(54)
`
`(76)
`
`Inventors: Gail K. Donaldson, Newark, DE (US);
`Andrew C. Eliot, Wilmington, DE
`(US); Dennis Flint, Newark, DE (US);
`Lori Ann Maggio-Hall, Wilmington,
`DE (US); Vasantha Nagarajan,
`Wilmington, DE (US)
`
`Correspondence Address:
`E I DU PONT DE NEMOURS AND
`COMPANY
`LEGAL PATENT RECORDS CENTER
`BARLEY MILL PLAZA 25/1128
`4417 LANCASTER PIKE
`WILMINGTON, DE 19805 (US)
`
`(21) Appl. No.:
`
`11/586,315
`
`(22)
`
`Filed:
`
`Oct. 25, 2006
`
`(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2007/0092957 A1
`Apr. 26, 2007
`Donaldson et al.
`(43) Pub. Date:
`
`US 20070092957Al
`
`FERMENTIVE PRODUCTION OF FOUR
`CARBON ALCOHOLS
`
`Related US. Application Data
`
`(60) Provisional application No. 60/730,290, filed on Oct.
`26, 2005.
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`(2006.01)
`C12P 7/04
`(2006.01)
`C12N 1/21
`(2006.01)
`C12N 1/18
`(52) US. Cl.
`................ 435/157; 435/252.3; 435/252.33;
`435/254.21; 435/254.22; 435/254.23
`
`(57)
`
`ABSTRACT
`
`Methods for the fermentative production of four carbon
`alcohols is provided. Specifically, butanol, preferably isobu-
`tanol is produced by the fermentative growth of a recombi-
`nant bacterium expressing an isobutanol biosynthetic path-
`way.
`
`BUTAMAX 1018
`
`

`

`Patent Application Publication Apr. 26, 2007
`
`US 2007/0092957 Al
`
`O
`
`.
`
`WW I 202%
`
`NH2
`
`02
`
`NH:
`
`NH3;
`
`0
`
`o
`
`o
`
`a
`0H 3'
`
`C02
`
`-,
`
`’bH
`
`b
`OH *
`
`2
`
`H2O
`
`H26.2H
`
`020"”?
`OH—:
`HO)<::fi\°H
`Hsrcm\3°2“
`2e:214%m
`k
`JY
`fl
`0
`
`0H
`
`O
`/\/I\
`
`S-CoA
`
`S-CoA
`
`

`

`US 2007/0092957 A1
`
`Apr. 26, 2007
`
`FERMENTIVE PRODUCTION OF FOUR CARBON
`ALCOHOLS
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`
`[0001] This application claims priority under 35 U.S.C.
`§ll9 from US. Provisional Application Ser. No. 60/730,
`290, filed Oct. 26, 2005.
`
`FIELD OF THE INVENTION
`
`[0002] The invention relates to the field of industrial
`microbiology and the production of alcohols. More specifi-
`cally, isobutanol is produced Via industrial fermentation of a
`recombinant microorganism.
`
`BACKGROUND OF THE INVENTION
`
`[0003] Butanol is an important industrial chemical, useful
`as a fuel additive, as a feedstock chemical in the plastics
`industry, and as a foodgrade extractant in the food and flavor
`industry. Each year 10 to 12 billion pounds of butanol are
`produced by petrochemical means and the need for this
`commodity chemical will likely increase.
`
`[0004] Methods for the chemical synthesis of isobutanol
`are known, such as oxo synthesis, catalytic hydrogenation of
`carbon monoxide (Ullmann’s Encyclopedia of Industrial
`Chemistry, 6Lb edition, 2003, Wiley-VCHVerlag GmbH and
`Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbet
`condensation of methanol with n-propanol (Carlini et al., J.
`Mol. Catal. A: Chem. 220:215-220 (2004)). These processes
`use starting materials derived from petrochemicals and are
`generally expensive and are not environmentally friendly.
`The production of isobutanol from plant-derived raw mate-
`rials would minimize green house gas emissions and would
`represent an advance in the art.
`
`Isobutanol is produced biologically as a by-product
`[0005]
`of yeast fermentation. It is a component of “fusel oil” that
`forms as a result of incomplete metabolism of amino acids
`by this group of fungi. Isobutanol is specifically produced
`from catabolism of L-valine. After the amine group of
`L-valine is harvested as a nitrogen source, the resulting
`ot-keto acid is decarboxylated and reduced to isobutanol by
`enzymes of the so-called Ehrlich pathway (Dickinson et al.,
`J. Biol. Chem. 273(40):25752-25756 (1998)). Yields offusel
`oil and/or its components achieved during beverage fermen-
`tation are typically low. For example, the concentration of
`isobutanol produced in beer fermentation is reported to be
`less than 16 parts per million (Garcia et al., Process Bio-
`chemistry 29:303-309 (1994)). Addition of exogenous L-va-
`line to the fermentation increases the yield of isobutanol, as
`described by Dickinson et al., supra, wherein it is reported
`that a yield of isobutanol of 3 g/L is obtained by providing
`L-valine at a concentration of 20 g/L in the fermentation.
