U.S. patent application number 13/528106 was filed with the patent office on 2012-11-15 for methods of increasing dihydroxy acid dehydratase activity to improve production of fuels, chemicals, and amino acids.
This patent application is currently assigned to Gevo, Inc.. Invention is credited to Lynne H. Albert, Aristos Aristidou, Thomas Buelter, Catherine Asleson Dundon, Reid M. Renny Feldman, Andrew Hawkins, Doug Lies, Peter Meinhold, Matthew Peters, Stephanie Porter-Scheinman, Christopher Smith, Jun Urano.
Application Number | 20120288910 13/528106 |
Document ID | / |
Family ID | 44066896 |
Filed Date | 2012-11-15 |
United States Patent
Application |
20120288910 |
Kind Code |
A1 |
Urano; Jun ; et al. |
November 15, 2012 |
METHODS OF INCREASING DIHYDROXY ACID DEHYDRATASE ACTIVITY TO
IMPROVE PRODUCTION OF FUELS, CHEMICALS, AND AMINO ACIDS
Abstract
The present invention is directed to recombinant microorganisms
comprising one or more dihydroxyacid dehydratase (DHAD)-requiring
biosynthetic pathways and methods of using said recombinant
microorganisms to produce beneficial metabolites derived from said
DHAD-requiring biosynthetic pathways. In various aspects of the
invention, the recombinant microorganisms may be engineered to
overexpress one or more polynucleotides encoding one or more Aft
proteins or homologs thereof. In some embodiments, the recombinant
microorganisms may comprise a cytosolically localized DHAD enzyme.
In additional embodiments, the recombinant microorganisms may
comprise a mitochondrially localized DHAD enzyme. In various
embodiments described herein, the recombinant microorganisms may be
microorganisms of the Saccharomyces clade, Crabtree-negative yeast
microorganisms, Crabtree-positive yeast microorganisms, post-WGD
(whole genome duplication) yeast microorganisms, pre-WGD (whole
genome duplication) yeast microorganisms, and non-fermenting yeast
microorganisms.
Inventors: |
Urano; Jun; (Irvine, CA)
; Dundon; Catherine Asleson; (Englewood, CO) ;
Meinhold; Peter; (Denver, CO) ; Feldman; Reid M.
Renny; (San Francisco, CA) ; Aristidou; Aristos;
(Englewood, CO) ; Hawkins; Andrew; (Parker,
CO) ; Buelter; Thomas; (Denver, CO) ; Peters;
Matthew; (Highlands Ranch, CO) ; Lies; Doug;
(Parker, CO) ; Porter-Scheinman; Stephanie;
(Conifer, CO) ; Smith; Christopher; (Englewood,
CO) ; Albert; Lynne H.; (Englewood, CO) |
Assignee: |
Gevo, Inc.
Englewood
CO
|
Family ID: |
44066896 |
Appl. No.: |
13/528106 |
Filed: |
June 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13246693 |
Sep 27, 2011 |
8273565 |
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13528106 |
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13228342 |
Sep 8, 2011 |
8071358 |
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13246693 |
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12953884 |
Nov 24, 2010 |
8017376 |
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13228342 |
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61263952 |
Nov 24, 2009 |
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61350209 |
Jun 1, 2010 |
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Current U.S.
Class: |
435/160 ;
435/254.2; 435/254.21; 435/254.22; 435/254.23 |
Current CPC
Class: |
C12P 7/16 20130101; C12P
13/02 20130101; C07K 14/395 20130101; C12P 13/06 20130101; C12Y
402/01009 20130101; Y02E 50/10 20130101; C12P 13/08 20130101; C12N
9/88 20130101 |
Class at
Publication: |
435/160 ;
435/254.2; 435/254.21; 435/254.23; 435/254.22 |
International
Class: |
C12P 7/16 20060101
C12P007/16; C12N 1/19 20060101 C12N001/19 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENTAL SUPPORT
[0002] This invention was made with government support under
Contract No. IIP-0823122, awarded by the National Science
Foundation, and under Contract No. EP-D-09-023, awarded by the
Environmental Protection Agency. The government has certain rights
in the invention.
Claims
1. A recombinant yeast microorganism comprising a recombinantly
overexpressed polynucleotide encoding a dihydroxy acid dehydratase
(DHAD), and recombinantly overexpressed one or more polynucleotides
encoding one or more activator of ferrous transport (Aft) proteins
which increase the dehydratase activity of DHAD.
2. The recombinant yeast microorganism of claim 1, wherein said
recombinant microorganism comprises an isobutanol producing
metabolic pathway, said isobutanol producing metabolic pathway
comprising the following substrate to product conversions: (a)
pyruvate to acetolactate; (b) acetolactate to
2,3-dihydroxyisovalerate; (c) 2,3-dihydroxyisovalerate to
.alpha.-ketoisovalerate; (d) .alpha.-ketoisovalerate to
isobutyraldehyde; and (e) isobutyraldehyde to isobutanol; and
wherein said DHAD catalyzes the conversion of
2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate.
3. The recombinant yeast microorganism of claim 2, wherein the
enzyme that catalyzes the conversion of pyruvate to acetolactate is
an acetolactate synthase.
4. The recombinant yeast microorganism of claim 2, wherein the
enzyme that catalyzes the conversion of acetolactate to
2,3-dihydroxyisovalerate is a ketol-acid reductoisomerase.
5. The recombinant yeast microorganism of claim 4, wherein said
ketol-acid reductoisomerase is an NADH-dependent ketol-acid
reductoisomerase.
6. The recombinant yeast microorganism of claim 2, wherein the
enzyme that catalyzes the conversion of .alpha.-ketoisovalerate to
isobutyraldehyde is a 2-keto acid decarboxylase.
7. The recombinant yeast microorganism of claim 2, wherein the
enzyme that catalyzes the conversion of isobutyraldehyde to
isobutanol is an alcohol dehydrogenase.
8. The recombinant yeast microorganism of claim 7, wherein said
alcohol dehydrogenase is an NADH-dependent alcohol
dehydrogenase.
9. The recombinant yeast microorganism of claim 1, wherein said
DHAD is localized in the mitochondria.
10. The recombinant yeast microorganism of claim 2, wherein said
recombinant yeast microorganism is engineered to inactivate one or
more endogenous pyruvate decarboxylase (PDC) genes.
11. The recombinant yeast microorganism of claim 2, wherein said
recombinant yeast microorganism is engineered to inactivate one or
more endogenous glycerol-3-phosphate dehydrogenase (GPD) genes.
12. The recombinant yeast microorganism of claim 1, wherein said
recombinant yeast microorganism is engineered to inactivate one or
more endogenous glutaredoxin genes selected from the group
consisting of GRX3 and GRX4.
13. The recombinant yeast microorganism of claim 1, wherein said
one or more polynucleotides encoding one or more Aft proteins is a
native polynucleotide.
14. The recombinant yeast microorganism of claim 1, wherein said
one or more polynucleotides encoding one or more Aft proteins is a
heterologous polynucleotide.
15. The recombinant yeast microorganism of claim 1, wherein said
Aft protein is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ
ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID
NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, and SEQ ID NO: 225.
16. The recombinant yeast microorganism of claim 1, wherein said
Aft protein is a constitutively active Aft protein.
17. The recombinant yeast microorganism of claim 16, wherein said
constitutively active Aft protein comprises a substitution at a
position corresponding to the cysteine 291 residue of the native S.
cerevisiae Aft1 protein of SEQ ID NO: 2.
18. The recombinant yeast microorganism of claim 16, wherein said
constitutively active Aft protein comprises a mutation substitution
at a position corresponding to the cysteine 187 residue of the
native S. cerevisiae Aft2 protein of SEQ ID NO: 4.
19. The recombinant yeast microorganism of claim 1, wherein the
overexpression of one or more polynucleotides encoding one or more
Aft proteins increases the specific activity of DHAD by at least
2-fold in the engineered recombinant yeast microorganism as
compared to a corresponding yeast microorganism that is not
engineered to overexpress one or more polynucleotides encoding one
or more Aft proteins.
20. The recombinant yeast microorganism of claim 1, wherein the
recombinant yeast microorganism is a yeast microorganism selected
from one of the following genera: Saccharomyces, Kluyveromyces,
Pachysolen, Zygosaccharomyces, Debaryomyces, Pichia,
Schizosaccharomyces, Candida, Issatchenkia, Hansenula, Yarrowia,
Tricosporon, Rhodotorula, and Myxozyma.
21. The recombinant yeast microorganism of claim 1, wherein the
recombinant yeast microorganism is a yeast microorganism selected
from one of the following species: Saccharomyces cerevisiae,
Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces
paradoxus, Saccharomyces castelli, Saccharomyces kluyveri,
Kluyveromyces thermotolerans, Kluyveromyces lactis, Kluyveromyces
marxianus, Kluyveromyces waltii, Pachysolen tannophilis,
Zygosaccharomyces bailli, Zygosaccharomyces rouxii, Debaryomyces
hansenii, Debaromyces carsonii, Pichia pastorius, Pichia anomala,
Pichia stipitis, Pichia castillae, Schizosaccharomyces pombe,
Candida utilis, Candida glabrata, Candida tropicalis, Candida
xestobii, Issatchenkia orientalis, Issatchenkia occidentalis,
Issatchenkia scutulata, Hansenula anomala, and Yarrowia
lipolytica.
22. A method of producing isobutanol comprising: (a) providing the
recombinant yeast microorganism of claim 2; and (b) cultivating the
recombinant yeast microorganism of claim 2 in a culture medium
containing a feedstock providing a carbon source, until a
recoverable quantity of the isobutanol is produced.
23. The method of claim 22, further comprising the step of
recovering the isobutanol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/246,693, filed Sep. 27, 2011, which is a divisional of U.S.
application Ser. No. 13/228,342, filed Sep. 8, 2011, now U.S. Pat.
No. 8,071,358, which is a divisional application of U.S.
application Ser. No. 12/953,884, filed Nov. 24, 2010, now U.S. Pat.
No. 8,017,376, which claims the benefit of U.S. Provisional
Application Ser. No. 61/263,952, filed Nov. 24, 2009, and U.S.
Provisional Application Ser. No. 61/350,209, filed Jun. 1, 2010,
each of which are herein incorporated by reference in their
entireties for all purposes.
TECHNICAL FIELD
[0003] Recombinant microorganisms and methods of producing such
organisms are provided. Also provided are methods of producing
beneficial metabolites including fuels, chemicals, and amino acids
by contacting a suitable substrate with recombinant microorganisms
and enzymatic preparations therefrom.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0004] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
GEVO.sub.--041.sub.--16US_SeqList.txt, date recorded: Jun. 19,
2012, file size: 658 kilobytes).
BACKGROUND
[0005] Dihydroxyacid dehydratase (DHAD) is an enzyme that catalyzes
the conversion of 2,3-dihydroxyisovalerate to
.alpha.-ketoisovalerate and of 2,3-dihydroxy-3-methylvalerate to
2-keto-3-methylvalerate. This enzyme plays an important role in a
variety of biosynthetic pathways, including pathways producing
valine, isoleucine, leucine and pantothenic acid (vitamin B5). DHAD
also catalyzes the conversion of 2,3-dihydroxyisovalerate to
.alpha.-ketoisovalerate as part of isobutanol biosynthetic pathways
disclosed in commonly owned and co-pending US Patent Publication
Nos. 2009/0226991 and 2010/0143997. In addition, biosynthetic
pathways for the production of 3-methyl-1-butanol and
2-methyl-1-butanol use DHAD to convert 2,3-dihydroxyisovalerate to
.alpha.-ketoisovalerate and 2,3-dihydroxy-3-methylvalerate to
2-keto-3-methylvalerate, respectively (Atsumi et al., 2008, Nature
451(7174): 86-9).
[0006] DHAD is an essential enzyme in all of these biosynthetic
pathways, hence, it is desirable that recombinant microorganisms
engineered to produce the above-mentioned compounds exhibit optimal
DHAD activity. The optimal level of DHAD activity will typically
have to be at levels that are significantly higher than those found
in non-engineered microorganisms in order to sustain commercially
viable productivities, yields, and titers. The present application
addresses this need by engineering recombinant microorganisms to
improve their DHAD activity.
SUMMARY OF THE INVENTION
[0007] The present inventors have discovered that overexpression of
the transcriptional activator genes AFT1 and/or AFT2 or homologs
thereof in a recombinant yeast microorganism improves DHAD
activity. Thus, the invention relates to recombinant yeast cells
engineered to provide increased heterologous or native expression
of AFT1 and/or AFT2 or homologs thereof. In general, cells that
overexpress AFT1 and/or AFT2 or homologs thereof exhibit an
enhanced ability to produce beneficial metabolites such as
isobutanol, 3-methyl-1-butanol, 2-methyl-1-butanol, valine,
isoleucine, leucine, and pantothenic acid.
[0008] One aspect of the invention is directed to a recombinant
microorganism comprising a DHAD-requiring biosynthetic pathway,
wherein said microorganism is engineered to overexpress one or more
polynucleotides encoding one or more Aft proteins or homologs
thereof. In one embodiment, the Aft protein is selected from SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ
ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:
20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ
ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:
209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO:
217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, and SEQ ID NO:
225. In another embodiment, one or more of the polynucleotides
encoding said one or more Aft proteins or homologs thereof is a
native polynucleotide. In yet another embodiment, one or more of
the polynucleotides encoding said one or more Aft proteins or
homologs thereof is a heterologous polynucleotide.
[0009] In a specific embodiment according to this aspect, the
invention is directed to a recombinant microorganism comprising a
DHAD-requiring biosynthetic pathway, wherein said microorganism has
been engineered to overexpress a polynucleotide encoding Aft1 (SEQ
ID NO: 2) and/or Aft2 (SEQ ID NO: 4) or a homolog thereof. In one
embodiment, the polynucleotide encoding the Aft protein or homolog
thereof is native to the recombinant microorganism. In another
embodiment, the polynucleotide encoding the Aft protein or homolog
thereof is heterologous to the recombinant microorganism.
[0010] Another aspect of the invention is directed to a recombinant
microorganism comprising a DHAD-requiring biosynthetic pathway,
wherein the activity of one or more Aft proteins or homologs
thereof is increased. In one embodiment, the Aft protein is
selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID
NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, and SEQ ID NO: 225. In one embodiment, the polynucleotide
encoding the Aft protein or homolog thereof is native to the
recombinant microorganism. In another embodiment, the
polynucleotide encoding the Aft protein or homolog thereof is
heterologous to the recombinant microorganism.
[0011] Another aspect of the invention is directed to a recombinant
microorganism comprising a DHAD-requiring biosynthetic pathway,
wherein said microorganism has been engineered to overexpress one
or more polynucleotides encoding one or more proteins or homologs
thereof regulated by an Aft protein or homolog thereof. In one
embodiment, the proteins regulated by an Aft protein or homolog
thereof are selected from FET3, FET4, FET5, FTR1, FTH1, SMF3, MRS4,
CCC2, COT1, ATX1, FRE1, FRE2, FRE3, FRE4, FRE5, FREE, FIT1, FIT2,
FIT3, ARN1, ARN2, ARN3, ARN4, ISU1, ISU2, TIS11, HMX1, AKR1, PCL5,
YOR387C, YHL035C, YMR034C, ICY2, PRY1, YDL124W, BNA2, ECM4, LAP4,
YOL083W, YGR146C, BIO5, YDR271C, OYE3, CTH1, CTH2, MRS3, MRS4,
HSP26, YAP2, VMR1, ECL1, OSW1, NFT1, ARA2, TAF1/TAF130/TAF145,
YOR225W, YKR104W, YBR012C, and YMR041C or homologs thereof. In a
specific embodiment, the protein regulated by an Aft protein or
homolog thereof is ENB1. In another specific embodiment, the
protein regulated by an Aft protein or homologs thereof is FET3. In
yet another specific embodiment, the protein regulated by an Aft
protein or homolog thereof is SMF3. In one embodiment, all genes
demonstrated to increase DHAD activity and/or the production of a
metabolite from a DHAD-requiring biosynthetic pathway are
overexpressed. Where none of the AFT regulon genes expressed alone
are effective in increasing DHAD activity and/or the production of
a metabolite from a DHAD-requiring biosynthetic pathway, then 1, 2,
3, 4, 5, or more of the genes in the AFT regulon may be
overexpressed together.
[0012] In various embodiments described herein, the DHAD-requiring
biosynthetic pathway may be selected from isobutanol,
3-methyl-1-butanol, 2-methyl-1-butanol, valine, isoleucine,
leucine, and/or pantothenic acid biosynthetic pathways. In various
embodiments described herein, the DHAD enzyme which acts as part of
an isobutanol, 3-methyl-1-butanol, 2-methyl-1-butanol, valine,
isoleucine, leucine, and/or pantothenic acid biosynthetic pathway
may be localized to the cytosol. In alternative embodiments, the
DHAD enzyme which acts as part of an isobutanol,
3-methyl-1-butanol, 2-methyl-1-butanol, valine, isoleucine,
leucine, and/or pantothenic acid biosynthetic pathway may be
localized to the mitochondria. In additional embodiments, a DHAD
enzyme which acts as part of an isobutanol, 3-methyl-1-butanol,
2-methyl-1-butanol, valine, isoleucine, leucine, and/or pantothenic
acid biosynthetic pathway is localized to the cytosol and the
mitochondria.
[0013] In one embodiment, the invention is directed to a
recombinant microorganism for producing isobutanol, wherein said
recombinant microorganism comprises an isobutanol producing
metabolic pathway and wherein said microorganism is engineered to
overexpress one or more polynucleotides encoding one or more Aft
proteins or homologs thereof. In one embodiment, the Aft protein is
selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID
NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO:
223, and SEQ ID NO: 225.
[0014] In a specific embodiment, the invention is directed to a
recombinant microorganism for producing isobutanol, wherein said
recombinant microorganism comprises an isobutanol producing
metabolic pathway and wherein said microorganism is engineered to
overexpress a polynucleotide encoding Aft1 (SEQ ID NO: 2) or a
homolog thereof. In another specific embodiment, the invention is
directed to a recombinant microorganism for producing isobutanol,
wherein said recombinant microorganism comprises an isobutanol
producing metabolic pathway and wherein said microorganism is
engineered to overexpress a polynucleotide encoding Aft2 (SEQ ID
NO: 4) or a homolog thereof. In yet another embodiment, the
invention is directed to a recombinant microorganism for producing
isobutanol, wherein said recombinant microorganism comprises an
isobutanol producing metabolic pathway and wherein said
microorganism is engineered to overexpress a polynucleotide
encoding Aft1 (SEQ ID NO: 2) or a homolog thereof and Aft2 (SEQ ID
NO: 4) or a homolog thereof.
[0015] In each of the aforementioned aspects and embodiments, the
Aft protein may be a constitutively active Aft protein or a homolog
thereof. In one embodiment, the constitutively active Aft protein
or homolog thereof comprises a mutation at a position corresponding
to the cysteine 291 residue of the native S. cerevisiae Aft1 (SEQ
ID NO: 2). In a specific embodiment, the cysteine 291 residue is
replaced with a phenylalanine residue. In another embodiment, the
constitutively active Aft protein or homolog thereof comprises a
mutation at a position corresponding to the cysteine 187 residue of
the native S. cerevisiae Aft2 (SEQ ID NO: 2). In a specific
embodiment, the cysteine 187 residue is replaced with a
phenylalanine residue.
[0016] In another embodiment, the invention is directed to a
recombinant microorganism for producing isobutanol, wherein said
recombinant microorganism comprises an isobutanol producing
metabolic pathway, wherein said microorganism has been engineered
to overexpress one or more polynucleotides encoding one or more
proteins or homologs thereof regulated by an Aft protein or homolog
thereof. In one embodiment, the proteins regulated by Aft or a
homolog thereof are selected from FET3, FET4, FET5, FTR1, FTH1,
SMF3, MRS4, CCC2, COT1, ATX1, FRE1, FRE2, FRE3, FRE4, FRE5, FREE,
FIT1, FIT2, FIT3, ARN1, ARN2, ARN3, ARN4, ISU1, ISU2, TIS11, HMX1,
AKR1, PCL5, YOR387C, YHL035C, YMR034C, ICY2, PRY1, YDL124W, BNA2,
ECM4, LAP4, YOL083W, YGR146C, 8105, YDR271C, OYE3, CTH1, CTH2,
MRS3, MRS4, HSP26, YAP2, VMR1, ECL1, OSW1, NFT1, ARA2,
TAF1/TAF130/TAF145, YOR225W, YKR104W, YBR012C, and YMR041C or
homologs thereof. In a specific embodiment, the protein regulated
by an Aft protein or homolog thereof is ENB1. In another specific
embodiment, the protein regulated by an Aft protein or homologs
thereof is FET3. In yet another specific embodiment, the protein
regulated by an Aft protein or homolog thereof is SMF3. In one
embodiment, all genes demonstrated to increase DHAD activity and/or
the production of a metabolite from a DHAD-requiring biosynthetic
pathway are overexpressed. Where none of the AFT regulon genes
expressed alone are effective in increasing DHAD activity and/or
the production of a metabolite from a DHAD-requiring biosynthetic
pathway, then 1, 2, 3, 4, 5, or more of the genes in the AFT
regulon may be overexpressed together.
[0017] In one embodiment, the isobutanol producing metabolic
pathway comprises at least one exogenous gene that catalyzes a step
in the conversion of pyruvate to isobutanol. In another embodiment,
the isobutanol producing metabolic pathway comprises at least two
exogenous genes that catalyze steps in the conversion of pyruvate
to isobutanol. In yet another embodiment, the isobutanol producing
metabolic pathway comprises at least three exogenous genes that
catalyze steps in the conversion of pyruvate to isobutanol. In yet
another embodiment, the isobutanol producing metabolic pathway
comprises at least four exogenous genes that catalyze steps in the
conversion of pyruvate to isobutanol. In yet another embodiment,
the isobutanol producing metabolic pathway comprises at five
exogenous genes that catalyze steps in the conversion of pyruvate
to isobutanol.
[0018] In one embodiment, one or more of the isobutanol pathway
genes encodes an enzyme that is localized to the cytosol. In one
embodiment, the recombinant microorganisms comprise an isobutanol
producing metabolic pathway with at least one isobutanol pathway
enzyme localized in the cytosol. In another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least two isobutanol pathway enzymes
localized in the cytosol. In yet another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least three isobutanol pathway enzymes
localized in the cytosol. In yet another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least four isobutanol pathway enzymes
localized in the cytosol. In an exemplary embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with five isobutanol pathway enzymes localized in
the cytosol. In a further exemplary embodiment, at least one of the
pathway enzymes localized to the cytosol is a cytosolically active
DHAD enzyme as disclosed herein.
[0019] In various embodiments described herein, the isobutanol
pathway genes encodes enzyme(s) selected from the group consisting
of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI),
dihydroxyacid dehydratase (DHAD), 2-keto-acid decarboxylase (KIVD),
and alcohol dehydrogenase (ADH).
[0020] Another aspect of the invention is directed to a recombinant
microorganism comprising a DHAD-requiring biosynthetic pathway,
wherein said microorganism has been engineered to overexpress a
polynucleotide encoding Grx3 and/or Grx4 or a homolog thereof. In
one embodiment, the polynucleotide encoding the Grx protein or
homolog thereof is native to the recombinant microorganism. In
another embodiment, the polynucleotide encoding the Grx protein or
homolog thereof is heterologous to the recombinant
microorganism.
[0021] In various embodiments described herein, the recombinant
microorganism may be engineered reduce the concentration of
reactive oxygen species (ROS) in the recombinant microorganism.
Thus, the recombinant microorganisms may be engineered to express
one or more proteins that reduce the concentration of reactive
oxygen species (ROS) in said cell. The proteins to be expressed for
reducing the concentration of reactive oxygen species may be
selected from catalases, superoxide dismutases, metallothioneins,
and methionine sulphoxide reductases. In a specific embodiment,
said catalase may be encoded by one of more of the genes selected
from the group consisting of the E. coli genes katG and katE, the
S. cerevisiae genes CTT1 and CTA1, or homologs thereof. In another
specific embodiment, said superoxide dismutase is encoded by one of
more of the genes selected from the group consisting of the E. coli
genes sodA, sodB, sodC, the S. cerevisiae genes SOD1 and SOD2, or
homologs thereof. In another specific embodiment, said
metallothionein is encoded by one of more of the genes selected
from the group consisting of the S. cerevisiae CUP1-1 and CUP1-2
genes or homologs thereof. In another specific embodiment, said
metallothionein is encoded by one or more genes selected from the
group consisting of the Mycobacterium tuberculosis MymT gene and
the Synechococcus PCC 7942 SmtA gene or homologs thereof. In
another specific embodiment, said methionine sulphoxide reductase
is encoded by one or more genes selected from the group consisting
of the S. cerevisiae genes MXR1 and MXR2, or homologs thereof.
[0022] In some embodiments, the recombinant microorganism may be
engineered to increase the level of available glutathione in the
recombinant microorganism. Thus, the recombinant microorganisms may
be engineered to express one or more proteins that increase the
level of available glutathione in the cell. In one embodiment, the
proteins are selected from glutaredoxin, glutathione reductase,
glutathione synthase, and combinations thereof. In a specific
embodiment, said glutaredoxin is encoded by one of more of the
genes selected from the group the S. cerevisiae genes GRX2, GRX4,
GRX6, and GRX7, or homologs thereof. In another specific
embodiment, said glutathione reductase is encoded by the S.
cerevisiae genes GLR1 or homologs thereof. In another specific
embodiment, said glutathione synthase is encoded by one of more of
the genes selected from the S. cerevisiae genes GSH1 and GSH2, or
homologs thereof. In some embodiments, two enzymes are expressed to
increase the level of available glutathione in the cell. In one
embodiment, the enzymes are .gamma.-glutamyl cysteine synthase and
glutathione synthase. In a specific embodiment, said glutathione
synthase is encoded by one of more of the genes selected from the
group the S. cerevisiae genes GSH1 and GSH2, or homologs
thereof.
[0023] In some embodiments, it may be desirable to overexpress one
or more functional components of the thioredoxin system, as
overexpression of the functional components of the thioredoxin
system can increase the amount of bioavailable thioredoxin. In one
embodiment, the functional components of the thioredoxin system may
be selected from a thioredoxin and a thioredoxin reductase. In a
specific embodiment, said thioredoxin is encoded by the S.
cerevisiae TRX1 and TRX2 genes or homologs thereof. In another
specific embodiment, said thioredoxin reductase is encoded by S.
cerevisiae TRR1 gene or homologs thereof. In additional
embodiments, the recombinant microorganism may further be
engineered to overexpress the mitochondrial thioredoxin system. In
one embodiment, the mitochondrial thioredoxin system is comprised
of the mitochondrial thioredoxin and mitochondrial thioredoxin
reductase. In a specific embodiment, said mitochondrial thioredoxin
is encoded by the S. cerevisiae TRX3 gene or homologs thereof. In
another specific embodiment, said mitochondrial thioredoxin
reductase is encoded by the S. cerevisiae TRR2 gene or homologs
thereof.
[0024] In various embodiments described herein, it may be desirable
to engineer the recombinant microorganism to overexpress one or
more mitochondrial export proteins. In a specific embodiment, said
mitochondrial export protein may be selected from the group
consisting of the S. cerevisiae ATM1, the S. cerevisiae ERV1, and
the S. cerevisiae BAT1, or homologs thereof.
[0025] In addition, the present invention provides recombinant
microorganisms that have been engineered to increase the inner
mitochondrial membrane electrical potential, .DELTA..psi..sub.M. In
one embodiment, this is accomplished via overexpression of an
ATP/ADP carrier protein, wherein said overexpression increases
ATP.sup.4- import into the mitochondrial matrix in exchange for
ADP.sup.3-. In a specific embodiment, said ATP/ADP carrier protein
is encoded by the S. cerevisiae AAC1, AAC2, and/or AAC3 genes or
homologs thereof. In another embodiment, the inner mitochondrial
membrane electrical potential, .DELTA..psi..sub.M is increased via
a mutation in the mitochondrial ATP synthase complex that increases
ATP hydrolysis activity. In a specific embodiment, said mutation is
an ATP1-111 suppressor mutation or a corresponding mutation in a
homologous protein.
[0026] In various embodiments described herein, it may further be
desirable to engineer the recombinant microorganism to express one
or more enzymes in the cytosol that reduce the concentration of
reactive nitrogen species (RNS) and/or nitric oxide (NO) in said
cytosol. In one embodiment, said one or more enzymes are selected
from the group consisting of nitric oxide reductases and
glutathione-S-nitrosothiol reductase. In a specific embodiment,
said nitric oxide reductase is encoded by one of more of the genes
selected from the group consisting of the E. coli gene norV and the
Fusarium oxysporum gene P-450dNIR, or homologs thereof. In another
specific embodiment, said glutathione-S-nitrosothiol reductase is
encoded by the S. cerevisiae gene SFA1 or homologs thereof. In one
embodiment, said glutathione-S-nitrosothiol reductase gene SFA1 is
overexpressed. In another specific embodiment, said one or more
enzymes is encoded by a gene selected from the group consisting of
the E. coli gene ytfE, the Staphylococcus aureus gene scdA, and
Neisseria gonorrhoeae gene dnrN, or homologs thereof.
[0027] Also provided herein are recombinant microorganisms that
demonstrate increased the levels of sulfur-containing compounds
within yeast cells, including the amino acid cysteine, such that
this sulfur is more available for the production of iron-sulfur
cluster-containing proteins in the yeast cell. In one embodiment,
the recombinant microorganism has been engineered to overexpress
one or more of the genes selected from the S. cerevisiae genes
MET1, MET2, MET3, MET5, MET8, MET10, MET14, MET16, MET17, HOM2,
HOM3, HOM3, CYS3, CYS4, SUL1, and SUL2, or homologs thereof. The
recombinant microorganism may additionally or optionally also
overexpress one or more of the genes selected from the S.
cerevisiae genes YCT1, MUP1, GAP1, AGP1, GNP1, BAP1, BAP2, TAT1,
and TAT2, or homologs thereof.
[0028] In various embodiments described herein, the recombinant
microorganism may exhibit at least about 5 percent greater
dihydroxyacid dehydratase (DHAD) activity as compared to the
parental microorganism. In another embodiment, the recombinant
microorganism may exhibit at least about 10 percent, at least about
15 percent, about least about 20 percent, at least about 25
percent, at least about 30 percent, at least about 35 percent, at
least about 40 percent, at least about 45 percent, at least about
50 percent, at least about 55 percent, at least about 60 percent,
at least about 65 percent, at least about 70 percent, at least
about 75 percent, at least about 80 percent, at least about 100
percent, at least about 200 percent, or at least about 500 percent
greater dihydroxyacid dehydratase (DHAD) activity as compared to
the parental microorganism.
[0029] In various embodiments described herein, the recombinant
microorganisms may be microorganisms of the Saccharomyces clade,
Saccharomyces sensu stricto microorganisms, Crabtree-negative yeast
microorganisms, Crabtree-positive yeast microorganisms, post-WGD
(whole genome duplication) yeast microorganisms, pre-WGD (whole
genome duplication) yeast microorganisms, and non-fermenting yeast
microorganisms.
[0030] In some embodiments, the recombinant microorganisms may be
yeast recombinant microorganisms of the Saccharomyces clade.
[0031] In some embodiments, the recombinant microorganisms may be
Saccharomyces sensu stricto microorganisms. In one embodiment, the
Saccharomyces sensu stricto is selected from the group consisting
of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S.
uvarum. S. carocanis and hybrids thereof.
[0032] In some embodiments, the recombinant microorganisms may be
Crabtree-negative recombinant yeast microorganisms. In one
embodiment, the Crabtree-negative yeast microorganism is classified
into a genera selected from the group consisting of Kluyveromyces,
Pichia, Issatchenkia, Hansenula, or Candida. In additional
embodiments, the Crabtree-negative yeast microorganism is selected
from Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala,
Pichia stipitis, Hansenula anomala, Candida utilis and
Kluyveromyces waltii.
[0033] In some embodiments, the recombinant microorganisms may be
Crabtree-positive recombinant yeast microorganisms. In one
embodiment, the Crabtree-positive yeast microorganism is classified
into a genera selected from the group consisting of Saccharomyces,
Kluyveromyces, Zygosaccharomyces, Debaryomyces, Candida, Pichia and
Schizosaccharomyces. In additional embodiments, the
Crabtree-positive yeast microorganism is selected from the group
consisting of Saccharomyces cerevisiae, Saccharomyces uvarum,
Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces
castelli, Saccharomyces kluyveri, Kluyveromyces thermotolerans,
Candida glabrata, Z. bailli, Z. rouxii, Debaryomyces hansenii,
Pichia pastorius, Schizosaccharomyces pombe, and Saccharomyces
uvarum.
[0034] In some embodiments, the recombinant microorganisms may be
post-WGD (whole genome duplication) yeast recombinant
microorganisms. In one embodiment, the post-WGD yeast recombinant
microorganism is classified into a genera selected from the group
consisting of Saccharomyces or Candida. In additional embodiments,
the post-WGD yeast is selected from the group consisting of
Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces
bayanus, Saccharomyces paradoxus, Saccharomyces castelli, and
Candida glabrata.
[0035] In some embodiments, the recombinant microorganisms may be
pre-WGD (whole genome duplication) yeast recombinant
microorganisms. In one embodiment, the pre-WGD yeast recombinant
microorganism is classified into a genera selected from the group
consisting of Saccharomyces, Kluyveromyces, Candida, Pichia,
Issatchenkia, Debaryomyces, Hansenula, Pachysolen, Yarrowia and
Schizosaccharomyces. In additional embodiments, the pre-WGD yeast
is selected from the group consisting of Saccharomyces kluyveri,
Kluyveromyces thermotolerans, Kluyveromyces marxianus,
Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis,
Pichia pastoris, Pichia anomala, Pichia stipitis, Isstachenkia
orientalis, Issatchenkia occidentalis, Debaryomyces hansenii,
Hansenula anomala, Pachysolen tannophilis, Yarrowia lipolytica, and
Schizosaccharomyces pombe.
[0036] In some embodiments, the recombinant microorganisms may be
microorganisms that are non-fermenting yeast microorganisms,
including, but not limited to those, classified into a genera
selected from the group consisting of Tricosporon, Rhodotorula,
Myxozyma, or Candida. In a specific embodiment, the non-fermenting
yeast is C. xestobii.
[0037] In another aspect, the present invention provides methods of
producing beneficial metabolites including fuels, chemicals, and
amino acids using a recombinant microorganism as described herein.
In one embodiment, the method includes cultivating the recombinant
microorganism in a culture medium containing a feedstock providing
the carbon source until a recoverable quantity of the metabolite is
produced and optionally, recovering the metabolite. In one
embodiment, the microorganism produces the metabolite from a carbon
source at a yield of at least about 5 percent theoretical. In
another embodiment, the microorganism produces the metabolite at a
yield of at least about 10 percent, at least about 15 percent,
about least about 20 percent, at least about 25 percent, at least
about 30 percent, at least about 35 percent, at least about 40
percent, at least about 45 percent, at least about 50 percent, at
least about 55 percent, at least about 60 percent, at least about
65 percent, at least about 70 percent, at least about 75 percent,
at least about 80 percent, at least about 85 percent, at least
about 90 percent, at least about 95 percent, or at least about 97.5
percent theoretical. The metabolite may be derived from any
DHAD-requiring biosynthetic pathway, including, but not limited to,
biosynthetic pathways for the production of isobutanol,
3-methyl-1-butanol, 2-methyl-1-butanol, valine, isoleucine,
leucine, and pantothenic acid.
[0038] In one embodiment, the recombinant microorganism is grown
under aerobic conditions. In another embodiment, the recombinant
microorganism is grown under microaerobic conditions. In yet
another embodiment, the recombinant microorganism is grown under
anaerobic conditions.
BRIEF DESCRIPTION OF DRAWINGS
[0039] Illustrative embodiments of the invention are illustrated in
the drawings, in which:
[0040] FIG. 1 illustrates an exemplary embodiment of an isobutanol
pathway.
[0041] FIG. 2 illustrates a phylogenetic tree of DHAD proteins.
Numbers at nodes indicate bootstrap values. Ec_ilvD is a known
4Fe-4S DHAD enzyme from Escherichia coli.
[0042] FIG. 3 illustrates a S. cerevisiae AFT1-1.sup.UP allelic
exchange construct.
[0043] FIG. 4 illustrates a S. cerevisiae AFT2-1.sup.UP allelic
exchange construct.
[0044] FIG. 5 illustrates a linear DNA fragment containing the K.
marxianus AFT, the L. lactis DHAD, and a G418 resistance
marker.
[0045] FIG. 6 illustrates a linear DNA fragment containing the L.
lactis DHAD and a G418 resistance marker.
DETAILED DESCRIPTION
[0046] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a polynucleotide" includes a plurality of such polynucleotides and
reference to "the microorganism" includes reference to one or more
microorganisms, and so forth.
[0047] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods, devices and
materials are described herein.
[0048] Any publications discussed above and throughout the text are
provided solely for their disclosure prior to the filing date of
the present application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior disclosure.
[0049] The term "microorganism" includes prokaryotic and eukaryotic
microbial species from the Domains Archaea, Bacteria and Eucarya,
the latter including yeast and filamentous fungi, protozoa, algae,
or higher Protista. The terms "microbial cells" and "microbes" are
used interchangeably with the term microorganism.
[0050] The term "genus" is defined as a taxonomic group of related
species according to the Taxonomic Outline of Bacteria and Archaea
(Garrity, G. M., Lilburn, T. G., Cole, J. R., Harrison, S. H.,
Euzeby, J., and Tindall, B. J. (2007) The Taxonomic Outline of
Bacteria and Archaea. TOBA Release 7.7, March 2007. Michigan State
University Board of Trustees.
[0051] The term "species" is defined as a collection of closely
related organisms with greater than 97% 16S ribosomal RNA sequence
homology and greater than 70% genomic hybridization and
sufficiently different from all other organisms so as to be
recognized as a distinct unit.
[0052] The terms "recombinant microorganism," "modified
microorganism" and "recombinant host cell" are used interchangeably
herein and refer to microorganisms that have been genetically
modified to express or over-express endogenous polynucleotides, or
to express heterologous polynucleotides, such as those included in
a vector, or which have an alteration in expression of an
endogenous gene. By "alteration" it is meant that the expression of
the gene, or level of a RNA molecule or equivalent RNA molecules
encoding one or more polypeptides or polypeptide subunits, or
activity of one or more polypeptides or polypeptide subunits is up
regulated or down regulated, such that expression, level, or
activity is greater than or less than that observed in the absence
of the alteration. For example, the term "alter" can mean
"inhibit," but the use of the word "alter" is not limited to this
definition.
