycerol dehydratase alpha subunit from Escherichia
blallae
freuridii
Glycerol dehydratase beta subunit from Cilrobacler
142
143
freuridii
Glycerol dehydratase gamma subunit from Cilrobacler
135
pasleuriarium
Glycerol dehydratase gamma subunit from Closlridium
[0038]
pasleuriarium
Glycerol dehydratase beta subunit from Closlridium
freuridii
3Description: from the Genbank annotation of the sequence and may not be
correct including the glycerol or diol designation, or may not include subunit
information.
tions or carry out the methods; and the like. The term about
also encompasses amounts that differ due to different equi
[0034]
vate.
[0043]
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[0044]
(SEQ ID NO:1)].
[0045]
lyase activity.
[0050]
[0046]
(SEQ ID NO:88)].
43:13037-13046).
example,
[0048]
from Klebsiella
axyloca
[GenBank Nos:
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tein).
ronments.
gene isolation are Well knoWn in the art (e.g., US. Pat. No.
[0057]
5,686,276).
[0053]
procedure.
[0058]
listed in Table 2.
US 2008/0274522 A1
Nov. 6, 2008
the Washes are identical to those above except for the tem
above.
[0062] 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
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eters that originally load With the softWare When ?rst initial
iZed.
[0070] As used herein the term coding sequence or
of percent identities include, but are not limited to: 24%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, or 95%, or any integer percentage from 24% to
as 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,
35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,
45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,
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 frag
ments not only have the above homologies but typically
encode a polypeptide having at least 50 amino acids, prefer
ably 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.
[0069] 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 inde
[0071]
tion.
[0073] 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 dis
closed herein. Expression may also refer to translation of
mRNA into a polypeptide.
[0074] As used herein the term transformation refers to
the transfer of a nucleic acid fragment into a host organism,
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formed organisms.
[0075] The terms plasmid and vector refer to an extra
chromosomal element often carrying genes Which are not part
formed.
pathWays:
[0083] PathWay
[0084] 1) I--->II--->III--->IV--->V (substrate to product
conversions b,c,d,e)
[0085] 2) I--->II--->VII--->IV--->V (substrate to prod
uct conversions b,g,h,e)
[0086] 3) I--->II--->VIII--->V (substrate to product con
versions b,i,j)
[0087]
versions k,l,m)
A detailed discussion of the substrate to product conversions
PathWay 1:
techniques used here are Well knoWn in the art and are
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O-phosphate
[0095]
butanol O-phosphate.
[0096] The skilled person Will appreciate that polypeptides
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[0105]
PathWay 3:
product. PathWay 2:
[0107]
by this pathWay.
O-pho sphate
provide activity.
[0110]
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possibly be engineered.
[0111] The skilled person Will appreciate that polypeptides
having butanediol dehydrogenase activity isolated from a
variety of sources Will be useful in the present invention
independent of sequence homology. As noted above a variety
of diol and glycerol dehydratases have been described in the
literature and Will be suitable for use in the present invention.
Accordingly, in one aspect of the invention preferred diol and
glycerol dehydratase enZymes are those that have at least
activity.
(m) 2-hydroxy-2-methyl-3 -phosphobutanoic acid to
2-butanone
[0127]
NO:166.