`However, the use of valine as a feed-stock would be cost
`prohibitive for industrial scale isobutanol production. The
`biosynthesis of isobutanol directly from sugars would be
`economically viable and would represent an advance in the
`art. There have been no reports of a recombinant microor-
`ganism designed to produce isobutanol.
`
`[0006] There is a need, therefore, for an environmentally
`responsible, cost-effective process for the production of
`isobutanol as a single product. The present
`invention
`
`addresses this need by providing a recombinant microbial
`production host that expresses an isobutanol biosynthetic
`pathway.
`
`SUMMARY OF THE INVENTION
`
`[0007] The invention provides a recombinant microorgan-
`ism having an engineered isobutanol biosynthetic pathway.
`The engineered microorganism may be used for the com-
`mercial production of isobutanol. Accordingly,
`in one
`embodiment the invention provides a recombinant microbial
`host cell comprising at least one DNA molecule encoding a
`polypeptide that catalyzes a substrate to product conversion
`selected from the group consisting of:
`
`[0008]
`
`i) pyruvate to acetolactate (pathway step a)
`
`ii) acetolactate to 2,3-dihydroxyisovalerate (path-
`[0009]
`way step b)
`
`iii) 2,3-dihydroxyisovalerate to ot-ketoisovalerate
`[0010]
`(pathway step c)
`
`iv) ot-ketoisovalerate to isobutyraldehyde, (path-
`[0011]
`way step d), and
`
`[0012]
`
`v) isobutyraldehyde to isobutanol; (pathway step e)
`
`wherein the at least one DNA molecule is heterologous to
`said microbial host cell and wherein said microbial host cell
`
`produces isobutanol.
`
`In another embodiment, the invention provides a
`[0013]
`recombinant microbial host cell comprising at
`least one
`DNA molecule encoding a polypeptide that catalyzes a
`substrate to product conversion selected from the group
`consisting of:
`
`[0014]
`
`i) pyruvate to acetolactate, (pathway step a)
`
`ii) acetolactate to 2,3-dihydroxyisovalerate, (path-
`[0015]
`way step b)
`
`iii) 2,3-dihydroxyisovalerate to ot-ketoisovalerate,
`[0016]
`(pathway step c)
`
`[0017]
`step f)
`
`iv) ot-ketoisovalerate to isobutyryl-CoA, (pathway
`
`v) isobutyryl-CoA to isobutyraldehyde, (pathway
`[0018]
`step g), and
`
`[0019]
`8)
`
`vi) isobutyraldehyde to isobutanol; (pathway step
`
`wherein the at least one DNA molecule is heterologous to
`said microbial host cell and wherein said microbial host cell
`
`produces isobutanol.
`
`In another embodiment, the invention provides a
`[0020]
`recombinant microbial host cell comprising at
`least one
`DNA molecule encoding a polypeptide that catalyzes a
`substrate to product conversion selected from the group
`consisting of:
`
`[0021]
`
`i) pyruvate to acetolactate, (pathway step a)
`
`ii) acetolactate to 2,3-dihydroxyisovalerate, (path-
`[0022]
`way step b)
`
`iii) 2,3-dihydroxyisovalerate to ot-ketoisovalerate,
`[0023]
`(pathway step c)
`
`[0024]
`
`iv) ot-ketoisovalerate to valine, (pathway step h)
`
`

`

`US 2007/0092957 A1
`
`Apr. 26, 2007
`
`[0025] V) Valine to isobutylamine, (pathway step i)
`
`[0026] Vi)
`step j), and
`
`isobutylamine to isobutyraldehyde, (pathway
`
`[0027] Vii) isobutyraldehyde to isobutanol: (pathway step
`6)
`
`wherein the at least one DNA molecule is heterologous to
`said microbial host cell and wherein said microbial host cell
`
`produces isobutanol.