[0053] The term "expression" with respect to a gene sequence refers
to transcription of the gene and, as appropriate, translation of
the resulting mRNA transcript to a protein. Thus, as will be clear
from the context, expression of a protein results from
transcription and translation of the open reading frame sequence.
The level of expression of a desired product in a host cell may be
determined on the basis of either the amount of corresponding mRNA
that is present in the cell, or the amount of the desired product
encoded by the selected sequence. For example, mRNA transcribed
from a selected sequence can be quantitated by qRT-PCR or by
Northern hybridization (see Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
Protein encoded by a selected sequence can be quantitated by
various methods, e.g., by ELISA, by assaying for the biological
activity of the protein, or by employing assays that are
independent of such activity, such as western blotting or
radioimmunoassay, using antibodies that recognize and bind the
protein. See Sambrook et al., 1989, supra. The polynucleotide
generally encodes a target enzyme involved in a metabolic pathway
for producing a desired metabolite. It is understood that the terms
"recombinant microorganism" and "recombinant host cell" refer not
only to the particular recombinant microorganism but to the progeny
or potential progeny of such a microorganism. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
[0054] The term "overexpression" refers to an elevated level (e.g.,
aberrant level) of mRNAs encoding for a protein(s) (e.g. an Aft
protein or homolog thereof), and/or to elevated levels of
protein(s) (e.g. Aft) in cells as compared to similar corresponding
unmodified cells expressing basal levels of mRNAs (e.g., those
encoding Aft proteins) or having basal levels of proteins. In
particular embodiments, Aft1 and/or Aft2, or homologs thereof, or
Aft regulon proteins, or homologs thereof, may be overexpressed by
at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold,
12-fold, 15-fold or more in microorganisms engineered to exhibit
increased Aft1 and/or Aft2, or Aft regulon mRNA, protein, and/or
activity.
[0055] The term "wild-type microorganism" describes a cell that
occurs in nature, i.e. a cell that has not been genetically
modified. A wild-type microorganism can be genetically modified to
express or overexpress a first target enzyme. This microorganism
can act as a parental microorganism in the generation of a
microorganism modified to express or overexpress a second target
enzyme. In turn, the microorganism modified to express or
overexpress a first and a second target enzyme can be modified to
express or overexpress a third target enzyme.
[0056] Accordingly, a "parental microorganism" functions as a
reference cell for successive genetic modification events. Each
modification event can be accomplished by introducing a nucleic
acid molecule in to the reference cell. The introduction
facilitates the expression or overexpression of a target enzyme. It
is understood that the term "facilitates" encompasses the
activation of endogenous polynucleotides encoding a target enzyme
through genetic modification of e.g., a promoter sequence in a
parental microorganism. It is further understood that the term
"facilitates" encompasses the introduction of heterologous
polynucleotides encoding a target enzyme in to a parental
microorganism.
[0057] The term "engineer" refers to any manipulation of a
microorganism that results in a detectable change in the
microorganism, wherein the manipulation includes but is not limited
to inserting a polynucleotide and/or polypeptide heterologous to
the microorganism and mutating a polynucleotide and/or polypeptide
native to the microorganism.
[0058] The term "mutation" as used herein indicates any
modification of a nucleic acid and/or polypeptide which results in
an altered nucleic acid or polypeptide. Mutations include, for
example, point mutations, deletions, or insertions of single or
multiple residues in a polynucleotide, which includes alterations
arising within a protein-encoding region of a gene as well as
alterations in regions outside of a protein-encoding sequence, such
as, but not limited to, regulatory or promoter sequences. A genetic
alteration may be a mutation of any type. For instance, the
mutation may constitute a point mutation, a frame-shift mutation,
an insertion, or a deletion of part or all of a gene. In addition,
in some embodiments of the modified microorganism, a portion of the
microorganism genome has been replaced with a heterologous
polynucleotide. In some embodiments, the mutations are
naturally-occurring. In other embodiments, the mutations are the
results of artificial selection pressure. In still other
embodiments, the mutations in the microorganism genome are the
result of genetic engineering.
[0059] The term "biosynthetic pathway", also referred to as
"metabolic pathway", refers to a set of anabolic or catabolic
biochemical reactions for converting one chemical species into
another. Gene products belong to the same "metabolic pathway" if
they, in parallel or in series, act on the same substrate, produce
the same product, or act on or produce a metabolic intermediate
(i.e., metabolite) between the same substrate and metabolite end
product.
[0060] As used herein, the term "isobutanol producing metabolic
pathway" refers to an enzyme pathway which produces isobutanol from
pyruvate.
[0061] The term "heterologous" as used herein with reference to
molecules and in particular enzymes and polynucleotides, indicates
molecules that are expressed in an organism other than the organism
from which they originated or are found in nature, independently of
the level of expression that can be lower, equal or higher than the
level of expression of the molecule in the native microorganism.
The term "heterologous" is also used synonymously herein with the
term "exogenous."
[0062] On the other hand, the term "native" or "endogenous" as used
herein with reference to molecules, and in particular enzymes and
polynucleotides, indicates molecules that are expressed in the
organism in which they originated or are found in nature,
independently of the level of expression that can be lower equal or
higher than the level of expression of the molecule in the native
microorganism. It is understood that expression of native enzymes
or polynucleotides may be modified in recombinant
microorganisms.
[0063] The term "feedstock" is defined as a raw material or mixture
of raw materials supplied to a microorganism or fermentation
process from which other products can be made. For example, a
carbon source, such as biomass or the carbon compounds derived from
biomass are a feedstock for a microorganism that produces a biofuel
in a fermentation process. However, a feedstock may contain
nutrients other than a carbon source.
[0064] The term "substrate" or "suitable substrate" refers to any
substance or compound that is converted or meant to be converted
into another compound by the action of an enzyme. The term includes
not only a single compound, but also combinations of compounds,
such as solutions, mixtures and other materials which contain at
least one substrate, or derivatives thereof. Further, the term
"substrate" encompasses not only compounds that provide a carbon
source suitable for use as a starting material, such as any biomass
derived sugar, but also intermediate and end product metabolites
used in a pathway associated with a recombinant microorganism as
described herein.
[0065] The term "C2-compound" as used as a carbon source for
engineered yeast microorganisms with mutations in all pyruvate
decarboxylase (PDC) genes resulting in a reduction of pyruvate
decarboxylase activity of said genes refers to organic compounds
comprised of two carbon atoms, including but not limited to ethanol
and acetate.
[0066] The term "fermentation" or "fermentation process" is defined
as a process in which a microorganism is cultivated in a culture
medium containing raw materials, such as feedstock and nutrients,
wherein the microorganism converts raw materials, such as a
feedstock, into products.
[0067] The term "volumetric productivity" or "production rate" is
defined as the amount of product formed per volume of medium per
unit of time. Volumetric productivity is reported in gram per liter
per hour (g/L/h).
[0068] The term "specific productivity" or "specific production
rate" is defined as the amount of product formed per volume of
medium per unit of time per amount of cells. Specific productivity
is reported in gram or milligram per liter per hour per OD
(g/L/h/OD).
[0069] The term "yield" is defined as the amount of product
obtained per unit weight of raw material and may be expressed as g
product per g substrate (g/g). Yield may be expressed as a
percentage of the theoretical yield. "Theoretical yield" is defined
as the maximum amount of product that can be generated per a given
amount of substrate as dictated by the stoichiometry of the
metabolic pathway used to make the product. For example, the
theoretical yield for one typical conversion of glucose to
isobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose
of 0.39 g/g would be expressed as 95% of theoretical or 95%
theoretical yield.
[0070] The term "titer" is defined as the strength of a solution or
the concentration of a substance in solution. For example, the
titer of a biofuel in a fermentation broth is described as g of
biofuel in solution per liter of fermentation broth (g/L).
[0071] "Aerobic conditions" are defined as conditions under which
the oxygen concentration in the fermentation medium is sufficiently
high for an aerobic or facultative anaerobic microorganism to use
as a terminal electron acceptor.
[0072] In contrast, "anaerobic conditions" are defined as
conditions under which the oxygen concentration in the fermentation
medium is too low for the microorganism to use as a terminal
electron acceptor. Anaerobic conditions may be achieved by sparging
a fermentation medium with an inert gas such as nitrogen until
oxygen is no longer available to the microorganism as a terminal
electron acceptor. Alternatively, anaerobic conditions may be
achieved by the microorganism consuming the available oxygen of the
fermentation until oxygen is unavailable to the microorganism as a
terminal electron acceptor.
[0073] "Aerobic metabolism" refers to a biochemical process in
which oxygen is used as a terminal electron acceptor to make
energy, typically in the form of ATP, from carbohydrates. Aerobic
metabolism occurs e.g. via glycolysis and the TCA cycle, wherein a
single glucose molecule is metabolized completely into carbon
dioxide in the presence of oxygen.
[0074] In contrast, "anaerobic metabolism" refers to a biochemical
process in which oxygen is not the final acceptor of electrons
contained in NADH. Anaerobic metabolism can be divided into
anaerobic respiration, in which compounds other than oxygen serve
as the terminal electron acceptor, and substrate level
phosphorylation, in which the electrons from NADH are utilized to
generate a reduced product via a "fermentative pathway."
[0075] In "fermentative pathways", NAD(P)H donates its electrons to
a molecule produced by the same metabolic pathway that produced the
electrons carried in NAD(P)H. For example, in one of the
fermentative pathways of certain yeast strains, NAD(P)H generated
through glycolysis transfers its electrons to pyruvate, yielding
ethanol. Fermentative pathways are usually active under anaerobic
conditions but may also occur under aerobic conditions, under
conditions where NADH is not fully oxidized via the respiratory
chain. For example, above certain glucose concentrations,
Crabtree-positive yeasts produce large amounts of ethanol under
aerobic conditions.
[0076] The term "byproduct" means an undesired product related to
the production of a biofuel or biofuel precursor. Byproducts are
generally disposed as waste, adding cost to a production
process.
[0077] The term "non-fermenting yeast" is a yeast species that
fails to demonstrate an anaerobic metabolism in which the electrons
from NADH are utilized to generate a reduced product via a
fermentative pathway such as the production of ethanol and CO.sub.2
from glucose. Non-fermentative yeast can be identified by the
"Durham Tube Test" (J. A. Barnett, R. W. Payne, and D. Yarrow.
2000. Yeasts Characteristics and Identification. 3.sup.rd edition.
p. 28-29. Cambridge University Press, Cambridge, UK.) or by
monitoring the production of fermentation productions such as
ethanol and CO.sub.2.
[0078] The term "polynucleotide" is used herein interchangeably
with the term "nucleic acid" and refers to an organic polymer
composed of two or more monomers including nucleotides, nucleosides
or analogs thereof, including but not limited to single stranded or
double stranded, sense or antisense deoxyribonucleic acid (DNA) of
any length and, where appropriate, single stranded or double
stranded, sense or antisense ribonucleic acid (RNA) of any length,
including siRNA. The term "nucleotide" refers to any of several
compounds that consist of a ribose or deoxyribose sugar joined to a
purine or a pyrimidine base and to a phosphate group, and that are
the basic structural units of nucleic acids. The term "nucleoside"
refers to a compound (as guanosine or adenosine) that consists of a
purine or pyrimidine base combined with deoxyribose or ribose and
is found especially in nucleic acids. The term "nucleotide analog"
or "nucleoside analog" refers, respectively, to a nucleotide or
nucleoside in which one or more individual atoms have been replaced
with a different atom or with a different functional group.
Accordingly, the term polynucleotide includes nucleic acids of any
length, DNA, RNA, analogs and fragments thereof. A polynucleotide
of three or more nucleotides is also called nucleotide oligomer or
oligonucleotide.
[0079] It is understood that the polynucleotides described herein
include "genes" and that the nucleic acid molecules described
herein include "vectors" or "plasmids." Accordingly, the term
"gene", also called a "structural gene" refers to a polynucleotide
that codes for a particular sequence of amino acids, which comprise
all or part of one or more proteins or enzymes, and may include
regulatory (non-transcribed) DNA sequences, such as promoter
sequences, which determine for example the conditions under which
the gene is expressed. The transcribed region of the gene may
include untranslated regions, including introns, 5'-untranslated
region (UTR), and 3'-UTR, as well as the coding sequence.
[0080] The term "operon" refers to two or more genes which are
transcribed as a single transcriptional unit from a common
promoter. In some embodiments, the genes comprising the operon are
contiguous genes. It is understood that transcription of an entire
operon can be modified (i.e., increased, decreased, or eliminated)
by modifying the common promoter. Alternatively, any gene or
combination of genes in an operon can be modified to alter the
function or activity of the encoded polypeptide. The modification
can result in an increase in the activity of the encoded
polypeptide. Further, the modification can impart new activities on
the encoded polypeptide. Exemplary new activities include the use
of alternative substrates and/or the ability to function in
alternative environmental conditions.
[0081] A "vector" is any means by which a nucleic acid can be
propagated and/or transferred between organisms, cells, or cellular
components. Vectors include viruses, bacteriophage, pro-viruses,
plasmids, phagemids, transposons, and artificial chromosomes such
as YACs (yeast artificial chromosomes), BACs (bacterial artificial
chromosomes), and PLACs (plant artificial chromosomes), and the
like, that are "episomes," that is, that replicate autonomously or
can integrate into a chromosome of a host cell. A vector can also
be a naked RNA polynucleotide, a naked DNA polynucleotide, a
polynucleotide composed of both DNA and RNA within the same strand,
a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or
RNA, a liposome-conjugated DNA, or the like, that are not episomal
in nature, or it can be an organism which comprises one or more of
the above polynucleotide constructs such as an agrobacterium or a
bacterium.
[0082] "Transformation" refers to the process by which a vector is
introduced into a host cell. Transformation (or transduction, or
transfection), can be achieved by any one of a number of means
including chemical transformation (e.g. lithium acetate
transformation), electroporation, microinjection, biolistics (or
particle bombardment-mediated delivery), or agrobacterium mediated
transformation.
[0083] The term "enzyme" as used herein refers to any substance
that catalyzes or promotes one or more chemical or biochemical
reactions, which usually includes enzymes totally or partially
composed of a polypeptide, but can include enzymes composed of a
different molecule including polynucleotides.
[0084] The term "protein," "peptide," or "polypeptide" as used
herein indicates an organic polymer composed of two or more amino
acidic monomers and/or analogs thereof. As used herein, the term
"amino acid" or "amino acidic monomer" refers to any natural and/or
synthetic amino acids including glycine and both D or L optical
isomers. The term "amino acid analog" refers to an amino acid in
which one or more individual atoms have been replaced, either with
a different atom, or with a different functional group.
Accordingly, the term polypeptide includes amino acidic polymer of
any length including full length proteins, and peptides as well as
analogs and fragments thereof. A polypeptide of three or more amino
acids is also called a protein oligomer or oligopeptide
[0085] The term "homolog," used with respect to an original enzyme
or gene of a first family or species, refers to distinct enzymes or
genes of a second family or species which are determined by
functional, structural or genomic analyses to be an enzyme or gene
of the second family or species which corresponds to the original
enzyme or gene of the first family or species. Most often, homologs
will have functional, structural or genomic similarities.
Techniques are known by which homologs of an enzyme or gene can
readily be cloned using genetic probes and PCR. Identity of cloned
sequences as homolog can be confirmed using functional assays
and/or by genomic mapping of the genes.
[0086] A protein has "homology" or is "homologous" to a second
protein if the amino acid sequence encoded by a gene has a similar
amino acid sequence to that of the second gene. Alternatively, a
protein has homology to a second protein if the two proteins have
"similar" amino acid sequences. (Thus, the term "homologous
proteins" is defined to mean that the two proteins have similar
amino acid sequences).
[0087] The term "analog" or "analogous" refers to nucleic acid or
protein sequences or protein structures that are related to one
another in function only and are not from common descent or do not
share a common ancestral sequence. Analogs may differ in sequence
but may share a similar structure, due to convergent evolution. For
example, two enzymes are analogs or analogous if the enzymes
catalyze the same reaction of conversion of a substrate to a
product, are unrelated in sequence, and irrespective of whether the
two enzymes are related in structure.
Enhancing DHAD Activity by Altering Aft1/Aft2 Activity and/or
Expression
[0088] The present inventors have found that altering the
expression of the AFT1 and/or AFT2 genes of S. cerevisiae
surprisingly increases DHAD activity and contributes to increased
isobutanol titers, productivity, and yield in strains comprising
DHAD as part of an isobutanol-producing metabolic pathway. The
observed increases in DHAD activity resulting from the increased
expression of AFT1 and/or AFT2 therefore has broad applicability to
any DHAD-requiring biosynthetic pathway, as DHAD activity is often
a rate-limiting component of such pathways.
[0089] Accordingly, one aspect of the invention is directed to a
recombinant microorganism comprising a DHAD-requiring biosynthetic
pathway, wherein said microorganism is engineered to overexpress
one or more polynucleotides encoding one or more Aft proteins or
homologs thereof.
[0090] As used herein, a "DHAD-requiring biosynthetic pathway"
refers to any metabolic pathway which utilizes DHAD to convert
2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate or
2,3-dihydroxy-3-methylvalerate to 2-keto-3-methylvalerate. Examples
of DHAD-requiring biosynthetic pathways include, but are not
limited to, isobutanol, 3-methyl-1-butanol, 2-methyl-1-butanol,
valine, isoleucine, leucine, and pantothenic acid (vitamin B5)
metabolic pathways. The metabolic pathway may naturally occur in a
microorganism (e.g., a natural pathway for the production of
valine) or arise from the introduction of one or more heterologous
polynucleotides through genetic engineering. In one embodiment, the
recombinant microorganisms expressing the DHAD-requiring
biosynthetic pathway are yeast cells. Engineered biosynthetic
pathways for synthesis of isobutanol are described in commonly
owned and co-pending applications U.S. Ser. No. 12/343,375
(published as US 2009/0226991), U.S. Ser. No. 12/696,645, U.S. Ser.
No. 12/610,784 (published as US 2010/0143997), U.S. Ser. No.
12/855,276, PCT/US09/62952 (published as WO/2010/051527), and
PCT/US09/69390 (published as WO/2010/075504), all of which are
herein incorporated by reference in their entireties for all
purposes. Additional DHAD-requiring biosynthetic pathways have been
described for the synthesis of valine, leucine, and isoleucine
(See, e.g., WO/2001/021772, and McCourt et al., 2006, Amino Acids
31: 173-210), pantothenic acid (See, e.g., WO/2001/021772),
3-methyl-1-butanol (See, e.g., WO/2008/098227, Atsumi et al., 2008,
Nature 451: 86-89, and Connor et al., 2008, Appl. Environ.
Microbiol. 74: 5769-5775), and 2-methyl-1-butanol (See, e.g.,
WO/2008/098227, WO/2009/076480, and Atsumi et al., 2008, Nature
451: 86-89).
[0091] As used herein, the terms "DHAD" or "DHAD enzyme" or
"dihydroxyacid dehydratase" are used interchangeably herein to
refer to an enzyme that catalyzes the conversion of
2,3-dihydroxyisovalerate to ketoisovalerate and/or the conversion
of 2,3-dihydroxy-3-methylvalerate to 2-keto-3-methylvalerate. DHAD
sequences are available from a vast array of microorganisms,
including, but not limited to, L. lactis, E. coli, S. cerevisiae,
B. subtilis, Streptococcus pneumoniae, and Streptococcus mutans. A
representative list of DHAD enzymes that can benefit from the
methods described herein, such as the increased expression of AFT1
and/or AFT2 or homologs thereof, include, but are not limited to
those, disclosed in 2010/0081154, as well as those disclosed in
commonly owned and co-pending U.S. patent application Ser. Nos.
12/855,276 and 61/407,815. Such DHAD enzymes may be cytosolically
localized or mitochondrially localized. A representative listing of
DHAD enzymes exhibiting cytosolic localization and activity are
disclosed in commonly owned and co-pending U.S. patent application
Ser. No. 12/855,276.
[0092] Without being bound by any theory, it is believed that
altered expression of an AFT gene (e.g. the AFT1 and/or AFT2 genes)
enhances cellular iron availability, which leads to an improvement
in the activity of the iron-sulfur (FeS) cluster-containing
protein, DHAD. The observation that increased expression of the AFT
genes improves DHAD activity is surprising, particularly in light
of recently published findings by Ihrig et al. (2010, Eukaryotic
Cell 9: 460-471). Notably, Ihrig et al. observed that the increased
expression of Aft1 in S. cerevisiae had little to no effect on the
activity of another FeS cluster-containing protein, Leu1
(isopropylmalate isomerase of the leucine biosynthesis pathway). In
contrast to observations made by Ihrig et al. with respect to the
FeS protein, Leu1, the present inventors unexpectedly observed that
increased expression of Aft1 and/or Aft2 resulted in a significant
increase in the activity of DHAD, also an iron-sulfur (FeS)
cluster-containing protein. Moreover, in strains comprising DHAD as
part of an isobutanol-producing metabolic pathway, the increased
expression of Aft1 produced significant increases in isobutanol
titer, productivity, and yield.
[0093] In S. cerevisiae, AFT1 and AFT2 encode for the transcription
factors, Aft1 and Aft2 ("activator of ferrous transport"),
respectively. It is hypothesized that Aft1 and Aft2 activate gene
expression when iron is scarce in wild-type S. cerevisiae.
Consequently, strains lacking both Aft1 and Aft2 exhibit reduced
expression of the iron regulon. As with many other paralogous
genes, AFT1 and AFT2 code for proteins that have significant
regions of identity and overlapping functions. The DNA-binding
domain of each protein is in a highly conserved N-terminal region,
and a conserved cysteine-to-phenylalanine mutation in either
protein generates a factor that activates the high expression of
the iron regulon irrespective of iron concentrations.
[0094] In yeast, homeostatic regulation of iron uptake occurs (Eide
et al., 1992, J. Biol. Chem. 267: 20774-81). Iron deprivation
induces activity of a high affinity iron uptake system. This
induction is mediated by increased transcript levels for genes
involved in the iron uptake system, and AFT1 is hypothesized to
play a critical role in this process (Yamaguchi-Iwai et al., 1995,
The EMBO Journal 14: 1231-9). Yamaguchi-Iwai et al. observed that
mutant strains lacking AFT1, due to gene deletion, are unable to
induce the high-affinity iron uptake system. On the other hand,
mutant strains carrying the AFT1.sup.UP allele exhibit a
gain-of-function phenotype in which iron uptake cannot be repressed
by available iron in the environment. The AFT1.sup.UP and
AFT2.sup.UP alleles described above act as gain of function point
mutations. AFT1.sup.UP is due to the mutation Cys.sup.291Phe
(Rutherford et al., 2005, Journal of Biological Chemistry 281:
10135-40). AFT2.sup.UP is due to the mutation Cys.sup.187Phe
(Rutherford et al., 2001, PNAS 98: 14322-27).
[0095] There are clear phenotypic differences in strains that
separately lack AFT1 or AFT2. An aft1 null strain exhibits low
ferrous iron uptake and grows poorly under low-iron conditions or
on a respiratory carbon source. No phenotype has been attributed to
an aft2 null strain. An aft1 aft2 double null strain is, however,
more sensitive to low-iron growth than a single aft1 null strain,
which is consistent with the functional similarity of these
factors. The partial redundancy of these factors allows AFT2 to
complement an aft1 null strain when it is overexpressed from a
plasmid. The properties of Aft1 and Aft2 that distinguish them from
each other have not been fully elucidated. Both factors mediate
gene regulation via an iron-responsive element that contains the
core sequence 5'-CACCC-3'. Without being bound to any theory, it is
likely that sequences adjacent to this element influence the
ability of each factor to mediate regulation via a particular
iron-responsive element. The differential regulation of individual
genes by Aft1 and Aft2 results in each factor generating a distinct
global transcriptional profile (Table 1) (Rutherford et al., 2004,
Eukaryotic Cell 3: 1-13; Conde e Silva et al., 2009, Genetics 183:
93-106).
TABLE-US-00001 TABLE 1 Genes Regulated by Metal-Responsive
Transcription Factors. Transcription Factor Description Gene
Name(s) Aft1 Transporters FET4, FET5, FTR1, FTH1, SMF3, MRS3, MRS4,
CCC2, COT1 Cu chaperone ATX1 Ferroxidase FET3, FET5
Metalloreductases FRE1, FRE2, FRE3, FRE4, FRE5, FRE6 Cell wall
proteins FIT1, FIT2, FIT3 Siderophore transport ARN1, ARN2, ARN3,
ARN4 Fe--S biosynthesis ISU1, ISU2 Other TIS11, HMX1, AKR1, PCL5,
YOR387c, YHL035c, YMR034c, ICY2, PRY1, YDL124w, CTH1, CTH2, Aft2
Transporters SMF3, MRS4, FTR1, COT1 Cu chaperone ATX1 Ferroxidase
FET3, FET5 Metalloreductases FRE1 Cell wall proteins FIT1, FIT3,
FIT2 Fe--S biosynthesis ISU1 Other BNA2, ECM4, LAP4, TIS11,
YOL083w, YGR146c, YHL035c
[0096] In S. cerevisiae, the Aft1 regulon consists of many genes
that are involved in the acquisition, compartmentalization, and
utilization of iron. These include genes involved in iron uptake
(FET3, FTR1, and FRE1, FRE2), siderophore uptake (ARN1-4 and
FIT1-3), iron transport across the vacuole membrane (FTH1), and
iron-sulfur cluster formation (ISU1 and ISU2). Aft1 binds to a
conserved promoter sequence in an iron-dependent manner and
activates transcription under low-iron conditions. The Aft2
regulator controls the expression of several distinct genes (Table
2) (Rutherford et al., 2004, Eukaryotic Cell 3: 1-13). The initial
step in iron acquisition requires reduction of ferric iron chelates
in the environment by externally directed reductases encoded by the
FRE1 and FRE2 genes, thereby generating the ferrous iron substrate
for the transport process (Dancis et al., 1992, PNAS 89: 3869-73;
Georgatsou and Alexandraki, 1994, Mol. Cell. Biol. 14: 3065-73).
FET3 encodes a multi-copper oxidase (Askwith et al., 1994, Cell 76:
403-10; De Silva et al., 1995, J. Biol. Chem. 270: 1098-1101) that
forms a molecular complex with the iron permease encoded by FTR1.
This complex, located in the yeast plasma membrane, mediates the
high-affinity transport of iron into the cell (Stearman et al.,
1996, Science 271: 1552-7). AFT genes may be found in yeast strains
other than S. cerevisiae. For example, in K. lactis, a homolog of
the S. cerevisiae AFT1 has been found and designated KI_AFT (Conde
e Silva et al., 2009, Genetics 183: 93-106). In this fungus, KI_Aft
has been found to activate transcription of genes regulated by Aft1
in S. cerevisiae. Thus, altering the regulation, activity, and/or
expression of AFT homologs in fungal strains other than S.
cerevisiae, is also within the scope of this invention. A person
skilled in the art will be able to utilize publicly available
sequences to construct relevant recombinant microorganisms with
altered expression of AFT homologs. A listing of a representative
number of AFT homologs known in the art and useful in the
construction of recombinant microorganisms engineered for increased
DHAD activity are listed Table 2. One skilled in the art, equipped
with this disclosure, will appreciate other suitable homologs for
the generation of recombinant microorganisms with increased DHAD
activity. Sequences of AFT genes found in sub-species or variants
of a given species may not be identical (See, e.g., >98%
identity amongst S. cerevisiae AFT1 genes of SEQ ID NOs: 1, 208,
210, 212, 214, 216, 218, 220, 222, and 224). While it is preferred
to overexpress an AFT gene native to the subspecies or variant, AFT
genes may be interchangeably expressed across subspecies or
variants of the same species.
TABLE-US-00002 TABLE 2 Representative Aft Homologs of Yeast Origin
Nucleic Acid Amino Acid Sequence Sequence Species Origin (Gene
Name) (SEQ ID NO) (SEQ ID NO) Saccharomyces cerevisiae S288c 1 2
(AFT1) Saccharomyces cerevisiae S288c 3 4 (AFT2) Candida glabrata
(AFT1) 5 6 Candida glabrata (AFT2) 7 8 Zygosaccharomyces rouxii
(AFT) 9 10 Ashbya gossypii (AFT) 11 12 Kluyveromyces lactis (AFT)
13 14 Vanderwaltozyma polyspora (AFT) 15 16 Lachancea
thermotolerans (AFT) 17 18 Debaromyces hanseii (AFT) 19 20
Saccharomyces bayanus* 21 22 Saccharomyces castelli* 23 24
Kluyveromyces waltii* 25 26 Saccharomyces kluyveri* 27 28
Kluyveromyces marxianus 29 30 Issatchenkia orientalis (AFT1-1) 31
32 Issatchenkia orientalis (AFT1-2) 33 34 Saccharomyces bayanus
(AFT2) 35 36 Saccharomyces castelli (AFT2) 37 38 S. cerevisiae W303
(AFT1) 208 209 S. cerevisiae DBVPG1106 (AFT1) 210 211 S. cerevisiae
NCYC361 (AFT1) 212 213 S. cerevisiae Y55 (AFT1) 214 215 S.
cerevisiae YJM981 (AFT1) 216 217 S. cerevisiae RM11_1A (AFT1) 218
219 S. cerevisiae UWOPS87_2421 (AFT1) 220 221 S. cerevisiae SK1
(AFT1) 222 223 S. cerevisiae YPS606 (AFT1) 224 225 *Byrne K. P.,
Wolfe, K. H. (2005) The Yeast Gene Order Browser: combining curated
homology and syntenic context reveals gene fate in polyploid
species. Genome Research, 15(10): 1456-61
[0097] Without being bound by any theory, it is believed that
increasing the expression of the gene AFT1 or a homolog thereof
will modulate the amount and availability of iron in the host cell.
Since Aft1 activates the expression of target genes in response to
changes in iron availability, overexpression of AFT1 increases the
machinery to import more iron into the cytosol and/or mitochondria.
A person skilled in the art, equipped with this disclosure, will
appreciate suitable methods for increasing the expression (i.e.
overexpressing) AFT1. For instance, in one embodiment, AFT1 or
homolog thereof may be overexpressed from a plasmid. In another
embodiment, one or more copies of the AFT1 gene or a homolog
thereof is inserted into the chromosome under the control of a
constitutive promoter. In addition, a skilled person in the art,
equipped with this disclosure, will recognize that the amount of
AFT1 overexpressed may vary from one yeast to the next. For
example, the optimal level of overexpression may be one, two,
three, four or more copies in a given yeast.
[0098] In additional embodiments, the native Aft1 or homolog
thereof may be replaced with a mutant version that is
constitutively active. In one embodiment, the native Aft1 is
replaced with a mutant version that comprises a modification or
mutation at a position corresponding to amino acid cysteine 291 of
the S. cerevisiae Aft1 (SEQ ID NO: 2). In an exemplary embodiment,
the cysteine 291 residue of the native S. cerevisiae Aft1 (SEQ ID
NO: 2) or homolog thereof is replaced with a phenylalanine
residue.
[0099] As will be understood by one of ordinary skill in the art,
modified Aft1 proteins and homologs thereof may be obtained by
recombinant or genetic engineering techniques that are routine and
well-known in the art. For example, mutant Aft1 proteins and
homologs thereof, can be obtained by mutating the gene or genes
encoding Aft1 or the homologs of interest by site-directed
mutagenesis. Such mutations may include point mutations, deletion
mutations and insertional mutations. For example, one or more point
mutations (e.g., substitution of one or more amino acids with one
or more different amino acids) may be used to construct mutant Aft1
proteins of the invention. The corresponding cysteine position of
Aft1 homologs may be readily identified by one skilled in the art.
Thus, given the defined region and the examples described in the
present application, one with skill in the art can make one or a
number of modifications which would result in the constitutive
expression of Aft1.
[0100] Without being bound by any theory, it is believed that
increasing the expression of the gene AFT2 or a homolog thereof
will modulate the amount and availability of iron in the host cell.
AFT2 overexpression is predicted to result in increased expression
of the machinery to import more iron into the cytosol and/or
mitochondria. A person skilled in the art, equipped with this
disclosure, will appreciate suitable methods for increasing the
expression (i.e. overexpression) of AFT2. For instance, in one
embodiment, AFT2 or homolog thereof may be overexpressed from a
plasmid. In another embodiment, one or more copies of the AFT2 gene
or a homolog thereof is inserted into the chromosome under the
control of a constitutive promoter. In addition, a skilled person
in the art, equipped with this disclosure, will recognize that the
amount of AFT2 overexpressed may vary from one yeast to the next.
For example, the optimal level of overexpression may be one, two,
three, four or more copies in a given yeast. Moreover, the
expression level may be tuned by using a promoter that achieves the
optimal expression level in a given yeast
[0101] In another embodiment, the native Aft2 or homolog thereof
may be replaced with a mutant version that is constitutively
active. In one embodiment, the native Aft2 is replaced with a
mutant version that comprises a modification or mutation at a
position corresponding to amino acid cysteine 187 of the S.
cerevisiae Aft2 (SEQ ID NO: 4). In an exemplary embodiment, the
cysteine 187 residue of the native S. cerevisiae Aft2 (SEQ ID NO:
4) or homolog thereof is replaced with a phenylalanine residue.
[0102] As will be understood by one of ordinary skill in the art,
modified Aft2 proteins and homologs thereof may be obtained by
recombinant or genetic engineering techniques that are routine and
well-known in the art. For example, mutant Aft2 proteins and
homologs thereof, can be obtained by mutating the gene or genes
encoding Aft2 or the homologs of interest by site-directed. Such
mutations may include point mutations, deletion mutations and
insertional mutations. For example, one or more point mutations
(e.g., substitution of one or more amino acids with one or more
different amino acids) may be used to construct mutant Aft2
proteins of the invention. The corresponding cysteine position of
Aft2 homologs may be readily identified by one skilled in the art.
Thus, given the defined region and the examples described in the
present application, one with skill in the art can make one or a
number of modifications which would result in the constitutive
expression of Aft2.
[0103] In various exemplary embodiments, increasing the expression
of both AFT1 and/or AFT2 will increase DHAD activity and the
production of beneficial metabolites from DHAD-requiring
biosynthetic pathways.
[0104] Embodiments in which the regulation, activity, and/or
expression of AFT1 and/or AFT2 are altered can also be combined
with increases in the extracellular iron concentration to provide
increased iron in the cytosol and/or mitochondria of the cell.
Increase in iron in either the cytosol or the mitochondria by this
method appears to make iron more available for the FeS
cluster-containing protein, DHAD. Without being bound by any
theory, it is believed that such an increase in iron leads to a
corresponding increase in DHAD activity.
[0105] As described herein, the increased activity of DHAD in a
recombinant microorganism is a favorable characteristic for the
production of beneficial metabolites including isobutanol,
3-methyl-1-butanol, 2-methyl-1-butanol, valine, isoleucine,
leucine, and pantothenic acid derived from DHAD-requiring
biosynthetic pathways. Without being bound by any theory, it is
believed that the increase in DHAD activity as observed by the
present inventors results from enhanced cellular iron levels as
mediated by the altered regulation, expression, and/or activity of
AFT1 and/or AFT2. Thus, in various embodiments described herein,
the present invention provides recombinant microorganisms with
increased DHAD activity as a result of alterations in AFT1 and/or
AFT2 regulation, expression, and/or activity. In one embodiment,
the alteration in AFT1 and/or AFT2 regulation, expression, and/or
activity increases the activity of a cytosolically-localized DHAD.
In another embodiment, the alteration in AFT1 and/or AFT2
regulation, expression, and/or activity increases the activity of a
mitochondrially-localized DHAD.
[0106] While particularly useful for the biosynthesis of
isobutanol, the altered regulation, expression, and/or activity of
AFT1 and/or AFT2 is also beneficial to any other fermentation
process in which increased DHAD activity is desirable, including,
but not limited to, the biosynthesis of isoleucine, valine,
leucine, pantothenic acid (vitamin B5), 2-methyl-1-butanol, and
3-methyl-1-butanol.
[0107] As described herein, the present inventors have observed
increased isobutanol titers, productivity, and yields in
recombinant microorganisms exhibiting increased expression of AFT1
and/or AFT2. Without being bound by any theory, it is believed that
the increases in isobutanol titer, productivity, and yield are due
to the observed increases in DHAD activity. Thus, in one
embodiment, the present invention provides a recombinant
microorganism for producing isobutanol, wherein said recombinant
microorganism comprises an isobutanol producing metabolic pathway,
and wherein the expression of AFT1 or a homolog thereof is
increased. In another embodiment, the present invention provides a
recombinant microorganism for producing isobutanol, wherein said
recombinant microorganism comprises an isobutanol producing
metabolic pathway, and wherein the expression of AFT2 or a homolog
thereof is increased. In yet another embodiment, the present
invention provides a recombinant microorganism for producing
isobutanol, wherein said recombinant microorganism comprises an
isobutanol producing metabolic pathway, and wherein the expression
of AFT1 and AFT2 or homologs thereof is increased.
[0108] In alternative embodiments, nucleic acids having a homology
to AFT1 and/or AFT2 of at least about 50%, of at least about 60%,
of at least about 70%, at least about 80%, or at least about 90%
similarity can be used for a similar purpose.
[0109] In one embodiment, the present invention provides a
recombinant microorganism for producing isobutanol, wherein said
recombinant microorganism comprises an isobutanol producing
metabolic pathway, and wherein the activity of Aft1 or a homolog
thereof is increased. In another embodiment, the present invention
provides a recombinant microorganism for producing isobutanol,
wherein said recombinant microorganism comprises an isobutanol
producing metabolic pathway, and wherein the activity of Aft2 or a
homolog thereof is increased. In yet another embodiment, the
present invention provides a recombinant microorganism for
producing isobutanol, wherein said recombinant microorganism
comprises an isobutanol producing metabolic pathway, and wherein
the activity of Aft1 and Aft2 or homologs thereof is increased.
[0110] In alternative embodiments, proteins having a homology to
Aft1 and/or Aft2 of at least about 50%, of at least about 60%, of
at least about 70%, at least about 80%, or at least about 90%
similarity can be used for a similar purpose.
[0111] In one embodiment, the isobutanol producing metabolic
pathway comprises at least one exogenous gene that catalyzes a step
in the conversion of pyruvate to isobutanol. In another embodiment,
the isobutanol producing metabolic pathway comprises at least two
exogenous genes that catalyze steps in the conversion of pyruvate
to isobutanol. In yet another embodiment, the isobutanol producing
metabolic pathway comprises at least three exogenous genes that
catalyze steps in the conversion of pyruvate to isobutanol. In yet
another embodiment, the isobutanol producing metabolic pathway
comprises at least four exogenous genes that catalyze steps in the
conversion of pyruvate to isobutanol. In yet another embodiment,
the isobutanol producing metabolic pathway comprises at five
exogenous genes that catalyze steps in the conversion of pyruvate
to isobutanol.