[0121]
[0128]
SEQ ID NOs: 8, 99, 105, 135, 138, 141, 146, and 164;
Medium subunit: SEQ ID NOs: 10, 101, 107, 136, 139, 142,
148, and 165; Small subunit: SEQ ID NOs:l2, 103, 109, 137,
140, 143, 150, and 166; Where at least 85%-90% identity is
more preferred and Where at least 95% identity based on the
PathWay 4:
(k) alpha-acetolactate to
2,3-dihydroxy-2-methylbutanoic acid
[0125] The substrate to product conversion of acetolactate
(I) to 2,3-dihydroxy-2-methylbutanoic acid (IX) is not knoWn
[0130]
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Corynebaclerium,
Brevibaclerium,
Pichia,
Candida,
[0134]
[0139]
Strain
% GC
B. lichenifarmis
46
B. subrilis
42
C. acelabulylicum
37
E. cali
50
R purida
A. eulraphus
61
61
Paenibacillus macerans
51
Rhadacaccus erylhrapalis
62
Zymomonas,
Escherichia,
Salmonella,
Rh0d0c0ccus,
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TABLE 3-continued
[0142]
Brevibacillus
50
Paenibacillus polymyxa
50
Strain
hosts by means well known in the art. Vectors useful for the
E. coli
lac, ara, tet, trp, IPL, IPR, T7, tac, and trc promoters (useful for
expression in Escherichia coli, Alcaligenes, and Pseudomo
nas); the amy, apr, and npr promoters, and various phage
promoters useful for expression in Bacillus sublilis, 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 syn
thetic P11 promoter (useful for expression in Laclobacillus
planlarum, Rud et al., Microbiology 152: 101 1-1019 (2006)).
[0144] 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.
[0148]
Rhodococcus erylhropolis
[0149]
[0151]
tions in B. sublilis are also well known in the art. For example,
the genes of a 2-butanone biosynthetic pathway may be iso
lated from various sources, as described above, cloned into a
modi?ed E. coli-Bacillus shuttle vector and transformed into
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that are known to one skilled in the art. For example, the
vectors.
B. licheniformis
[0152]
72:334-345 (2006)).
pathway.
Expression of a 2-Butanone Biosynthetic Pathway in
Pseudomonas putida
(2003)).
Lactobacillus plantarum
acidilactici
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[0161]
are not limited to, corn grain, corn cobs, crop residues such as
70:161-183 (1996)).
Fermentation Media
[0166]
medium.
[0167] Suitable pH ranges for the fermentation are betWeen
initial condition.
[0168] Fermentations may be performed under aerobic or
[0169]
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[0170]
[0174]
[0172]
predetermined time.
[0173] Additionally, the temperature loWering and incuba
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being draWn off must be balanced against the cell groWth rate
in the fermentation. Methods of modulating nutrients and
erWise noted.
(Woodlands, Tex.).
[0183]
TABLE 4
Cloninq and Screeninq Primers
SEQ
Primer
ID
Gene
Name
Sequence
budB
B1
CACCATGGACAAACAGTA
TCCGGTACGCC
15 budB
forward
budB
B2
CGAAGGGCGATAGCTTTA
16 budB
NO: Description
CCAATCC
budA
B3
budA
B4
evaporation, or pervaporation.
EXAMPLES
and, Without departing from the spirit and scope thereof, can
make various changes and modi?cations of the invention to
budC
B6
TTAGTTAAATACCAT
2O budC
reverse
pddA
B7
CACCATGAGATCGA
AAAGATTTG
pddC
B8
CTTAGAGAAGTTAATCGT
CGCC
sadh
B9
sadh
B10
pddA
pddC
B14
sadh
B15
sadh
B16
reverse
24 sadh
reverse
GATCGAATTCGTTTAAACT
TAGTTTTCTACCGCACG
25 budABC
forward
GATCGCATGCAAGCTTTC
26 budABC
GATCGAATTCGTTTAAACA
GATCGGATTCTTAATCGT
CGCC
22 pddABC
CGTCGTGTCATGCCCGG
AAGGAGGTCTGATTCATG
AGATCG
forward
23 sadh
forward
ATATAGTCGGAATTCC
B13
21 pddABC
CACCATGAAAGCCCTCCA
GTACACC
B12
reverse
19 budC
forward
budC
18 budA
CACCATGAAAAAAGTCGC
ACTTGTTACC
B11
GATACTGTTTGTCCATGT
B5
budA
1'7 budA
forward
budC
CACCATGAATCATTCTGC
TGAATGCACCTGCG
GACC
General Methods
reverse
reverse
2'7 pddABC
forward
28 pddABC
reverse
GATCGGATCCAAAGGAGG
TCGGGCGCATGAAAGCC
C
29 sadh
forward
GATCTCTAGAAAGCTTTC
3O sadh
AGCCCGGGACGACC
reverse