`
`In another embodiment, the inVention pr0Vides a
`[0045]
`method for the production of isobutanol comprising:
`
`l) pr0Viding a recombinant microbial host cell
`[0046]
`comprising at
`least one DNA molecule encoding a
`polypeptide that catalyzes a substrate to product con-
`Version selected from the group consisting of:
`
`[0047]
`
`i) pyruVate to acetolactate, (pathway step a)
`
`ii) acetolactate to 2,3-dihydroxyis0Valerate, (path-
`[0048]
`way step b)
`
`In another embodiment, the inVention pr0Vides a
`[0028]
`method for the production of isobutanol comprising:
`
`iii) 2,3-dihydroxyis0Valerate to ot-ketois0Valerate,
`[0049]
`(pathway step c)
`
`l) pr0Viding a recombinant microbial host cell
`[0029]
`comprising at
`least one DNA molecule encoding a
`polypeptide that catalyzes a substrate to product con-
`Version selected from the group consisting of:
`
`[0030]
`
`i) pyruVate to acetolactate (pathway step a)
`
`ii) acetolactate to 2,3-dihydroxyis0Valerate (path-
`[0031]
`way step b)
`
`iii) 2,3-dihydroxyis0Valerate to ot-ketois0Valerate
`[0032]
`(pathway step c)
`
`iV) ot-ketois0Valerate to isobutyraldehyde, (path-
`[0033]
`way step d), and
`
`[0034] V) isobutyraldehyde to isobutanol; (pathway step e)
`
`wherein the at least one DNA molecule is heterologous to
`said microbial host cell; and
`
`2) contacting the host cell of (i) with a ferment-
`[0035]
`able carbon substrate in a fermentation medium under
`
`conditions whereby isobutanol is produced.
`
`In another embodiment, the inVention pr0Vides a
`[0036]
`method for the production of isobutanol comprising:
`
`l) pr0Viding a recombinant microbial host cell
`[0037]
`comprising at
`least one DNA molecule encoding a
`polypeptide that catalyzes a substrate to product con-
`Version selected from the group consisting of:
`
`[0038]
`
`i) pyruVate to acetolactate, (pathway step a)
`
`ii) acetolactate to 2,3-dihydroxyis0Valerate, (path-
`[0039]
`way step b)
`
`iii) 2,3-dihydroxyis0Valerate to ot-ketois0Valerate,
`[0040]
`(pathway step c)
`
`[0041]
`step f)
`
`iV) ot-ketois0Valerate to isobutyryl-CoA, (pathway
`
`[0042] V) isobutyryl-CoA to isobutyraldehyde, (pathway
`step g), and
`
`[0043] Vi) isobutyraldehyde to isobutanol; (pathway step
`8)
`
`wherein the at least one DNA molecule is heterologous to
`said microbial host cell; and
`
`2) contacting the host cell of (i) with a ferment-
`[0044]
`able carbon substrate in a fermentation medium under
`
`conditions whereby isobutanol is produced.
`
`[0050]
`
`iV) ot-ketois0Valerate to Valine, (pathway step h)
`
`[0051] V) Valine to isobutylamine, (pathway step i)
`
`[0052] Vi)
`step j), and
`
`isobutylamine to isobutyraldehyde, (pathway
`
`[0053] Vii) isobutyraldehyde to isobutanol: (pathway step
`8)
`
`wherein the at least one DNA molecule is heterologous to
`said microbial host cell; and
`
`2) contacting the host cell of (i) with a ferment-
`[0054]
`able carbon substrate in a fermentation medium under
`
`conditions whereby isobutanol is produced.
`
`In an alternate embodiment the inVention pr0Vides
`[0055]
`an isobutanol constraining fermentation medium produced
`by the methods of the inVention.
`
`BRIEF DESCRIPTION OF THE FIGURES AND
`
`SEQUENCE DESCRIPTIONS
`
`[0056] The inVention can be more fully understood from
`the following detailed description, figure, and the accompa-
`nying sequence descriptions, which form a part of this
`application.
`
`FIG. 1 shows four different isobutanol biosynthetic
`[0057]
`pathways. The steps labeled “a”, “b”, “c”, “d”, “e”, “f”, “ ”,
`“h”, “i”, “j” and “k” represent the substrate to product
`conVersions described below.
`
`[0058] The following sequences conform with 37 C.F.R.
`l.821-l.825 (“Requirements for Patent Applications Con-
`taining Nucleotide Sequences and/or Amino Acid Sequence
`Disclosuresithe 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 sym-
`bols and format used for nucleotide and amino acid
`
`sequence data comply with the rules set forth in 37 C.F.R.
`§l.822.
`
`[0059] A Sequence Listing is pr0Vided herewith on Com-
`pact Disk. The contents of the Compact Disk containing the
`Sequence Listing are hereby incorporated by reference in
`compliance with 37 CFR l.52(e). The Compact Disks are
`submitted in triplicate and are identical to one another. The
`disks are labeled “Copy lisequence Listing”, “Copy
`Zisequence Listing”, and CRF. The disks contain the
`following file: CL3243 Seq List ConV.ST25 haVing the
`following size: 368,000 bytes and which was created Oct.
`23, 2006.