[0112] In one embodiment, one or more of the isobutanol pathway
genes encodes an enzyme that is localized to the cytosol. In one
embodiment, the recombinant microorganisms comprise an isobutanol
producing metabolic pathway with at least one isobutanol pathway
enzyme localized in the cytosol. In another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least two isobutanol pathway enzymes
localized in the cytosol. In yet another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least three isobutanol pathway enzymes
localized in the cytosol. In yet another embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with at least four isobutanol pathway enzymes
localized in the cytosol. In an exemplary embodiment, the
recombinant microorganisms comprise an isobutanol producing
metabolic pathway with five isobutanol pathway enzymes localized in
the cytosol. In a further exemplary embodiment, at least one of the
pathway enzymes localized to the cytosol is a cytosolically active
DHAD enzyme as disclosed herein.
[0113] In various embodiments described herein, the isobutanol
pathway genes encodes enzyme(s) selected from the group consisting
of acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI),
dihydroxyacid dehydratase (DHAD), 2-keto-acid decarboxylase (KIVD),
and alcohol dehydrogenase (ADH).
[0114] As described above, the transcription factors Aft1 and Aft2
regulate genes involved in the acquisition, compartmentalization,
and utilization of iron. Thus, in additional aspects, the present
invention provides methods of increasing DHAD activity and the
production of beneficial metabolites produced from DHAD-requiring
biosynthetic pathways as a result of alterations in the regulation,
expression, and/or activity of genes regulated by Aft1 and Aft2. In
one embodiment, the gene(s) regulated by Aft1 and Aft2 is selected
from the group consisting of FET3, FET4, FET5, FTR1, FTH1, SMF3,
MRS4, CCC2, COT1, ATX1, FRE1, FRE2, FRE3, FRE4, FRE5, FRE6, FIT1,
FIT2, FIT3, ARN1, ARN2, ARN3, ARN4, ISU1, ISU2, TIS11, HMX1, AKR1,
PCL5, YOR387C, YHL035C, YMR034C, ICY2, PRY1, YDL124W, BNA2, ECM4,
LAP4, YOL083W, YGR146C, BIO5, YDR271C, OYE3, CTH1, CTH2, MRS3,
MRS4, HSP26, YAP2, VMR1, ECL1, OSW1, NFT1, ARA2,
TAF1/TAF130/TAF145, YOR225W, YKR104W, YBR012C, and YMR041C or a
homolog thereof. While particularly useful for the biosynthesis of
isobutanol, the altered regulation, expression, and/or activity of
genes regulated by Aft1 and Aft2 is also beneficial to any other
fermentation process in which increased DHAD activity is desirable,
including, but not limited to, the biosynthesis of isoleucine,
valine, leucine, pantothenic acid (vitamin B5), 1-butanol,
2-methyl-1-butanol, and 3-methyl-1-butanol.
[0115] In one embodiment, all genes demonstrated to increase DHAD
activity and/or the production of a metabolite from a
DHAD-requiring biosynthetic pathway are overexpressed. Where none
of the AFT regulon genes expressed alone are effective in
increasing DHAD activity and/or the production of a metabolite from
a DHAD-requiring biosynthetic pathway, then 1, 2, 3, 4, 5, or more
of the genes in the AFT regulon are overexpressed together.
[0116] As described herein, the present inventors have observed
increased isobutanol titers, productivity, and yields in
recombinant microorganisms exhibiting increased expression of the
transcription factors AFT1 and/or AFT2, which regulate the
expression of genes involved in the acquisition,
compartmentalization, and utilization of iron. Thus, in one
embodiment, the present invention provides a recombinant
microorganism for producing isobutanol, wherein said recombinant
microorganism comprises an isobutanol producing metabolic pathway,
and wherein the expression and/or activity of one or more genes
selected from the group consisting of FET3, FET4, FET5, FTR1, FTH1,
SMF3, MRS4, CCC2, COT1, ATX1, FRE1, FRE2, FRE3, FRE4, FRE5, FREE,
FIT1, FIT2, FIT3, ARN1, ARN2, ARN3, ARN4, ISU1, ISU2, TIS11, HMX1,
AKR1, PCL5, YOR387C, YHL035C, YMR034C, ICY2, PRY1, YDL124W, BNA2,
ECM4, LAP4, YOL083W, YGR146C, B/05, YDR271C, OYE3, CTH1, CTH2,
MRS3, MRS4, HSP26, YAP2, VMR1, ECL1, OSW1, NFT1, ARA2,
TAF1/TAF130/TAF145, YOR225W, YKR104W, YBRO12C, and YMR041c or a
homolog thereof is increased.
Enhancing DHAD Activity by Increased GRX3/GRX4 Activity and/or
Expression
[0117] As described herein, increasing the expression of the genes
GRX3 and/or GRX4 will generally modulate the amount and
availability of iron in the yeast cytosol or mitochondria.
Accordingly, one aspect of the invention is directed to a
recombinant microorganism comprising a DHAD-requiring biosynthetic
pathway, wherein said microorganism has been engineered to
overexpress a polynucleotide encoding Grx3 and/or Grx4 or a homolog
thereof. In one embodiment, the polynucleotide encoding the Grx
protein or homolog thereof is native to the recombinant
microorganism. In another embodiment, the polynucleotide encoding
the Grx protein or homolog thereof is heterologous to the
recombinant microorganism.
[0118] Grx3 and Grx4 are monothiol glutaredoxins that have been
shown to be involved in cellular Fe content modulation and delivery
in yeast. Glutaredoxins are glutathione-dependent thiol-disulfide
oxidoreductases that function in maintaining the cellular redox
homeostasis. S. cerevisiae has two dithiol glutaredoxins (Grx1 and
Grx2) and three monothiol glutaredoxins (Grx3, Grx4, and Grx5). The
monothiol glutaredoxins are believed to reduce mixed disulfides
formed between a protein and glutathione in a process known as
deglutathionylation. In contrast, dithiol glutaredoxins can
participate in deglutathionylation as well as in the direct
reduction of disulfides. Grx5, the most studied monothiol
glutaredoxin, is localized to the mitochondrial matrix, where it
participates in the maturation of Fe--S clusters. Grx3 and Grx4 are
predominantly localized to the nucleus. These proteins can
substitute for Grx5 when overexpressed and targeted to the
mitochondrial matrix; no information on their natural function has
been reported. In addition to the reported interaction between Grx3
and Aft1, iron inhibition of Aft1 requires glutathione. It has been
shown that iron sensing is dependent on the presence of the
redundant Grx3 and Grx4 proteins. One report indicated that removal
of both Grx3 and Grx4 resulted in constitutive expression of the
genes regulated by Aft1/Aft2. This result suggested that the cells
accumulated Fe at levels greater than normal.
[0119] In one embodiment, Grx3 is overexpressed from a plasmid or
by inserting multiple copies of the gene into the chromosome under
the control of a constitutive promoter. In another embodiment, Grx4
is overexpressed from a plasmid or by inserting multiple copies of
the gene into the chromosome under the control of a constitutive
promoter. In another embodiment, Grx3 and Grx4 are overexpressed
from a plasmid or by inserting multiple copies of the gene into the
chromosome under the control of a constitutive promoter. In another
embodiment, Grx3, Grx4, or Grx3 and Grx4 are deleted or attenuated.
In another embodiment, Grx3 and Aft1 are overexpressed from a
plasmid or by inserting multiple copies of the gene into the
chromosome under the control of a constitutive promoter. In another
embodiment, Grx4 and Aft1 are overexpressed from a plasmid or by
inserting multiple copies of the gene into the chromosome under the
control of a constitutive promoter. In another embodiment, Grx3 and
Aft2 are overexpressed from a plasmid or by inserting multiple
copies of the gene into the chromosome under the control of a
constitutive promoter. In another embodiment, Grx4 and Aft2 are
overexpressed from a plasmid or by inserting multiple copies of the
gene into the chromosome under the control of a constitutive
promoter. These embodiments can also be combined with increases in
the extracellular iron concentration to provide increased iron in
the cytosol or mitochondria of the cell. One or both of: Aft1, Aft2
is overexpressed either alone or in combination with: Grx3 or Grx4.
Such overexpression can be accomplished by plasmid or by inserting
multiple copies of the gene into the chromosome under the control
of a constitutive promoter.
[0120] As described herein, the increased activity of DHAD in a
recombinant microorganism is a favorable characteristic for the
production of beneficial metabolites including isobutanol,
3-methyl-1-butanol, 2-methyl-1-butanol, valine, isoleucine,
leucine, and pantothenic acid from DHAD-requiring metabolic
pathways. Thus, in various embodiments described herein, the
present invention provides recombinant microorganisms with
increased DHAD activity as a result of alterations in GRX3 and/or
GRX4 regulation, expression, and/or activity. In one embodiment,
the alteration in GRX3 and/or GRX4 regulation, expression, and/or
activity increases the activity of a cytosolically-localized DHAD.
In another embodiment, the alteration in GRX3 and/or GRX4
regulation, expression, and/or activity increases the activity of a
mitochondrially-localized DHAD.
[0121] While particularly useful for the biosynthesis of
isobutanol, the altered regulation, expression, and/or activity of
GRX3 and/or GRX4 is also beneficial to any other fermentation
process in which increased DHAD activity is desirable, including,
but not limited to, the biosynthesis of isoleucine, valine,
leucine, pantothenic acid (vitamin B5), 1-butanol,
2-methyl-1-butanol, and 3-methyl-1-butanol.
[0122] In one embodiment, the present invention provides a
recombinant microorganism for producing isobutanol, wherein said
recombinant microorganism comprises an isobutanol producing
metabolic pathway, and wherein the expression of GRX3 or a homolog
thereof is increased. In another embodiment, the present invention
provides a recombinant microorganism for producing isobutanol,
wherein said recombinant microorganism comprises an isobutanol
producing metabolic pathway, and wherein the expression of GRX4 or
a homolog thereof is increased. In yet another embodiment, the
present invention provides a recombinant microorganism for
producing isobutanol, wherein said recombinant microorganism
comprises an isobutanol producing metabolic pathway, and wherein
the expression of GRX3 and GRX4 or homologs thereof is
increased.
[0123] In alternative embodiments, nucleic acids having a homology
to GRX3 and/or GRX4 of at least about 50%, of at least about 60%,
of at least about 70%, at least about 80%, or at least about 90%
similarity can be used for a similar purpose.
[0124] In one embodiment, the present invention provides a
recombinant microorganism for producing isobutanol, wherein said
recombinant microorganism comprises an isobutanol producing
metabolic pathway, and wherein the activity of Grx3 or a homolog
thereof is increased. In another embodiment, the present invention
provides a recombinant microorganism for producing isobutanol,
wherein said recombinant microorganism comprises an isobutanol
producing metabolic pathway, and wherein the activity of Grx4 or a
homolog thereof is increased. In yet another embodiment, the
present invention provides a recombinant microorganism for
producing isobutanol, wherein said recombinant microorganism
comprises an isobutanol producing metabolic pathway, and wherein
the activity of Grx3 and Grx4 or homologs thereof is increased.
[0125] In alternative embodiments, proteins having a homology to
Grx3 and/or Grx4 of at least about 50%, of at least about 60%, of
at least about 70%, at least about 80%, or at least about 90%
similarity can be used for a similar purpose.
Altering the Iron-Sulfur Cluster Domain and/or Redox Active
Domain
[0126] In general, the yeast cytosol demonstrates a different redox
potential than a bacterial cell, as well as the yeast mitochondria.
As a result, isobutanol pathway enzymes such as DHAD which exhibit
an iron sulfur (FeS) domain and/or redox active domain, may require
the redox potential of the native environments to be folded or
expressed in a functional form. Expressing the protein in the yeast
cytosol, which can harbor unfavorable redox potential, has the
propensity to result in an inactive protein, even if the protein is
expressed. The present inventors have identified a number of
different strategies to overcome this problem, which can arise when
an isobutanol pathway enzyme such as DHAD which is suited to a
particular environment with a specific redox potential is expressed
in the yeast cytosol.
[0127] In one embodiment, the present invention provides DHAD
enzymes that exhibit a properly folded iron-sulfur cluster domain
and/or redox active domain in the cytosol. Such DHAD enzymes may
either be native or heterologous DHAD homologs or functional
analogs or comprise a mutated or modified iron-sulfur cluster
domain and/or redox active domain, allowing for a DHAD enzyme to be
expressed in the yeast cytosol in a functional form. Thus, if an
enzyme in the isobutanol production pathway was identified that was
fully soluble and active in the cytosol of said recombinant
microorganism, such enzyme can be used without addition of
chaperone proteins not already present in the cytosol or without
increased expression of chaperone proteins already present in the
cytosol. However, some DHAD proteins may need the assistance of
additional chaperones or increased chaperone levels to exhibit
optimal cytosolic activity.
[0128] Therefore, in various embodiments described herein, the
recombinant microorganisms may further comprise a nucleic acid
encoding a chaperone protein, wherein said chaperone protein
assists the folding of a protein exhibiting cytosolic activity.
Addition of the chaperone protein can lead to improved activity,
solubility, and/or correct folding of the DHAD enzyme. In one
embodiment, the chaperone may be a native protein. In another
embodiment, the chaperone protein may be an exogenous protein. In
some embodiments, the chaperone protein may be selected from the
group consisting of: endoplasmic reticulum oxidoreductin 1 (Ero1,
accession no. NP.sub.--013576.1), including variants of Ero1 that
have been suitably altered to reduce or prevent its normal
localization to the endoplasmic reticulum; thioredoxins (which
includes Trx1, accession no. NP.sub.--013144.1; and Trx2, accession
no. NP.sub.--011725.1), thioredoxin reductase (Trr1, accession no.
NP.sub.--010640.1); glutaredoxins (which includes Grx1, accession
no. NP.sub.--009895.1; Grx2, accession no. NP.sub.--010801.1; Grx3,
accession no. NP.sub.--010383.1; Grx4, accession no.
NP.sub.--01101.1; Grx5, accession no. NP.sub.--015266.1; Grx6,
accession no. NP.sub.--010274.1; Grx7, accession no.
NP.sub.--009570.1; Grx8, accession no. NP.sub.--013468.1);
glutathione reductase Glr1 (accession no. NP.sub.--015234.1); Jac1
(accession no. NP.sub.--011497.1), including variants of Jac1 that
have been suitably altered to reduce or prevent its normal
mitochondrial localization; Hsp60 and Hsp10 proteins (e.g., yeast
Hsp 60 and Hsp10 proteins, or other eukaryotic Hsp60 and Hsp10
homologs), bacterial chaperonin homologs (e.g., GroEL and GroES
proteins from Lactococcus lactis); homologs or active variants
thereof, and combinations thereof.
[0129] As described herein, it is preferred that the DHAD enzymes
are properly assembled and folded, thus allowing for said DHADs to
exhibit maximal activity in the cytosol. In yeast, the DHAD Ilv3 is
involved in biosynthesis of the amino acids leucine, isoleucine and
valine. Ilv3 is typically localized to the mitochondria, where the
chaperonin proteins Hsp60 and Hsp10 aid in the proper folding of
the protein (Dubaquie et. al. The EMBO Journal 1998 17: 5868-5876).
In wild-type yeast cells, Ilv3 is found in the soluble fraction of
cell lysates. In extracts from an hsp60 temperature-sensitive
mutant, at the non-permissive temperature, there is no detectable
soluble Ilv3. All of the protein is found in the insoluble
fraction, in a presumably inactivated state. In an hsp10
temperature-sensitive mutant, at the non-permissive temperature,
about half of the Ilv3 is found in the insoluble portion,
indicating that Hsp10 is also important for proper folding of Ilv3,
but that Hsp60 is required. (Dubaquie et. al. The EMBO Journal 1998
17: 5868-5876).
[0130] Thus, in one embodiment of the present invention, wherein
the yeast DHAD encoded by ILV3 gene is used in the cytosol of a
isobutanol-producing recombinant microorganism (e.g., a yeast
microorganism), Hsp60 and/or Hsp10 from the same yeast, homologs
thereof from other microorganisms, or active variants thereof can
be overexpressed in said microorganism to increase the activity,
solubility, and/or correct folding of DHAD encoded by ILV3 gene to
increase the productivity, titer, and/or yield of isobutanol
produced. Alternatively, if said microorganism is a yeast and it
naturally expresses chaperonin proteins homologous to Hsp60 and/or
Hsp10 in its cytosol, DHAD encoded by ILV3 can be expressed in said
yeast without the overexpression of the Hsp60 and/or the Hsp10
proteins. In another embodiment, wherein the DHAD derived from an
organism other than yeast is used for isobutanol production,
chaperonin homologs, or active variants thereof derived from said
non-yeast organism or related non-yeast organism can be
overexpressed together with the DHAD derived from said non-yeast
organism. In one embodiment, said non-yeast organism is an
eukaryotic organism. In another embodiment, said non-yeast organism
is a prokaryotic organism. In a further embodiment, said non-yeast
organism is a bacterium (e.g., E. coli., or Lactococcus lactis).
For example, the Lactococcus lactis GroEL and GroES chaperonin
proteins are expressed in the yeast cytosol in conjunction with the
IIvD from Lactococcus lactis. Overexpression of these genes may be
accomplished by methods as described herein.
[0131] Also disclosed herein are recombinant microorganisms
comprising one or more genes encoding an iron-sulfur cluster
assembly protein. Iron-sulfur cluster assembly for insertion into
yeast apo-iron-sulfur proteins begins in yeast mitochondria. To
assemble in yeast the active iron-sulfur proteins containing the
cluster, either the apo-iron-sulfur protein is imported into the
mitochondria from the cytosol and the iron-sulfur cluster is
inserted into the protein and the active protein remains localized
in the mitochondria; or the iron-sulfur clusters or precursors
thereof are exported from the mitochondria to the cytosol and the
active protein is assembled in the cytosol or other cellular
compartments.
[0132] Targeting of yeast mitochondrial iron-sulfur proteins or
non-yeast iron-sulfur proteins to the yeast cytosol can result in
such proteins not being properly assembled with their iron-sulfur
clusters. This present invention overcomes this problem by
co-expression and cytosolic targeting in yeast of proteins for
iron-sulfur cluster assembly and cluster insertion into
apo-iron-sulfur proteins, including iron-sulfur cluster assembly
and insertion proteins from organisms other than yeast, together
with the apo-iron-sulfur protein to provide assembly of active
iron-sulfur proteins in the yeast cytosol.
[0133] In some embodiments, the present invention provides methods
of using Fe--S cluster containing protein in the eukaryotic cytosol
for improved isobutanol production in a microorganism, comprising
overexpression of a Fe--S cluster-containing protein in the
isobutanol production pathway in an microorganism. In a preferred
embodiment, said microorganism is a yeast microorganism. In one
embodiment, said Fe--S cluster-containing protein is a endogenous
protein. In another embodiment, said Fe--S cluster-containing
protein is an exogenous protein. In one embodiment, said Fe--S
cluster-containing protein is derived from a eukaryotic organism.
In another embodiment, said Fe--S cluster-containing protein is
derived from a prokaryotic organism. In one embodiment, said Fe--S
cluster-containing protein is DHAD. In one embodiment, said Fe--S
cluster is a 2Fe-2S cluster. In another embodiment, said Fe--S
cluster is a 4Fe-4S cluster.
[0134] All known DHAD enzymes contain an iron sulfur cluster, which
is assembled in vivo by a multi-component pathway. DHADs contain
one of at least two types of iron sulfur clusters, a 2Fe-2S cluster
as typified by the spinach enzyme (Flint and Emptage, JBC 1988
263(8): 3558) or a 4Fe-4S cluster as typified by the E. coli enzyme
(Flint et. al., JBC 1993 268(20): 14732). In eukaryotic cells,
iron-sulfur cluster proteins can be found in either the cytosol or,
more commonly, in the mitochondria. Within the mitochondria, a set
of proteins, collectively similar to the ISC and/or SUF systems of
E. coli, are present and participate in the assembly, maturation,
and proper insertion of Fe--S clusters into mitochondrial target
proteins. (Lill and Muhlenhoff, 2008, Annu. Rev. Biochem.,
77:669-700). In addition, a cytosolic iron sulfur assembly system
is present and is collectively termed the CIA machinery. The CIA
system promotes proper Fe--S cluster maturation and loading into
cytosolically-localized iron sulfur proteins such as Leu1.
Importantly, function of the CIA system is dependent on a critical
(but still uncharacterized) factor exported from the mitochondria.
In the yeast S. cerevisiae, the native DHAD, encoded by ILV3, is a
mitochondrially-localized protein, where it is presumably properly
recognized and activated by Fe--S cluster insertion by the
endogenous machinery. Accordingly, ectopic expression of a DHAD in
the yeast cytosol might be not expected to be functional due to its
presence in a non-native compartment and the concomitant lack of
appropriate Fe--S cluster assembly machinery.
[0135] The E. coli DHAD (encoded by ilvD) is sensitive to oxygen,
becoming quickly inactivated when isolated under aerobic conditions
(Flint et. al., JBC 1993 268(20): 14732; Brown et. al. Archives
Biochem. Biophysics 1995 319(1): 10). It is thought that this
oxygen sensitivity is due to the presence of a labile 4Fe-4S
cluster, which is unstable in the presence of oxygen and reactive
oxygen species, such as oxygen radicals and hydrogen peroxide. In
yeast and other eukaryotes, the mitochondrial environment is
reducing, i.e. it is a low oxygen environment, in contrast to the
more oxygen-rich environment of the cytosol. The redox state of the
cytosol is thus expected to be a problem for expressing
mitochondrially localized DHADs, which are natively located in the
mitochondria, or in expressing DHADs from many bacterial species
which typically have an intracellular reducing environment. The
spinach DHAD has been shown to be more oxygen resistant than the E.
coli enzyme in in vitro assays (Flint and Emptage, JBC 1988
263(8):3558), which may be due to its endogenous localization to
the plastid, where it would normally encounter a relatively
high-oxygen environment. It has been suggested that DHADs with
2Fe-2S clusters are inherently more resistant to oxidative damage
and they are therefore an attractive possibility for inclusion in
the cytosolically localized isobutanol pathway.
[0136] An additional complication to the oxygen sensitivity of
DHADs is that the iron sulfur clusters must be properly assembled
and inserted into the enzyme such that an active enzyme results.
There are several types of machinery that produce iron sulfur
clusters and properly assemble them into proteins, including the
NIF system found in bacteria and in some eukaryotes, the ISC system
found in bacteria and mitochondria, the SUF system found in
bacteria and plastids, and the CIA system found in the cytosol of
eukaryotes.
[0137] Thus, the methods of using Fe--S cluster in the eukaryotic
cytosol for improved enzymatic activity in isobutanol production
pathway as described above may further comprise the co-expression a
heterologous Fe--S cluster-containing DHAD with the NIF assembly
system in the yeast cytosol to aid in assembling said heterologous
DHADs. The NIF system found in the parasite Entamoeba histolytica
has been shown to complement the double deletion of the E. coli ISC
and SUF assembly systems (Ali et. al. JBC 2004 279(16): 16863). The
critical components of the Entamoeba assembly system comprise only
two genes, NifS and NifU. In one embodiment, these two components
are overexpressed in the yeast cytosol to increase activity and/or
stability of cytosolic DHADs. In one embodiment, the NIF system is
the E. hisotlytica NIF system; in another embodiment, the NIF
system is from other organisms (e.g. Lactococcus lactis). An
advantage of using the E. hisotlytica assembly system is that it
has already been demonstrated to work in a heterologous organism,
E. coli.
[0138] A 2Fe-2S cluster-containing DHAD can be used in the present
invention. In one embodiment, the 2Fe-2S cluster DHADs includes all
known 2Fe-2S cluster dehydratase enzymes identified biochemically.
In another embodiment, the 2Fe-2S cluster DHADs include those
predicted to be 2Fe-2S cluster dehydratases containing some version
of the consensus motif for 2Fe-2S cluster proteins, e.g., the motif
CX.sub.4CX.sub.2CX.sub..about.30C (SEQ ID NO: 39, Lill and
Muhlenhoff, 2008, Annu. Rev. Biochem., 77:669-700). For example,
based on the extremely highly conserved DHAD gene sequences shared
amongst plant species, the inventors have synthesized a likely
2Fe-2S DHAD from Arabidopsis (and rice, Oryza sativa japonica)
which can be used to improve isobutanol production in vivo in the
cytosolic isobutanol pathway.
[0139] Alternatively, a DHAD may be determined to be a 2Fe-2S
protein or a 4Fe-4S protein based on a phylogenetic tree, such as
FIG. 2. Sequences not present on the example phylogenetic tree
disclosed here could be added to the tree by one skilled in the
art. Furthermore, once a new sequence was added to the DHAD
phylogenetic tree, one skilled in the art may be able to determine
if it is a 2Fe-2S or a 4Fe-4S cluster containing protein based on
the phylogenetic relationship to known 2Fe-2S or a 4Fe-4S cluster
containing DHADs.
[0140] In another embodiment, a 4Fe-4S cluster-containing DHAD
could substitute for the 2Fe-2S cluster-containing DHAD in the
cytosol. In one embodiment, said 4Fe-4S cluster DHAD is engineered
to be oxygen resistant, and therefore more active in the cytosol of
cells grown under aerobic conditions.
[0141] In one embodiment of this invention, the apo-iron-sulfur
protein DHAD enzyme encoded by the E. coli ilvD gene is expressed
in yeast together with E. coli iron-sulfur cluster assembly and
insertion genes comprising either the cyaY, iscS, iscU, iscA, hscB,
hscA, fdx and isuX genes or the sufA, sufB, sufC, sufD, sufS and
sufE genes. This strategy allows for both the apo-iron-sulfur
protein (DHAD) and the iron-sulfur cluster assembly and insertion
components (the products of the isc or suf genes) to come from the
same organism, causing assembly of the active DHAD iron-sulfur
protein in the yeast cytosol. As a modification of this embodiment,
for those E. coli iron-sulfur cluster assembly and insertion
components that localize to or are predicted to localize to the
yeast mitochondria upon expression in yeast, the genes for these
components are engineered to eliminate such targeting signals to
ensure localization of the components in the yeast cytoplasm. Thus,
in some embodiments, one or more genes encoding an iron-sulfur
cluster assembly protein may be mutated or modified to remove a
signal peptide, whereby localization of the product of said one or
more genes to the mitochondria is prevented. In certain
embodiments, it may be preferable to overexpress one or more genes
encoding an iron-sulfur cluster assembly protein.
[0142] In additional embodiments, iron-sulfur cluster assembly and
insertion components from other than E. coli can be co-expressed
with the E. coli DHAD protein to provide assembly of the active
DHAD iron-sulfur cluster protein. Such iron-sulfur cluster assembly
and insertion components from other organisms can consist of the
products of the Helicobacter pylori nifS and nifU genes or the
Entamoeba histolytica nifS and nifU genes. As a modification of
this embodiment, for those non-E. coli iron-sulfur cluster assembly
and insertion components that localize to or are predicted to
localize to the yeast mitochondria upon expression in yeast, the
genes for these components can be engineered to eliminate such
targeting signals to ensure localization of the components in the
yeast cytoplasm.
[0143] As a further modification of this embodiment, in addition to
co-expression of these proteins in aerobically-grown yeast, these
proteins may be co-expressed in anaerobically-grown yeast to lower
the redox state of the yeast cytoplasm to improve assembly of the
active iron-sulfur protein.
[0144] In another embodiment, the above iron-sulfur cluster
assembly and insertion components can be co-expressed with DHAD
apo-iron-sulfur enzymes other than the E. coli IlvD gene product to
generate active DHAD enzymes in the yeast cytoplasm. As a
modification of this embodiment, for those DHAD enzymes that
localize to or are predicted to localize to the yeast mitochondria
upon expression in yeast, then the genes for these enzymes can be
engineered to eliminate such targeting signals to ensure
localization of the enzymes in the yeast cytoplasm.
[0145] In additional embodiments, the above methods used to
generate active DHAD enzymes localized to yeast cytoplasm may be
combined with methods to generate active acetolactate synthase,
KARI, KIVD and ADH enzymes in the same yeast for the production of
isobutanol by yeast.
[0146] In another embodiment, production of active iron-sulfur
proteins other than DHAD enzymes in yeast cytoplasm can be
accomplished by co-expression with iron-sulfur cluster assembly and
insertion proteins from organisms other than yeast, with proper
targeting of the proteins to the yeast cytoplasm if necessary and
expression in anaerobically growing yeast if needed to improve
assembly of the active proteins.
[0147] In another embodiment, the iron-sulfur cluster assembly
protein encoding genes may be derived from eukaryotic organisms,
including, but not limited to yeasts and plants. In one embodiment,
the iron-sulfur cluster protein encoding genes are derived from a
yeast organism, including, but not limited to S. cerevisiae. In
specific embodiments, the yeast-derived genes encoding iron-sulfur
cluster assembly proteins are selected from the group consisting of
Cfd1 (accession no. NP.sub.--012263.1), Nbp35 (accession no.
NP.sub.--011424.1), Nar1 (accession no. NP.sub.--014159.1), Cia1
(accession no. NP.sub.--010553.1), and homologs or variants
thereof. In a further embodiment, the iron-sulfur cluster assembly
protein encoding genes may be derived from plant nuclear genes
which encode proteins translocated to chloroplasts or plant genes
found in the chloroplast genome itself.
[0148] In certain embodiments described herein, it may be desirable
to reduce or eliminate the activity and/or proteins levels of one
or more iron-sulfur cluster containing cytosolic proteins. This
modification increases the capacity of a yeast to incorporate
[Fe--S] clusters into cytosolically expressed proteins wherein said
proteins can be native proteins that are expressed in a non-native
compartment or heterologous proteins. This is achieved by deletion
of a highly expressed native cytoplasmic [Fe--S]-dependent protein.
More specifically, the gene LEU1 is deleted coding for the
3-isopropylmalate dehydratase which catalyses the conversion of
3-isopropylmalate into 2-isopropylmaleate as part of the leucine
biosynthetic pathway in yeast. Leu1p contains an 4Fe-4S cluster
which takes part in the catalysis of the dehydratase. Some DHAD
enzymes also contain a 4Fe-4S cluster involved in its dehydratase
activity. Therefore, although the two enzymes have different
substrate preferences the process of incorporation of the Fe--S
cluster is generally similar for the two proteins. Given that Leu1p
is present in yeast at 10000 molecules per cell (Ghaemmaghami S. et
al. Nature 2003 425: 737), deletion of LEU1 therefore ensures that
the cell has enough spare capacity to incorporate [Fe--S] clusters
into at least 10000 molecules of cytosolically expressed DHAD.
Taking into account the specific activity of DHAD (E. coli DHAD is
reported to have a specific activity of 63 U/mg (Flint, D. H. et
al., JBC 1993 268: 14732), the LEU1 deletion yeast strain would
generally exhibit an increased capacity for DHAD activity in the
cytosol as measured in cell lysate.
[0149] In alternative embodiments, it may be desirable to further
overexpress an additional enzyme that converts
2,3-dihydroxyisovalerate to ketoisovalerate in the cytosol. In a
specific embodiment, the enzyme may be selected from the group
consisting of 3-isopropylmalate dehydratase (Leu1p) and
imidazoleglycerol-phosphate dehydrogenase (His3p) or other
dehydratases listed in Table 3.
TABLE-US-00003 TABLE 3 Dehydratases with putative activity towards
2,3-dihydroxyisovalerate. Gene Species Native Substrate Comments
dgoD E. coli D-galactonate Acid-sugar rspA E. coli D-mannonate,
dehydratases D-altronate yfaW E. coli L-rhamnonate fucD X.
campestris L-fuconate LGD1 H. jecorina L-galactonate pdd K. oxytoca
diols Other non-Fe--S ENO1/2, S. cerevisiae 2-phosphoglycerate
dehydratases ERR1/2/3 HIS3 S. cerevisiae Imidazoleglycerol-
phosphate
[0150] Because in some embodiments, DHAD activity may be limited in
the cytosol, alternative dehydratases that convert
dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) and are
physiologically localized to the yeast cytosol may be utilized.
Leu1p and His3p and other enzymes encoded by genes listed in Table
3 are dehydratases that potentially may exhibit affinity for DHIV.
Leu1p is an Fe--S binding protein that is involved in leucine
biosynthesis and is also normally localized to the cytosol. His3p
is involved in histidine biosynthesis and is similar to Leu1p, it
is generally localized to the cytosol or predicted to be localized
to the cytosol. This modification overcomes the problem of a DHAD
that is limiting isobutanol production in the cytosol of yeast. The
use of an alternative dehydratase that has activity in the cytosol
with a low activity towards DHIV may thus be used in place of the
DHAD in the isobutanol pathway. As described herein, such enzyme
may be further engineered to increase activity with DHIV.
[0151] Increased Mitochondrial Export of Essential Components for
Iron Sulfur Protein Assembly in the Cytosol
[0152] As noted herein, the third step in an exemplary isobutanol
biosynthetic pathway is the conversion of dihydroxyisovalerate
(DHIV) to ketoisovalerate (KIV) by a dihydroxyacid dehydratase
(DHAD). DHADs often require iron sulfur clusters for activity, and
the native yeast DHAD acquires its iron sulfur cluster via the
mitochondrial ISC machinery, remaining within the mitochondria as
an active enzyme. However, isobutanol production by the engineered
pathway requires DHAD to be functionally expressed within the
cytosol, and such a DHAD presumably requires iron sulfur clusters
to be added in the cytosol. One of the inventions disclosed herein
addresses possible genetic or chemical approaches to increase the
functional activity of cytosol DHADs. The present invention
provides ways to increase the export of an essential compound that
is generated in mitochondria, thereby increasing the amount of the
compound available for use by the cytosolic iron sulfur assembly
machinery (e.g. CIA) to effectively increase the functional
expression of cytosolic DHADs.
Overexpressing Mitochondrial Iron Sulfur Cluster (ISC)
Machinery
[0153] The compound generated within the mitochondrial matrix that
is essential for iron sulfur protein assembly in the cytosol is
subsequently exported through the ABC transporter, Atm1, and is
chaperoned across the intermembrane space of the mitochondria to
the cytosol by Erv1 (reviewed in Lill and Muhlenhoff, 2008, Annu.
Rev. Biochem., 77:669-700). Sc_BAT1 was identified as a third
putative component of the mitochondrial export machinery required
for the export of an unknown compound essential for cytosolic
iron-sulfur cluster biosynthesis from the mitochondrial matrix to
the cytosol by a genetic selection of suppressors of a Sc_atm1
temperature sensitive allele (Kispal et al, 1996, JBC,
271:24458-24464). It is also suggested that a further strong
indication for a direct functional relationship between Atm1p and
Bat1p is the leucine auxotrophy associated with the deletion of the
ATM1 gene.
[0154] To facilitate export of the essential compound, the present
invention provides in an embodiment recombinant microorganisms that
have been engineered to overexpress one or more mitochondrial
export proteins. In various embodiments described herein, the
mitochondrial export protein may be selected from the group
consisting of the S. cerevisiae ATM1, the S. cerevisiae ERV1, and
the S. cerevisiae BAT1, or homologs thereof. Such manipulations can
increase the export of the essential compound out of the
mitochondria to increase the amount available for use by the
cytosolic iron sulfur assembly machinery (e.g. CIA) to effectively
increase the functional expression of cytosolic DHADs.
Increasing Inner Mitochondrial Membrane Electrical Potential
[0155] In one embodiment, the present invention provides
recombinant microorganisms that have further been engineered to
increase inner mitochondrial membrane potential,
.DELTA..psi..sub.M. As described herein, although yeast cells
require a function mitochondrial compartment, they are viable
without the mitochondrial genome (mtDNA). However, loss of mtDNA
has been linked to destabilization of the nuclear genome (Veatch et
al., 2009, Cell, 137(7):1179-1181). Nuclear genome stability was
restored in yeast lacking mtDNA when a suppressor mutation
(ATP1-111) was introduced (Veatch et al., 2009, Cell,
137(7):1179-1181, Francis et al, 2007, J. Bioenerg. Biomembr.
39(2):149-157). The mutation has been shown to increase ATP
hydrolysis activity of the mitochondrial ATP synthase, and similar
mutations in the ATP synthase complex have also been shown to
increase the electrical potential across the inner membrane of
mitochondria, A.DELTA..psi..sub.M, in cells lacking mtDNA (Smith et
al., 2005, Euk Cell, 4(12):2057-2065; Kominsky et al., 2002,
Genetics, 162:1595-1604). Generation of .DELTA..psi..sub.M is
required for efficient import of proteins into the mitochondrial
matrix, including those involved in assembly and export of a
complex required for the assembly of iron sulfur clusters into
proteins in the cytosol. The link between .DELTA..PSI..sub.M and
iron sulfur cluster assembly in the cytosol is supported by
microarray data that indicate that the transcriptional profile of
cells lacking mtDNA (decreased .DELTA..psi..sub.M) is similar to
yeast grown under iron depletion conditions (Veatch et al., 2009,
Cell, 137(7):1179-1181). Introduction of the ATP1-111 suppressor
mutation restores the transcriptional profile to one resembling a
wild-type cell's transcriptional profile (Veatch et al., 2009,
Cell, 137(7):1179-1181). Taken together, these data indicate that
.DELTA..psi..sub.M must be sufficient to support assembly of
cytosolic iron sulfur proteins, particularly those involved in
nuclear genome stability (Veatch et al., Cell 2009,
137(7):1247-1258).
[0156] Thus, the present invention aims to generate the highest
possible .DELTA..psi..sub.M in a yeast with an intact mitochondrial
genome, allowing for the maximization the export of the complex
required for assembly of cytosolic iron sulfur proteins, which can
in turn increase the amount available for use by the cytosolic iron
sulfur assembly machinery (e.g. CIA) to effectively increase the
functional expression of cytosolic DHADs. .DELTA..omega..sub.M can
be maximized several different ways, including, but not limited to:
(1) Introducing mutations in the mitochondrial ATP synthase complex
that increase ATP hydrolysis activity, or active variants thereof;
(2) Overexpressing an ATP/ADP carrier protein that leads to an
increase ATP.sup.4- import into the mitochondrial matrix in
exchange for ADP.sup.3-, contributing to generation of
.DELTA..psi..sub.M; (3) Removal and/or overexpression of additional
gene(s) involved in generation of .DELTA..psi..sub.M; and (4)
Addition of chemical reagents that lead to an increase in
.DELTA..psi..sub.M.
[0157] In various embodiments described herein, the recombinant
microorganism may comprise a mutation in the mitochondrial ATP
synthase complex that increases ATP hydrolysis activity. In one
embodiment, said mutant mitochondrial is an ATP synthase which can
increase ATP hydrolysis activity is from a eukaryotic organism
(e.g., a yeast ATP1, ATP2, ATP3). In another embodiment, said
mutant mitochondrial ATP synthase is from a prokaryotic organism
(e.g., bacteria). Non-limiting examples of said mutant
mitochondrial ATP synthase include, mutant ATPase from the ATP1-111
strain in Francis et al., J Bioenerg Biomembr, 2007,
39(2):127-144), a mutant ATPase from the atp2-227 strain in Smith
et al., 2005, Euk Cell, 4(12):2057-2065, or a mutant ATPase from
the yme1 strain in Kominsky et al., 2002, Genetics, 162:1595-1604).
