U.S. patent application number 10/808717 was filed with the patent office on 2004-10-07 for increased bacterial coa and acetyl-coa pools.
This patent application is currently assigned to Rice University. Invention is credited to Bennett, George Nelson, San, Ka-Yiu, Vadali, Ravishankar V..
Application Number | 20040199941 10/808717 |
Document ID | / |
Family ID | 33102174 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040199941 |
Kind Code |
A1 |
San, Ka-Yiu ; et
al. |
October 7, 2004 |
Increased bacterial CoA and acetyl-CoA pools
Abstract
Methods of increasing the cellular pool of A-CoA and thus
driving the metabolic pathways in the direction of A-CoA containing
metabolites by overexpressing rate limiting enzymes in A-CoA
synthesis. Methods of increasing intracellular levels of CoA and
A-CoA through genetic engineering of bacterial strains in
conjunction with supplementation with precursor molecules.
Inventors: |
San, Ka-Yiu; (Houston,
TX) ; Bennett, George Nelson; (Houston, TX) ;
Vadali, Ravishankar V.; ( Indianapolis, IN) |
Correspondence
Address: |
JENKENS & GILCHRIST
1401 MCKINNEY
SUITE 2600
HOUSTON
TX
77010
US
|
Assignee: |
Rice University
Houston
TX
|
Family ID: |
33102174 |
Appl. No.: |
10/808717 |
Filed: |
March 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60457093 |
Mar 24, 2003 |
|
|
|
60457635 |
Mar 26, 2003 |
|
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Current U.S.
Class: |
800/281 ;
435/468 |
Current CPC
Class: |
C12Y 102/04001 20130101;
C12Y 207/01033 20130101; C12P 7/62 20130101; C12N 9/0008 20130101;
C12N 9/1205 20130101; C12P 1/00 20130101 |
Class at
Publication: |
800/281 ;
435/468 |
International
Class: |
A01H 001/00; C12N
015/82 |
Goverment Interests
[0002] The present invention has been developed with funds from the
National Science Foundation and United States Department of
Agriculture. Therefore, the United States Government may have
certain rights in the invention.
Claims
What is claimed is:
1. A method of manipulating the metabolism of a cell, comprising
elevated expression of one or more enzymes involved in A-CoA
metabolism, wherein said one or more enzymes are involved in one or
more rate limiting steps of A-CoA synthesis.
2. The method of claim 1, wherein the enzymes are selected from the
group consisting of pyruvate dehydrogenase, pyruvate formate lyase,
pyruvate oxidoreductase, pantothenate kinase, phosphopantetheine
adenylyltransferase and combinations thereof.
3. The method of claim 2, where the cell expresses one of the group
consisting of i) overexpresses pantothenate kinase; ii)
overexpresses pantothenate kinase and pyruvate dehydrogenase; iii)
overexpresses pantothenate kinase where the panK gene is under the
control of the lac promoter and additionally overexpresses the ATF2
gene under the control of the ptb promoter; and iii) overexpresses
pantothenate kinase expression plasmid where the panK gene is under
the control of the lac promoter and additionally overexpressing the
ATF2 gene under the control of the ptb promoter, and pyruvate
dehydrogenase.
4. A method of increasing the A-CoA flux in a cell comprising
elevated expression of one or more enzymes involved in A-CoA
metabolism, wherein said one or more enzymes are involved in one or
more rate limiting steps of A-CoA synthesis.
5. The method of claim 4, wherein the enzymes are selected from the
group consisting of pyruvate dehydrogenase, pyruvate formate lyase,
pyruvate oxidoreductase, pantothenate kinase, phosphopantetheine
adenylyltransferase and combinations thereof.
6. A method of manipulating the metabolism of a cell, comprising
deletion of one or more A-CoA utilizing pathways.
7. The method of claim 6, wherein said one or more A-CoA utilizing
pathways are selected from the group consisting of acetate
formation pathway, citrate synthase formation pathway, fatty acid
biosynthesis pathway, malonate formation pathway, and acetoacetate
formation pathway.
8. A method of increasing the A-CoA pools in a cell comprising
deletion of one or more A-CoA utilizing pathways.
9. The method of claim 8, wherein said one or more A-CoA utilizing
pathways are selected from the group consisting of acetate
formation pathway, citrate synthase formation pathway, fatty acid
biosynthesis pathway, malonate formation pathway, and acetoacetate
formation pathway.