`
`

`

`US 2007/0092957 A1
`
`Apr. 26, 2007
`
`TABLE 1
`
`TABLE 1-c0ntinued
`
`Summafl of Gene and Protein SEQ ID Numbers
`
`Summfl of Gene and Protein SEQ ID Numbers
`
`Description
`
`
`
`SEQ ID
`NO:
`Nucleic
`acid
`
`1
`
`78
`
`179
`
`80
`
`182
`
`184
`
`83
`
`87
`
`89
`
`91
`
`92
`
`SEQ ID
`NO:
`Pe 3tide
`
`2
`
`Description
`
`78
`
`80
`
`4
`
`81
`
`88 90
`
`83
`
`85
`
`86
`
`193
`
`SEQ ID
`NO:
`Nucleic
`acid
`
`SEQ ID
`NO:
`Peptide
`
`219
`
`221
`
`223
`
`225
`
`227
`
`229
`231
`
`233
`235
`
`237
`
`239
`
`241
`
`243
`
`220
`
`222
`
`224
`
`226
`
`228
`
`230
`232
`
`234
`236
`
`238
`
`240
`
`242
`
`244
`
`Klebsiella pneumoniae budB
`(acetolactate synthase)
`Bacillus subrilis alsS
`(acetolactate synthase)
`Laclacaccus lacris als
`(acetolactate synthase)
`E. cali ilVC (acetohydroxy acid
`reductoisomerase)
`S. cerevisiae ILV5
`(acetohydroxy acid
`reductoisomerase)
`M. maripaludis ilVC
`(Ketol-acid reductoisomerase)
`B. sublilis ilVC
`(acetohydroxy aci
`reductoisomerase)
`E. cali ilVD (acet01ydroxy acid
`dehydratase)
`S. cerevisiae ILV3
`(Dihydroxyacid de 1ydratase)
`M. maripaludis ilV)
`(Dihydroxy-acid dehydratase)
`B. sublilis ilVD
`(dihydroxy-acid de 1ydratase)
`Laclacaccus lacris {lVD (branched-
`chain OL-kCtO acid ecarboxylase),
`codon optimized
`Laclacaccus lacris {lVD (branched-
`chain OL-kCtO acid ecarboxylase),
`Laclacaccus lacris {ch
`(branched-chain alpha-ketoacid
`decarboxylase)
`Salmonella lyphimurium
`(indolepyruvate decarboxylase)
`Claslridium acelabulylicum pdc
`(Pyruvate decarboxylase)
`E. cali thD (branched-chain alcohol
`ehydrogenase)
`S. cerevisiae YPR1
`(2-methylbutyraldehyde reductase)
`S. cerevisiae ADH6
`(NADPH- ependent cinnamyl alcohol
`ehydrogenase)
`Claslridium acelabulylicum bdhA
`(NADH- e3endent butanol
`ehydrogenase A)
`Claslridium acelabulylicum bth
`Butanol
`eiydrogenase
`B. sublilis DdeA
`(31311016 -c1ain keto acid
`eiydrogenase E1 subunit)
`B. sublilis DdeB
`(31311016 -c1ain alpha-keto acid
`eiydrogenase E1 subunit)
`B. sublilis DkdB
`3ranc 1e -c1ain alpha-keto acid
`1ydrogenase E2 subunit)
`sublilis 3dV
`3ranc 1e -chain alpha-keto acid
`eiydrogenase E3 subunit)
`P purida bidAl
`({eto aci
`ehydrogenase E1-alpha
`5
`s
`311.111)
`P purida 3(dA2
`(
`.{eto aci
`
`ehydrogenase E1-beta
`s
`uauni )
`P. purida DidB
`(ransacylase E2)
`
`P. purida 1pdV
`(lipoamide dehydrogenase)
`C. beijerinckii ald
`(coenzyme A acylating aldehyde
`dehydrogenase)
`C. acelabulylicum adhe1
`(aldehyde dehydrogenase)
`C. acelabulylicum adhe
`(alcohol-aldehyde dehydrogenase)
`P. purida nahO
`(acetaldehyde dehydrogenase)
`T. lhermaphilus
`(acetaldehyde dehydrogenase)
`E. cali thA
`(valine-pyruvate transaminase)
`B. lichem'farmis thA
`(valine-pyruvate transaminase)
`E. cali ilVE
`(branched chain amino acid
`aminotransferase)
`S. cerevisiae BAT2
`(branched chain amino acid
`aminotransferase)
`M. lhermaaulalraphicum
`(branched chain amino acid
`aminotransferase)
`S. Gaelicalar
`(valine dehydrogenase)
`3.. subrilis bcd
`(leucine dehydrogenase)
`S. viridifaciens
`(valine decarboxyase)
`A. denimficans aptA
`(omega-amino acid:pyruvate
`transaminase)
`R. eulrapha
`(alanine-pyruvate transaminase)
`S. aneidensis
`(beta alanine-pyruvate transaminase)
`P. pulida
`(beta alanine-pyruvate transaminase)
`S. Cinnamanensis icm
`(isobutyrl-CoA mutase)
`S. Cinnamanensis icmB
`(isobutyrl-CoA mutase)
`S. Gaelicalar SCO5415
`(isobutyrl-CoA mutase)
`S. Gaelicalar SCO4800
`(isobutyrl-CoA mutase)
`S. avermililis icmA
`(isobutyrl-COA mutase)
`266
`265
`S. avermililis icmB
`(isobutyrl-CoA mutase)
`
`246
`
`248
`
`250
`252
`
`254
`256
`
`258
`
`260
`
`262
`
`264
`
`245
`
`247
`
`249
`251
`
`253
`255
`
`257
`
`259
`
`261
`
`263
`
`SEQ ID NOszll-38, 40-69, 72-75, 85-138, 144,
`[0060]
`145, 147-157, 159-176 are the nucleotide sequences of
`oligonucleotide cloning, screening or sequencing primers
`used in the Examples described herein.