In another embodiment, active variants, or homologs of the mutant
mitochondrial ATP synthases described above can be applied. In one
embodiment, an ATP synthase having a homology to any of ATP1, ATP2,
and ATP3 of at least about 70%, at least about 80%, or at least
about 90% similarity can be used for a similar purpose.
[0158] In one embodiment, the inner mitochondrial membrane
electrical potential can be increased by overexpressing an ATP/ADP
carrier protein. Overexpression of the ATP/ADP carrier protein
increases ATP.sup.4- import into the mitochondrial matrix in
exchange for ADP.sup.3-. Non-limiting examples of ATP/ADP carrier
proteins include the S. cerevisiae_AAC1 or the S. cerevisiae_AAC3,
and active variants or homologs thereof. In one embodiment, an
ATP/ADP carrier protein having a homology to either the S.
cerevisiae_AAC1 or S. cerevisiae_AAC3 of at least about 70%, at
least about 80%, or at least about 90% similarity can be used for a
similar purpose.
[0159] In another embodiment, the inner mitochondrial membrane
electrical potential can be increased by removal and/or
overexpression of additional gene(s) involved in the generation of
.DELTA..psi..sub.M. A person skilled in the art will be familiar
with proteins encoded by such genes. Non-limiting examples include
the protein complexes in the mitochondrial electron transport chain
which are responsible for establishing H.sup.+ ions gradient. For
examples, complexes on the inner membrane of mitochondria that are
involved in conversion of NADH to NAD.sup.+ (Complex I, NADH
dehydrogenase), succinate to fumarate (Complex II, cytochrome
bc.sub.1 complex), and oxygen to water (Complex IV, cytochrome c
oxidase), which are responsible for the transfer of H.sup.+ ions.
In another embodiment, enzymes in the citric acid cycle in the
matrix of mitochondria can be overexpressed to increase NADH and
succinate production, such that more H.sup.+ ions are available.
These enzymes include, citrate synthase, aconitase, isocitrate
dehydrogenase, .alpha.-Ketoglutarate dehydrogenase, succinyl-CoA
synthetase, succinate dehydrogenase, fumarase, and malate
dehydrogenase.
[0160] In yet another embodiment, the inner mitochondrial membrane
electrical potential can be increased by the addition of chemical
reagents that lead to an increase in .DELTA..psi..sub.M. In one
embodiment, said chemical reagents are substrates in the citric
acid cycle in the matrix of mitochondria, wherein when added into
the culture, more NADH and succinate can be produced which in turn
increase .DELTA..psi..sub.M in the mitochondria. Non-limiting
examples of said substrates include, oxaloacetate, acetyl CoA,
citrate, cis-Aconitate, isocitrate, oxalosuccinate,
.alpha.-Ketoglutarate, succinyl-CoA, succinate, fumarate and
L-Malate.
Enhancing Cytosolic DHADs Activity by Increasing Cytosol Sulfur
Levels
[0161] Also provided herein are methods of increasing the levels of
sulfur-containing compounds within yeast cells, including the amino
acid cysteine, such that this sulfur is more available for the
production of iron-sulfur cluster-containing proteins in the yeast
cytosol or mitochondria. Specifically, by increasing the
concentration of sulfur-containing compounds in the cell such, the
activity of a functional DHAD is enhanced in the yeast cytosol or
mitochondria.
[0162] Accordingly, the present invention provides in an embodiment
recombinant microorganisms that have been engineered to overexpress
one or more genes to increase biosynthesis of cysteine or uptake of
exogenous cysteine by the cell in order to increase the amount and
availability of sulfur-containing compounds for the production of
active iron-sulfur cluster-containing proteins in the yeast cytosol
or mitochondria. In one embodiment, the recombinant microorganisms
have been engineered to increase the expression of one or more
proteins to increase cysteine biosynthesis by the cell, including,
but not limited to MET3, MET14, MET16, MET10, MET5, MET1, MET5,
MET2, MET17, HOM3, HOM2, HOM3, CYS3, CYS4, SUL1, SUL2, active
variants thereof, homologs thereof, and combination thereof, to
increase cysteine biosynthesis by the cell. In another embodiment,
the recombinant microorganisms have been engineered to increase the
expression of one or more transport proteins, including, but not
limited to YCT1, MUP1, GAP1, AGP1, GNP1, BAP1, BAP2, TAT1, active
variants thereof, homologs thereof, and combination thereof.
[0163] As noted above, increasing uptake of exogenous cysteine by
the cell will increase the amount and availability of
sulfur-containing compounds for the production of active
iron-sulfur cluster containing proteins in the cytosol or
mitochondria of the cell. Addition of increased exogenous cysteine
to yeast cells, separately from or in addition to increased
expression of the transport protein-encoding genes as described
above, can also increase the level and availability of
sulfur-containing compounds within the cell such that the sulfur is
more available for the production of iron-sulfur cluster-containing
proteins in the cell cytosol or mitochondria.
[0164] Sulfur is a necessary element for the biogenesis of
iron-sulfur cluster (FeS cluster)-containing protein in vivo.
Sulfur is a component of the FeS clusters that are incorporated
into such proteins and is also a component of compounds such as
glutathiones, which are essential for FeS cluster biogenesis in
many organisms as well as being involved in cellular redox
homeostasis. The direct source of the sulfur for these processes in
many organisms is the amino acid cysteine. The sulfur from cysteine
is mobilized into FeS clusters during FeS cluster biogenesis using
cysteine desulfurase proteins identified in many organisms such as
IscS, SufS (together with SufE), NifS and Nfs1 (together with
Isd11). Additionally, glutathione biosynthesis requires
cysteine.
[0165] Increased expression of Fe--S cluster-containing proteins in
organisms such as the budding yeast S. cerevisiae results in an
increased demand for sulfur, in the form of cysteine, in the cell.
Such an increased demand for cysteine may possibly be met by
natural induction of the endogenous cysteine biosynthetic pathway
but maximal natural induction of this pathway may be insufficient
to provide enough cysteine for the proper assemble and maintenance
of increased levels of FeS cluster-containing proteins in the cell.
Such cells with an increased demand for cysteine may also induce
cysteine and/or sulfate transport pathways to bring in exogenous
cysteine for or sulfate, which is the sulfur donor for cysteine
biosynthesis. However, maximal natural induction of these transport
systems may also be insufficient to meet the sulfur requirement of
such cells.
[0166] Assembly of active FeS cluster-containing proteins in the
native yeast cytosol requires the production and export to the
cytosol by the mitochondria of an unidentified sulfur-containing
compound derived from the mitochondrial FeS cluster biogenesis
pathway and the amino acid cysteine and requiring glutathione for
export. Overexpression of an FeS cluster-containing protein in the
yeast cytosol or the localization of a previously non-cytosolic FeS
cluster-containing protein to the yeast cytosol may result in the
decreased availability of this unidentified sulfur-containing
compound in the yeast cytosol and low activity of the cytosolic FeS
cluster-containing protein or proteins. Increased availability of
cysteine to the cell may prevent this limitation by providing
increased sulfur for the biosynthesis of this compound and
sufficient glutathione for its export from the mitochondria.
[0167] Sulfur for the assembly of FeS cluster-containing proteins
expressed in the yeast cytosol may also be provided by localization
of cysteine desulfurase proteins to the yeast cytosol. Expression
of such proteins in the yeast cytosol may result in an increased
demand for cysteine by such cells, especially in the cytosol.
Additionally, damage to the FeS cluster of FeS cluster-containing
proteins expressed in the yeast cytosol, due to the toxic nature of
the yeast cytosol or due to reactive oxygen or nitrogen species,
may require additional sulfur derived from cysteine for repair or
regeneration of the damaged clusters. As well, additional sulfur
derived from cysteine may modulate the redox balance of the yeast
cytosol through the production of increased levels of compounds
such as glutathione which may positively affect the assembly or
activity of FeS cluster-containing proteins in the yeast
cytosol.
[0168] Increased cellular sulfur in the form of cysteine can be
provided by increasing the biosynthesis of cysteine in the cell or
by increasing cellular uptake of exogenous cysteine. Increasing the
cellular level of cysteine in these ways is expected to increase
the level of other sulfur-containing compounds in the cell that
derive their sulfur from cysteine or the cysteine biosynthesis
pathway. Cysteine biosynthesis in S. cerevisiae involves the uptake
of exogenous sulfate by transport proteins encoded by the SUL1
and/or SUL2 genes and the action of the proteins encoded by the
MET3, MET14, MET16, MET10, MET5, MET1, METE, MET2, MET17, HOM3,
HOM2, HOM3, CYS4 and CYS4 genes. Exogenous cysteine is taken up
into S. cerevisiae by the high-affinity transport system encoded by
the YCT1 gene but also by the broader-specificity transport
proteins encoded by the MUP1, GAP1, AGP1, GNP1, BAP1, BAP2, TAT1
and TAT2 genes.
[0169] Thus, in an additional aspect, the invention is directed to
methods of increasing the levels of sulfur-containing compounds
within the yeast cytosol and/or mitochondria, such that sulfur is
more available for the production of iron-sulfur cluster-containing
proteins in the cytosol or mitochondria. In one embodiment, the
levels of sulfur-containing compounds within the yeast cytosol
and/or mitochondria are increased. In another embodiment, an
increase in sulfur-containing compounds in the yeast cytosol or
mitochondria leads to an increase in activity of a cytosolically
expressed FeS cluster-containing protein DHAD, which catalyzes the
reaction of 2,3-dihydroxyisovalerate to 2-ketoisovalerate. In
another embodiment, an increase in sulfur-containing compounds in
the yeast cytosol or mitochondria leads to an increase in activity
of a cytosolically expressed DHAD. In another embodiment, an
increase in sulfur-containing compounds in the yeast cytosol and/or
mitochondria leads to an increase in activity of a cytosolically
expressed DHAD and a subsequent increase in the productivity,
titer, and/or yield of isobutanol produced by the DHAD-containing
strain. In another embodiment, an increase in sulfur-containing
compounds in the yeast cytosol or mitochondria leads to an increase
in activity of a mitochondrially expressed FeS cluster-containing
protein DHAD, which catalyzes the reaction of
2,3-dihydroxyisovalerate to 2-ketoisovalerate. In another
embodiment, an increase in sulfur-containing compounds in the yeast
cytosol or mitochondria leads to an increase in activity of a
mitochondrially expressed DHAD. In another embodiment, an increase
in sulfur-containing compounds in the yeast cytosol and/or
mitochondria leads to an increase in activity of a mitochondrially
expressed DHAD and a subsequent increase in the productivity,
titer, and/or yield of isobutanol produced by the DHAD-containing
strain.
[0170] In another embodiment, the genes YCT1, MUP1, GAP1, AGP1,
GNP1, BAP1, BAP2, TAT1, and TAT2, active variants thereof, homologs
thereof or combination thereof are overexpressed from a plasmid or
by inserting multiple copies of the gene or genes into the
chromosome under the control of a constitutive promoter. This
embodiment can also be combined with providing increased
extracellular cysteine to the yeast cells to provide increased
sulfur-containing compounds in the cytosol and/or mitochondria of
the cells. Overexpression of these genes may be accomplished by
methods as described above.
[0171] In another embodiment, providing increased extracellular
cysteine to the yeast cells in the absence of any additional
engineered expression of transport proteins will provide increased
sulfur containing compounds in the cytosol and/or mitochondria of
the cells for the improved production of active FeS
cluster-containing proteins in the yeast cytosol or mitochondria,
which leads to increased isobutanol productivity, titer, and/or
yield by the cell.
Enhancing Cytosolic DHAD Activity by Mitigating Oxidative Species
or Oxidative Stress
[0172] The present application also describes methods of protecting
enzymes in a DHAD-requiring biosynthetic pathway (specifically
DHAD) in a microorganism to increase the production of beneficial
metabolites by mitigating oxidative species or oxidative stress
induced damage in the cytosol of said microorganism. Non-limiting
examples of oxidative species include, nitric oxide (NO), reactive
nitrogen species (RNS), reactive oxygen species (ROS), hydroxyl
radical species, organic hydroperoxide, hypochlorous acids, and
combinations thereof. As used herein, the phrase "reactive oxygen
species" or "ROS" refers to free radicals that contain the oxygen
atom. ROS are very small molecules that include oxygen ions and
peroxides and can be either inorganic or organic. They are highly
reactive due to the presence of unpaired valence shell electrons.
During times of environmental stress (e.g. UV or heat exposure) ROS
levels can increase dramatically, which can result in significant
damage to cell structures. This cumulates into a situation known as
oxidative stress. ROS are also generated by exogenous sources such
as ionizing radiation.
[0173] Oxidative stress is caused by an imbalance between the
production of reactive oxygen and a biological system's ability to
readily detoxify the reactive intermediates or easily repair the
resulting damage. All forms of life maintain a reducing environment
within their cells. This reducing environment is preserved by
enzymes that maintain the reduced state through a constant input of
metabolic energy. Disturbances in this normal redox state can cause
toxic effects through the production of peroxides and free radicals
that damage all components of the cell, including proteins, lipids,
and DNA.
[0174] In chemical terms, oxidative stress is a large rise
(becoming less negative) in the cellular reduction potential, or a
large decrease in the reducing capacity of the cellular redox
couples, such as glutathione. The effects of oxidative stress
depend upon the size of these changes, with a cell being able to
overcome small perturbations and regain its original state.
However, more severe oxidative stress can cause cell death and even
moderate oxidation can trigger apoptosis, while more intense
stresses may cause necrosis.
[0175] A particularly destructive aspect of oxidative stress is the
production of reactive oxygen species, which include free radicals
and peroxides, and/or other reactive species. Some of the less
reactive of these species (such as superoxide) can be converted by
oxidoreduction reactions with transition metals or other redox
cycling compounds (including quinones) into more aggressive radical
species that can cause extensive cellular damage. The major portion
of long term effects is inflicted by damage on DNA. Most of these
oxygen-derived species are produced at a low level by normal
aerobic metabolism and the damage they cause to cells is constantly
repaired. However, under the severe levels of oxidative stress that
cause necrosis, the damage causes ATP depletion, preventing
controlled apoptotic death and causing the cell to simply fall
apart. Non-limiting example of oxidants include, superoxide anion
(.O.sub.2--, formed in many autoxidation reactions and by the
electron transport chain), hydrogen peroxide (H.sub.2O.sub.2,
formed by disputation of .O.sub.2-- or by direct reduction of
O.sub.2), organic hydroperoxide (ROOH, formed by radical reactions
with cellular components such as lipids and/or nucleobases), oxygen
centered organic radicals (e.g., RO. alkoxy and ROO., peroxy
radicals, formed in the presence of oxygen by radical addition to
double bonds or hydrogen abstraction), hypochlorous acid (HOCl,
formed from H.sub.2O.sub.2 by myeloperoxidase, and peroxynitrite
(ONOO--, formed in a rapid reaction between .O.sub.2-- and
NO.).
[0176] Biological defenses against oxidative damage include
protective proteins that remove reactive oxygen species, molecules
that sequester metal ions, and enzymes that repair damaged cellular
components. Oxidative stress can be defined as a disturbance in the
prooxidant-antioxidant balance in favor of prooxidants. One such
class of prooxidants are reactive oxygen species, or ROS. ROS are
highly reactive species of oxygen, such as superoxide (O2..sup.-),
hydrogen peroxide (H.sub.2O.sub.2), and hydroxyl radicals (OH.),
produced within the cell, usually as side products of aerobic
respiration. By some reports, as much as 2% of the oxygen that
enters the respiratory chain is converted to superoxide through a
one-electron reduction of oxygen. A small amount of superoxide
radical is always released from the enzyme when oxygen is reduced
by electron carriers such as flavoproteins or cytochromes. This is
because the electrons are transferred to oxygen one at a time. The
hydroxyl radical and hydrogen peroxide are derived from the
superoxide radical.
[0177] Many microbes possess native enzymes to detoxify these ROS.
One example of such a system is superoxide dismutase (SOD) plus
catalase. SOD catalyzes a reaction where one superoxide radical
transfers its extra electron to the second radical, which is then
reduced to hydrogen peroxide. Catalase catalyzes the transfer of
two electrons from one hydrogen peroxide molecule to the second,
oxidizing the first to oxygen and reducing the second to two
molecules of water. If the hydrogen peroxide is not disposed of,
then it can oxidize transition metals, such as free iron(II) in the
Fenton reaction, and form the free hydroxyl radical, OH. No known
mechanisms exists to detoxify hydroxyl radicals, and thus
protection from toxic forms of oxygen must rely on eliminating
superoxide and hydrogen peroxide.
[0178] In yeast, to counteract damage of oxidative stress, there
are several antioxidant systems with an apparent functional
redundancy. For example, there are detoxifying enzymes such as
catalases, cytochrome c peroxidase, glutathione peroxidases,
glytaredoxins and peroxiredoxins, and many isoforms in distinct
cellular compartments (Jamieson et al., 1998, Yeast. 14:1511-1527;
Grant et al., 2001, Mol. Microbiol 39:533-541; Collinson et al.,
2003, J. Biol. Chem. 278:22492-22497; Park et al., 2000, J. Biol.
Chem. 275:5723-5732).
[0179] As described above, an enzyme involved in the isobutanol
production pathway, dihydroxyacid dehydratase (DHAD), contains an
iron-sulfur (FeS) cluster domain. This iron-sulfur (FeS) cluster
domain is sensitive to damage by ROS, which can lead to inactive
enzyme. Both 2Fe-2S and 4Fe-4S DHAD enzymes may be susceptible to
inactivation by ROS, however direct evidence exists for
inactivation of 4Fe-4S cluster containing proteins, such as
homoaconitase and isopropylmalate dehydratase in yeast and DHAD and
fumarase from E. coli. Therefore, to achieve a functional DHAD
expressed in the yeast cytosol in an environment where a
substantial amount of ROS may exist from respiration, it may be
beneficial to protect the DHAD enzyme from ROS inactivation or
oxidative stress through expression of on or more enzymes that
reduce or eliminate ROS from the cell.
[0180] To mitigate the potential harmful effects of reactive oxygen
species (ROS) or oxidative stress on DHAD in the yeast cytosol, the
present inventors have devised several strategies to protect or
repair the DHAD from ROS damage. In various embodiments described
herein, the invention provides recombinant microorganisms that have
been engineered to express one or more proteins in the cytosol that
reduce the concentration of reactive oxygen species (ROS) in said
cytosol.
[0181] In one embodiment, enzymes that reduce or eliminate the
amount of ROS in the cytosol are expressed and targeted to the
yeast cytosol. Specifically, enzymes such as catalase, superoxide
dismutase (SOD), cytochrome c peroxidase, glutathione peroxidases,
glytaredoxins, peroxiredoxins, metallothioneins, and methionine
sulphoxide reductases, or any isoforms thereof are expressed, such
that they lead to reduction in ROS such as hydrogen peroxide,
superoxide, peroxide radicals, and other ROS in the yeast
cytosol.
[0182] In one embodiment, a catalase is expressed to reduce the
concentration of ROS in the cytosol. In another embodiment, a
superoxide dismutase (SOD) is expressed to reduce the concentration
of ROS in the cytosol. Usually, microbes that grow by aerobic
respiration possess one or both of SOD and catalase. For example,
the bacterium E. coli and the yeast Saccharomyces cerevisiae each
possesses at least one native SOD and catalase (e.g., SOD1 or SOD2
from yeast). In E. coli, the genes katG and katE encode catalase
enzymes, and the genes sodA, sodB and sodC encode SodA, SodB, and
SodC superoxide dismutase enzymes. respectively. In S. cerevisiae,
the genes CTT1 and CTA1 encode catalase CTT1 and CTA1 enzymes, and
the genes SOD1 and SOD2 encode SOD1 and SOD2 superoxide dismutase
enzymes. Many other organisms possess catalase and SOD enzymes and
these genes may also be useful for reduction of ROS in the yeast
cytosol. In one embodiment, SOD homologs from species other than E.
coli or yeast can be expressed in yeast cytosol to reduce oxidative
stress. In one embodiment, said other species is a plant or a
fungus. For example, SOD1 from N. crassa (fungus) may be
functionally expressed in the yeast cytosol. In various embodiments
described herein, active variants or homologs of the
above-described catalases and SODs can be functionally expressed in
the yeast cytosol. In another embodiment, protein having a homology
to any one of the catalases or SODs described above possessing at
least about 70%, at least about 80%, or at least about 90%
similarity can be functionally expressed in the yeast cytosol.
[0183] In one embodiment, the catalase genes from E. coli are
expressed in and targeted to the cytosol of yeast to reduce the
amount of ROS and increase the activity of DHAD also expressed in
and targeted to the yeast cytosol. In another embodiment, the
catalase genes from S. cerevisiae are overexpressed in and targeted
to the cytosol of yeast to reduce the amount of ROS and increase
the activity of DHAD also expressed in and targeted to the yeast
cytosol. In one embodiment, the SOD genes from E. coli are
expressed in and targeted to the cytosol of yeast to reduce the
amount of ROS and increase the activity of DHAD also expressed in
and targeted to the yeast cytosol. In another embodiment, the SOD
genes from S. cerevisiae are expressed in and targeted to the
cytosol of yeast to reduce the amount of ROS and increase the
activity of DHAD also expressed in and targeted to the yeast
cytosol. In another embodiment, promoters of native genes are
altered, such that the level of SOD or catalase in the S.
cerevisiae cytosol is increased. In yet another embodiment,
expression of SOD or catalase in the yeast cytosol is mediated by a
plasmid. In yet another embodiment, expression of SOD or catalase
in the yeast cytosol is mediated by expression of one or more
copies of the gene from the chromosome. Other homologs of catalase
or SOD may be identified by one skilled in the art through tools
such as BLAST and sequence alignment. These other homologs may be
expressed in a similar manner described above to achieve a
functional catalase or SOD in the yeast cytosol.
[0184] In another embodiment, a methionine sulphoxide reductase
enzyme is expressed to reduce the amount of ROS and protect DHAD
from ROS damage and inactivation. In one embodiment, the methionine
sulphoxide reductase may be derived from a eukaryotic organism
(e.g., a yeast, fungus, or plant). In another embodiment, the
methionine sulphoxide reductases may be derived from a prokaryotic
organism (e.g., E. coli). The principal enzymatic mechanism for
reversing protein oxidation acts on the oxidation product of just
one amino acid residue, methionine. This specificity for Met
reflects the fact that Met is particularly susceptible to oxidation
compared with other amino acids. Methionine sulphoxide reductases
(MSRs) are conserved across nearly all organisms from bacteria to
humans, and have been the focus of considerable attention in recent
years. Two MSR activities have been characterized in the yeast
Saccharomyces cerevisiae: MsrA (encoded by MXR1) reduces the S
stereoisomer of methionine sulphoxide (MetO), while MsrB (encoded
by the YCL033c ORF), which we term here MXR2) reduces the R
stereoisomer of MetO. Consistent with defense against oxidative
damage, mutants deficient in MSR activity are hypersensitive to
pro-oxidants such as H.sub.2O.sub.2, paraquat and Cr, while MSR
overexpression enhances resistance. Besides methionine residues,
iron-sulfur (FeS) clusters are exquisitely ROS-sensitive components
of many cellular proteins. It has been reported that MSR activity
helps to preserve the function of cellular FeS clusters.
[0185] In one embodiment, the methionine sulphoxide reductase genes
from S. cerevisiae are expressed in and targeted to the cytosol of
yeast to reduce the amount of ROS and increase the activity of DHAD
also expressed in and targeted to the yeast cytosol. Specifically,
the S. cerevisiae methionine sulphoxide reductase genes MsrA
(encoded by MXR1) and MsrB (encoded by the YCL033c ORF) are
expressed in and targeted to the cytosol of yeast to reduce the
amount of ROS and increase the activity of DHAD also expressed in
and targeted to the yeast cytosol. The resulting methionine
sulphoxide reductase expressing strain will generally demonstrate
improved isobutanol productivity, titer, and/or yield compared to
the parental strain that does not comprise methionine sulphoxide
reductase genes that are expressed in and targeted to the cytosol.
Methionine sulphoxide reductases from other organisms, such as
bacteria, may be identified by sequence homology using tools such
as BLAST and pairwise sequence alignments by one skilled in the
art.
[0186] In yet another embodiment, expression or overexpression of
glutathione synthesis enzymes, for example GSH1, leads to increased
glutathione in the cell and protection of the DHAD enzyme in the
yeast cytosol. In one embodiment, said enzymes are derived from a
bacteria (e.g., E. coli.). In another embodiment, said enzymes are
derived from yeast (e.g., S. cerevisiae). In yet another
embodiment, said enzymes are derived from a yeast species different
from the yeast used for isobutanol production.
[0187] In one embodiment, one or more metallothionein proteins are
expressed in the yeast cytosol to mitigate oxidative stress.
Metallothioneins are a family of proteins found in many organisms
including yeast and mammals. The biologic function of
metallothionein (MT) has been a perplexing topic ever since the
discovery of this protein. Many studies have suggested that MT
plays a role in the homeostasis of essential metals such as zinc
and copper, detoxification of toxic metals such as cadmium, and
protection against oxidative stress. MT contains high levels of
sulfur. The mutual affinity of sulfur for transition metals makes
the binding of these metals to MT thermodynamically stable. Under
physiologic conditions, zinc-MT is the predominant form of the
metal-binding protein. However, other metals such as copper (Cu)
are also bound by MT. Oxidation of the thiolate cluster by a number
of mild cellular oxidants causes metal release and formation of
MT-disulfide (or thionin if all metals are released from MT, but
this is unlikely to occur in vivo), which have been demonstrated in
vivo. MT-disulfide can be reduced by glutathione in the presence of
selenium catalyst, restoring the capacity of the protein to bind
metals like Zn and Cu. This MT redox cycle may play a crucial role
in MT biologic function. It may link to the homeostasis of
essential metals, detoxification of toxic metals and protection
against oxidative stress. In fact, MT has been shown to substitute
for superoxide dismutase in yeast cells in the presence of Cu to
protect cells and proteins from oxidative stress.
[0188] In one embodiment, said metallothuineins are derived from a
eukaryotic organism (e.g., a yeast, fungus, or plant). In another
embodiment, said metallothuineins are derived from a prokaryotic
organism (e.g., E. coli, Mycobacterium tuberculosis). For example,
the metallothionein genes CUP1-1 and CUP1-2 encoding
metallothionein CUP1 from S. cerevisiae, active variants thereof,
homologs thereof, or combination thereof are expressed in and
targeted to the cytosol of yeast to reduce the amount of ROS and
increase the activity of DHAD also expressed in and targeted to the
yeast cytosol. In another embodiment, S. cerevisiae metallothionein
genes CUP1-1 and CUP1-2 are expressed in and targeted to the
cytosol of yeast to reduce the amount of ROS and increase the
activity of DHAD also expressed in and targeted to the yeast
cytosol. In another embodiment, Mycobacterium tuberculosis
metallothionein gene MymT encoding metallothionein is expressed in
and targeted to the cytosol of yeast to reduce the amount of ROS
and increase the activity of DHAD that is also expressed in and
targeted to the yeast cytosol. In another embodiment, Synechococcus
PCC 7942 metallothionein gene SmtA is expressed in and targeted to
the cytosol of yeast to reduce the amount of ROS and increase the
activity of DHAD that is also expressed in and targeted to the
yeast cytosol. The resulting metallothionein expressing strain has
improved isobutanol productivity, titer, and/or yield compared to
the parental strain. Metallothioneins from other organisms, such as
bacteria, may be identified by sequence homology using tools such
as BLAST and pairwise sequence alignments by one skilled in the
art.
[0189] In another embodiment, one or more proteins in the
thioredoxin system and/or the glutathione/glutaredoxin system,
active variants thereof, homologs thereof, or combination thereof
are expressed in the yeast cytosol to mitigate oxidative stress. In
one embodiment, said proteins in the thioredoxin system and/or the
glutathione/glutaredoxin system are derived from a eukaryotic
organism (e.g., a yeast, fungus, or plant). In another embodiment,
said proteins in the thioredoxin system and/or the
glutathione/glutaredoxin system are derived from a prokaryotic
organism (e.g., E. coli). The thioredoxin system and the
glutathione/glutaredoxin system help maintain the reduced
environment of the cell and play significant roles in defending the
cell against oxidative stress. Glutathione is the major protective
small molecule against oxidative stress in Saccharomyces
cerevisiae. Glutathione, the tripeptide
.gamma.-glutamyl-cysteinyl-glycine, makes up the major free thiol
pool present in millimolar concentrations in aerobic cells. The
biosynthesis of glutathione requires .gamma.-glutamyl cysteine
synthase (termed Gsh1p) glutathione synthase (Gsh2p) and ATP.
Glutathione is essential for viability of yeast but not of bacteria
such as E. coli. Yeast cells lacking Gsh1p (genotype gsh1.DELTA.)
are able to survive in the presence of an external source of
glutathione. Deletion of the GSH1 gene encoding the enzyme that
catalyzes the first step of glutathione biosynthesis leads to
growth arrest, which can be relieved by either glutathione or
reducing agents such as dithiothreitol. Evidence suggests that
glutathione, in addition to its protective role against oxidative
damage, performs a novel and specific function in the maturation of
cytosolic Fe/S proteins. Therefore, increasing the levels of
glutathione in the yeast cytosol is predicted to protect or
increase the steady-state levels of active FeS cluster containing
proteins expressed in the yeast cytosol. Specifically, increasing
glutathione within the yeast cytosol may increase the amount of
active DHAD enzyme expressed in the yeast cytosol, thereby leading
to an increase in the titer, productivity, and/or yield of
isobutanol produced from the pathway within which DHAD participates
(e.g. the isobutanol pathway in FIG. 1).
[0190] Thioredoxins and glutaredoxins are small heat-stable
proteins with redox-active cysteines that facilitate the reduction
of other proteins by catalyzing cysteine thiol-disulfide exchange
reactions. The glutathione/glutaredoxin system consists of
glutaredoxin, glutathione (produced by glutathione synthase),
glutathione reductase and NADPH (as an electron donor). Thus, to
increase the effective levels of available glutathione, one or a
combination of each of the following enzymes is functionally
overexpressed in the yeast cytosol: glutaredoxin (encoded in S.
cerevisiae by GRX2, GRX4, GRX6, and GRX7), glutathione reductase
(encoded in S. cerevisiae by GLR1); and glutathione synthase
(encoded in S. cerevisiae by GSH1 and GSH2). In one embodiment,
homologs thereof, active variants thereof, or combination thereof
can be expressed in the yeast cytosol to mitigate oxidative
stress.
[0191] In another embodiment, the .gamma.-glutamyl cysteine
synthase and glutathione synthase genes from S. cerevisiae are
expressed in and targeted to the cytosol of yeast to increase the
amount of glutathione and increase the activity of DHAD also
expressed in and targeted to the yeast cytosol. In another
embodiment, S. cerevisiae .gamma.-glutamyl cysteine synthase and
glutathione synthase genes Gsh1 and Gsh2 are expressed in and
targeted to the cytosol of yeast to increase the amount of
glutathione and increase the activity of DHAD also expressed in and
targeted to the yeast cytosol. The resulting .gamma.-glutamyl
cysteine synthase and glutathione synthase expressing strain has
improved isobutanol productivity, titer, and/or yield compared to
the parental strain. Homologous genes encoding .gamma.-glutamyl
cysteine synthase and glutathione synthase from other organisms,
such as other yeast strains, may be identified by sequence homology
using tools such as BLAST and pairwise sequence alignments by one
skilled in the art.
[0192] Thioredoxins contain two conserved cysteines that exist in
either a reduced form as in thioredoxin-(SH).sub.2) or in an
oxidized form as in thioredoxin-S.sub.2) when they form an
intramolecular disulfide bridge. Thioredoxins donate electrons from
their active center dithiol to protein disulfide bonds
(Protein-S.sub.2) that are then reduced to dithiols
(Protein-(SH).sub.2). The resulting oxidized thioredoxin disulfide
is reduced directly by thioredoxin reductase with electrons donated
by NADPH. Hence the thioredoxin reduction system consists of
thioredoxin, thioredoxin reductase, and NADPH. Oxidized
glutaredoxins, on the other hand, are reduced by the tripeptide
glutathione (gamma-Glu-Cys-Gly, known as GSH) using electrons
donated by NADPH. Hence the glutathione/glutaredoxin system
consists of glutaredoxin, glutathione, glutathione reductase and
NADPH.
[0193] S. cerevisiae contains a cytoplasmic thioredoxin system
comprised of the thioredoxins Trx1p and Trx2p and the thioredoxin
reductase Trr1p, and a complete mitochondrial thioredoxin system
comprised of the thioredoxin Trx3p and the thioredoxin reductase
Trr2p. Evidence suggests that the cytoplasmic thioredoxin system
may have overlapping function with the glutathione/glutaredoxin
system. The mitochondrial thioredoxin system, on the other hand,
does not appear to be able to substitute for either the cytoplasmic
thioredoxin or glutathione/glutaredoxin systems. Instead, the
mitochondrial thioredoxin proteins, thioredoxin (Trx3p) and
thioredoxin reductase (Trr2p) have been implicated in the defense
against oxidative stress generated during respiratory
metabolism.
[0194] Overexpression of the essential cytosolic functional
components of the thioredoxin system is thus predicted to increase
the amount of bioavailable cytosolic thioredoxin, resulting in a
significant increase in cellular redox buffering potential and
concomitant increase in stable, active cytosolic FeS clusters and
DHAD activity. Thus, one or more of the following genes are
expressed either singly or in combination, thereby resulting in a
functional increase in available thioredoxin: a thioredoxin
(encoded in S. cerevisiae by TRX1 and TRX2) and a thioredoxin
reductase (encoded in S. cerevisiae by TRR1). Separately, or in
combination with the aforementioned genes, the mitochondrial
thioredoxin system (encoded by thioredoxin gene TRX3 and
thioredoxin reductase gene TRR2) are overexpressed, and, although
functional in the mitochondria, provide an added or synergistic
effect on FeS cluster assembly or stability, as assayed by
increased DHAD activity and/or output of isobutanol in a
fermentation. Overexpression of these genes may be accomplished by
methods as described above. In one embodiment, active variants of
any one of the aforementioned thioredoxins or thioredoxin
reductases, homologs thereof, or combination thereof are expressed
in the yeast cytosol to mitigate oxidative stress.
Enhancing Cytosolic DHAD Activity by Mitigating Stress Mediated by
Reactive Nitrogen Species (RNS)
[0195] Nitric oxide and reactive nitrogen species are highly
reactive, short-lived molecules that can be generated during
periods of cellular stress. The exact mechanisms by which these
molecules are created, or their downstream targets, is not
completely understood and is the subject of intense investigation.
However, the functional groups present in many proteins--for
example, FeS clusters--are readily attacked and inactivated by
NO/RNS. Loss of these labile functional groups usually results in
an inactive enzyme.
[0196] Nitric oxide and reactive nitrogen species are highly
reactive, short-lived molecules that can be generated during normal
cellular function, respiration, and during periods of cellular or
redox stress. RNS are produced in eukaryotic cells starting with
the reaction of nitric oxide (.NO) with superoxide (O2.--) to form
peroxynitrite (ONOO--):
.NO(nitric oxide)+O2-(super oxide).fwdarw.ONOO-(peroxynitrite)
[0197] Peroxynitrite itself is a highly reactive species which can
directly react with various components of the cell. Alternatively
peroxynitrite can react with other molecules to form additional
types of RNS including nitrogen dioxide (.NO.sub.2) and dinitrogen
trioxide (N.sub.2O.sub.3) as well as other types of chemically
reactive radicals. Important reactions involving RNS include:
ONOO--+H+.fwdarw.ONOOH(peroxynitrous
acid).fwdarw..NO.sub.2(nitrogen dioxide)+.OH(hydroxyl radical)
ONOO--+CO.sub.2(carbon
dioxide).fwdarw.ONOOCO.sub.2-(nitrosoperoxycarbonate)
ONOOCO.sub.2--.fwdarw..NO.sub.2(nitrogen
dioxide)+O.dbd.C(O.)O-(carbonate radical)
.NO+.NO.sub.2 is in equilibrium with N.sub.2O.sub.3(dinitrogen
trioxide)
[0198] NO exhibits other types of interaction that are candidates
for mediating aspects of its physiological action. Notably, in a
process known as nitrosylation, or nitrosation, NO can modify free
sulfhydryl (thiol) groups of cysteines in proteins to produce
nitrosothiols, SNOs. Transfer of the NO adduct from one sulfhydryl
to another transnitrosylation) is likely to play a signal
transduction role (reviewed in Stamler et al., 2001). Study of this
post-translational modification, which is proposed to be a
widespread mediator of signaling, is a relatively new field, and
the list of proteins that are modified through nitrosylation is
expanding rapidly. Because NO is highly reactive, transport of an
NO signal in tissues can be facilitated through reaction with
glutathione and movement of the resulting S-nitrosoglutathione
(GSNO), which can subsequently signal by modifying thiol groups on
target proteins by transnitrosylation (Lipton et al., 2001; Foster
et al., 2003). The discovery of GSNO reductase (GSNOR), which
reduces GSNO to restore GSH and to eliminate the NO adduct as
NH.sup.4+ (Jensen et al., 1998), revealed the importance of the
control of this NO metabolite.
[0199] The exact mechanisms by which the aforementioned molecules
are generated, or their downstream targets, are not completely
understood and are the subject of intense investigation. However,
the functional groups present in many proteins--for example, FeS
clusters--are readily attacked by NO/RNS. The enzyme dihydroxyacid
dehydratase (DHAD) contains an iron-sulfur (FeS) cluster cofactor
that is sensitive to damage by NO or RNS. As an example of the
biological sensitivity of this class of enzyme to attack by NO/RNS,
inactivation of the E. coli DHAD (encoded by ilvD) and subsequent
bacterial cell death resulting from macrophage-generated NO is a
major component of the mammalian humoral immune response.
[0200] The present invention provides methods of mitigating the
potentially harmful effects of oxidative and nitrosative stress
(e.g., NO and/or or RNS) on enzymes involved in the production of
isobutanol in the yeast cytosol. Specifically, the enzyme
dihydroxyacid dehydratase (DHAD) contains an iron-sulfur (Fe--S)
cluster that is sensitive to damage by NO and/or RNS, leading to
inactive enzyme. Strategies of mitigating such harmful effects
include, but are not limited to, increasing repair of iron-sulfur
clusters damaged by oxidative and nitrosative stress conditions;
reducing nitric oxide levels by introduction of a nitric oxide
reductase (NOR) activity in the cell; reducing the levels of SNO's
by overexpression of a GSNO-reductase; or combination thereof.