`
`SEQ ID NO:39 is the nucleotide sequence of the
`[0061]
`cscBKA gene cluster described in Example 16.
`
`SEQ ID NO:70 is the nucleotide sequence of the
`[0062]
`glucose isomerase promoter 1.6GI described in Example 13.
`
`SEQ ID NO:71 is the nucleotide sequence of the
`[0063]
`1.5GI promoter described in Example 13.
`
`96 98
`
`94
`
`200
`
`202
`
`158
`
`205
`
`207
`
`209
`
`211
`
`213
`
`215
`
`217
`
`195
`
`197
`
`10
`
`199
`
`201
`
`203
`
`204
`
`206
`
`208
`
`210
`
`212
`
`214
`
`216
`
`218
`
`
`
`
`
`
`
`
`
`
`
`
`
`A A
`
`9302
`
`

`

`US 2007/0092957 A1
`
`Apr. 26, 2007
`
`SEQ ID NO:76 is the nucleotide sequence of the
`[0064]
`GPD promoter described in Example 17.
`
`SEQ ID NO:77 is the nucleotide sequence of the
`[0065]
`CYC1 terminator described in Example 17.
`
`SEQ ID NO:79 is the nucleotide sequence of the
`[0066]
`FBA promoter described in Example 17.
`
`SEQ ID NO:81 is the nucleotide sequence of
`[0067]
`ADH1 promoter described in Example 17.
`
`SEQ ID NO:82 is the nucleotide sequence of
`[0068]
`ADH1 terminator described in Example 17.
`
`SEQ ID NO:84 is the nucleotide sequence of GPM
`[0069]
`promoter described in Example 17.
`
`SEQ ID NO:139 is the amino acid sequence of
`[0070]
`sucrose hydrolase (CscA).
`
`SEQ ID NO:140 is the amino acid sequence of
`[0071]
`D-fructokinase (CscK).
`
`SEQ ID NO:141 is the amino acid sequence of
`[0072]
`sucrose permease (CscB).
`
`SEQ ID NO:142 is the nucleotide sequence of
`[0073]
`plasmid pFP988DssPspac described in Example 20.
`
`SEQ ID NO:143 is the nucleotide sequence of
`[0074]
`plasmid pFP988DsngroE described in Example 20.
`
`SEQ ID NO:146 is the nucleotide sequence of the
`[0075]
`pFP988Dss vector fragment described in Example 20.
`
`SEQ ID NO:177 is the nucleotide sequence of the
`[0076]
`pFP988 integration vector described in Example 21.
`
`SEQ ID NO:267 is the nucleotide sequence of
`[0077]
`plasmid pC194 described in Example 21.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`[0078] The present invention relates to methods for the
`production of isobutanol using recombinant microorgan-
`isms. The present invention meets a number of commercial
`and industrial needs. Butanol
`is an important
`industrial
`commodity chemical with a variety of applications, where
`its potential as a fuel or fuel additive is particularly signifi-
`cant. Although only a four-carbon alcohol, butanol has an
`energy content similar to that of gasoline and can be blended
`with any fossil fuel. Butanol is favored as a fuel or fuel
`additive as it yields only CO2 and little or no SOx or NOx
`when burned in the standard internal combustion engine.
`Additionally butanol is less corrosive than ethanol, the most
`preferred fuel additive to date.
`
`In addition to its utility as a biofuel or fuel additive,
`[0079]
`butanol has the potential of impacting hydrogen distribution
`problems in the emerging fuel cell industry. Fuel cells today
`are plagued by safety concerns associated with hydrogen
`transport and distribution. Butanol can be easily reformed
`for its hydrogen content and can be distributed through
`existing gas stations in the purity required for either fuel
`cells or vehicles.
`
`[0080] Finally the present invention produces isobutanol
`from plant derived carbon sources, avoiding the negative
`environmental impact associated with standard petrochemi-
`cal processes for butanol production.
`
`[0081] The following definitions and abbreviations are to
`be used for the interpretation of the claims and the specifi-
`cation.
`
`invention” as
`[0082] The term “invention” or “present
`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.
`
`[0083] The term “isobutanol biosynthetic pathway” refers
`to an enzyme pathways to produce isobutanol.
`
`[0084] The terms “acetolactate synthase” and “acetolac-
`tate synthetase” are used interchangeably herein to refer to
`an enzyme that catalyzes the conversion of pyruvate to
`acetolactate and C02. Preferred acetolactate synthases are
`known by the EC number 2.2.1.6 9 (Enzyme Nomenclature
`1992, Academic Press, San Diego). These enzymes are
`available from a number of sources,
`including, but not
`limited to, Bacillus subtilis (GenBank Nos: CAB15618
`(SEQ ID NO:178), Z99122 (SEQ ID NO:78), NCBI
`(National Center for Biotechnology Information) amino acid
`sequence, NCBI nucleotide sequence, respectively), Kleb—
`siella pneumoniae (GenBank Nos: AAA25079 (SEQ ID
`NO:2), M73842 (SEQ ID NO:1)), and Lactococcus lactis
`(GenBank Nos: AAA25161 (SEQ ID NO:180), L16975
`(SEQ ID NO:179)).
`
`[0085] The terms “acetohydroxy acid isomeroreductase”
`and “acetohydroxy acid reductoisomerase” are used inter-
`changeably herein to refer to an enzyme that catalyzes the
`conversion of acetolactate to 2,3-dihydroxyisovalerate using
`NADPH (reduced nicotinamide adenine dinucleotide phos-
`phate) as an electron donor. Preferred acetohydroxy acid
`isomeroreductases are known by the EC number 1.1.1.86
`and sequences are available from a vast array of microor-
`ganisms,
`including, but not
`limited to, Escherichia coli
`(GenBank Nos: NPi418222 (SEQ ID NO:4), NCi000913
`(SEQ ID NO:3)), Saccharomyces cerevisiae (GenBank Nos:
`NPi013459 (SEQ ID NO:181), NCi001144 (SEQ ID
`NO:80)), Melhanococcus maripaludis
`(GenBank Nos:
`CAF30210 (SEQ ID NO:183), BX957220 (SEQ ID
`NO:182)), and Bacillus. subtilis (GenBank Nos: CAB14789
`(SEQ ID NO:185), Z99118 (SEQ ID NO:184)).
`
`[0086] The term “acetohydroxy acid dehydratase” refers
`to an enzyme that catalyzes the conversion of 2,3-dihy-
`droxyisovalerate to ot-ketoisovalerate. Preferred acetohy-
`droxy acid dehydratases are known by the EC number
`4.2.1.9. These enzymes are available from a vast array of
`microorganisms, including, but not limited to, E. coli (Gen-
`Bank Nos: YPi026248 (SEQ ID NO:6), NCi000913 (SEQ
`ID NO:5)), S. cerevisiae (GenBank Nos: NPi012550 (SEQ
`ID NO:186), NC 001142 (SEQ ID NO:83)), M. maripaludis
`(GenBank Nos: CAF29874 (SEQ ID NO:188), BX957219
`(SEQ ID NO:187)), and B.
`subtilis
`(GenBank Nos:
`CAB14105 (SEQ ID NO:190), Z99115 (SEQ ID NO:189)).
`
`[0087] The term “branched-chain ot-keto acid decarboxy-
`lase” refers to an enzyme that catalyzes the conversion of
`ot-ketoisovalerate to isobutyraldehyde and C02. Preferred
`branched-chain ot-keto acid decarboxylases are known by
`the EC number 4.1.1.72 and are available from a number of
`
`sources, including, but not limited to, Lactococcus lactis
`(GenBank Nos: AAS49166 (SEQ ID NO:193), AY548760
`(SEQ ID NO:192); CAG34226 (SEQ ID NO:8), AJ746364
`
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`

`US 2007/0092957 A1
`
`Apr. 26, 2007
`
`(SEQ ID NO: 191), Salmonella typhimurium (GenBank Nos:
`NPi461346 (SEQ ID NO:195), NCi003197 (SEQ ID
`NO:194)), and Closlridium acetobuzylicum (GenBank Nos:
`NPi149189 (SEQ ID NO:197), NCi001988 (SEQ ID
`NO:196)).