[0201] Strategies disclosed herein are intended to protect or
repair DHAD from NO/RNS damage. Accordingly, in one embodiment, the
present invention provides recombinant microorganisms that have
been engineered to express one or more enzymes in the cytosol that
reduce the concentration of reactive nitrogen species (RNS) and/or
nitric oxide in said cytosol.
[0202] In one embodiment, the present invention provides
recombinant microorganisms that have been engineered to express a
nitric oxide reductase that reduce the concentration of reactive
nitrogen species (RNS) and/or nitric oxide in said cytosol. To
reduce nitric oxide levels in the yeast cytosol, one or more nitric
oxide reductases (NORs) or active variants thereof can be
introduced into the cell by overexpression. Genes present in
several microbial species have been shown to encode a nitric oxide
reductase activity. For example, in E. coli the gene for a
flavorubredoxin, norV, encodes a flavo-diiron NO reductase that is
one of the most highly induced genes when E. coli cells are exposed
to NO or GSNO. Previous work has identified a gene present in the
microbe Fusarium oxysporum as encoding a cytochrome P-450 55A1
(P-450dNIR) that encodes a nitric oxide reductase (Nakahara et al.,
1993, J. Biol. Chem. 268:8350-8355). When expressed in a eukaryotic
cell, this gene product appears to be cytosolically localized and
exhibits effects consistent with its reducing intracellular NO
levels (Dijkers et al., 2009, Molecular Biology of the Cell, 20:
4083-4090). Thus, in one embodiment, homologs of any
above-described nitric oxide reductases, active variants thereof,
or combinations thereof are expressed in the yeast cytosol to
mitigate nitric oxide.
[0203] In contrast to E. coli and F. oxysporum, S. cerevisiae lacks
an endogenous NOR activity (and no homologs of either NOR protein
is found in the S. cerevisiae genome). Thus, to provide such an
activity, the F. oxysporum NOR gene is synthesized or amplified
from genomic DNA, or the E. coli norV gene is amplified from
genomic DNA, and either (or both) cloned into a suitable yeast
expression vector. Such a vector could either be high copy (e.g., 2
micron origin) or low copy (CEN/ARSH), or a single or multiple
copies of the gene could be stably integrated into the genome of a
host organism, specifically a yeast containing a cytosolic
isobutanol pathway. In each case, methods to clone a gene into a
plasmid so that it is expressed at a desired level under the
control of a known yeast promoter (including those steps required
to transform a host yeast cell) are well known to those skilled in
the art. In those cases where the NOR gene is expressed from an
episomal plasmid, it can be advantageous to simultaneous
overexpress a desired DHAD gene, either from the same or from
another plasmid, thereby allowing one to assay the resulting output
in DHAD activity. Similar approaches are undertaken to express the
NOR gene in the presence of a plasmid(s) encoding an isobutanol
production pathway, where the results of NOR expression are
manifested in changes in isobutanol productivity, titer, or yield.
It is understood by one skilled in the art that expression of all
genes, both NOR and genes encoding the isobutanol pathway may be
integrated into the genome of a host organism in a single or
multiple copies of the gene(s), specifically a yeast containing a
cytosolic isobutanol pathway.
[0204] In another embodiment, the present invention provides
recombinant microorganisms that have been engineered to express a
glutathione-S-nitrosothiol reductase (GSNO-reductase) that reduces
the concentration of reactive nitrogen species (RNS) and/or nitric
oxide in said cytosol. To reduce the levels of SNO's, one or more
GSNO-reductases or active variants thereof can be introduced into
the cell by overexpression. In S. cerevisiae, the gene SFA1 has
been shown to encode a formaldehyde dehydrogenase that possesses
GSNO reductase activity (Liu et al., 2001, Nature 410:490-494).
Sfa1p is a member of the class III alcohol dehydrogenases
(EC:1.1.1.284), which are bifunctional enzymes containing both
alcohol dehydrogenase and glutathione-dependent formaldehyde
dehydrogenase activities. The glutathione-dependent formaldehyde
dehydrogenase activity of Sfa1 p is required for the detoxification
of formaldehyde, and the alcohol dehydrogenase activity of Sfa1p
can catalyze the final reactions in phenylalanine and tryptophan
degradation. Sfa1p is also able to act as a hydroxymethylfurfural
(HMF) reductase and catabolize HMF, a compound formed in the
production of certain biofuels. Sfa1p has been localized to the
cytoplasm and the mitochondria, and can act on a variety of
substrates, including S-hydroxymethylglutathione,
phenylacetaldehyde, indole acetaldehyde, octanol,
10-hydroxydecanoic acid, 12-hydroxydodecanoic acid, and
S-nitrosoglutathione.
[0205] Sfa1 protein levels are reported as being low-to-moderate
from proteome-wide analyses (Ghaemmaghami et al., 2003, Nature
425(6959):737-41). Thus, in an analogous fashion to the approach
described for overexpression of NOR, the gene SFA1 is
overexpressed, thereby decoupling it from its normal regulatory
control and permitting significant increase in Sfa1 activity in the
cell, which results in measurable increases in DHAD activity and/or
fermentation output, as described above. Overexpression of these
genes may be accomplished by methods as described above. In one
embodiment, homologs of SFA1, active variants thereof, or
combinations thereof are expressed in the yeast cytosol to mitigate
stresses brought on by reactive nitrogen species.
[0206] In additional embodiments, alternative enzymes may be
expressed and targeted to the yeast cytosol containing the
isobutanol pathway to mitigate the effects of reactive nitrogen
species. Specifically, the enzyme YtfE encoded by E. coli ytfE,
homologs thereof, active variants thereof, may be expressed, such
that they lead to reduction in NO/RNS in the yeast cytosol and/or a
concomitant increase in DHAD function. Such an increase is detected
by in vitro assay of DHAD activity, and/or by an increase in
productivity, titer, or yield of isobutanol produced by isobutanol
pathway-containing cells.
[0207] To increase repairment of iron-sulfur clusters, in one
embodiment, the gene ytfE from E. coli is expressed in the yeast
cytosol which contains a functional isobutanol pathway and DHAD
such that DHAD activity and/or isobutanol productivity, titer, or
yield are increased from the yeast cells. In E. coli, the gene ytfE
has been shown to play an important role in maintaining active
Fe--S clusters. A recent report (Justino et al., (2009).
Escherichia coli Di-iron YtfE protein is necessary for the repair
of stress-damaged Iron-Sulfur Clusters. JBC 282(14): 10352-10359)
showed that .DELTA.ytfE strains have several phenotypes, including
enhanced susceptibility to nitrosative stress and are defective in
the activity of several iron-sulfur-containing proteins. For
example, the damage of the [4Fe-4S].sup.2+ clusters of aconitase B
and fumarase A caused by exposure to hydrogen peroxide and nitric
oxide stress occurs at higher rates in the absence of ytfE. The
ytfE null mutation also abolished the recovery of aconitase and
fumarase activities, which is observed in wild-type E. coli once
the stress is scavenged. Notably, upon the addition of purified
holo-YtfE protein to mutant cell extracts, the enzymatic activities
of fumarase and aconitase were fully recovered, and at rates
similar to the wild-type strain. Thus, YtfE is critical for the
repair of iron-sulfur clusters damaged by oxidative and nitrosative
stress conditions, and presents an attractive candidate for
overexpression in a host cell that normally lacks this activity,
such as S. cerevisiae, where Fe--S cluster proteins are also being
overexpressed as part of the isobutanol pathway.
[0208] To provide such an activity, the E. coli ytfE gene can be
amplified from genomic DNA by PCR with appropriate primers, and
cloned into a suitable yeast expression vector. Such a vector could
either be high copy (e.g., 2 micron origin) or low copy (CEN/ARS),
or a single or multiple copies of the gene could be stably
integrated into the genome of a host organism. In each case,
methods to clone a gene into a plasmid so that it is expressed at a
desired level under the control of a known yeast promoter
(including those steps required to transform a host yeast cell) are
well known to those skilled in the art. In those cases where the
ytfE gene is expressed from an episomal plasmid, it can be
advantageous to simultaneous overexpress a desired DHAD gene,
either from the same or from another plasmid, thereby allowing one
to assay the resulting output in DHAD activity. Similar approaches
are undertaken to express the ytfE gene in the presence of a
plasmid(s) encoding an isobutanol production pathway, where the
results of ytfE expression are manifested in changes in isobutanol
productivity, titer, or yield. More specifically, ytfE is expressed
in the yeast cytosol which contains a functional isobutanol pathway
and DHAD such that DHAD activity and/or isobutanol productivity,
titer, or yield are increased from the yeast cells.
[0209] In addition, functional homologs of E. coli ytfE have been
identified and characterized. For example, genes from two
pathogenic prokaryotes--scdA from Staphylococcus aureus, and dnrN
from Neisseria gonorrhoeae, have been shown to have properties
similar to that of ytfE (Overton, T. W., et al (2008). Widespread
distribution in pathogenic bacteria of di-iron proteins that repair
oxidative and nitrosative damage to iron-sulfur centers. J.
Bacteriology 190(6): 2004-2013). Thus, similar approaches to
overexpress either of these genes are employed, as described for E.
coli ytfE, above. Overexpression of these genes may be accomplished
by methods as described above.
The Microorganism in General
[0210] The recombinant microorganisms provided herein can express a
plurality of heterologous and/or native target enzymes involved in
pathways for the production of beneficial metabolites such as
isobutanol, 3-methyl-1-butanol, 2-methyl-1-butanol, valine,
isoleucine, leucine, and pantothenic acid from a suitable carbon
source.
[0211] Accordingly, "engineered" or "modified" microorganisms are
produced via the introduction of genetic material into a host or
parental microorganism of choice and/or by modification of the
expression of native genes, thereby modifying or altering the
cellular physiology and biochemistry of the microorganism. Through
the introduction of genetic material and/or the modification of the
expression of native genes the parental microorganism acquires new
properties, e.g. the ability to produce a new, or greater
quantities of, an intracellular metabolite. As described herein,
the introduction of genetic material into and/or the modification
of the expression of native genes in a parental microorganism
results in a new or modified ability to produce beneficial
metabolites such as isobutanol, 3-methyl-1-butanol,
2-methyl-1-butanol, valine, isoleucine, leucine, and pantothenic
acid from a suitable carbon source. The genetic material introduced
into and/or the genes modified for expression in the parental
microorganism contains gene(s), or parts of genes, coding for one
or more of the enzymes involved in a biosynthetic pathway for the
production of one or more metabolites selected from isobutanol,
3-methyl-1-butanol, 2-methyl-1-butanol, valine, isoleucine,
leucine, and pantothenic acid and may also include additional
elements for the expression and/or regulation of expression of
these genes, e.g. promoter sequences.
[0212] In addition to the introduction of a genetic material into a
host or parental microorganism, an engineered or modified
microorganism can also include alteration, disruption, deletion or
knocking-out of a gene or polynucleotide to alter the cellular
physiology and biochemistry of the microorganism. Through the
alteration, disruption, deletion or knocking-out of a gene or
polynucleotide the microorganism acquires new or improved
properties (e.g., the ability to produce a new metabolite or
greater quantities of an intracellular metabolite, improve the flux
of a metabolite down a desired pathway, and/or reduce the
production of byproducts).
[0213] Recombinant microorganisms provided herein may also produce
metabolites in quantities not available in the parental
microorganism. A "metabolite" refers to any substance produced by
metabolism or a substance necessary for or taking part in a
particular metabolic process. A metabolite can be an organic
compound that is a starting material (e.g., glucose or pyruvate),
an intermediate (e.g., 2-ketoisovalerate), or an end product (e.g.,
isobutanol) of metabolism. Metabolites can be used to construct
more complex molecules, or they can be broken down into simpler
ones. Intermediate metabolites may be synthesized from other
metabolites, perhaps used to make more complex substances, or
broken down into simpler compounds, often with the release of
chemical energy.
[0214] The disclosure identifies specific genes useful in the
methods, compositions and organisms of the disclosure; however it
will be recognized that absolute identity to such genes is not
necessary. For example, changes in a particular gene or
polynucleotide comprising a sequence encoding a polypeptide or
enzyme can be performed and screened for activity. Typically such
changes comprise conservative mutations and silent mutations. Such
modified or mutated polynucleotides and polypeptides can be
screened for expression of a functional enzyme using methods known
in the art.
[0215] Due to the inherent degeneracy of the genetic code, other
polynucleotides which encode substantially the same or functionally
equivalent polypeptides can also be used to clone and express the
polynucleotides encoding such enzymes.
[0216] As will be understood by those of skill in the art, it can
be advantageous to modify a coding sequence to enhance its
expression in a particular host. The genetic code is redundant with
64 possible codons, but most organisms typically use a subset of
these codons. The codons that are utilized most often in a species
are called optimal codons, and those not utilized very often are
classified as rare or low-usage codons. Codons can be substituted
to reflect the preferred codon usage of the host, a process
sometimes called "codon optimization" or "controlling for species
codon bias."
[0217] Optimized coding sequences containing codons preferred by a
particular prokaryotic or eukaryotic host (Murray et al., 1989,
Nucl Acids Res. 17: 477-508) can be prepared, for example, to
increase the rate of translation or to produce recombinant RNA
transcripts having desirable properties, such as a longer
half-life, as compared with transcripts produced from a
non-optimized sequence. Translation stop codons can also be
modified to reflect host preference. For example, typical stop
codons for S. cerevisiae and mammals are UAA and UGA, respectively.
The typical stop codon for monocotyledonous plants is UGA, whereas
insects and E. coli commonly use UAA as the stop codon (Dalphin et
al., 1996, Nucl Acids Res. 24: 216-8). Methodology for optimizing a
nucleotide sequence for expression in a plant is provided, for
example, in U.S. Pat. No. 6,015,891, and the references cited
therein.
[0218] Those of skill in the art will recognize that, due to the
degenerate nature of the genetic code, a variety of DNA compounds
differing in their nucleotide sequences can be used to encode a
given enzyme of the disclosure. The native DNA sequence encoding
the biosynthetic enzymes described above are referenced herein
merely to illustrate an embodiment of the disclosure, and the
disclosure includes DNA compounds of any sequence that encode the
amino acid sequences of the polypeptides and proteins of the
enzymes utilized in the methods of the disclosure. In similar
fashion, a polypeptide can typically tolerate one or more amino
acid substitutions, deletions, and insertions in its amino acid
sequence without loss or significant loss of a desired activity.
The disclosure includes such polypeptides with different amino acid
sequences than the specific proteins described herein so long as
they modified or variant polypeptides have the enzymatic anabolic
or catabolic activity of the reference polypeptide. Furthermore,
the amino acid sequences encoded by the DNA sequences shown herein
merely illustrate embodiments of the disclosure.
[0219] In addition, homologs of enzymes useful for generating
metabolites are encompassed by the microorganisms and methods
provided herein.
[0220] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences have at
least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine
the percent identity of two amino acid sequences, or of two nucleic
acid sequences, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second amino acid or nucleic acid sequence for optimal
alignment and non-homologous sequences can be disregarded for
comparison purposes). In one embodiment, the length of a reference
sequence aligned for comparison purposes is at least 30%, typically
at least 40%, more typically at least 50%, even more typically at
least 60%, and even more typically at least 70%, 80%, 90%, 100% of
the length of the reference sequence. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position (as used herein amino acid or
nucleic acid "identity" is equivalent to amino acid or nucleic acid
"homology"). The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
[0221] When "homologous" is used in reference to proteins or
peptides, it is recognized that residue positions that are not
identical often differ by conservative amino acid substitutions. A
"conservative amino acid substitution" is one in which an amino
acid residue is substituted by another amino acid residue having a
side chain (R group) with similar chemical properties (e.g., charge
or hydrophobicity). In general, a conservative amino acid
substitution will not substantially change the functional
properties of a protein. In cases where two or more amino acid
sequences differ from each other by conservative substitutions, the
percent sequence identity or degree of homology may be adjusted
upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well known to those of skill
in the art (See, e.g., Pearson W. R., 1994, Methods in Mol Biol 25:
365-89.
[0222] The following six groups each contain amino acids that are
conservative substitutions for one another: 1) Serine (S),
Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3)
Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0223] Sequence homology for polypeptides, which is also referred
to as percent sequence identity, is typically measured using
sequence analysis software. See commonly owned and co-pending
application US 2009/0226991. A typical algorithm used comparing a
molecule sequence to a database containing a large number of
sequences from different organisms is the computer program BLAST.
When searching a database containing sequences from a large number
of different organisms, it is typical to compare amino acid
sequences. Database searching using amino acid sequences can be
measured by algorithms described in commonly owned and co-pending
application US 2009/0226991.
[0224] It is understood that a range of microorganisms can be
modified to include a recombinant metabolic pathway suitable for
the production of beneficial metabolites from DHAD-requiring
biosynthetic pathways. In various embodiments, microorganisms may
be selected from yeast microorganisms. Yeast microorganisms for the
production of a metabolite such as isobutanol, 3-methyl-1-butanol,
2-methyl-1-butanol, valine, isoleucine, leucine, and pantothenic
acid may be selected based on certain characteristics:
[0225] One characteristic may include the property that the
microorganism is selected to convert various carbon sources into
beneficial metabolites such as isobutanol, 3-methyl-1-butanol,
2-methyl-1-butanol, valine, isoleucine, leucine, and pantothenic
acid. The term "carbon source" generally refers to a substance
suitable to be used as a source of carbon for prokaryotic or
eukaryotic cell growth. Examples of suitable carbon sources are
described in commonly owned and co-pending application US
2009/0226991. Accordingly, in one embodiment, the recombinant
microorganism herein disclosed can convert a variety of carbon
sources to products, including but not limited to glucose,
galactose, mannose, xylose, arabinose, lactose, sucrose, and
mixtures thereof.
[0226] The recombinant microorganism may thus further include a
pathway for the production of isobutanol, 3-methyl-1-butanol,
2-methyl-1-butanol, valine, isoleucine, leucine, and/or pantothenic
acid from five-carbon (pentose) sugars including xylose. Most yeast
species metabolize xylose via a complex route, in which xylose is
first reduced to xylitol via a xylose reductase (XR) enzyme. The
xylitol is then oxidized to xylulose via a xylitol dehydrogenase
(XDH) enzyme. The xylulose is then phosphorylated via an
xylulokinase (XK) enzyme. This pathway operates inefficiently in
yeast species because it introduces a redox imbalance in the cell.
The xylose-to-xylitol step uses NADH as a cofactor, whereas the
xylitol-to-xylulose step uses NADPH as a cofactor. Other processes
must operate to restore the redox imbalance within the cell. This
often means that the organism cannot grow anaerobically on xylose
or other pentose sugar. Accordingly, a yeast species that can
efficiently ferment xylose and other pentose sugars into a desired
fermentation product is therefore very desirable.
[0227] Thus, in one aspect, the recombinant is engineered to
express a functional exogenous xylose isomerase. Exogenous xylose
isomerases functional in yeast are known in the art. See, e.g.,
Rajgarhia et al, US2006/0234364, which is herein incorporated by
reference in its entirety. In an embodiment according to this
aspect, the exogenous xylose isomerase gene is operatively linked
to promoter and terminator sequences that are functional in the
yeast cell. In a preferred embodiment, the recombinant
microorganism further has a deletion or disruption of a native gene
that encodes for an enzyme (e.g. XR and/or XDH) that catalyzes the
conversion of xylose to xylitol. In a further preferred embodiment,
the recombinant microorganism also contains a functional, exogenous
xylulokinase (XK) gene operatively linked to promoter and
terminator sequences that are functional in the yeast cell. In one
embodiment, the xylulokinase (XK) gene is overexpressed.
[0228] In one embodiment, the microorganism has reduced or no
pyruvate decarboxylase (PDC) activity. PDC catalyzes the
decarboxylation of pyruvate to acetaldehyde, which is then reduced
to ethanol by ADH via an oxidation of NADH to NADH+. Ethanol
production is the main pathway to oxidize the NADH from glycolysis.
Deletion of this pathway increases the pyruvate and the reducing
equivalents (NADH) available for the DHAD-requiring biosynthetic
pathway. Accordingly, deletion of PDC genes can further increase
the yield of desired metabolites.
[0229] In another embodiment, the microorganism has reduced or no
glycerol-3-phosphate dehydrogenase (GPD) activity. GPD catalyzes
the reduction of dihydroxyacetone phosphate (DHAP) to
glycerol-3-phosphate (G3P) via the oxidation of NADH to NAD+.
Glycerol is then produced from G3P by Glycerol-3-phosphatase (GPP).
Glycerol production is a secondary pathway to oxidize excess NADH
from glycolysis. Reduction or elimination of this pathway would
increase the pyruvate and reducing equivalents (NADH) available for
the DHAD-requiring biosynthetic pathway. Thus, deletion of GPD
genes can further increase the yield of desired metabolites.
[0230] In yet another embodiment, the microorganism has reduced or
no PDC activity and reduced or no GPD activity. PDC-minus/GPD-minus
yeast production strains are described in co-pending applications
U.S. Ser. No. 12/343,375 (published as US 2009/0226991), U.S. Ser.
No. 12/696,645, and U.S. Ser. No. 12/820,505, which claim priority
to U.S. Provisional Application 61/016,483, all of which are herein
incorporated by reference in their entireties for all purposes.
[0231] In one embodiment, the yeast microorganisms may be selected
from the "Saccharomyces Yeast Clade", as described in commonly
owned and co-pending application US 2009/0226991.
[0232] The term "Saccharomyces sensu stricto" taxonomy group is a
cluster of yeast species that are highly related to S. cerevisiae
(Rainieri et al., 2003, J. Biosci Bioengin 96: 1-9). Saccharomyces
sensu stricto yeast species include but are not limited to S.
cerevisiae, S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus,
S. uvarum, S. carocanis and hybrids derived from these species
(Masneuf et al., 1998, Yeast 7: 61-72).
[0233] An ancient whole genome duplication (WGD) event occurred
during the evolution of the hemiascomycete yeast and was discovered
using comparative genomic tools (Kellis et al., 2004, Nature 428:
617-24; Dujon et al., 2004, Nature 430:35-44; Langkjaer et al.,
2003, Nature 428: 848-52; Wolfe et al., 1997, Nature 387: 708-13).
Using this major evolutionary event, yeast can be divided into
species that diverged from a common ancestor following the WGD
event (termed "post-WGD yeast" herein) and species that diverged
from the yeast lineage prior to the WGD event (termed "pre-WGD
yeast" herein).
[0234] Accordingly, in one embodiment, the yeast microorganism may
be selected from a post-WGD yeast genus, including but not limited
to Saccharomyces and Candida. The favored post-WGD yeast species
include: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S.
castelli, and C. glabrata.
[0235] In another embodiment, the yeast microorganism may be
selected from a pre-whole genome duplication (pre-WGD) yeast genus
including but not limited to Saccharomyces, Kluyveromyces, Candida,
Pichia, Issatchenkia, Debaryomyces, Hansenula, Yarrowia and,
Schizosaccharomyces. Representative pre-WGD yeast species include:
S. kluyveri, K. thermotolerans, K. marxianus, K. waltii, K. lactis,
C. tropicalis, P. pastoris, P. anomala, P. stipitis, I. orientalis,
I. occidentalis, I. scutulata, D. hansenii, H. anomala, Y.
lipolytica, and S. pombe.
[0236] A yeast microorganism may be either Crabtree-negative or
Crabtree-positive as described in described in commonly owned and
co-pending application US 2009/0226991. In one embodiment the yeast
microorganism may be selected from yeast with a Crabtree-negative
phenotype including but not limited to the following genera:
Kluyveromyces, Pichia, Issatchenkia, Hansenula, and Candida.
Crabtree-negative species include but are not limited to: K.
lactis, K. marxianus, P. anomala, P. stipitis, I. orientalis, I.
occidentalis, I. scutulata, H. anomala, and C. utilis. In another
embodiment, the yeast microorganism may be selected from a yeast
with a Crabtree-positive phenotype, including but not limited to
Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces,
Pichia and Schizosaccharomyces. Crabtree-positive yeast species
include but are not limited to: S. cerevisiae, S. uvarum, S.
bayanus, S. paradoxus, S. castelli, S. kluyveri, K. thermotolerans,
C. glabrata, Z. bailli, Z. rouxii, D. hansenii, P. pastorius, and
S. pombe.
[0237] Another characteristic may include the property that the
microorganism is that it is non-fermenting. In other words, it
cannot metabolize a carbon source anaerobically while the yeast is
able to metabolize a carbon source in the presence of oxygen.
Nonfermenting yeast refers to both naturally occurring yeasts as
well as genetically modified yeast. During anaerobic fermentation
with fermentative yeast, the main pathway to oxidize the NADH from
glycolysis is through the production of ethanol. Ethanol is
produced by alcohol dehydrogenase (ADH) via the reduction of
acetaldehyde, which is generated from pyruvate by pyruvate
decarboxylase (PDC). In one embodiment, a fermentative yeast can be
engineered to be non-fermentative by the reduction or elimination
of the native PDC activity. Thus, most of the pyruvate produced by
glycolysis is not consumed by PDC and is available for the
isobutanol pathway. Deletion of this pathway increases the pyruvate
and the reducing equivalents available for the DHAD-requiring
biosynthetic pathway. Fermentative pathways contribute to low yield
and low productivity of desired metabolites such as isobutanol.
Accordingly, deletion of PDC may increase yield and productivity of
desired metabolites such as isobutanol.
[0238] In some embodiments, the recombinant microorganisms may be
microorganisms that are non-fermenting yeast microorganisms,
including, but not limited to those, classified into a genera
selected from the group consisting of Tricosporon, Rhodotorula,
Myxozyma, or Candida. In a specific embodiment, the non-fermenting
yeast is C. xestobii.
Isobutanol-Producing Yeast Microorganisms
[0239] As described herein, in one embodiment, a yeast
microorganism is engineered to convert a carbon source, such as
glucose, to pyruvate by glycolysis and the pyruvate is converted to
isobutanol via an isobutanol producing metabolic pathway (See,
e.g., WO/2007/050671, WO/2008/098227, and Atsumi et al., 2008,
Nature 45: 86-9). Alternative pathways for the production of
isobutanol have been described in WO/2007/050671 and in Dickinson
et al., 1998, J Biol Chem 273:25751-6.
[0240] Accordingly, in one embodiment, the isobutanol producing
metabolic pathway to convert pyruvate to isobutanol can be
comprised of the following reactions:
[0241] 1. 2 pyruvate.fwdarw.acetolactate+CO.sub.2
[0242] 2. acetolactate+NAD(P)H
2,3-dihydroxyisovalerate+NAD(P).sup.+
[0243] 3. 2,3-dihydroxyisovalerate.fwdarw.alpha-ketoisovalerate
[0244] 4.
alpha-ketoisovalerate.fwdarw.isobutyraldehyde+CO.sub.2
[0245] 5. isobutyraldehyde+NAD(P)H.fwdarw.isobutanol+NADP
[0246] These reactions are carried out by the enzymes 1)
Acetolactate Synthase (ALS), 2) Keto-acid Reducto-Isomerase (KARI),
3) Dihydroxy-acid dehydratase (DHAD), 4) Keto-isovalerate
decarboxylase (KIVD), and 5) an Alcohol dehydrogenase (ADH) (FIG.
1). In another embodiment, the yeast microorganism is engineered to
overexpress these enzymes. For example, these enzymes can be
encoded by native genes. Alternatively, these enzymes can be
encoded by heterologous genes. For example, ALS can be encoded by
the alsS gene of B. subtilis, alsS of L. lactis, or the ilvK gene
of K. pneumonia. For example, KARI can be encoded by the ilvC genes
of E. coli, C. glutamicum, M. maripaludis, or Piromyces sp E2. For
example, DHAD can be encoded by the ilvD genes of E. coli, C.
glutamicum, or L. lactis. For example, KIVD can be encoded by the
kivD gene of L. lactis. ADH can be encoded by ADH2, ADH6, or ADH7
of S. cerevisiae.
[0247] In one embodiment, pathway steps 2 and 5 may be carried out
by KARI and ADH enzymes that utilize NADH (rather than NADPH) as a
co-factor. Such enzymes are described in commonly owned and
co-pending applications U.S. Ser. No. 12/610,784 and PCT/US09/62952
(published as WO/2010/051527), which are herein incorporated by
reference in their entireties for all purposes. The present
inventors have found that utilization of NADH-dependent KARI and
ADH enzymes to catalyze pathway steps 2 and 5, respectively,
surprisingly enables production of isobutanol under anaerobic
conditions. Thus, in one embodiment, the recombinant microorganisms
of the present invention may use an NADH-dependent KARI to catalyze
the conversion of acetolactate (+NADH) to produce
2,3-dihydroxyisovalerate. In another embodiment, the recombinant
microorganisms of the present invention may use an NADH-dependent
ADH to catalyze the conversion of isobutyraldehyde (+NADH) to
produce isobutanol. In yet another embodiment, the recombinant
microorganisms of the present invention may use both an
NADH-dependent KARI to catalyze the conversion of acetolactate
(+NADH) to produce 2,3-dihydroxyisovalerate, and an NADH-dependent
ADH to catalyze the conversion of isobutyraldehyde (+NADH) to
produce isobutanol.
[0248] In another embodiment, the yeast microorganism may be
engineered to have increased ability to convert pyruvate to
isobutanol. In one embodiment, the yeast microorganism may be
engineered to have increased ability to convert pyruvate to
isobutyraldehyde. In another embodiment, the yeast microorganism
may be engineered to have increased ability to convert pyruvate to
keto-isovalerate. In another embodiment, the yeast microorganism
may be engineered to have increased ability to convert pyruvate to
2,3-dihydroxyisovalerate. In another embodiment, the yeast
microorganism may be engineered to have increased ability to
convert pyruvate to acetolactate.
[0249] Furthermore, any of the genes encoding the foregoing enzymes
(or any others mentioned herein (or any of the regulatory elements
that control or modulate expression thereof)) may be optimized by
genetic/protein engineering techniques, such as directed evolution
or rational mutagenesis, which are known to those of ordinary skill
in the art. Such action allows those of ordinary skill in the art
to optimize the enzymes for expression and activity in yeast.
[0250] In addition, genes encoding these enzymes can be identified
from other fungal and bacterial species and can be expressed for
the modulation of this pathway. A variety of organisms could serve
as sources for these enzymes, including, but not limited to,
Saccharomyces spp., including S. cerevisiae and S. uvarum,
Kluyveromyces spp., including K. thermotolerans, K. lactic, and K.
marxianus, Pichia spp., Hansenula spp., including H. polymorphs,
Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp.
stipitis, Torulaspora pretoriensis, Schizosaccharomyces spp.,
including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora
spp., or Ustilago spp. Sources of genes from anaerobic fungi
include, but not limited to, Piromyces spp., Orpinomyces spp., or
Neocallimastix spp. Sources of prokaryotic enzymes that are useful
include, but not limited to, Escherichia. coli, Zymomonas mobilis,
Staphylococcus aureus, Bacillus spp., Clostridium spp.,
Corynebacterium spp., Pseudomonas spp., Lactococcus spp.,
Enterobacter spp., and Salmonella spp.
Methods in General
[0251] Identification of an Aft Protein in a Microorganism
[0252] Any method can be used to identify genes that encode for
proteins with Aft activity. Aft1 and Aft2 enhance cellular iron
availability. Generally, genes that are homologous or similar to a
known AFT gene, e.g. S. cerevisiae AFT1 (encoding for SEQ ID NO: 2)
or S. cerevisiae AFT2 (encoding for SEQ ID NO: 4) can be identified
by functional, structural, and/or genetic analysis. In most cases,
homologous or similar AFT genes and/or homologous or similar Aft
proteins will have functional, structural, or genetic similarities.
Techniques known to those skilled in the art may be suitable to
identify homologous genes and homologous enzymes. Generally,
analogous genes and/or analogous enzymes can be identified by
functional analysis and will have functional similarities.
Techniques known to those skilled in the art may be suitable to
identify analogous genes and analogous enzymes. For example, to
identify homologous or analogous genes, proteins, or enzymes,
techniques may include, but not limited to, cloning a AFT gene by
PCR using primers based on a published sequence of a gene/enzyme or
by degenerate PCR using degenerate primers designed to amplify a
conserved region among AFT genes. Further, one skilled in the art
can use techniques to identify homologous or analogous genes,
proteins, or enzymes with functional homology or similarity. For
instance, the computer program BLAST may be used for such a
purpose. To identify homologous or similar genes and/or homologous
or similar proteins, analogous genes and/or analogous proteins,
techniques also include comparison of data concerning a candidate
gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC. The
candidate gene or enzyme may be identified within the above
mentioned databases in accordance with the teachings herein.
Identification of PDC and GPD in a Yeast Microorganism
[0253] Any method can be used to identify genes that encode for
enzymes with pyruvate decarboxylase (PDC) activity or
glycerol-3-phosphate dehydrogenase (GPD) activity. Suitable methods
for the identification of PDC and GPD are described in co-pending
applications U.S. Ser. No. 12/343,375 (published as US
2009/0226991), U.S. Ser. No. 12/696,645, and U.S. Ser. No.
12/820,505, which claim priority to US Provisional Application
61/016,483, all of which are herein incorporated by reference in
their entireties for all purposes.
Genetic Insertions and Deletions
[0254] Any method can be used to introduce a nucleic acid molecule
into yeast and many such methods are well known. For example,
transformation and electroporation are common methods for
introducing nucleic acid into yeast cells. See, e.g., Gietz et al.,
1992, Nuc Acids Res. 27: 69-74; Ito et al., 1983, J. Bacteriol.
153: 163-8; and Becker et al., 1991, Methods in Enzymology 194:
182-7.
[0255] In an embodiment, the integration of a gene of interest into
a DNA fragment or target gene of a yeast microorganism occurs
according to the principle of homologous recombination. According
to this embodiment, an integration cassette containing a module
comprising at least one yeast marker gene and/or the gene to be
integrated (internal module) is flanked on either side by DNA
fragments homologous to those of the ends of the targeted
integration site (recombinogenic sequences). After transforming the
yeast with the cassette by appropriate methods, a homologous
recombination between the recombinogenic sequences may result in
the internal module replacing the chromosomal region in between the
two sites of the genome corresponding to the recombinogenic
sequences of the integration cassette. (Orr-Weaver et al., 1981,
PNAS USA 78: 6354-58).
[0256] In an embodiment, the integration cassette for integration
of a gene of interest into a yeast microorganism includes the
heterologous gene under the control of an appropriate promoter and
terminator together with the selectable marker flanked by
recombinogenic sequences for integration of a heterologous gene
into the yeast chromosome. In an embodiment, the heterologous gene
includes an appropriate native gene desired to increase the copy
number of a native gene(s). The selectable marker gene can be any
marker gene used in yeast, including but not limited to, HIS3,
TRP1, LEU2, URA3, bar, ble, hph, and kan. The recombinogenic
sequences can be chosen at will, depending on the desired
integration site suitable for the desired application.
[0257] In another embodiment, integration of a gene into the
chromosome of the yeast microorganism may occur via random
integration (Kooistra at al., 2004, Yeast 21: 781-792).
[0258] Additionally, in an embodiment, certain introduced marker
genes are removed from the genome using techniques well known to
those skilled in the art. For example, URA3 marker loss can be
obtained by plating URA3 containing cells in FOA (5-fluoro-orotic
acid) containing medium and selecting for FOA resistant colonies
(Boeke et al., 1984, Mol. Gen. Genet. 197: 345-47).
[0259] The exogenous nucleic acid molecule contained within a yeast
cell of the disclosure can be maintained within that cell in any
form. For example, exogenous nucleic acid molecules can be
integrated into the genome of the cell or maintained in an episomal
state that can stably be passed on ("inherited") to daughter cells.
Such extra-chromosomal genetic elements (such as plasmids,
mitochondrial genome, etc.) can additionally contain selection
markers that ensure the presence of such genetic elements in
daughter cells. Moreover, the yeast cells can be stably or
transiently transformed. In addition, the yeast cells described
herein can contain a single copy, or multiple copies of a
particular exogenous nucleic acid molecule as described above.
Reduction of Enzymatic Activity
[0260] Yeast microorganisms within the scope of the invention may
have reduced enzymatic activity such as reduced
glycerol-3-phosphate dehydrogenase activity. The term "reduced" as
used herein with respect to a particular enzymatic activity refers
to a lower level of enzymatic activity than that measured in a
comparable yeast cell of the same species. The term reduced also
refers to the elimination of enzymatic activity than that measured
in a comparable yeast cell of the same species. Thus, yeast cells
lacking glycerol-3-phosphate dehydrogenase activity are considered
to have reduced glycerol-3-phosphate dehydrogenase activity since
most, if not all, comparable yeast strains have at least some
glycerol-3-phosphate dehydrogenase activity. Such reduced enzymatic
activities can be the result of lower enzyme concentration, lower
specific activity of an enzyme, or a combination thereof. Many
different methods can be used to make yeast having reduced
enzymatic activity. For example, a yeast cell can be engineered to
have a disrupted enzyme-encoding locus using common mutagenesis or
knock-out technology. In addition, certain point-mutation(s) can be
introduced which results in an enzyme with reduced activity.
[0261] Alternatively, antisense technology can be used to reduce
enzymatic activity. For example, yeast can be engineered to contain
a cDNA that encodes an antisense molecule that prevents an enzyme
from being made. The term "antisense molecule" as used herein
encompasses any nucleic acid molecule that contains sequences that
correspond to the coding strand of an endogenous polypeptide. An
antisense molecule also can have flanking sequences (e.g.,
regulatory sequences). Thus antisense molecules can be ribozymes or
antisense oligonucleotides. A ribozyme can have any general
structure including, without limitation, hairpin, hammerhead, or
axhead structures, provided the molecule cleaves RNA.
[0262] Yeast having a reduced enzymatic activity can be identified
using many methods. For example, yeast having reduced
glycerol-3-phosphate dehydrogenase activity can be easily
identified using common methods, which may include, for example,
measuring glycerol formation via liquid chromatography.
Overexpression of Heterologous Genes
[0263] Methods for overexpressing a polypeptide from a native or
heterologous nucleic acid molecule are well known. Such methods
include, without limitation, constructing a nucleic acid sequence
such that a regulatory element promotes the expression of a nucleic
acid sequence that encodes the desired polypeptide. Typically,
regulatory elements are DNA sequences that regulate the expression
of other DNA sequences at the level of transcription. Thus,
regulatory elements include, without limitation, promoters,
enhancers, and the like. For example, the exogenous genes can be
under the control of an inducible promoter or a constitutive
promoter. Moreover, methods for expressing a polypeptide from an
exogenous nucleic acid molecule in yeast are well known. For
example, nucleic acid constructs that are used for the expression
of exogenous polypeptides within Kluyveromyces and Saccharomyces
are well known (see, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529,
for Kluyveromyces and, e.g., Gellissen et al., Gene 190(1):87-97
(1997) for Saccharomyces). Yeast plasmids have a selectable marker
and an origin of replication. In addition certain plasmids may also
contain a centromeric sequence. These centromeric plasmids are
generally a single or low copy plasmid. Plasmids without a
centromeric sequence and utilizing either a 2 micron (S.
cerevisiae) or 1.6 micron (K. lactis) replication origin are high
copy plasmids. The selectable marker can be either prototrophic,
such as HIS3, TRP1, LEU2, URA3 or ADE2, or antibiotic resistance,
such as, bar, ble, hph, or kan.