`
`[0088] The term “branched-chain alcohol dehydrogenase”
`refers to an enzyme that catalyzes the conversion of isobu-
`tyraldehyde to isobutanol. Preferred branched-chain alcohol
`dehydrogenases are known by the EC number 1.1.1.265, but
`may also be classified under other alcohol dehydrogenases
`(specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize
`NADH (reduced nicotinamide adenine dinucleotide) and/or
`NADPH as electron donor and are available from a number
`
`limited to, S. cerevisiae
`including, but not
`of sources,
`(SEQ ID NO:199),
`(GenBank Nos: NPi010656
`NCi001136 (SEQ ID NO:198); NPi014051 (SEQ ID
`NO:201) NCi001145 (SEQ ID NO:200)), E. coli (GenBank
`Nos: NPi417484 (SEQ ID NO:10), NCi000913 (SEQ ID
`NO:9)), and C. acelobulylicum (GenBank Nos: NPi349892
`(SEQ ID NO:203), NCi003030 (SEQ ID NO:202);
`NPi349891 (SEQ ID NO:204), NCi003030 (SEQ ID
`NO: 158)).
`
`[0089] The term “branched-chain keto acid dehydroge-
`nase” refers to an enzyme that catalyzes the conversion of
`ot-ketoisovalerate to isobutyryl-CoA (isobutyryl-coenzyme
`A), using NAD+ (nicotinamide adenine dinucleotide) as
`electron acceptor. Preferred branched-chain keto acid dehy-
`drogenases are known by the EC number 1.2.4.4. These
`branched-chain keto acid dehydrogenases are comprised of
`four subunits and sequences from all subunits are available
`from a vast array of microorganisms,
`including, but not
`limited to, B. sublilis (GenBank Nos: CAB14336 (SEQ ID
`NO:206), Z99116 (SEQ ID NO:205); CAB14335 (SEQ ID
`NO:208), Z99116 (SEQ ID NO:207); CAB14334 (SEQ ID
`NO:210), Z99116 (SEQ ID NO:209); and CAB14337 (SEQ
`ID NO:212), Z99116 (SEQ ID NO:211)) and Pseudomonas
`pulida (GenBank Nos: AAA65614 (SEQ ID NO:214),
`M57613 (SEQ ID NO:213); AAA65615 (SEQ ID NO:216),
`M57613 (SEQ ID NO:215); AAA65617 (SEQ ID NO:218),
`M57613 (SEQ ID NO:217); and AAA65618 (SEQ ID
`NO:220), M57613 (SEQ ID NO:219)).
`
`[0090] The term “acylating aldehyde dehydrogenase”
`refers to an enzyme that catalyzes the conversion of isobu-
`tyryl-CoA to isobutyraldehyde, using either NADH or
`NADPH as electron donor. Preferred acylating aldehyde
`dehydrogenases are known by the EC numbers 1.2.1.10 and
`1.2.1.57. These enzymes are available from multiple
`sources, including, but not limited to, Closlridium beijer—
`inckz'i
`(GenBank Nos: AAD31841 (SEQ ID NO:222),
`AF157306 (SEQ ID NO:221)), C. acelobulylicum (Gen-
`Bank Nos: NPi149325 (SEQ ID NO:224), NCi001988
`(SEQ ID NO:223); NPi149199 (SEQ ID NO:226),
`NCi001988 (SEQ ID NO:225)), P. pulida (GenBank Nos:
`AAA89106 (SEQ ID NO:228), U13232 (SEQ ID NO:227)),
`and Thermus lhermophilus (GenBank Nos: YPi145486
`(SEQ ID NO:230), NCi006461 (SEQ ID NO:229)).
`
`[0091] The term “transaminase” refers to an enzyme that
`catalyzes the conversion of ot-ketoisovalerate to L-valine,
`using either alanine or glutamate as amine donor. Preferred
`transaminases are known by the EC numbers 2.6.1.42 and
`2.6.1.66. These enzymes are available from a number of
`sources. Examples of
`sources
`for
`alanine-dependent
`
`enzymes include, but are not limited to, E. coli (GenBank
`Nos: YPi026231 (SEQ ID NO:232), NCi000913 (SEQ ID
`NO:231)) and Bacillus
`licheniformis
`(GenBank Nos:
`YPi093743 (SEQ ID NO:234), NCi006322 (SEQ ID
`NO:233)). Examples of sources for glutamate-dependent
`enzymes include, but are not limited to, E. coli (GenBank
`Nos: YPi026247 (SEQ ID NO:236), NCi000913 (SEQ ID
`NO:235)), S. cerevisiae (GenBank Nos: NPi012682 (SEQ
`ID NO:238), NCi001142 (SEQ ID NO:237)) and Metha—
`nobacterium lhermoaulolrophicum (GenBank
`Nos:
`NPi276546 (SEQ ID NO:240), NCi000916 (SEQ ID
`NO:239)).
`
`[0092] The term “valine dehydrogenase” refers to an
`enzyme that catalyzes the conversion of ot-ketoisovalerate to
`L-valine, using NAD(P)H as electron donor and ammonia as
`amine donor. Preferred valine dehydrogenases are known by
`the EC numbers 1.4.1.8 and 1.4.1.9 and are available from
`
`a number of sources, including, but not limited to, Strepto—
`myces coelicolor (GenBank Nos: NPi628270 (SEQ ID
`NO:242), NCi003888 (SEQ ID NO:241)) and B. sublilis
`(GenBank Nos: CAB14339 (SEQ ID NO:244), Z99116
`(SEQ ID NO:243)).
`
`[0093] The term “valine decarboxylase” refers to an
`enzyme that catalyzes the conversion of L-valine to isobu-
`tylamine and C02. Preferred valine decarboxylases are
`known by the EC number 4.1.1.14. These enzymes are found
`in Streptomycetes, such as for example, Slreplomyces viri-
`difaciens (GenBank Nos: AAN10242 (SEQ ID NO:246),
`AY116644 (SEQ ID NO:245)).
`
`[0094] The term “omega transaminase” refers to an
`enzyme that catalyzes the conversion of isobutylamine to
`isobutyraldehyde using a suitable amino acid as amine
`donor. Preferred omega transaminases are known by the EC
`number 2.6.1.18 and are available from a number of sources,
`including, but not
`limited to, Alcaligenes denilrificans
`(AAP92672 (SEQ ID NO:248), AY330220 (SEQ ID
`NO:247)), Ralslonia eulropha (GenBank Nos: YPi294474
`(SEQ ID NO:250), NCi007347 (SEQ ID NO:249)),
`Shewanella oneidensis (GenBank Nos: NPi719046 (SEQ
`ID NO:252), NCi004347 (SEQ ID NO:251)), andP. pulida
`(GenBank Nos: AAN66223 (SEQ ID NO:254), AE016776
`(SEQ ID NO:253)).
`
`[0095] The term “isobutyryl-CoA mutase” refers to an
`enzyme that catalyzes the conversion of butyryl-CoA to
`isobutyryl-CoA. This enzyme uses coenzyme B12 as cofac-
`tor. Preferred isobutyryl-CoA mutases are known by the EC
`number 5.4.99.13. These enzymes are found in a number of
`Streptomycetes, including, but not limited to, Slreplomyces
`cinnamonensis
`(GenBank Nos: AAC08713
`(SEQ ID
`NO:256), U67612 (SEQ ID NO:255); CAB59633 (SEQ ID
`NO:258), AJ246005 (SEQ ID NO:257)), S. coelicolor (Gen-
`Bank Nos: CAB70645 (SEQ ID NO:260), AL939123 (SEQ
`ID NO:259); CAB92663 (SEQ ID NO:262), AL939121
`(SEQ ID NO:261)), and Slreplomyces avermililis (GenBank
`Nos: NPi824008 (SEQ ID NO:264), NCi003155 (SEQ ID
`NO:263); NPi824637 (SEQ ID NO:266), NCi003155
`(SEQ ID NO:265)).
`
`[0096] The term “a facultative anaerobe” refers to a micro-
`organism that can grow in both aerobic and anaerobic
`environments.
`
`[0097] The term “carbon substrate” or “fermentable car-
`bon substrate” refers to a carbon source capable of being
`
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`

`US 2007/0092957 A1
`
`Apr. 26, 2007
`
`metabolized by host organisms of the present invention and
`particularly carbon sources selected from the group consist-
`ing of monosaccharides, oligosaccharides, polysaccharides,
`and one-carbon substrates or mixtures thereof.
`
`[0098] 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.
`
`[0099] As used herein the term “coding sequence” refers
`to a DNA sequence that codes for a specific amino acid
`sequence. “Suitable regulatory sequences” refer to nucle-
`otide
`sequences
`located
`upstream (5'
`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.
`
`[0100] 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 bound-
`aries of regulatory sequences have not been completely
`defined, DNA fragments of different
`lengths may have
`identical promoter activity.
`
`[0101] The term “operably linked” refers to the associa-
`tion 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.
`
`[0102] 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.
`
`[0103] 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 “trans-
`formed” organisms.
`
`[0104] The terms “plasmid”, “vector” and “cassette” 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 frag-
`ments. Such elements may be autonomously replicating
`sequences, genome integrating sequences, phage or nucle-
`otide 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