[0264] In another embodiment, heterologous control elements can be
used to activate or repress expression of endogenous genes.
Additionally, when expression is to be repressed or eliminated, the
gene for the relevant enzyme, protein or RNA can be eliminated by
known deletion techniques.
[0265] As described herein, any yeast within the scope of the
disclosure can be identified by selection techniques specific to
the particular enzyme being expressed, over-expressed or repressed.
Methods of identifying the strains with the desired phenotype are
well known to those skilled in the art. Such methods include,
without limitation, PCR, RT-PCR, and nucleic acid hybridization
techniques such as Northern and Southern analysis, altered growth
capabilities on a particular substrate or in the presence of a
particular substrate, a chemical compound, a selection agent and
the like. In some cases, immunohistochemistry and biochemical
techniques can be used to determine if a cell contains a particular
nucleic acid by detecting the expression of the encoded
polypeptide. For example, an antibody having specificity for an
encoded enzyme can be used to determine whether or not a particular
yeast cell contains that encoded enzyme. Further, biochemical
techniques can be used to determine if a cell contains a particular
nucleic acid molecule encoding an enzymatic polypeptide by
detecting a product produced as a result of the expression of the
enzymatic polypeptide. For example, transforming a cell with a
vector encoding acetolactate synthase and detecting increased
acetolactate concentrations compared to a cell without the vector
indicates that the vector is both present and that the gene product
is active. Methods for detecting specific enzymatic activities or
the presence of particular products are well known to those skilled
in the art. For example, the presence of acetolactate can be
determined as described by Hugenholtz and Starrenburg, 1992, Appl.
Micro. Biot. 38:17-22.
Methods for the Overexpression of AFT Genes
[0266] Overexpression of the AFT1 and AFT2 genes may be
accomplished by any number of methods. In one embodiment,
overexpression of the AFT1 and AFT2 genes may be accomplished with
the use of plasmid vectors that function in yeast. In exemplary
embodiments, the expression of AFT1, AFT2, and/or homologous genes
may be increased by overexpressing the genes on a CEN plasmid or
alternative plasmids with a similar copy number. In one embodiment,
AFT1 or a homolog thereof is overexpressed on a CEN plasmid or
alternative plasmids with a similar copy number. In another
embodiment, AFT2 or a homolog thereof is overexpressed on a CEN
plasmid or alternative plasmids with a similar copy number. In yet
another embodiment, AFT1 and AFT2 or homologs thereof are
overexpressed on a CEN plasmid or alternative plasmids with a
similar copy number.
[0267] In further embodiments, expression of genes from single or
multiple copy integrations into the chromosome of the cell may be
useful. Use of a number of promoters, such as TDH3, TEF1, CCW12,
PGK1, and ENO2, may be utilized. As would be understood in the art,
the expression level may be fine-tuned by using a promoter that
achieves the optimal expression (e.g. optimal overexpression) level
in a given yeast. Different levels of expression of the genes may
be achieved by using promoters with different levels of activity,
either in single or multiple copy integrations or on plasmids. An
example of such a group of promoters is a series of truncated PDC1
promoters designed to provide different strength promoters.
Alternatively promoters that are active under desired conditions,
such as growth on glucose, may be used. For example a promoter from
one of the glycolytic genes, the PDC1 promoter, and a promoter from
one of the ADH genes in S. cerevisiae may all be useful. Also,
embodiments are exemplified using the yeast S. cerevisiae. However,
other yeasts, such as those from the genera listed herein may also
be used.
[0268] As described herein, overexpression of the Aft1 protein or a
homolog thereof may be obtained by expressing a constitutively
active Aft1 or a homolog thereof. In one embodiment, the
constitutively active Aft1 or a homolog thereof comprises a
mutation at a position corresponding to the cysteine 291 residue of
the native S. cerevisiae Aft1 (SEQ ID NO: 2). In a specific
embodiment, the cysteine 291 residue is replaced with a
phenylalanine residue.
[0269] As described herein, overexpression of the Aft2 protein or a
homolog thereof may be obtained by expressing a constitutively
active Aft2 or a homolog thereof. In one embodiment, the
constitutively active Aft2 or a homolog thereof comprises a
mutation at a position corresponding to the cysteine 187 residue of
the native S. cerevisiae Aft2 (SEQ ID NO: 2). In a specific
embodiment, the cysteine 187 residue is replaced with a
phenylalanine residue.
Increase of Enzymatic Activity
[0270] Yeast microorganisms of the invention may be further
engineered to have increased activity of enzymes. The term
"increased" as used herein with respect to a particular enzymatic
activity refers to a higher level of enzymatic activity than that
measured in a comparable yeast cell of the same species. For
example, overexpression of a specific enzyme can lead to an
increased level of activity in the cells for that enzyme. Increased
activities for enzymes involved in glycolysis or the isobutanol
pathway would result in increased productivity and yield of
isobutanol.
[0271] Methods to increase enzymatic activity are known to those
skilled in the art. Such techniques may include increasing the
expression of the enzyme by increased copy number and/or use of a
strong promoter, introduction of mutations to relieve negative
regulation of the enzyme, introduction of specific mutations to
increase specific activity and/or decrease the Km for the
substrate, or by directed evolution. See, e.g., Methods in
Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press
(2003).
Methods of Using Recombinant Microorganisms for High-Yield
Fermentations
[0272] For a biocatalyst to produce a beneficial metabolite most
economically, it is desirable to produce said metabolite at a high
yield. Preferably, the only product produced is the desired
metabolite, as extra products (i.e. by-products) lead to a
reduction in the yield of the desired metabolite and an increase in
capital and operating costs, particularly if the extra products
have little or no value. These extra products also require
additional capital and operating costs to separate these products
from the desired metabolite.
[0273] In one aspect, the present invention provides a method of
producing a beneficial metabolite derived from a DHAD-requiring
biosynthetic pathway. In one embodiment, the method includes
cultivating a recombinant microorganism comprising a DHAD-requiring
biosynthetic pathway in a culture medium containing a feedstock
providing the carbon source until a recoverable quantity of the
beneficial metabolite is produced and optionally, recovering the
metabolite. In an exemplary embodiment, said recombinant
microorganism has been engineered to overexpress a polynucleotide
encoding Aft1 (SEQ ID NO: 2) and/or Aft2 (SEQ ID NO: 4) or a
homolog thereof. The beneficial metabolite may be derived from any
DHAD-requiring biosynthetic pathway, including, but not limited to,
biosynthetic pathways for the production of isobutanol,
3-methyl-1-butanol, 2-methyl-1-butanol, valine, isoleucine,
leucine, and pantothenic acid. In a specific embodiment, the
beneficial metabolite is isobutanol.
[0274] In a method to produce a beneficial metabolite from a carbon
source, the yeast microorganism is cultured in an appropriate
culture medium containing a carbon source. In certain embodiments,
the method further includes isolating the beneficial metabolite
from the culture medium. For example, isobutanol may be isolated
from the culture medium by any method known to those skilled in the
art, such as distillation, pervaporation, or liquid-liquid
extraction
[0275] In one embodiment, the recombinant microorganism may produce
the beneficial metabolite from a carbon source at a yield of at
least 5 percent theoretical. In another embodiment, the
microorganism may produce the beneficial metabolite from a carbon
source at a yield of at least about 10 percent, at least about 15
percent, about least about 20 percent, at least about 25 percent,
at least about 30 percent, at least about 35 percent, at least
about 40 percent, at least about 45 percent, at least about 50
percent, at least about 55 percent, at least about 60 percent, at
least about 65 percent, at least about 70 percent, at least about
75 percent, at least about 80 percent, at least about 85 percent,
at least about 90 percent, at least about 95 percent, or at least
about 97.5% theoretical. In a specific embodiment, the beneficial
metabolite is isobutanol.
[0276] This invention is further illustrated by the following
examples that should not be construed as limiting. The contents of
all references, patents, and published patent applications cited
throughout this application, as well as the Figures and the
Sequence Listing, are incorporated herein by reference for all
purposes.
EXAMPLES
General Materials and Methods for Examples
[0277] Media:
[0278] Media used were standard yeast medium (for example Sambrook,
J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3rd ed.
2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press
and Guthrie, C. and Fink, G. R. eds. Methods in Enzymology Part B:
Guide to Yeast Genetics and Molecular and Cell Biology 350:3-623
(2002)). YP medium contains 1% (w/v) yeast extract, 2% (w/v)
peptone. YPD is YP containing 2% (w/v) glucose.
[0279] S. cerevisiae Transformations:
[0280] The yeast strain of interest was grown on YPD medium. The
strain was re-suspended in 100 mM lithium acetate. Once the cells
were re-suspended, a mixture of DNA (final volume of 15 .mu.L with
sterile water), 72 .mu.L 50% w/v PEG, 10 .mu.L 1 M lithium acetate,
and 3 .mu.L of denatured salmon sperm DNA (10 mg/mL) was prepared
for each transformation. In a 1.5 mL tube, 15 .mu.L of the cell
suspension was added to the DNA mixture (100 .mu.L), and the
transformation suspension was vortexed for 5 short pulses. The
transformation was incubated for 30 min at 30.degree. C., followed
by incubation for 22 min at 42.degree. C. The cells were collected
by centrifugation (18,000 rcf, 10 sec, 25.degree. C.). The cells
were resuspended in 1 mL YPD and after an overnight recovery
shaking at 30.degree. C. and 250 rpm, the cells were spread over
YPD+0.2 g/L G418+0.1 g/L hygromycin selective plates. Transformants
were then single colony purified onto selective plates containing
appropriate antibiotics.
[0281] Preparation of Yeast Lysate:
[0282] Cells were thawed on ice and resuspended in lysis buffer (50
mM Tris pH 8.0, 5 mM MgSO.sub.4) such that the result was a 20%
cell suspension by mass. 1000 .mu.L of glass beads (0.5 mm
diameter) were added to a 1.5 mL microcentrifuge tube and 875 .mu.L
of cell suspension was added. Yeast cells were lysed using a Retsch
MM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 6.times.1 min
each at full speed with 1 min incubations on ice between each
bead-beating step. The tubes were centrifuged for 10 min at 23,500
rcf at 4.degree. C. and the supernatant was removed for use. The
lysates were held on ice until assayed.
[0283] DHAD Assay:
[0284] Each sample was diluted in DHAD assay buffer (50 mM Tris pH
8, 5 mM MgSO.sub.4) to a 1:10 and a 1:40 to 1:100 dilution. Three
samples of each lysate were assayed, along with no lysate controls.
10 .mu.L of each sample (or DHAD assay buffer) was added to 0.2 mL
PCR tubes. Using a multi-channel pipette, 90 .mu.L of the substrate
was added to each tube (substrate mix was prepared by adding 4 mL
DHAD assay buffer to 0.5 mL 100 mM DHIV). Samples were put in a
thermocycler (Eppendorf Mastercycler) at 35.degree. C. for 30 min
followed by a 5 min incubation at 95.degree. C. Samples were cooled
to 4.degree. C. on the thermocycler, then centrifuged at 3000 rcf
for 5 min. Finally, 75 .mu.L of supernatant was transferred to new
PCR tubes and submitted to analytics for analysis by Liquid
Chromatography, method 2. DHAD activity units were calculated as
.mu.mol KIV produced/min/mg total cell lysate protein in the
assay.
[0285] Protein Concentration Determination: Yeast lysate protein
concentration was determined using the BioRad Bradford Protein
Assay Reagent Kit (Cat #500-0006, BioRad Laboratories, Hercules,
Calif.) and using BSA for the standard curve. Briefly, 10 .mu.L
standard or lysate were added into a microcentrifuge tube. The
samples were diluted to fit in the linear range of the standard
curve (1:40). 500 .mu.L of 1:4 diluted and filtered Bio-Rad protein
assay dye was added to the blank and samples and then vortexed.
Samples were incubated at room temperature for 6 min, transferred
into cuvettes and the OD.sub.595 was determined in a
spectrophotometer. The linear regression of the standards was then
used to calculate the protein concentration in each sample.
[0286] Gas Chromatography:
[0287] Analysis of volatile organic compounds including isobutanol,
was performed on a HP 5890/6890/7890 gas chromatograph fitted with
an HP 7673 Autosampler, a ZB-FFAP column (Phenomenex; 30 m length,
0.32 mm ID, 0.25 .mu.M film thickness) or equivalent connected to a
flame ionization detector (FID). The temperature program was as
follows: 200.degree. C. for the injector, 300.degree. C. for the
detector, 100.degree. C. oven for 1 min, 70.degree. C./min gradient
to 230.degree. C., and then hold for 2.5 min. Analysis was
performed using authentic standards (>99%, obtained from
Sigma-Aldrich) and a 5-point calibration curve with 1-pentanol as
the internal standard.
[0288] Liquid Chromatography, Method 1:
[0289] Analysis of organic acid metabolites, specifically pyruvate,
acetate, 2,3-dihydroxy-isovalerate, and 2,3-butanediol, was
performed on an HP-1200 High Performance Liquid Chromatography
system equipped with two Rezex RFQ 150.times.4.6 mm columns in
series. Organic acid metabolites were detected using an HP-1100 UV
detector (210 nm) and refractive index. The column temperature was
60.degree. C. This method was isocratic with 0.0180 N
H.sub.2SO.sub.4 in Milli-Q water as mobile phase. Flow was set to
1.1 mL/min. Injection volume was 20 .mu.L and run time was 16 min.
Analysis was performed using authentic standards (>99%, obtained
from Sigma-Aldrich, with the exception of DHIV
(2,3-dihydroxy-3-methyl-butanoate, CAS 1756-18-9), which was custom
synthesized at Caltech (Cioffi, E. et al. Anal Biochem 104 pp. 485
(1980)), and a 5-point calibration curve.
[0290] Liquid Chromatography, Method 2:
[0291] Analysis of 2-keto-isovalerate (KIV), the product indicating
DHAD activity, was measured using liquid chromatography. DNPH
reagent (12 mM 2,4-Dinitrophenyl Hydrazine, 20 mM Citric Acid pH
3.0, 80% Acetonitrile, 20% MilliQ H.sub.2O) was added to each
sample in a 1:1 ratio. Samples were incubated for 30 min at
70.degree. C. in a thermo-cycler (Eppendorf, Mastercycler).
Analysis of KIV was performed on an HP-1200 High Performance Liquid
Chromatography system equipped with an Eclipse XDB C-18 reverse
phase column (Agilent) and a C-18 reverse phase column guard
(Phenomenex). KIV was detected using an HP-1100 UV detector (360
nm). The column temperature was 50.degree. C. This method was
isocratic with 70% acetonitrile 2.5% phosphoric acid (4%), 27.5%
water as mobile phase. Flow was set to 3 mL/min. Injection size was
10 .mu.L and run time was 2 min.
Example 1
Overexpression of AFT1 Increases DHAD Activity and Isobutanol
Productivity, Titer, and Yield in Fermentation Vessels
[0292] The purpose of this example is to demonstrate that
overexpression of AFT1 increases DHAD activity, isobutanol titer,
productivity, and yield.
[0293] Media:
[0294] Medium used for the fermentation was YP+80 g/L glucose+0.2
g/L G418+0.1 g/L hygromycin+100 .mu.M CuSO.sub.4.5H.sub.2O+1% v/v
ethanol. The medium was filter sterilized using a 1 L bottle top
Corning PES 0.22 .mu.m filter (431174). Medium was pH adjusted to
6.0 in the fermenter vessels using 6N KOH.
[0295] Vessel Preparation and Operating Conditions:
[0296] Batch fermentations were conducted using six 2 L top drive
motor DasGip vessels with a working volume of 0.9 L per vessel.
Vessels were sterilized, along with the appropriate dissolved
oxygen probes and pH probes, for 60 min at 121.degree. C. pH probes
were calibrated prior to sterilization, however, dissolved oxygen
probes were calibrated post sterilization in order to allow for
polarization.
[0297] Process Control Parameters:
[0298] Initial volume, 900 mL. Temperature, 30.degree. C. pH 6.0,
pH was controlled using 6N KOH and 2N H.sub.2SO.sub.4 (Table
4).
TABLE-US-00004 TABLE 4 Process control parameters. Growth phase
Oxygen transfer rate 10 mM/h Air overlay 5.0slph Agitation 700 rpm
Dissolved oxygen Not controlled Fermentation phase Oxygen transfer
rate 0.5 mM/h to 1.8 mM/h* Air overlay 5.0slph Agitation 300
rpm/400 rpm* Dissolved oxygen Not controlled *Oxygen transfer rate
increased from 0.5 mM/h to 1.8 mM/h by increase in agitation from
300 rpm to 400 rpm 56 h post inoculation.
[0299] Fermentation:
[0300] The fermentation was run for 119 h. Vessels were sampled 3
times daily. Sterile 5 mL syringes were used to collect 3 mL of
fermenter culture via a sterile sample port. The sample was placed
in a 2 mL microfuge tube and a portion was used to measure cell
density (OD.sub.600) on a Genesys 10 spectrophotometer (Thermo
Scientific). The remaining sample was filtered through a 0.22 .mu.m
pore-size Corning filter. The supernatant from each vessel was
refrigerated in a 96-well, deep well plate, and stored at 4.degree.
C. prior to gas and liquid chromatography analysis (see General
Methods).
[0301] Off-Gas Measurements:
[0302] On-line continuous measurement of each fermenter vessel
off-gas by mass spectrometry analysis was performed for oxygen,
isobutanol, ethanol, carbon dioxide, and nitrogen throughout the
experiment. Fermentor off-gas was analyzed by Prima dB mass
spectrometer (Thermo, Waltham, Mass.) for nitrogen, oxygen, argon,
carbon dioxide, isobutanol, ethanol, and isobutyraldehyde. A
reference stream of similar composition to the inlet fermentor air
was also analyzed. The mass spectrometer cycles through the
reference air and fermentor off-gas streams (one by one) and
measures percent concentration of these gases after an 8.3 min
settling time to ensure representative samples. Equation 1 is a
derived value expression input into the mass spectrometer software
to determine OTR using percent oxygen and percent nitrogen from the
reference air (% O.sub.2in and % N.sub.2in) and fermentor off-gas
(% O.sub.2out and % N.sub.2out). Nitrogen is not involved in
cellular respiration, and therefore, can be used to compensate for
outlet oxygen dilution caused by the formation of CO.sub.2. The
inlet flow is calculated from Equation 2 based on the ideal gas law
and is standardized to 1.0 sLph flow rate and 1.0 L fermentor
working volume to yield a derived value OTR in mmol/L/h from the
mass spectrometer. This derived value OTR is then multiplied by
actual inlet flow rate (sLph) and divided by actual working volume
(L) in fermentation spreadsheets to obtain an OTR for specific
operating conditions.
OTR = [ % O 2 i n - ( % O 2 out * % N 2 i n % N 2 out ) ] * Flow i
n . Equation 1 Flow i n = 1 L h * [ 0.83 atm 0.08206 L atm mol K *
294 K * 1 L ] * 1000 mmol mol . Equation 2 ##EQU00001##
[0303] See the General Methods for a description of how the yeast
transformations were performed, as well as a description of how the
yeast lysate was prepared. The DHAD assay and protein concentration
assay are also described in the general methods section. Strains,
plasmids, and the gene/protein sequences used in Example 1 are
described in Tables 5, 6, and 7, respectively.
TABLE-US-00005 TABLE 5 Genotype of strain disclosed in Example 1.
GEVO Number Genotype GEVO2843 S. cerevisiae CEN.PK2, MATa ura3 leu2
his3 trp1 pdc1.DELTA.::[P.sub.CUP1:Bs_alsS1_coSc:T.sub.CYC1:
P.sub.PGK1: Ll_kivD2: P.sub.ENO2: Sp_HIS5]
pdc5.DELTA.::[LEU2-bla-P.sub.TEF1: ILV3.DELTA.N: P.sub.TDH3:
Ec_ilvC_coSc.sup.Q110V] pdc6.DELTA.::[URA3: bla; P.sub.TEF1:
Ll_kivD2: P.sub.TDH3: Dm_ADH] {evolved for C2
supplement-independence, glucose tolerance and faster growth}
TABLE-US-00006 TABLE 6 Plasmids disclosed in Example 1. Plasmid
Name Relevant Genes/Usage Genotype pGV2227 Plasmid pGV2227 is a
P.sub.TDH3:Ec_ilvC_coSc.sup.Q110V 2micron plasmid
P.sub.TEF1:Ll_ilvD_coSc expressing KARI, DHAD,
P.sub.PGK1-Ll_kivD2_coEc KIVD, and ADH P.sub.ENO2_Ll_adhA 2.mu.
ori, bla, G418R pGV2196 Empty CEN plasmid P.sub.TDH3: empty
P.sub.TEF1: empty P.sub.PGK1: empty CEN ori, bla, HygroR pGV2472
CEN plasmid expressing P.sub.TDH3:Sc_AFT1 AFT1 P.sub.TEF1: empty
P.sub.PGK1:empty CEN ori, bla, HygroR
TABLE-US-00007 TABLE 7 Nucleotide and amino acid sequences of genes
and proteins disclosed in Examples. Protein Source Gene (SEQ ID NO)
Protein (SEQ ID NO) AFT S. cerevisiae Sc_AFT1 (SEQ ID NO: 1)
Sc_Aft1 (SEQ ID NO: 2) S. cerevisiae Sc_AFT2 (SEQ ID NO: 3) Sc_Aft2
(SEQ ID NO: 4) K. lactis Kl_AFT (SEQ ID NO: 13) Kl_Aft (SEQ ID NO:
14) K. marxianus Km_AFT (SEQ ID NO: 29) Km_Aft (SEQ ID NO: 30) I.
orientalis Io_AFT1-2 (SEQ ID NO: 33) Io_Aft1-2 (SEQ ID NO: 34) ALS
B. subtilis Bs_alsS1_coSc (SEQ ID NO: 40) Bs_AlsS1 (SEQ ID NO: 41)
KARI E. coli Ec_ilvC_coSc.sup.Q110V (SEQ ID NO: 42)
Ec_IlvC.sup.Q110V (SEQ ID NO: 43) E. coli Ec_ilvC_coSc.sup.P2D1A1
(SEQ ID NO: 44) Ec_IlvC.sup.P2D1A1 (SEQ ID NO: 45) KIVD L. lactis
Ll_kivd2_coEc (SEQ ID NO: 46) Ll_Kivd2 (SEQ ID NO: 47) DHAD L.
lactis Ll_ilvD_coSc (SEQ ID NO: 48) Ll_IlvD (SEQ ID NO: 49) S.
cerevisiae Sc_ILV3.DELTA.N20 (SEQ ID NO: 50) Sc_Ilv3.DELTA.N20 (SEQ
ID NO: 51) S. mutans Sm_ilvD_coSc (SEQ ID NO: 52) Sm_IlvD (SEQ ID
NO: 53) N. crassa Nc_ILVD2_coSc(SEQ ID NO: 54) Nc_IlvD2 (SEQ ID NO:
55) ADH D. melanogaster Dm_ADH (SEQ ID NO: 56) Dm_Adh (SEQ ID NO:
57) L. lactis Ll_adhA (SEQ ID NO: 58) Ll_AdhA (SEQ ID NO: 59) L.
lactis Ll_adhA.sup.RE1 (SEQ ID NO: 60) Ll_AdhA.sup.RE1 (SEQ ID NO:
61) TFC1 S. cerevisiae TFC1 (SEQ ID NO: 202) Tfc1 (SEQ ID NO:
203)
[0304] GEVO2843 was co-transformed with two plasmids (Table 8).
GEVO3342 contains plasmids pGV2227 and pGV2196; GEVO3343 contains
plasmids pGV2227 and pGV2472.
TABLE-US-00008 TABLE 8 Indicates the strains containing plasmids
transformed together into strain GEVO2843. GEVO Plasmid 1 Plasmid 2
3342 pGV2227 (DHAD) pGV2196 (no AFT1) 3343 pGV2227 (DHAD) pGV2472
(AFT1)
[0305] DHAD Assay Results:
[0306] The in vitro DHAD enzymatic activity of lysates from the
microaerobic fermentation of GEVO3342 and GEVO3343 were carried out
as described above. Overexpression of AFT1 from a CEN plasmid
resulted in a three-fold increase in specific DHAD activity (U/mg
total cell lysate protein). Data is presented as specific DHAD
activity (U/mg total cell lysate protein) averages from technical
triplicates with standard deviations. DHAD activity for GEVO3342
(control) was 0.066.+-.0.005 U/mg and DHAD activity for GEVO3343
(AFT1 over-expressed) was 0.215.+-.0.008 U/mg at the end of the
fermentation (119 h).
[0307] Isobutanol Results:
[0308] Isobutanol titers, rates and yields were calculated based on
the experiment run in batch fermentors. Table 9 shows the increase
in isobutanol titer, rate and yield in the strain overexpressing
the AFT1 gene. The overexpression of AFT1 from a CEN plasmid
(GEVO3343) resulted in an increase in isobutanol titer, an increase
in isobutanol yield, and an increase in isobutanol rate.
TABLE-US-00009 TABLE 9 Isobutanol titer, rate and yield for
replicate fermentation experiments. GEVO3342 GEVO3342 GEVO3343
GEVO3343 control plasmid Aft1 gene on a CEN plasmid Titer (g/L)
3.66 3.96 5.69 5.80 Rate (g/L/h) 0.03 0.03 0.05 0.05 Yield 19 20 34
34 (% theor.)
[0309] Change in Metabolic By-Products:
[0310] The strain transformed with the AFT1 gene expressed on the
CEN plasmid (GEVO3343) produced less pyruvate, acetate, DHIV
(dihydroxyisovalerate)/DH2 MB (2,3-dihydroxy-2-methylbutanoic
acid), and 2,3-butanediol than the strain with the control plasmid
(GEVO3342) during the fermentation. There was a six fold decrease
in pyruvate, one fold decrease in acetate, one and a half fold
decrease in DHIV/DH2 MB, and six fold decrease in
2,3-butanediol.
Example 2
Overexpression of AFT2 Increases DHAD Activity
[0311] The purpose of this example is to demonstrate that
overexpression of AFT2 increases DHAD activity. Methods of strain
construction and cloning techniques are described in Example 1.
Strain GEVO2843 is described in Table 5.
TABLE-US-00010 TABLE 10 Plasmids disclosed in Example 2. Plasmid
Name Relevant Genes/Usage Genotype pGV2247 Plasmid pGV2247 is a 2
P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1A1 micron plasmid expressing
P.sub.TEF1:Ll_ilvD_coSc KARI, DHAD, KIVD, and
P.sub.PGK1-Ll_kivD2_coEc ADH P.sub.ENO2: Ll_adhA 2.mu. ori, bla,
G418R pGV2196 Empty CEN plasmid P.sub.TDH3: empty P.sub.TEF1: empty
P.sub.PGK1: empty CEN ori, bla, HygroR pGV2627 CEN plasmid
expressing P.sub.TDH3:empty AFT2 P.sub.TEF1: empty
P.sub.PGK1:Sc_AFT2 CEN ori, bla, HygroR
Methods
[0312] Methods for yeast transformations and the preparation of
yeast lysates are described in the general methods. The DHAD assay,
the liquid chromatography, method 2, assay, and assays for
measuring protein concentration are described in the general
methods.
[0313] Results for DHAD Activity:
[0314] Data is presented as specific DHAD activity (U/mg total cell
lysate protein) averages from biological and technical triplicates
with standard deviations. DHAD activity in GEVO2843 (Table 5)
transformed with pGV2247+pGV2196 (no AFT2) was 0.358.+-.0.009 U/mg,
DHAD activity for pGV2247+pGV2627 (contains AFT2) was
0.677.+-.0.072 U/mg. The overexpression of AFT2 increased the
amount of DHAD activity in the strain.
Example 3
Overexpression of AFT1 Increases DHAD Activity for DHAD Enzymes
from Multiple Organisms
[0315] The purpose of this example is to demonstrate that
overexpression of AFT1 increases DHAD activity for DHAD enzymes
from multiple organisms.
[0316] Strains and plasmids used in Example 4 are described in
Tables 11 and 12, respectively.
TABLE-US-00011 TABLE 11 Genotype of strains disclosed in Example 3.
GEVO Number Genotype Plasmid GEVO3626 Saccharomyces cerevisiae MATa
ura3 leu2 his3 trp1 gpd1::T.sub.Kl.sub.--.sub.URA3 None
gpd2::T.sub.Kl.sub.--.sub.URA3pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:-
LEU2:T.sub.LEU2-
P.sub.ADH1:Bs_alsS1_coSc:T.sub.CYC1:P.sub.PGK1:Ll_kivD2_coEc:P.sub.ENO2:S-
p_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3873 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2603
gpd2::T.sub.Kl.sub.--.sub.URA3
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3874 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2603
gpd2::T.sub.Kl.sub.--.sub.URA3
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3875 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2607
gpd2::T.sub.Kl.sub.--.sub.URA3
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3876 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2608
gpd2::T.sub.Kl.sub.--.sub.URA3
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3877 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2608
gpd2::T.sub.Kl.sub.--.sub.URA3
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3878 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2608
gpd2::T.sub.Kl.sub.--.sub.URA3
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3879 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2603 +
gpd2::T.sub.Kl.sub.--.sub.URA3 pGV2472
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3880 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2603 +
gpd2::T.sub.Kl.sub.--.sub.URA3 pGV2472
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3881 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2603 +
gpd2::T.sub.Kl.sub.--.sub.URA3 pGV2472
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3928 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2607 +
gpd2::T.sub.Kl.sub.--.sub.URA3 pGV2472
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3929 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2607 +
gpd2::T.sub.Kl.sub.--.sub.URA3 pGV2472
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3930 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2608 +
gpd2::T.sub.Kl.sub.--.sub.URA3 pGV2472
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3931 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2608 +
gpd2::T.sub.Kl.sub.--.sub.URA3 pGV2472
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
GEVO3932 Saccharomyces cerevisiae MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3 pGV2608 +
gpd2::T.sub.Kl.sub.--.sub.URA3 pGV2472
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1: Ll_kivD2_coEc:P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3
pdc6::P.sub.TEF:Ll_ilvD_coSc_P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1-A1:
P.sub.ENO2:Ll_adhA:P.sub.FBA1:Sc_TRP1 {evolved for C2
supplement-independence, glucose tolerance and faster growth}
TABLE-US-00012 TABLE 12 Plasmids disclosed in Example 3. Plasmid
Name Relevant Genes/Usage Genotype pGV2603 Plasmid pGV2603 is a 2
micron P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1A1-his* plasmid expressing
KARI, Ll_IlvD P.sub.TEF1:Ll_ilvD_coSc DHAD, KIVD, and ADH
P.sub.ENO2_Ll_adhA.sup.RE1 2.mu. ori, bla, G418R pGV2607 Plasmid
pGV2607 is a 2 micron P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1A1 plasmid
expressing KARI, Nc_IlvD2 P.sub.TEF1:Nc_ilvD2_coSc DHAD, KIVD, and
ADH P.sub.ENO2_Ll_adhA.sup.RE1 2.mu. ori, bla, G418R pGV2608
Plasmid pGV2608 is a 2 micron P.sub.TDH3:Ec_ilvC_coSc.sup.P2D1A1
plasmid expressing KARI, Sm_IlvD P.sub.TEF1:Sm_ilvD_coSc DHAD,
KIVD, and ADH P.sub.ENO2_Ll_adhA.sup.RE1 2.mu. ori, bla, G418R
pGV2472 CEN plasmid expressing AFT1 P.sub.TDH3: Sc_AFT1 P.sub.TEF1:
empty P.sub.PGK1: empty CEN ori, bla, HygroR *Contains 6-his tags
as compared to Ec_ilvC_coSc.sup.P2D1A1
[0317] Shake Flask Fermentations:
[0318] Fermentations were performed to compare the DHAD enzyme
activity of strains GEVO3879, GEVO3880, GEVO3881, GEVO3928,
GEVO3929, GEVO3930, GEVO3931 and GEVO3932, which overexpress AFT1
from S. cerevisiae from plasmid pGV2472, with strains GEVO3873,
GEVO3874, GEVO3875, GEVO3876, GEVO3877, and GEVO3878, which do not
overexpress AFT1. Strains GEVO3873, GEVO3874, GEVO3879, GEVO3880
and GEVO3881 express the Lactococcus lactis IlvD protein (Ll_IIvD)
from the Ll_iIvD gene on pGV2603. Strains GEVO3875, GEVO3928 and
GEVO3929 express the Neurospora crassa IlvD2 protein (Nc_IlvD2)
from the Nc_ilvD2 gene on pGV2607. Strains GEVO3876, GEVO3877,
GEVO3878, GEVO3930, GEVO3931 and GEVO3932 express the Streptococcus
mutans IlvD protein (Sm_IlvD) from the Sm_ilvD gene on pGV2608.
These plasmids were all present in the same host background strain,
GEVO3626.
[0319] Strains containing plasmid pGV2472 were maintained and grown
in media containing both 0.2 g/L G418 and 0.1 g/L hygromycin while
strains lacking pGV2472 were maintained and grown in media
containing 0.2 g/L G418. Yeast strains were inoculated from cell
patches or from purified single colonies from YPD supplemented with
0.2 g/L G418 medium agar plates or from YPD supplemented with 0.2
g/L G418 and 0.1 g/L hygromycin medium agar plates into 3 mL of
growth medium in 14 mL round-bottom snap-cap tubes to provide three
replicates of strains carrying each plasmid or plasmid combination.
The growth media used were YPD+0.2 g/L G418+1% v/v ethanol medium
for strains lacking pGV2472 and YPD+0.2 g/L G418+0.1 g/L
hygromycin+1% v/v ethanol medium for strains containing pGV2472.
The cultures were incubated for up to 24 h shaking at an angle at
250 rpm at 30.degree. C. Separately for each tube culture, these
overnight cultures were used to inoculate 50 mL of medium in a 250
mL baffled flask with a sleeve closure to an OD.sub.600 of 0.1. The
media used were YP+50 g/L glucose+0.2 g/L G418+1% v/v ethanol
medium for strains lacking pGV2472 and YP+50 g/L glucose+0.2 g/L
G418+0.1 g/L hygromycin+1% v/v ethanol medium for strains
containing pGV2472. These flask cultures were incubated for up to
24 h shaking at 250 rpm at 30.degree. C. The cells from these flask
cultures were harvested separately for each flask culture by
centrifugation at 3000 rcf for 5 min and each cell pellet was
resuspended separately in 5 mL of YP medium supplemented with 80
g/L glucose, 1% v/v stock solution of 3 g/L ergosterol and 132 g/L
Tween 80 dissolved in ethanol, 200 mM MES buffer, pH 6.5, and 0.2
g/L G418. Each cell suspension was used to inoculate 50 mL of YP
medium supplemented with 80 g/L glucose, 1% v/v stock solution of 3
g/L ergosterol and 132 g/L Tween 80 dissolved in ethanol, 200 mM
MES buffer, pH 6.5, and 0.2 g/L G418 in a 250 mL non-baffled flask
with a vented screw-cap to an OD.sub.600 of approximately 5. These
fermentations were incubated shaking at 250 rpm at 30.degree. C.
After 73 h of incubation, the cells from half of each fermentation
culture were harvested by centrifugation at 3000 rcf for 5 min at
4.degree. C. Each cell pellet was resuspended in 25 mL of cold
MilliQ water and then harvested by centrifugation at 3000 rcf for 5
min at 4.degree. C. The supernatant was removed from each pellet
and the tubes containing the pellets were frozen at -80.degree.
C.
[0320] Cell lysate production, total protein quantification, DHAD
assays and liquid chromatography, method 2, were performed as
described in the general methods.
[0321] Overexpression of S. cerevisiae AFT1 Increased the DHAD
Activity of Strains Expressing Different DHAD Enzymes:
[0322] Overexpression of S. cerevisiae AFT1 increased the DHAD
enzyme activity of strains expressing the L. lactis IlvD, N. crassa
IlvD2 and S. mutans IIvD DHADs by at least 2.5-fold (Table 13).
DHAD enzyme activities of the strains expressing the different
DHADs were similar in the absence of AFT1 overexpression but were
at different increased enzyme activity levels in the strains
expressing the different DHADs together with AFT1 overexpression.
This demonstrates that AFT1 overexpression increases the activity
of multiple DHAD enzymes from several different organisms.
TABLE-US-00013 TABLE 13 DHAD enzyme activity results from shake
flask fermentations demonstrating increased DHAD activity from S.
cerevisiae expressing DHAD enzymes from L. lactis, N. crassa and S.
mutans and overexpressing AFT1. DHAD Enzyme Activity (.mu.mol
KIV/min/mg lysate) Expressed DHAD No AFT1 Overexpression AFT1
Overexpression Ll_IlvD 0.27 .+-. 0.02 1.26 .+-. 0.16 Nc_IlvD2 0.29
.+-. 0.05 1.14 .+-. 0.15 Sm_IlvD 0.34 .+-. 0.05 0.85 .+-. 0.08
Example 4
Simultaneous Overexpression of AFT1 and AFT2 Increases DHAD
Activity
[0323] The purpose of this example is to demonstrate that
overexpression of S. cerevisiae AFT1 (Sc_AFT1) and S. cerevisiae
AFT2 (Sc_AFT2) increases DHAD activity.
[0324] Standard molecular biology methods for cloning and plasmid
construction were generally used, unless otherwise noted (Sambrook,
J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed.
2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press). Cloning techniques included gel purification of DNA
fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002,
Zymo Research Corp, Orange, Calif.).
[0325] S. cerevisiae Transformations:
[0326] Co-transformations with the CEN and 2.mu. plasmids into S.
cerevisiae strains are described below. Briefly, the S. cerevisiae
strain GEVO2843 (Table 5) was grown on YPD medium. From the plate,
the strain was re-suspended in 100 mM lithium acetate. Once the
cells were re-suspended, a mixture of DNA (final volume of 15 .mu.L
with sterile water), 72 .mu.L 50% w/v PEG, 10 .mu.L 1 M lithium
acetate, and 3 .mu.L of denatured salmon sperm DNA (10 mg/mL) was
prepared for each transformation. In a 1.5 mL tube, 15 .mu.L of the
cell suspension was added to the DNA mixture (100 .mu.L), and the
transformation suspension was vortexed for 5 short pulses. The
transformation was incubated for 30 min at 30.degree. C., followed
by incubation for 22 min at 42.degree. C. The cells were collected
by centrifugation (18,000 rcf, 10 sec, 25.degree. C.). The cells
were resuspended in 1 mL YPD and after an overnight recovery
shaking at 30.degree. C. and 250 rpm, the cells were spread over
YPD supplemented with 0.2 g/L G418 and 0.1 g/L hygromycin selective
plates. Transformants were then single colony purified onto G418
and hygromycin selective plates.
[0327] Shake Flask Fermentation:
[0328] Fermentations for the AFT1/AFT2 transformant strains were
performed. Starter cultures with each transformed strain were
inoculated into 3 mL YPD with 0.1 g/L hygromycin, 0.2 g/L G418, 1%
v/v EtOH and incubated shaking at 250 rpm at 30.degree. C.
Pre-cultures for the fermentations were inoculated to 0.05
OD.sub.600 into 50 mL YPD (8% w/v glucose) with 200 mM MES, 0.1 g/L
hygromycin, 0.2 g/L G418, 1% v/v stock solution of 3 g/L ergosterol
and 132 g/L Tween 80 dissolved in ethanol, and 20 .mu.M CuSO.sub.4
at pH 6.5 in 250 mL baffled flasks, shaking at 250 rpm at
30.degree. C. Fermentation cultures were inoculated to 4.0-5.0
OD.sub.600 into 50 mL YPD (8% w/v glucose) with 200 mM MES, 0.1 g/L
hygromycin, 0.2 g/L G418, 1% v/v stock solution of 3 g/L ergosterol
and 132 g/L Tween 80 dissolved in ethanol, and 20 .mu.M CuSO.sub.4
at pH 6.5 in 250 mL unbaffled flasks, shaking at 75 rpm at
30.degree. C. All cultures were done in biological triplicate.
[0329] Preparation of Yeast Lysate:
[0330] 50 mL of cells were spun down at 4.degree. C., 3000 rcf for
5 min from the 72 hr timepoint of the fermentation. The medium was
decanted and the cells were resuspended in 10 mL of cold MilliQ
water. The cells were centrifuged a second time at 4.degree. C.,
3000 rcf for 5 min. The medium was again decanted and the cells
were centrifuged at 4.degree. C., 3000 rcf for 5 min. Remaining
media was removed and the cell pellet was frozen at -80.degree. C.
Cells were thawed on ice and resuspended in lysis buffer (50 mM
Tris pH 8.0, 5 mM MgSO.sub.4) such that the result was a 20% cell
suspension by mass. 1000 .mu.L of glass beads (0.5 mm diameter)
were added to a 1.5 mL microcentrifuge tube and 875 .mu.L of cell
suspension was added. Yeast cells were lysed using a Retsch MM301
mixer mill (Retsch Inc. Newtown, Pa.), mixing 6.times.1 min each at
full speed with 1 min incubations on ice between each bead-beating
step. The tubes were centrifuged for 10 min at 23,500 rcf at
4.degree. C. and the supernatant was removed for use. The lysates
were held on ice until assayed.
[0331] DHAD Assay:
[0332] each sample was diluted in DHAD assay buffer (50 mM Tris pH
8, 5 mM MgSO.sub.4) to a 1:10 and 1:100 dilution. Three samples of
each lysate were assayed, along with no lysate controls. 10 .mu.L
of each sample (or DHAD assay buffer) was added to 0.2 mL PCR
tubes. Using a multi-channel pipette, 90 .mu.L of the substrate was
added to each tube (substrate mix was prepared by adding 4 mL DHAD
assay buffer to 0.5 mL 100 mM DHIV). Samples were put in a
thermocycler (Eppendorf Mastercycler) at 35.degree. C. for 30 min
followed by a 5 min incubation at 95.degree. C. Samples were cooled
to 4.degree. C. on the thermocycler, then centrifuged at 3000 rcf
for 5 min. Finally, 75 .mu.L of supernatant was transferred to new
PCR tubes and submitted to analytics for analysis by Liquid
Chromatography, method 2. Yeast lysate protein concentration was
determined as described under General Methods.
[0333] Liquid Chromatography, method 2:
[0334] DNPH reagent (4:1 of 15 mM 2,4-Dinitrophenyl Hydrazine:100
mM Citric Acid pH 3.0) was added to each sample in a 1:1 ratio.
Samples were incubated for 30 min at 70.degree. C. in a
thermo-cycler (Eppendorf, Mastercycler). Analysis of
keto-isovalerate and isobutyraldehyde was performed on an Agilent
1200 High Performance Liquid Chromatography system equipped with an
Eclipse XDB C-18 reverse phase column (Agilent) and a C-18 reverse
phase column guard (Phenomenex). Ketoisovalerate and
isobutyraldehyde were detected using an Agilent 1100 UV detector
(360 nm). The column temperature was 50.degree. C. This method was
isocratic with 70% acetonitrile 2.5% phosphoric acid (0.4%), 27.5%
water as mobile phase. Flow was set to 3 mL/min. Injection size was
10 .mu.L and run time was 2 min.
[0335] Results for DHAD Activity:
[0336] Data is presented as specific DHAD activity (U/mg total cell
lysate protein) averages from biological and technical triplicates
with standard deviations. DHAD activity in GEVO2843 transformed
with pGV2247 (Table 10)+pGV2196 (empty vector, Table 6) was
0.358.+-.0.009 U/mg. DHAD activity for GEVO2843 transformed with
pGV2247+pGV2626 (CEN plasmid that contains Sc_AFT1 and Sc_AFT2;
Genotype: P.sub.TDH3:SC_AFT1, P.sub.TEF1: empty,
P.sub.PGK1:Sc_AFT2, CEN ori, bla, HygroR) was 0.902.+-.0.032 U/mg.
The simultaneous overexpression of Sc_AFT1 and Sc_AFT2 increased
the amount of DHAD activity in the strain.
Example 5
AFT1 Expression Increases DHAD Activity Independently of DHAD
Protein Levels
[0337] The following example illustrates that overexpression of the
AFT1 gene in Saccharomyces cerevisiae leads to increased DHAD
activity independently of DHAD protein levels.
TABLE-US-00014 TABLE 14 Genotype of strains disclosed in Example 5.
GEVO No. Genotype GEVO3882 MATa ura3 leu2 his3 trp1
gpd1::T.sub.Kl.sub.--.sub.URA3
gpd2::T.sub.Kl.sub.--.sub.URA3tma29::T.sub.Kl.sub.--.sub.URA3
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1:Ll_kivD2_coEc: P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3
pdc6::T.sub.Kl.sub.--.sub.URA3.sub.--.sub.short:P.sub.FBA1:Kl_URA3:T.sub.-
Kl.sub.--.sub.URA3 {evolved for C2 supplement-independence, glucose
tolerance and faster growth} [pGV2603] GEVO3901 MATa ura3 leu2 his3
trp1 gpd1::T.sub.Kl.sub.--.sub.URA3
gpd2::T.sub.Kl.sub.--.sub.URA3tma29::T.sub.Kl.sub.--.sub.URA3
pdc1::P.sub.PDC1:Ll_kivD2_coSc5:P.sub.FBA1:LEU2:T.sub.LEU2:P.sub.ADH1:Bs_-
alsS1_coSc:T.sub.CYC1:P.sub.PGK1:Ll_kivD2_coEc: P.sub.ENO2:Sp_HIS5
pdc5::T.sub.Kl.sub.--.sub.URA3
pdc6::P.sub.TDH3:Sc_AFT1:P.sub.ENO2:Ll_adhA.sup.RE1:T.sub.Kl.sub.--.sub.U-
RA3.sub.--.sub.short:P.sub.FBA1-Kl_URA3:T.sub.Kl.sub.--.sub.URA3
{evolved for C2 supplement-independence, glucose tolerance and
faster growth} [pGV2603]
[0338] Media:
[0339] Medium used was standard yeast medium (for example Sambrook,
J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3rd ed.
2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press
and Guthrie, C. and Fink, G. R. eds. Methods in Enzymology Part B:
Guide to Yeast Genetics and Molecular and Cell Biology 350:3-623
(2002)). YP medium contains 1% (w/v) yeast extract, 2% (w/v)
peptone. YPD is YP containing 2% (w/v) glucose.
[0340] Fermentations in Benchtop Fermentors:
[0341] Fermentations in benchtop fermentors were performed to
compare the DHAD enzyme activity and DHAD protein level of GEVO3882
(no AFT1 overexpression) to GEVO3901 (AFT1 overexpression). For
these fermentations, 1 mL from thawed frozen stocks of the strains
were transferred to 500 mL baffled flasks containing 80 mL of YP
medium supplemented with 80 g/L glucose, 5 g/L ethanol, 0.5 g/L
MgSO.sub.4 and 0.2 g/L G418 and incubated for 24 h at 30.degree. C.
in an orbital shaker at 250 rpm. The flask culture for each strain
was transferred to duplicate 2-L top drive motor fermentor vessels
with a working volume of 0.9 L of YP medium supplemented with 80
g/L glucose, 5 g/L ethanol, 0.5 g/L MgSO.sub.4 and 0.2 g/L G418 per
vessel for a starting OD.sub.600 of 0.5. Fermentors were operated
at 30.degree. C. and pH 6.0 controlled with 6N KOH and 2N
H.sub.2SO.sub.4 in a 2-phase aerobic condition based on oxygen
transfer rate (OTR). Initially, fermentors were operated at a
growth phase OTR of 10 mM/h by fixed agitation of 700 rpm and an
air overlay of 5 sL/h. Cultures were grown for 20 h to
approximately 10-130D.sub.600 then immediately switched to a
production aeration OTR=0.5 mM/h by reducing agitation from 700 rpm
to 300 rpm for the period of 20 h to 70.5 h.
[0342] Sample Collection:
[0343] Samples from each fermentor were collected at 15.5 h, 20 h,
27 h, 48.5 h and 70.5 h to measure optical density at 600 nm
(OD.sub.600). A volume of culture equal to 150 OD600 was then
collected from each fermentor at each time point using 60 mL
sterile syringes via a sterile sample port on each vessel and
placed on ice in 500 mL centrifuge bottles. The samples were
centrifuged at 4000 rcf for 10 min at 4.degree. C. to pellet the
cells. The cell pellets were then separately resuspended in 60 mL
cold deionized water for DHAD enzyme assays or cold deionized water
containing Yeast/Fungal Protease Arrest (GBiosciences) for DHAD
protein quantification and separated into 10 mL aliquots which were
centrifuged at 4000 rcf for 10 min at 4.degree. C. to pellet the
cells. The supernatant was removed from each pellet and the
resulting cell pellets were stored frozen at -80.degree. C. until
used to prepare cell lysates.
[0344] Cell Lysate Production:
[0345] Cell lysates were prepared for each frozen sample pellet in
lysis buffer U1, which contains 0.1 M sodium phosphate, pH 7.0, 5
mM MgCl.sub.2 and 1 mM DTT, for DHAD enzyme assays or lysis buffer
U1 containing Yeast/Fungal Protease Arrest (GBiosciences) for DHAD
protein quantification. Each cell pellet was individually suspended
to 20% (w/v) in the appropriate lysis buffer and 1 mL of that cell
suspension was added together with 1000 .mu.L of 0.5 mm diameter
glass beads to a 1.5 mL microcentrifuge tube. The yeast cells were
lysed using a Retsch MM301 mixer mill (Retsch Inc., Newtown, Pa.)
by mixing for six 1-min cycles at full speed with 1-min incubations
on ice between each cycle. The tubes were then centrifuged for 10
min at 23,500 rcf at 4.degree. C. and the supernatant was removed.
Samples for DHAD enzyme assays were held on ice until assayed on
the same day and samples for DHAD protein quantification were
frozen at -20.degree. C. Yeast lysate protein concentration was
determined as described under General Methods.
[0346] DHAD Assay:
[0347] Each cell lysate sample was diluted 1:10 in DHAD assay
buffer (50 mM Tris, pH 8, 5 mM MgSO.sub.4). Three samples of
diluted lysate were assayed, along with three controls of DHAD
assay buffer containing no lysate. 10 .mu.L of each sample or
control was added to 0.2 mL PCR tubes. Using a multi-channel
pipette, 90 .mu.L of substrate mix, prepared by adding 4 mL DHAD
assay buffer to 0.5 mL 100 mM DHIV, was added to each tube. These
tubes were placed in an Eppendorf Mastercycler thermocycler and
incubated at 35.degree. C. for 30 min followed by incubation at
95.degree. C. for 5 min then cooled to 4.degree. C. in the
thermocycler and centrifuged at 3000 rcf for 5 min. 75 .mu.L of
supernatant from each tube was transferred to separate new PCR
tubes and submitted for liquid chromatography analysis for
keto-isovalerate quantification. The DHAD activity was calculated
as .mu.mol KIV produced/min/mg total cell lysate protein in the
assay.
[0348] Liquid Chromatography for Keto-Isovalerate
Quantification:
[0349] 100 .mu.L of DNPH reagent, containing 12 mM
2,4-dinitrophenyl hydrazine, 10 mM citric acid, pH 3.0, 80%
Acetonitrile and 20% MilliQ H.sub.2O, was added to 100 .mu.L of
each sample. The mixtures were then incubated for 30 min at
70.degree. C. in an Eppendorf Mastercycler thermocycler. Analysis
of keto-isovalerate (KIV) was performed on an HP-1200 High
Performance Liquid Chromatography system equipped with an Eclipse
XDB C-18 reverse phase column (Agilent) and a C-18 reverse phase
column guard (Phenomenex). Keto-isovalerate (KIV) was detected
using an HP-1100 UV detector at 210 nm. The column temperature was
50.degree. C. This method was isocratic with 70% acetonitrile to
water as mobile phase with 2.5% dilute phosphoric acid (4%). Flow
was set to 3 mL/min. Injection size was 10 .mu.L and the run time
was 2 min.
[0350] DHAD Protein Quantification:
[0351] Cell lysate samples were prepared for gel electrophoresis by
mixing with appropriate volumes of 4.times.LDS loading buffer
(Invitrogen) and 10.times. reducing agent solution (Invitrogen) and
MilliQ water, followed by incubation at 70.degree. C. for 10 min.
Prepared samples were run on 4-12% acrylamide Bis-Tris gels
(Invitrogen) at 200V for 55 min on the Novex Gel Midi System
(Invitrogen) and protein was subsequently transferred from the gel
to PVDF membrane with the Novex Semi-Dry Blotter (Invitrogen). Gel
electrophoresis and protein transfer were performed according to
the manufacturer's recommendations. PVDF membranes with transferred
proteins were blocked in 2% ECL Advance Blocking Agent (GE
Healthcare) diluted in filtered TBST (150 mM NaCl, 10 mM Tris-HCl,
pH 7.5, 0.5% v/v Tween 20) for 1 h at room temperature under mild
agitation. Membranes were then probed with a 1:500 dilution of
rabbit anti-LI_IIvD or a 1:500 dilution of rabbit anti-Sc_Ilv3
serum for 1 h at room temperature under mild agitation. Membranes
were washed with filtered TBST for 15 min, followed by three 5 min
washes with additional filtered TBST. Membranes were then incubated
with a 1:5000 dilution of goat anti-rabbit AlexaFluor 633-tagged
secondary antibody (Invitrogen) for 1 h at room temperature under
mild agitation while protected from light. Membranes were washed
with TBST as described above while protected from light and then
were dried and scanned on a Storm 860 fluorescence imaging system
(Molecular Dynamics) using the 635 nm laser at 300V and 100 .mu.m
resolution. ImageQuant software (GE Healthcare) was used to perform
standardized densitometry to quantify relative levels of protein
expression, reported as integrated band intensity from the
blots.
[0352] Overexpression of AFT1 Increases DHAD Activity Without
Increasing DHAD Protein Levels:
[0353] DHAD enzyme activity and DHAD protein levels from benchtop
fermentor fermentations are summarized in Tables 15 and 16.
AFT1-overexpressing strain GEVO3901 contains at least 1.5-fold
higher DHAD enzyme activity at all fermentation sample time points
compared with strain GEVO3882 with no AFT1 overexpression (Table
15). The ratio of DHAD enzyme activity in GEVO3901 overexpressing
AFT1 compared to DHAD enzyme activity in strain GEVO3882 with no
AFT1 overexpression was higher during the growth phase of the
fermentation (3.7 at 15.5 h, 3.8 at 20 h) than during the
production phase of the fermentation (2.8 at 27 h, 1.5 at 48.5 h
and 1.8 at 70.5 h).
[0354] DHAD protein levels from AFT1-overexpressing strain GEVO3901
were not substantially different from strain GEVO3882 with no AFT1
overexpression at any of the fermentation sample time points (Table
16). Neither the LI_IIvD nor the Sc_Ilv3 DHAD protein levels were
substantially different from GEVO3901 overexpressing AFT1 compared
with GEVO3882 without AFT1 overexpression at any fermentation
sample time point.
TABLE-US-00015 TABLE 15 DHAD enzyme activity results from
fermentation samples demonstrating increased DHAD activity with
AFT1 overexpression. DHAD Enzyme Activity (.mu.mol KIV/min/mg
lysate protein) Time of No AFT1 Overexpression AFT1 Overexpression
Sample (GEVO3882) (GEVO3901) 15.5 h 0.060 .+-. 0.007 0.224 .+-.
0.009 20.5 h 0.076 .+-. 0.003 0.286 .+-. 0.064 27 h 0.119 .+-.
0.049 0.338 .+-. 0.020 48.5 h 0.262 .+-. 0.026 0.386 .+-. 0.078
70.5 h 0.367 .+-. 0.021 0.652 .+-. 0.083
TABLE-US-00016 TABLE 16 DHAD protein level determinations from
fermentation samples demonstrating no increase in DHAD protein
levels with AFT1 overexpression. Ll_IlvD DHAD Protein Level Sc_Ilv3
DHAD Protein Level (Integrated Band Intensity) (Integrated Band
intensity) Time of No AFT1 AFT1 No AFT1 AFT1 Over- Sample
Overexpression Overexpression Overexpression expression 15.5 h
11941 .+-. 870 11144 .+-. 821 206 .+-. 47 227 .+-. 20 20.5 h 10339
.+-. 830 10634 .+-. 749 225 .+-. 108 260 .+-. 52 27 h 10057 .+-.
636 10065 .+-. 816 256 .+-. 37 244 .+-. 74 48.5 h 9803 .+-. 114
9956 .+-. 273 158 .+-. 6 180 .+-. 41 70.5 h 10010 .+-. 341 11212
.+-. 1922 181 .+-. 15 268 .+-. 25
Example 6
Mutating Sc_AFT1 or Sc_AFT2 to Sc_AFT1.sup.UP or Sc_AFT2.sup.UP
Alleles
[0355] A point mutation in Sc_Aft1 and Sc_Aft2 causes depression of
transcriptional activation in the presence of iron.
Sc_Aft1-1.sup.UP mutation changes Cys291Phe (Yamaguchi-Iwia et al.
1995 EMBO Journal 14: 1231-9). The Sc_Aft2-1.sup.UP mutation
changes Cys187Phe (Rutherford et al. 2001 PNAS 98: 14322-7). The
purpose of this example is to demonstrate that mutating the
endogenous copy of Sc_AFT1 or Sc_AFT2 into the Sc_AFT1-1.sup.up or
Sc_AFT2-1.sup.up mutant alleles generally mimics the overexpression
of Sc_AFT1 or Sc_AFT2 by increasing DHAD activity and isobutanol
titers in yeast strains carrying an isobutanol producing metabolic
pathway.
[0356] In this example, Sc_AFT1 and Sc_AFT2 are replaced in the
genome by Sc_AFT1-1.sup.UP and Sc_AFT2-1.sup.UP alleles, either
individually or together. FIGS. 3 and 4 show the constructs for the
allelic replacement for Sc_AFT1-1.sup.UP (SEQ ID NO: 62) and
Sc_AFT2-1.sup.UP (SEQ ID NO: 63). These constructs are synthesized
by DNA2.0. The constructs are transformed into GEVO2843 (Table 5)
either with pGV2227 (Table 6) or pGV2196 (empty vector control,
Table 6) to yield GEVO6209 and GEVO6210 (Table 17).
[0357] Yeast Transformations:
[0358] Transformations of either the linear Sc_AFT1-1.sup.UP or the
Sc_AFT2-1.sup.UP constructs or pGV2227(or pGV2196) into GEVO2483
are described below. Briefly, the S. cerevisiae strain GEVO2843 is
grown on YPD medium. The strain is re-suspended in 100 mM lithium
acetate. Once the cells are re-suspended, a mixture of DNA (final
volume of 15 .mu.L with sterile water), 72 .mu.L 50% w/v PEG, 10
.mu.L 1 M lithium acetate, and 3 .mu.L of denatured salmon sperm
DNA (10 mg/mL) is prepared for each transformation. In a 1.5 mL
tube, 15 .mu.L of the cell suspension is added to the DNA mixture
(100 .mu.L), and the transformation suspension is vortexed for 5
short pulses. The transformation is incubated for 30 min at
30.degree. C., followed by incubation for 22 min at 42.degree. C.
The cells are collected by centrifugation (18,000 rcf, 10 sec,
25.degree. C.). The cells are resuspended in 1 mL YPD and after an
overnight recovery shaking at 30.degree. C. and 250 rpm, the
transformants are spread over YPD supplemented with 0.2 g/L G418
selective plates. Transformants are then single colony purified
onto G418 selective plates. GEVO2483 containing pGV2227 or pGV2196
and transformed with the linear AFT.sup.UP constructs are plated
onto YPD with 0.2 g/L G418 and 0.1 g/L hygromycin.
TABLE-US-00017 TABLE 17 Genotype of strains disclosed in Example 6.
GEVO Number Genotype GEVO6209 S. cerevisiae CEN.PK2, MATa ura3 leu2
his3 trp1 pdc1.DELTA.::P.sub.CUP1:[Bs_alsS1_coSc:T.sub.CYC1:
P.sub.PGK1: Ll_kivD2_coEc: P.sub.ENO2: Sp_HIS5]
pdc5.DELTA.::[LEU2:bla:P.sub.TEF1: ILV3.DELTA.N20: P.sub.TDH3:
Ec_ilvC_coSc.sup.Q110V] pdc6.DELTA.::[URA3: bla; P.sub.TEF1:
Ll_kivD2_coEC: P.sub.TDH3: Dm_ADH] aft1
.DELTA.::[P.sub.AFT1:AFT1-1.sup.UP:P.sub.ENO2:G418] {evolved for C2
supplement-independence, glucose tolerance and faster growth}.
GEVO6210 S. cerevisiae CEN.PK2, MATa ura3 leu2 his3 trp1
pdc1.DELTA.::P.sub.CUP1:[Bs_alsS1_coSc:T.sub.CYC1: P.sub.PGK1:
Ll_kivD2_coEc: P.sub.ENO2: Sp_HIS5]
pdc5.DELTA.::[LEU2:bla:P.sub.TEF1: ILV3.DELTA.N20: P.sub.TDH3:
Ec_ilvC_coSc.sup.Q110V] pdc6.DELTA.::[URA3: bla; P.sub.TEF1:
Ll_kivD2_coEC: P.sub.TDH3: Dm_ADH] aft2 .DELTA.::
[P.sub.AFT2:AFT2-1.sup.UP: P.sub.ENO2:G418] {evolved for C2
supplement-independence, glucose tolerance and faster growth}
[0359] Strains that grow on 0.2 g/L G418 and 0.1 g/L hygromycin are
further screened by PCR to determine if the integration has
replaced Sc_AFT1 or Sc_AFT2.
[0360] For AFT1:
[0361] The primer AFT1UP forward (SEQ ID NO: 64) is used with the
primer pENO2R (SEQ ID NO: 65) to yield a 599 base pair product that
will not be present in the parental strain. The primer AFT1UP
forward is used with primer AFT1termR (SEQ ID NO: 66) to ensure
that the parental Sc_AFT1 does not remain in the strain. If
integrated correctly, these primers give an approximately 2210 base
pair product; if the parental Sc_AFT1 remains in the strain the
product size is 584 base pairs. Finally, the Sc_AFT1-1.sup.UP gene
is amplified using the AFT1UPfullF (SEQ ID NO: 67) and pENO2R
primers. This product is submitted for sequencing using the
AFT1UPsequence1 (SEQ ID NO: 68) and AFT1UPsequence2 (SEQ ID NO: 69)
primers to ensure that the proper mutation is in the genome.
[0362] For AFT2:
[0363] Primer AFT2Upforward (SEQ ID NO: 70) is used with primer
pENO2R to yield an approximately 350 base pair product that will
not be present in the parental strain. Primer AFT2UP forward is
used with primer AFT2termR (SEQ ID NO: 71) to ensure that the
parental Sc_AFT2 does not remain in the strain. If integrated
correctly these primers give an approximately 1819 base pair
product. If the parental Sc_AFT2 remains in the strain the product
size is 195 base pairs. Finally, the Sc_AFT2-1.sup.UP gene is
amplified using the AFT2UPfullF (SEQ ID NO: 72) and pENO2R primers.
This product is submitted for sequencing using the AFT2UPsequence1
(SEQ ID NO: 73) and AFT2UPsequence2 (SEQ ID NO: 74) primers to
ensure that the proper mutation is in the genome.
[0364] Preparation of Yeast Cells:
[0365] Yeast strains are grown in 50 mL YPD with 0.2 g/L G418 (if
carrying the AFT.sup.UP allele) to mid-log phase (1-30D.sub.600). A
volume of cells so that 200D.sub.600 of cells are acquired are spun
down at 4.degree. C., 3000 rcf for 5 min. The medium is decanted
and the cells are resuspended in 10 mL of cold MilliQ water. The
cells are centrifuged a second time at 4.degree. C., 3000 rcf for 5
min. The medium is again decanted and the cells are centrifuged at
4.degree. C., 3000 rcf for 5 min. The remaining medium is removed
and the cell pellet is frozen at -80.degree. C.
[0366] DHAD Assays are performed as described in the general
methods section. Yeast lysate protein concentration was determined
as described in the general methods section.
[0367] Gas Chromatography, Liquid chromatography method 1 and
liquid chromatography method 2 are performed as described in the
general methods section.
[0368] Shake-Flask Fermentation:
[0369] Fermentations for the AFT1-1.sup.UP and AFT2-1.sup.UP
transformant strains are performed. Starter cultures with each
transformed strain are inoculated into 3 mL YPD with 0.2 g/L G418
and 1% v/v EtOH and incubated shaking at 250 rpm at 30.degree. C.
Pre-cultures for the fermentations are inoculated to 0.05
OD.sub.600 into 50 mL YPD (8% w/v glucose) with 200 mM MES, 0.2 g/L
G418, 1% v/v stock solution of 3 g/L ergosterol and 132 g/L Tween
80 dissolved in ethanol, and 20 .mu.M CuSO.sub.4 at pH 6.5 in 250
mL baffled flasks, shaking at 250 rpm at 30.degree. C. Fermentation
cultures are inoculated to 5.0 OD.sub.600 into 50 mL YPD (8% w/v
glucose) with 200 mM MES, 0.2 g/L G418, 1% v/v stock solution of 3
g/L ergosterol and 132 g/L Tween 80 dissolved in ethanol, and 20
.mu.M CuSO.sub.4 at pH 6.5 in 250 mL unbaffled flasks, shaking at
75 rpm at 30.degree. C. All cultures are done in biological
triplicate. Samples are collected at 24, 48 and 72 h and analyzed
using the liquid chromatography, method 1, and gas chromatography
protocols.
[0370] Results for DHAD Activity:
[0371] Data is presented as specific DHAD activity (U/mg total cell
lysate protein) averages from biological and technical triplicates
with standard deviations. DHAD activity in GEVO2843 transformed
with pGV2227 is generally expected to be lower than that of
GEVO2843+pGV2227 transformed with either the Sc_AFT1-1.sup.UP or
Sc_AFT2-1.sup.UP allele.
[0372] Results for Isobutanol Fermentation:
[0373] Data is presented as specific isobutanol titer
(g/L/O.sub.D600); averages from biological and technical
triplicates with standard deviations. Isobutanol titers in GEVO2843
transformed with pGV2227 is generally expected to be lower than
that of GEVO2843+pGV2227 transformed with either the
Sc_AFT1-1.sup.UP or Sc_AFT2-1.sup.UP allele.
Example 7
Overexpression of AFT1 in S. cerevisiae Carrying an Isobutanol
Producing Metabolic Pathway Increases AFT Regulon Genes as Measured
by mRNA
[0374] The purpose of this example is to demonstrate that
overexpression of AFT1 in strains expressing an isobutanol
producing metabolic pathway increases the expression of genes in
the AFT regulon in fermentation vessels. This in turn increases
DHAD activity and isobutanol titer, productivity, and yield.
[0375] Media:
[0376] Medium used was standard yeast medium (for example Sambrook,
J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3rd ed.
2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press
and Guthrie, C. and Fink, G. R. eds. Methods in Enzymology Part B:
Guide to Yeast Genetics and Molecular and Cell Biology 350:3-623
(2002)). YP medium contains 1% (w/v) yeast extract, 2% (w/v)
peptone. YPD is YP containing 2% (w/v) glucose. Medium used for the
fermentation was YP with 80 g/L glucose, 0.2 g/L G418, 0.1 g/L
hygromycin, 100 .mu.M CuSO.sub.4.5H.sub.2O and 1% v/v ethanol. The
medium was filter sterilized using a 1 L bottle top Corning PES
0.22 .mu.m filter (431174). Medium was pH adjusted to 6.0 in the
fermenter vessels using 6N KOH.
[0377] Fermentation Vessel Preparation and Operating
Conditions:
[0378] Batch fermentations were conducted using six 2 L top drive
motor DasGip vessels with a working volume of 0.9 L per vessel.
Vessels were sterilized, along with the appropriate dissolved
oxygen probes and pH probes, for 60 min at 121.degree. C. pH probes
were calibrated prior to sterilization, however, dissolved oxygen
probes were calibrated post sterilization in order to allow for
polarization.
[0379] Process Control Parameters:
[0380] Initial volume, 900 mL. Temperature, 30.degree. C. pH 6.0,
pH was controlled using 6N KOH and 2N H.sub.2SO.sub.4 (Table
20).
TABLE-US-00018 TABLE 18 Process Control Parameters. Growth phase
Oxygen transfer rate 10 mM/h Air overlay 5.0 slph Agitation 700 rpm
Dissolved oxygen Not controlled Fermentation phase Oxygen transfer
rate 0.5 mM/h to 1.8 mM/h* Air overlay 5.0 slph Agitation 300
rpm/400 rpm* Dissolved oxygen Not controlled *Oxygen transfer rate
increased from 0.5 mM/h to 1.8 mM/h by increase in agitation from
300 rpm to 400 rpm 56 h post inoculation.
[0381] Fermentation:
[0382] The fermentation was run for 119 h. Vessels were sampled 3
times daily. Sterile 5 mL syringes were used to collect 3 mL of
fermenter culture via a sterile sample port. The sample was placed
in a 2 mL microfuge tube and a portion was used to measure cell
density (OD.sub.600) on a Genesys 10 spectrophotometer (Thermo
Scientific). An additional 2 mL portion was taken in the same
manner as described above, for use in qRT-PCR analysis. This sample
was spun in a microcentrifuge for 1 min at 14,000 rpm.
[0383] Yeast Transformations:
[0384] Co-transformations with the CEN and 2p plasmids are
described below. Briefly, the S. cerevisiae strain GEVO2843 (Table
5) was grown on YPD medium. The strain was re-suspended in 100 mM
lithium acetate. Once the cells were re-suspended, a mixture of DNA
(final volume of 15 .mu.L with sterile water), 72 .mu.L 50% w/v
PEG, 10 .mu.L 1 M lithium acetate, and 3 .mu.L of denatured salmon
sperm DNA (10 mg/mL) was prepared for each transformation. In a 1.5
mL tube, 15 .mu.L of the cell suspension was added to the DNA
mixture (100 .mu.L), and the transformation suspension was vortexed
for 5 short pulses. The transformation was incubated for 30 min at
30.degree. C., followed by incubation for 22 min at 42.degree. C.
The cells were collected by centrifugation (18,000 rcf, 10 sec,
25.degree. C.). The cells were resuspended in 1 mL YPD and after an
overnight recovery shaking at 30.degree. C. and 250 rpm, the cells
were spread over YPD supplemented with 0.2 g/L G418 and 0.1 g/L
hygromycin selective plates. Transformants were then single colony
purified onto G418 and hygromycin selective plates.
[0385] RNA Preparation:
[0386] RNA was isolated using the YeaStar RNAKit.TM. (Zymo Research
Corp. Orange, Calif.). Cells were resuspended in 80 .mu.l of YR
Digestion Buffer, 1 .mu.l RNAsin (Promega, Madison, Wis.) and 5
.mu.l of Zymolyase.TM. (provided with YeaStar RNAKit). The pellet
was completely resuspended by repeated pipetting. The suspension
was incubated at 37.degree. C. for 60 min. Following the
incubation, 160 .mu.l of YR Lysis Buffer was added to the
suspension, which was then mixed thoroughly by vortexing. The
mixture was centrifuged at 7,000 g for 2 min in a microcentrifuge,
and the supernatant was transferred to a Zymo-Spin Column in a
collection tube. The column was centrifuged at 10,000 g for 1 min
in a microcentrifuge. To the column, 200 .mu.l RNA Wash Buffer was
added, and the column was centrifuged for 1 min at full speed in a
microcentrifuge. The flow-through was discarded and 200 .mu.l RNA
Wash Buffer was added to the column. The column was centrifuged for
1 min at 14,000 g in a microcentrifuge. The Zymo-Spin Column was
transferred to a new RNase-free 1.5 mL centrifuge tube, and 60
.mu.l of DNase/RNase-free water was added directly to the column
membrane and let stand for 1 min at room temperature. The RNA was
eluted by centrifugation for 1 min at full speed in the
microcentrifuge. Concentrations were determined by measuring the
OD.sub.260 with the NanoDrop spectrophotometer (Thermo Scientific,
Waltham, Mass. 02454). RNA was stored at -80.degree. C. until
use.
[0387] qRT-PCR Analysis:
[0388] RNA prepared from the fermentation samples (at a dilution of
5 ng/.mu.l) was used as a template for one-step quantitative RT-PCR
using the qScript One-Step SYBR Green qRT-PCR kit (Quanta
Biosciences.TM. Gaithersburg, Md.). Each PCR reaction contained 10
ng of RNA, 0.5 .mu.L of 10 .mu.M forward primer, 0.5 .mu.L of 10
.mu.M reverse primer, 6.1 .mu.L of sterile water, and 10 .mu.L of
the One-Step SYBR Green Master Mix, 0.5 .mu.L RNAsin, and 0.4 .mu.L
of qScript One-Step Reverse Transcriptase. qRT-PCR was done in
triplicate for each sample. For the purpose of normalizing the
experimental samples, qRT-PCR was also done for the TFC1
housekeeping gene. Primers used to target the AFT regulon genes and
for the TFC1 gene are presented in Table 19. The reactions were
incubated in an Eppendorf Mastercycler ep thermocycler (Eppendorf,
Hamburg, Germany) using the following conditions: 50.degree. C. for
10 min, 95.degree. C. for 5 min, 40 cycles of 95.degree. C. for 15
sec and 60.degree. C. for 45 sec (amplification), then 95.degree.
C. for 15 sec, 60.degree. C. for 15 sec, and a 20 min slow ramping
up of the temperature until it reaches 95.degree. C. (melting curve
analysis). The fluorescence emitted by the SYBR dye was measured at
the 60.degree. C. incubation step during each of the 40 cycles, as
well as during the ramping up to 95.degree. C. for melting curve
analysis of the PCR product.
TABLE-US-00019 TABLE 19 Primers used for qRT-PCR analysis to target
the AFT regulon. Target Primer Sequence TFC1 2649
TCCAGGCGGTATTGACAGCAGG (SEQ ID NO: 75) 2650
CAATCTGCAACATCAGGTACCACGG (SEQ ID NO: 76) AFT1 2962
ACGCCAACATCTTCGCAACACTC (SEQ ID NO: 77) 2963 TGCCGGCAGTGGCAAGATTTC
(SEQ ID NO: 78) AFT2 2966 CCTCTTCAAGATCCCATGCATGTCC (SEQ ID NO: 79)
2967 TGTAACCGCACAGAGTAGGCTGC (SEQ ID NO: 80) FET3 2972
TGGCCACTGAAGGTAACGCCG (SEQ ID NO: 81) 2973 CCGGTAGGAATGAAGGCATGCTG
(SEQ ID NO: 82) ENB1 2976 TGGCGCTGAGATTGTGGTCGG (SEQ ID NO: 83)
2977 TGAAGCGTGCACTAGCGTCC (SEQ ID NO: 84) SMF3 2978
TGCCGGGCAAATCGTTTCTGAG (SEQ ID NO: 85) 2979
CTTGTGGCCCAAGGTGGTAAAGACC (SEQ ID NO: 86)
[0389] Standard molecular biology methods for cloning and plasmid
construction were generally used, unless otherwise noted (Sambrook,
J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed.
2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press).
[0390] Cloning techniques included gel purification of DNA
fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002,
Zymo Research Corp, Orange, Calif.).
[0391] GEVO2843 (Table 5) was co-transformed with two plasmids.
GEVO3342 (Table 8) has plasmids pGV2227 (Table 6) and pGV2196
(empty vector, Table 6); GEVO3343 (Table 8) has plasmids pGV2227
(Table 6) and pGV2472 (Table 6-contains Sc_AFT1).
[0392] In Table 20, the fold change data was normalized to the
strain without Sc_AFT1 overexpression at 24 h. Thus, all data
points for the strain without Sc_AFT1 overexpression at 24 h have
been set to one. The overexpression of Sc_AFT1 in S. cerevisiae
strains increased predicted Sc_AFT1 target genes, ENB1 (SEQ ID NO:
123) and FET3 (SEQ ID NO: 91). SMF3 (SEQ ID NO: 159) is predicted
to be more dependent on Sc_AFT2 for expression and SMF3 had a much
weaker response to the overexpression of Sc_AFT1, as can be seen in
Table 20.
TABLE-US-00020 TABLE 20 Fold change in mRNA expression between
strains with and without Sc_AFT1 overexpressed. Expression at 24 h
Expression at 119 h Without With Without With over- over- over-
over- qRT-PCR expression expression expression expression target of
Sc_AFT1 of Sc_AFT1 of Sc_AFT1 of Sc_AFT1 AFT1 1.00 16.17 0.83 7.29
AFT2 1.00 1.02 0.86 0.79 ENB1 1.00 18.00 0.83 7.59 FET3 1.00 31.89
0.92 10.16 SMF3 1.00 5.37 1.23 3.23
[0393] Overexpression of Sc_AFT1 increased gene expression of
targeted genes in the AFT regulon. As shown in Example 1, the
increased expression of Sc_AFT1 in these strains also caused
increased isobutanol titers, production rates and yields and DHAD
activity in fermentations. Thus, it is likely that one or more
genes in the AFT regulon impacts DHAD activity and isobutanol
production.
Example 8
Overexpression of Specific Genes in the AFT1 and AFT2 Requlons
[0394] The purpose of this example is to demonstrate that a
specific gene or genes from the AFT1 or AFT2 regulon are important
for an increase in DHAD activity and isobutanol production.
[0395] Standard molecular biology methods for cloning and plasmid
construction are generally used, unless otherwise noted (Sambrook,
J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed.
2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press).
[0396] Media:
[0397] Medium used is described in the general methods section.
Cloning techniques include gel purification of DNA fragments (using
the Zymoclean Gel DNA Recovery Kit, Cat# D4002, Zymo Research Corp,
Orange, Calif.).
[0398] AFT1 and AFT2 regulon genes presented in Table 21 are
synthesized by DNA 2.0 (Menlo Park, Calif., USA) removing any HpaI
or SacI restriction sites within the genes. The synthesized AFT
regulon genes are cloned behind the PGK1 promoter in pGV2196 (empty
vector-Table 6) creating a series of 50 plasmids that are
co-transformed with pGV2227 (Table 6) into S. cerevisiae strain
GEVO2843 (Table 5). Isobutanol production from strain GEVO2843
containing pGV2227 has been shown to be limited by DHAD activity.
Thus, this provides a suitable background for detecting increases
in DHAD activity and subsequent increases in the production of a
metabolite from a DHAD-requiring biosynthetic pathway, such as an
isobutanol producing metabolic pathway.
TABLE-US-00021 TABLE 21 Genes in the AFT1 and AFT2 Regulon For
Screening DHAD Activity Gene Protein Gene name (SEQ ID NO) (SEQ ID
NO) FIT3 SEQ ID NO: 87 SEQ ID NO: 88 FIT1 SEQ ID NO: 89 SEQ ID NO:
90 FET3 SEQ ID NO: 91 SEQ ID NO: 92 FRE1 SEQ ID NO: 93 SEQ ID NO:
94 FTR1 SEQ ID NO: 95 SEQ ID NO: 96 FIT2 SEQ ID NO: 97 SEQ ID NO:
98 COT1 SEQ ID NO: 99 SEQ ID NO: 100 OYE3 SEQ ID NO: 101 SEQ ID NO:
102 TIS11/CTH2 SEQ ID NO: 103 SEQ ID NO: 104 VMR1 SEQ ID NO: 105
SEQ ID NO: 106 AKR1 SEQ ID NO: 107 SEQ ID NO: 108 BIO5 SEQ ID NO:
109 SEQ ID NO: 110 YOR387C SEQ ID NO: 111 SEQ ID NO: 112 YDR271C
SEQ ID NO: 113 SEQ ID NO: 114 YMR034C SEQ ID NO: 115 SEQ ID NO: 116
FRE2 SEQ ID NO: 117 SEQ ID NO: 118 ARN1 SEQ ID NO: 119 SEQ ID NO:
120 ATX1 SEQ ID NO: 121 SEQ ID NO: 122 ENB1/ARN4 SEQ ID NO: 123 SEQ
ID NO: 124 SIT1/ARN3 SEQ ID NO: 125 SEQ ID NO: 126 ARN2 SEQ ID NO:
127 SEQ ID NO: 128 TAF1/TAF130/TAF145 SEQ ID NO: 129 SEQ ID NO: 130
FRE5 SEQ ID NO: 131 SEQ ID NO: 132 FRE6 SEQ ID NO: 133 SEQ ID NO:
134 FRE3 SEQ ID NO: 135 SEQ ID NO: 136 BNA2 SEQ ID NO: 137 SEQ ID
NO: 138 ECM4/GTO2 SEQ ID NO: 139 SEQ ID NO: 140 HSP26 SEQ ID NO:
141 SEQ ID NO: 142 YAP2/CAD1 SEQ ID NO: 143 SEQ ID NO: 144
LAP4/APE1/YSC1/API SEQ ID NO: 145 SEQ ID NO: 146 ECL1 SEQ ID NO:
147 SEQ ID NO: 148 OSW1 SEQ ID NO: 149 SEQ ID NO: 150 NFT1 SEQ ID
NO: 151 SEQ ID NO: 152 YBR012C SEQ ID NO: 153 SEQ ID NO: 154
YOL083W SEQ ID NO: 155 SEQ ID NO: 156 ARA2 SEQ ID NO: 157 SEQ ID
NO: 158 SMF3 SEQ ID NO: 159 SEQ ID NO: 160 MRS4 SEQ ID NO: 161 SEQ
ID NO: 162 ISU1/NUA1 SEQ ID NO: 163 SEQ ID NO: 164 FET4 SEQ ID NO:
165 SEQ ID NO: 166 FET5 SEQ ID NO: 167 SEQ ID NO: 168 FTH1 SEQ ID
NO: 169 SEQ ID NO: 170 CCC2 SEQ ID NO: 171 SEQ ID NO: 172 FRE4 SEQ
ID NO: 173 SEQ ID NO: 174 ISU2 SEQ ID NO: 175 SEQ ID NO: 176 HMX1
SEQ ID NO: 177 SEQ ID NO: 178 PCL5 SEQ ID NO: 179 SEQ ID NO: 180
ICY2 SEQ ID NO: 181 SEQ ID NO: 182 PRY1 SEQ ID NO: 183 SEQ ID NO:
184 YDL124w SEQ ID NO: 185 SEQ ID NO: 186
[0399] Yeast Transformations are performed as described in the
general methods section.
[0400] Preparation of Yeast Cells for Enzyme Assays:
[0401] Yeast strains are grown in 50 mL YPD with 0.2 g/L G418 and
0.1 g/L hygromycin to mid-log phase (1-30D.sub.600). A volume of
cells so that 200D.sub.600 of cells are acquired are spun down at
4.degree. C., 3000 rcf for 5 min. The medium is decanted and the
cells are resuspend in 10 mL of cold MilliQ water. The cells are
centrifuged a second time at 4.degree. C., 3000 rcf for 5 min. The
medium is again decanted and the cells are centrifuged at 4.degree.
C., 3000 rcf for 5 min. The remaining media is removed and the cell
pellet is frozen at -80.degree. C.
[0402] Preparation of Yeast Lysate for Enzyme Assays:
[0403] Cell pellets are thawed on ice. Y-PER Plus reagent (Thermo
Scientific #78999) is added to each pellet at a ratio of 12.5 .mu.L
of reagent per one OD of cells and the cells resuspended by
vortexing. The suspension is gently agitated for 20 min at room
temperature. After 20 min, a volume equal to the Y-PER Plus volume
of universal lysis buffer (0.1 M Sodium Phosphate, pH 7.0, 5 mM
MgCl.sub.2, 1 mM DTT) is added. The suspension is shaken for
another 40 min. Samples are centrifuged at 5300 g for 10 min at
room temperature. The clarified lysates are transferred to a fresh
tube and kept on ice until assayed.
[0404] DHAD Assays are performed as described in the general
methods section.
[0405] Yeast lysate protein concentration was determined as
described in the general methods section.
[0406] Gas Chromatography, liquid chromatography method 1 and
liquid chromatography method 2 are performed as described in the
general methods section.
[0407] Shake-Flask Fermentation:
[0408] Fermentations with the AFT regulon gene transformant strains
are performed. Starter cultures with each transformed strain are
inoculated into 3 mL YPD supplemented with 0.2 g/L G418 and 1% v/v
EtOH and incubated shaking at 250 rpm at 30.degree. C. Pre-cultures
for the fermentations are inoculated to 0.05 OD.sub.600 into 50 mL
YPD (8% w/v glucose) with 200 mM MES, 0.2 g/L G418, 1% v/v stock
solution of 3 g/L ergosterol and 132 g/L Tween 80 dissolved in
ethanol, and 20 .mu.M CuSO.sub.4 at pH 6.5 in 250 mL baffled
flasks, shaking at 250 rpm at 30.degree. C. Fermentation cultures
are inoculated to 5.0 OD.sub.600 into 50 mL YPD (8% w/v glucose)
with 200 mM MES, 0.2 g/L G418, 1% v/v stock solution of 3 g/L
ergosterol and 132 g/L Tween 80 dissolved in ethanol, and 20 .mu.M
CuSO.sub.4 at pH 6.5 in 250 mL unbaffled flasks, shaking at 75 rpm
at 30.degree. C. All cultures are done in biological triplicate.
Samples are collected at 24, 48 and 72 h and analyzed using the
liquid chromatography, method 1, and gas chromatography
protocols.
[0409] Results for DHAD Activity:
[0410] Data is presented as specific DHAD activity (U/mg total cell
lysate protein) averages from biological and technical triplicates
with standard deviations. DHAD activity in GEVO2843 transformed
with pGV2227+pGV2196 (empty vector) is generally expected to be
lower than that of GEVO2843 transformed with either AFT1 or AFT2
genes. In addition, GEVO2843 transformed with pGV2227 and clones
containing AFT regulon genes that are important for increasing DHAD
activity will generally have similar or higher DHAD activity to
GEVO2843 transformed with pGV2227 and the AFT1 or AFT2 genes.
[0411] Results for Isobutanol Fermentation:
[0412] Data is presented as specific isobutanol titer
(g/L/OD.sub.600); averages from biological and technical
triplicates with standard deviations. Isobutanol titers in GEVO2843
transformed with pGV2227+pGV2196 (empty vector) are generally
expected to be lower than that of GEVO2843 transformed with either
AFT1 or AFT2 genes. In addition, GEVO2843 transformed with pGV2227
and clones containing AFT regulon genes that are important for
increasing DHAD activity will generally have similar or higher
isobutanol titers to GEVO2843 transformed with pGV2227 and AFT1 or
AFT2.
Example 9
Overexpression of the Kluyveromyces lactis AFT Increases DHAD
Activity in K. lactis
[0413] The purpose of this example is to demonstrate that
overexpression of AFT from K. lactis increases DHAD activity in K.
lactis.
[0414] Standard molecular biology methods for cloning and plasmid
construction were generally used, unless otherwise noted (Sambrook,
J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed.
2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press).
[0415] Cloning techniques included gel purification of DNA
fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002,
Zymo Research Corp, Orange, Calif.).
[0416] Strains and plasmids used in Example 9 are described in
Tables 22 and 23, respectively.
TABLE-US-00022 TABLE 22 Genotype of strains disclosed in Example 9.
GEVO Number Genotype K. lactis MATalpha uraA1 trp1 leu2 lysA1 ade1
lac4-8 [pKD1] GEVO1287 K. lactis MATalpha uraA1 trp1 leu2 lysA1
ade1 lac4-8 [pKD1] + GEVO4378 pGV2273 K. lactis MATalpha uraA1 trp1
leu2 lysA1 ade1 lac4-8 [pKD1] + GEVO6169 pGV2273 Random integrant
of KL_AFT and G418. Linear fragment from plasmid pGV2962 - cut:
SalI, BglII, PfoI
TABLE-US-00023 TABLE 23 Plasmids disclosed in Example 9. Plasmid
Name Relevant Genes/Usage Genotype pGV2273 Plasmid pGV2273 is a
1.6micron vector P.sub.TDH3: Ec_ilvC_coSc.sup.P2D1-A1 that
expresses KARI, KIVD, DHAD and P.sub.TEF1: Ll_ilvD_coSc ADH,
encodes hygromycin resistance. P.sub.PGK1: Ll_kivD2_coEc
P.sub.ENO2: Ll_adhA 1.6.mu. ori, bla, HygroR pGV2796 A CEN plasmid
carrying used as a P.sub.TEF1: Ll_ilvD_coSc backbone for creating
pGV2962 and P.sub.TPI1: G418 pGV2963. P.sub.ENO2: Ll_adhA.sup.REI
CEN ori, bla pGV2962 A CEN plasmid carrying Ll_ilvD, Kl_AFT
P.sub.TEF1: Ll_ilvD_coSc genes, and G418 resistance. The plasmid
P.sub.TPI1: G418 was used to create linearization fragments
P.sub.ENO2: KL_AFT for integration into K. lactis. CEN ori, bla
[0417] K. lactis Strains:
[0418] K. lactis strain GEVO1287 was transformed with pGV2273 to
form GEVO4378. KL_AFT was PCR amplified from template DNA from
strain GEVO4378 using primers oGV3432 (SEQ ID NO: 189) (contains
KpnI) and oGV3433 (SEQ ID NO: 190) (contains AvII). Plasmid pGV2796
and the KL_AFT PCR product were cut with KpnI and AvrII and ligated
together to form plasmid pGV2962. The linear fragment containing
KI_AFT:G418 was obtained by the restriction digest of pGV2962 with
restriction enzymes, Sail, Bg/II and PfoI. The linear KI_AFT:G418
(SEQ ID NO: 201) fragment was randomly integrated by transformation
into GEVO4378 to make GEVO6169.
[0419] Yeast Transformations--K. lactis:
[0420] K. lactis strain GEVO1287 or GEVO4378 was inoculated into a
3 mL YPD culture and incubated overnight at 250 rpm and 30.degree.
C. A 50 mL YPD culture in a baffled 250 mL shake flask was
inoculated and shaken at 30.degree. C. until the K. lactis strain
GEVO1287 reached an OD.sub.600 of 0.83 and K. lactis strain
GEVO4378 reached an OD.sub.600 of 0.79. Cells were made chemically
competent by the following procedure. Cells were collected by
centrifugation at 2700 rcf for 2 min. To wash, cells were
re-suspended with 50 mL of sterile milliQ water and again
centrifuged at 2700 rcf for 2 min. The wash was repeated by
re-suspending cells with 25 mL sterile milliQ water, cells were
collected by centrifugation at 2700 rcf for 2 min. Finally the
cells were resuspend with 1 mL 100 mM lithium acetate (LiOAc) and
transferred to sterile 1.5 mL microcentrifuge tube. Cells were then
collected by centrifugation in microfuge (set to max speed) for 10
sec. The supernatant was removed and the cells were re-suspended
with 4 times the pellet volume of 100 mM LiOAc. Once the cells were
prepared, a mixture of DNA (approximately 1 ug for linear DNA
fragment and about 500 ng of plasmid DNA, wasbrought to 15 .mu.L
with sterile water), 72 .mu.L 50% w/v PEG, 10 .mu.L 1 M lithium
acetate, and 3 .mu.L of denatured salmon sperm DNA (10 mg/mL) was
prepared for each transformation. In a 1.5 mL tube, 15 .mu.L of the
cell suspension was added to the DNA mixture (100 .mu.L), and the
transformation suspension was vortexed for 5 short pulses. The
transformation was incubated for 30 min at 30.degree. C., followed
by incubation for 22 min at 42.degree. C. The cells were collected
by centrifugation (18,000 rcf, 10 sec, 25.degree. C.). The cells
were resuspended in 1 mL YPD and, after an overnight recovery
shaking at 30.degree. C. and 250 rpm, 200 .mu.L of the GEVO1287
transformation was spread over YPD supplemented with 0.1 g/L
hygromycin. 200 .mu.L of the GEVO4378 transformation was spread
over YPD supplemented with 0.1 g/L hygromycin and 0.2 g/L G418.
Transformants were selected at 30.degree. C. Transformants were
then single colony purified onto either hygromycin and G418 or
hygromycin selective plates.
[0421] Preparation of Yeast Lysate:
[0422] K. lactis strains GEVO4378 and GEVO6169 were inoculated into
3 mL of YPD with 0.1 g/L hygromycin and incubated at 30.degree. C.
at 250 rpm overnight culture. After approximately 18 h a 50 mL YPD
or YPD+0.1 g/L hygromycin culture in a baffled 250 mL shake flask
was inoculated and shaken at 250 rpm until the culture reached
approximately 2-30D.sub.600. 200D.sub.600 of cells were harvested
in 15 mL Falcon tubes and centrifuged at 4.degree. C., 3000 rcf for
5 min. The medium was decanted and the cells were re-suspended in 2
mL of ice-cold MilliQ water. The cells were centrifuged a second
time at 4.degree. C., 3000 rcf for 5 min. The supernatant was again
decanted, and the cells were centrifuged at 4.degree. C., 3000 rcf
for 5 min. The remaining medium was removed. The cell pellet was
frozen at -80.degree. C. The cell pellets were thawed on ice and
750 .mu.L of lysis buffer (0.1 M Sodium Phosphate, pH 7.0, 5 mM
MgCl.sub.2, 1 mM DTT) was used to re-suspend each pellet. 800 .mu.L
of re-suspended cell pellet was added to a 1.5 mL centrifuge tube
with 1 mL of 0.5 mm glass beads. The tubes containing the glass
beads and cell suspension were put into the two bead beater blocks
chilled to -20.degree. C. The Retsch MM301 bead beater was set to 1
min and 300 1/sec frequency. To lyse the cells, the cell
suspensions were beat 6 times for 1 min each, with 2 min of cooling
the tubes and the bead beater blocks on ice in between beatings.
After bead beating, the tubes were centrifuged at 4.degree. C. at
21,500 g for 10 min in a tabletop centrifuge. The supernatant was
transferred into 1.5 mL tubes and placed on ice for use in the DHAD
assay. Yeast lysate protein concentration was determined as
described under General Methods.
[0423] DHAD Assay:
[0424] The assay was performed in triplicate for each sample. In
addition, a no lysate control with lysis buffer was included. To
assay each sample, 10 .mu.L of a 1:10 dilution of lysate in lysis
buffer (0.1 M Sodium Phosphate, pH 7.0, 5 mM MgCl.sub.2, 1 mM DTT)
was mixed with 90 .mu.L of assay buffer (5 .mu.L of 0.1 M
MgSO.sub.4, 10 .mu.L of 0.1 M DHIV, and 75 .mu.L 50 mM Tris pH
7.5), and incubated in a thermocycler for 30 min at 30.degree. C.,
then at 95.degree. C. for 5 min. Insoluble material was removed
from the samples by centrifugation at 3000 rcf for 5 min. The
supernatants are transferred to fresh PCR tubes and submitted to
analytics for analysis by liquid chromatography, method 2.
[0425] Liquid Chromatography, Method 2:
[0426] DNPH reagent (4:1 of 15 mM 2,4-Dinitrophenyl Hydrazine:100
mM Citric Acid pH 3.0) was added to each sample in a 1:1 ratio.
Samples were incubated for 30 min at 70.degree. C. in a
thermo-cycler (Eppendorf, Mastercycler). Analysis of
keto-isovalerate was performed on an Agilent 1200 High Performance
Liquid Chromatography system equipped with an Eclipse XDB C-18
reverse phase column (Agilent) and a C-18 reverse phase column
guard (Phenomenex). Ketoisovalerate were detected using an Agilent
1100 UV detector (360 nm). The column temperature was 50.degree. C.
This method was isocratic with 70% acetonitrile 2.5% phosphoric
acid (0.4%), 27.5% water as mobile phase. Flow was set to 3 mL/min.
Injection size was 10 .mu.L and run time was 2 min.
[0427] DHAD Assay Results:
[0428] The in vitro DHAD enzymatic activity of lysates from the
microaerobic fermentation of K. lactis strains was determined as
described above. All values are the specific DHAD activity (U/mg
total cell lysate protein) as averages from technical triplicates.
In K. lactis, overexpression of the KI_AFT gene resulted in an
increase in DHAD activity (U/mg total cell lysate protein).
GEVO4378 without KI_AFT overexpression had an activity of
0.053.+-.0.009 U/mg while GEVO6169, overexpressing KI_AFT had a
specific activity of 0.131.+-.0.012 U/mg.
Example 10
Overexpression of the Kluyveromyces marxianus AFT
[0429] The purpose of this example is to demonstrate that
overexpression of K. marxianus AFT (Km_AFT) is generally expected
to increase DHAD activity in K. marxianus.
[0430] Standard molecular biology methods for cloning and plasmid
construction are generally used, unless otherwise noted (Sambrook,
J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3 ed.
2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press). Cloning techniques include gel purification of DNA
fragments (using the Zymoclean Gel DNA Recovery Kit, Cat# D4002,
Zymo Research Corp, Orange, Calif.).
[0431] Strains used in Example 10 are described in Table 24.
TABLE-US-00024 TABLE 24 Genotype of strains disclosed in Example
10. GEVO Number Genotype K. marxianus K. marxianus, NRRL-Y7571
GEVO1068 K. marxianus ura3.DELTA. GEVO1947 K. marxianus ura3.DELTA.
GEVO6222 Random integration of:
P.sub.KmPDC:Ll_ilvD:P.sub.TPI:G418:P.sub.PGK1:Km_AFT:T:.sub.ScAFT
K. marxianus .DELTA.ura3 GEVO6223 Random integration of:
P.sub.KmPDC:Ll_ilvD:P.sub.TPI:G418:P.sub.PGK1
[0432] In this example, the K. marxianus URA3 gene was deleted by
transformation of GEVO1068 with a PCR fragment (SEQ ID NO: 191) of
K. marxianus URA3 carrying a deletion of 348 base pairs that was
amplified from pGV1799 (SEQ ID NO: 192) using primers oGV394 (SEQ
ID NO: 193) and oGV395 (SEQ ID NO: 194). The K. marxianus ura3
deletion strain transformants were selected by plating on 5-FOA
(5-fluoroorotic acid) plates (For 500 mL: 10 g agar, 400 mL
dH.sub.2O, 0.5 g 5-FOA (in 5 mL DMSO), 50 mL 10Xa.a (14 g yeast
synthetic drop-out supplement (US Biological) dissolved in 1 L
water), 3.35 g YNB, 10 g glucose, 10 mL 50.times.HIS (0.95 g
histidine/250 mL H.sub.2O), 10 mL 50.times.TRP (1.9 g in 500 mL
H.sub.2O), 10 mL 10.times.LEU (4.75 g Leucine/250 mL H.sub.2O),
3.15 mL 25.times.URA (0.475 g uracil/250 mL H.sub.2O). The 5-FOA
resistant colonies were confirmed for the correct phenotype
(auxotrophic for uracil). PCR demonstrated a partial deletion of
approximately 200 by in the ura3 gene and this strain was named
GEVO1947.
[0433] A linear DNA fragment containing Km_AFT, LI_ilvD, and a G418
resistance marker (SEQ ID NO: 195, FIG. 5) is synthesized by
DNA2.0. The fragment is randomly integrated by transformation into
K. marxianus strain GEVO1947 to obtain GEVO6222. A linear fragment
containing LI_ilvD and a G418 marker is also synthesized by DNA2.0
(SEQ ID NO: 196, FIG. 6) and is randomly integrated by transforming
K. marxianus strain GEVO1947 to obtain GEVO6223.
[0434] Transformations are carried out as follows: K. marxianus
strain GEVO1947 is incubated in 50 mL of YPD medium (1% (w/v) yeast
extract, 2% (w/v) peptone, 2% (w/v) glucose) shaking at 250 RPM at
30.degree. C. until the culture is at an OD.sub.600 of
approximately 5. The cells are collected in a sterile 50 mL conical
tube by centrifugation (1600 rcf, 5 min at room temperature). The
cells are then resuspended in 10 mL of electroporation buffer (10
mM Tris-HCl, 270 mM sucrose, 1 mM MgCl.sub.2, pH 7.5), and
collected at 1600 rcf for 5 min at room temperature. The cells are
then resuspended in 10 mL IB (YPD medium, 25 mM DTT, 20 mM HEPES,
pH 8.0; prepared fresh by diluting 100 .mu.L of 2.5M DTT and 200
.mu.L of 1 M HEPES, pH 8.0 into 10 mL of YPD) and are incubated for
30 min, 250 RPM, 30.degree. C. (tube standing vertical). The cells
are collected at 1600 rcf for 5 min at room temperature and
resuspended in 10 mL of chilled electroporation buffer. The cells
are then pelleted at 1600 rcf for 5 min at 4.degree. C. The cells
are then resuspended in 1 mL of chilled electroporation buffer and
transferred to a microfuge tube. The cells are collected by
centrifugation at >10,000 rcf for 20 sec at 4.degree. C. The
cells are then resuspended in an appropriate amount of chilled
electroporation buffer for a final biomass concentration of
300D.sub.600/mL. 400 .mu.L of cell suspension is added to a chilled
electroporation cuvette (0.4 cm gap) and 50 .mu.L of DNA (SEQ ID
NO: 195 or SEQ ID NO: 196 or water control) is added and mixed by
pipetting up and down, and the cuvette is incubated on ice for
15-30 min. The samples are then electroporated at 1.8 kV, 1000 Ohm,
25 .mu.F. The samples are transferred to a 50 mL tube with 1 mL YPD
medium, and the samples are incubated for 4 h at 250 rpm at
30.degree. C. 200 .mu.L of each transformation culture are spread
onto YPD plates containing 0.2 g/L G418 and the plates are
incubated at 30.degree. C. until individual colonies develop.
[0435] K. marxianus strain GEVO6222 is verified by colony PCR for
the integration of Km_AFT using primers PGK1F (SEQ ID NO: 197) and
KmAFTR (SEQ ID NO: 198) (yielding an approximately 325 base pair
product) and integration of LI_ilvD using primers oGV2107 (SEQ ID
NO: 199) and oGV2108 (SEQ ID NO: 200) (yielding an approximately
104 base pair product). K. marxianus strain GEVO6223 is verified by
colony PCR for the integration of LI_ilvD using primers oGV2107 and
oGV2108.
[0436] Next, K. marxianus strains GEVO1947, GEVO6222 and GEVO6223
are inoculated into 3 mL of YPD medium (1% (w/v) yeast extract, 2%
(w/v) peptone, 2% (w/v) glucose) and incubated at 30.degree. C. at
250 rpm. After approximately 18 h, a 50 mL YPD culture in a baffled
250 mL shake flask is inoculated and shaken at 250 rpm until the
culture reaches approximately 2-30D.sub.600. Cell pellets are
prepared by taking 200D units of culture [OD.sub.600nm.times.
volume (mL)=20] and centrifuging the appropriate volume at 3000 rpm
and 4.degree. C. for 5 min. The medium is decanted and the cells
are resuspended in 2 mL of ice-cold MilliQ water. The cells are
centrifuged a second time at 4.degree. C., 3000 rcf for 5 min. The
supernatant is again decanted, and the cells are centrifuged at
4.degree. C., 3000 rcf for 5 min. The remaining medium is removed.
The cell pellet is frozen at -80.degree. C. To prepare lysate, the
cell pellets are thawed on ice and 750 .mu.L of lysis buffer (0.1 M
Sodium Phosphate, pH 7.0, 5 mM MgCl.sub.2, 1 mM DTT) is used to
re-suspend each pellet. 800 .mu.L of re-suspended cell pellet is
added to a 1.5 mL centrifuge tube with 1 mL of 0.5 mm glass beads.
The tubes containing the glass beads and cell suspension are put
into the two bead beater blocks chilled to -20.degree. C. A Retsch
MM301 bead beater is set to 1 min and 300 l/sec frequency. To lyse
the cells, the cell suspensions are beat 6 times for 1 min each,
with 2 min of cooling the tubes and the bead beater blocks on ice
in between beatings. After bead beating, the tubes are centrifuged
at 4.degree. C. at 21,500 g for 10 min in a tabletop centrifuge.
The supernatant is transferred into 1.5 mL tubes and placed on ice
for use in the DHAD activity assay. Yeast lysate protein
concentration is determined as described under General Methods.
[0437] DHAD assays are performed as described in the general
methods section Liquid chromatography method 2 is performed as
described in the general methods section.
[0438] Results for DHAD Activity:
[0439] Data is presented as specific DHAD activity (U/mg total cell
lysate protein) averages from biological and technical triplicates
with standard deviations. DHAD activity in GEVO6223, containing
DHAD is generally expected to be lower than that of GEVO6222
containing both Km_AFT and DHAD.
Example 11
Construction of Issatchenkia orientalis Strain with Isobutanol
Pathway Genes Integrated into the Genome
[0440] The purpose of this example is to demonstrate that
overexpression of Issatchenkia orientalis AFT1-2 (herein referred
to as lo_AFT1-2) increases DHAD activity in I. orientalis.
[0441] An I. orientalis strain derived from PTA-6658 (US
2009/0226989) was grown overnight and transformed using the lithium
acetate method as described in Gietz, et al (1992, Nucleic Acids
Research 20: 1524). The strain was transformed with homologous
integration constructs using native I. orientalis promoters to
drive protein expression. Issatchenkia orientalis strains used are
described in Table 25.
TABLE-US-00025 TABLE 25 Genotype of strains disclosed in Example
11. Strain Number Genotype GEVO6155 ura3/ura3
gpd1.DELTA.::P.sub.lo.sub.--.sub.PDC: Ll_adhA.sup.RE1:
T.sub.ScCYC1: P.sub.lo.sub.--.sub.TDH3: Ec_ilvC.sup.P2D1-A1:
T.sub.ScGAL10: loxP: lo_URA3: loxP: P.sub.lo.sub.--.sub.ENO1:
Ll_ilvD-1/ gpd1.DELTA.:: P.sub.lo.sub.--.sub.PDC:Ll_adhA.sup.RE1:
T.sub.ScCYC1: P.sub.lo.sub.--.sub.TDH3: Ec_ilvC.sup.P2D1-A1:
T.sub.ScGAL10: loxP: Sc_MEL5: loxP:
P.sub.lo.sub.--.sub.ENO1:Ll_ilvD-1
TMA29/tma29.DELTA.::P.sub.lo.sub.--.sub.PDC1:Ll_adhA.sup.RE1:P.sub.lo.sub-
.--.sub.TDH3:Ec_ilvC.sup.P2D1-A1: loxP: lo_URA3: loxP: P.sub.lo
ENO1: Ll_ilvD-4 GEVO6162 ura3/ura3
gpd1.DELTA.::P.sub.lo.sub.--.sub.PDC: Ll_adhA.sup.RE1:
T.sub.ScCYC1: P.sub.lo.sub.--.sub.TDH3: Ec_ilvC.sup.P2D1-A1:
T.sub.ScGAL10: loxP: lo_URA3: loxP: P.sub.lo.sub.--.sub.ENO1:
Ll_ilvD-1/ gpd1.DELTA.:: P.sub.lo.sub.--.sub.PDC:Ll_adhA.sup.RE1:
T.sub.ScCYC1: P.sub.lo.sub.--.sub.TDH3: Ec_ilvC.sup.P2D1-A1:
T.sub.ScGAL10: loxP: Sc_MEL5: loxP:
P.sub.lo.sub.--.sub.ENO1:Ll_ilvD-1 (SEQ ID NO: 204)
TMA29/tma29.DELTA.:: P.sub.lo.sub.--.sub.PDC1: Ll_adhA.sup.RE1:
P.sub.lo.sub.--.sub.TDH3: Ec_ilvC.sup.P2D1-A1: loxP: lo_URA3: loxP:
P.sub.ENO1:Ll_ilvD-4 (SEQ ID NO: 206): P.sub.PYK1:lo_AFT1-2
GEVO6203 ura3/ura3 gpd1.DELTA.::P.sub.lo.sub.--.sub.PDC:
Ll_adhA.sup.RE1: T.sub.ScCYC1: P.sub.lo.sub.--.sub.TDH3:
Ec_ilvC.sup.P2D1-A1: T.sub.ScGAL10: loxP: lo_URA3: loxP:
P.sub.lo.sub.--.sub.ENO1: Ll_ilvD/ gpd1.DELTA.::
P.sub.lo.sub.--.sub.PDC:Ll_adhA.sup.RE1: T.sub.ScCYC1:
P.sub.lo.sub.--.sub.TDH3: Ec_ilvC.sup.P2D1-A1: T.sub.ScGAL10: loxP:
Sc_MEL5: loxP: P.sub.lo.sub.--.sub.ENO1:Ll_ilvD
TMA29/tma29.DELTA.:: P.sub.lo.sub.--.sub.PDC1: Ll_adhA.sup.RE1:
P.sub.lo.sub.--.sub.TDH3:Ec_ilvC.sup.P2D1-A1: loxP: lo_URA3: loxP:
P.sub.ENO1:Ll_ilvD: P.sub.PYK1:lo_AFT1-2
[0442] Three strains were used to demonstrate that the
overexpression of I. orientalis AFT1-2 increases DHAD activity in
I. orientalis. GEVO6155 does not contain the heterologous AFT1-2
expression construct, while both GEVO6162 and GEVO6203 have the
heterologous AFT1-2 construct integrated into the genome. All three
strains were cultured in two different conditions and then tested
for DHAD activity.
[0443] In the first condition, cultures were started for each
strain (GEVO6155, GEVO6162, and GEVO6203) in 12 mL YP medium (1%
(w/v) yeast extract, 2% (w/v) peptone) containing 5% (w/v) glucose
and incubated at 30.degree. C. and 250 RPM for 9 h. The OD.sub.600
of the 12 mL cultures was determined and the appropriate volume of
each culture was used to inoculate 50 mL of YP medium containing 8%
glucose in separate 250 mL baffled flasks to an OD.sub.600 of 0.01.
The flasks were incubated at 30.degree. C. and 250 RPM for 18 h. A
total of 800D.sub.600 of cells were harvested and the cell
suspension was transferred to 50 mL Falcon tubes. Cells were
pelleted at 3000 rcf for 5 min at 4.degree. C., and washed twice in
2 mL cold, sterile water. The cell pellets were stored at
-80.degree. C. until analysis by DHAD assay.
[0444] In the second condition, cultures were inoculated at a
starting OD.sub.600 of 0.1 and were incubated at 30.degree. C. with
250 rpm shaker speed for 20 h and then the shaker speed was reduced
to 75 rpm for an additional 28 h prior to sampling. Cells were
washed twice with cold sterile water and stored at -80.degree. C.
until analysis.
[0445] To determine DHAD activity in whole cell lysates, the frozen
cell pellets were thawed on ice and resuspended in 750 .mu.L lysis
buffer (100 mM NaPa.sub.4 pH 7.0, 5 mM MgCl.sub.2 and 1 mM DTT).
One mL of glass beads (0.5 mm diameter) were added to a 1.5 mL
microcentrifuge tube and the entire cell suspension for each strain
was added to separate tubes containing glass beads. Yeast cells
were lysed using a Retsch MM301 bead beater (Retsch Inc. Newtown,
Pa.), bead beating six times for 1 min each at full speed with 1
min icing in between each bead beating step. The tubes were
centrifuged for 10 min at 23,500.times.g at 4.degree. C. and the
supernatant was removed. Supernatants were held on ice until
assayed. Yeast lysate protein concentration was determined as
described under General Methods.
[0446] DHAD assays were performed in triplicate for each sample. In
addition, an assay on a no lysate control with lysis buffer was
performed. To assay each sample, 10 .mu.L of lysate in assay buffer
was mixed with 90 .mu.L of assay buffer (5 .mu.L of 0.1 M
MgSO.sub.4, 10 .mu.L of 0.1 M DHIV, and 75 .mu.L 50 mM Tris pH
7.5), and incubated in a thermocycler (Eppendorf, Mastercycler) for
30 min at 30.degree. C., then at 95.degree. C. for 5 min. Insoluble
material was removed from the samples by centrifugation at 3000 rcf
for 5 min. The supernatants were transferred to fresh PCR tubes.
100 .mu.L DNPH reagent (12 mM 2,4-dinitrophenyl hydrazine, 10 mM
citric acid, pH 3.0, in 80% acetonitrile, 20% MilliQ H.sub.2O) was
added to 50 .mu.L of each sample and 50 .mu.L of MilliQ H.sub.2O.
Samples were incubated for 30 min at 70.degree. C. in a
thermocycler.
[0447] Analysis of keto-isovalerate (KIV) was performed on an
Agilent 1200 High Performance Liquid Chromatography system equipped
with an Eclipse XDB C-18 reverse phase column (Agilent) and a C-18
reverse phase column guard (Phenomenex). Ketoisovalerate was
detected using an Agilent 1100 UV detector (360 nm). The column
temperature was 50.degree. C. This method was isocratic with 70%
acetonitrile 2.5% phosphoric acid (0.4%), 27.5% water as mobile
phase. Flow was set to 3 mL/min. Injection size was 10 .mu.L and
run time was 2 min. KIV was quantified on a 3-point linear
calibration curve.
[0448] The in vitro DHAD enzymatic activity of lysates from the
samples of I. orientalis strains were carried out as described
above. DHAD activity (U/mg total cell lysate protein) is reported
as averages from biological triplicate samples. In I. orientalis,
overexpression of the I. orientalis AFT1-2 gene resulted in an
increase in DHAD activity (U/mg total cell lysate protein). The
cultures harvested at 18 h (samples inoculated at 0.01) had DHAD
activity values as follows: GEVO6155 had an activity of
0.039.+-.0.004 U/mg while GEVO6162 had an activity of
0.082.+-.0.005 U/mg and GEVO6203 had an activity of 0.060.+-.0.011
U/mg. The cultures harvested at 48 h (cultures inoculated at 0.1)
had DHAD activity values as follows: GEVO6155 had an activity of
0.085.+-.0.014 U/mg while GEVO6162 had an activity of
0.155.+-.0.020 U/mg and GEVO6203 had an activity of 0.140.+-.0.033
U/mg. Therefore, this example demonstrates that overexpression of
lo_AFT1-2 increases DHAD activity in I. orientalis.
Example 12
Overexpression of Fe--S Assembly Machinery
[0449] To ascertain the effects of overexpressing a cytosolic
2Fe-2S or 4Fe-4S cluster-containing DHAD with candidate assembly
machinery, the following steps, or equivalent steps can be carried
out. First, the coding sequence for the open reading frame of the
DHAD from spinach or other 2Fe-2S or 4Fe-4S cluster-containing DHAD
is cloned into the high-copy (2 micron origin) S. cerevisiae
expression vector pGV2074, such that expression of the coding
sequence is directed by the PGK1 promoter sequence, yielding
plasmid pGV2074-1. Next, the NifU and NifS genes from Entamoeba
histolytica or the homologous NIF genes from Lactococcus lactis are
successively introduced into the aforementioned vector, eventually
yielding a single plasmid (pGV2074-2) where the expression of all 3
genes is directed by strong constitutive S. cerevisiae promoter
sequences. Plasmids pGV2074-1 and pGV2074-2 are transformed into S.
cerevisiae strain GEVO2244 (relevant genotype, iIv3.DELTA.) and
transformants selected by resistance to Hygromycin B (0.1 g/L). At
least 3 individual colonies arising from each transformation are
cultured, a cell lysate produced, and the DHAD activity present
therein measured, all according to previously-described
methods.
[0450] The foregoing detailed description has been given for
clearness of understanding only and no unnecessary limitations
should be understood there from as modifications will be obvious to
those skilled in the art.
[0451] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
[0452] The disclosures, including the claims, figures and/or
drawings, of each and every patent, patent application, and
publication cited herein are hereby incorporated herein by
reference in their entireties.
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=US20120288910A1).
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=US20120288910A1).
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