10. A method for the biosynthesis of one or more target compounds
comprising increasing the intracellular levels of A-CoA and
directing the increased A-CoA levels towards the biosynthesis of
said one or more target compounds.
11. The method of claim 10, wherein the intracellular levels of
A-CoA are increased by elevated expression of one or more enzymes
involved in A-CoA metabolism.
12. The method of claim 10, wherein the intracellular levels of
A-CoA are increased by deletion of one or more A-CoA utilizing
pathways.
13. The method of claim 10 wherein said one or more target
compounds are selected from the group consisting of succinate,
isoamyl alcohol, isoamyl acetate, esters, PHBs and polyketides.
14. A method of producing isoamyl acetate in a cell comprising
expressing at elevated levels one or more enzymes involved in A-CoA
metabolism, wherein said cell displays increased flux through the
A-CoA node.
15. The method of claim 14 wherein said one or more enzymes are
involved in one or more rate limiting steps of A-CoA synthesis.
16. The method of claim 15, wherein the one or more enzymes are
selected from the group consisting of pyruvate dehydrogenase,
pyruvate formate lyase, pyruvate oxidoreductase, pantothenate
kinase, phosphopantetheine adenylyltransferase and combinations
thereof.
17. A microorganism which expresses one or more enzymes involved in
A-CoA metabolism at elevated levels, wherein said microorganism
displays increased flux through the A-CoA node.
18. The microorganism of claim 17, wherein said one or more enzymes
are involved in one or more rate limiting steps of A-CoA
synthesis.
19. The microorganism of claim 18, wherein the one or more enzymes
are selected from the group consisting of pyruvate dehydrogenase,
pyruvate formate lyase, pyruvate oxidoreductase, pantothenate
kinase, phosphopantetheine adenylyltransferase and combinations
thereof.
20. The microorganism of claim 17, wherein said microorganism is
selected from the goup consisting of ATCC ______, ______, ______,
______, ______, ______.
21. A method of increasing CoA pools, comprising producing
increased levels of pantothenate kinase (PanK) activity in a cell
together with providing increased pantothenic acid levels,
sufficient to increase the pool of CoA in the cell.
22. The method of claim 21, wherein producing increased levels of
PanK activity is achieved by transforming the cell with a vector
that overexpresses the PanK gene and increased pantothenic acid is
provided in a medium used to grow the cells.
23. The method of claim 21, wherein producing increased levels of
PanK activity is achieved by manipulating the host genome to
overexpress the PanK gene and increased pantothenic acid is
provided in the cell medium.
24. A method of increasing synthesis of CoA containing compounds
from a bacterial cell, comprising producing increased levels of
pantothenate kinase (PanK) activity in a cell together with
providing increased pantothenic acid levels, sufficient to increase
the pool of CoA in the cell and drive the synthesis of CoA
containing compounds.
25. The method of claim 24, wherein producing increased levels of
PanK activity is achieved by transforming the cell with a vector
that overexpresses the PanK gene and increased pantothenic acid is
provided in a medium used to grow the cells.
26. The method of claim 24, wherein producing increased levels of
PanK activity is achieved by manipulating the host genome to
overexpresses the PanK gene and increased pantothenic acid is
provided in the cell medium.
Description
PRIOR RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/457,093, filed Mar. 24, 2003 and U.S.
Provisional Application No. 60/457,635, filed Mar. 26, 2003.
REFERENCE TO APPENDIX
[0003] A Sequence Listing, including SEQ ID NO: 1 and 2, is
submitted with this application.
FIELD OF THE INVENTION
[0004] The invention relates to methods of increasing intracellular
levels or flux of CoA and A-CoA through genetic engineering of
bacterial strains in conjunction with supplementation with
precursor molecules. The invention further relates to methods of
increasing the cellular pool or flux of A-CoA and thus driving the
metabolic pathways in the direction of A-CoA containing metabolites
and A-CoA derivatives.
BACKGROUND OF THE INVENTION
[0005] Coenzyme A (CoA) and its thioester derivative Acetyl CoA
(A-CoA) are essential intermediates in numerous biosynthetic and
energy yielding metabolic pathways as well as regulators of several
key metabolic reactions. A-CoA is an important intracellular
metabolite in central carbon metabolism and is a precursor in the
enzymatic synthesis of many useful compounds. A-CoA is formed
during the enzymatic oxidation of pyruvate or fatty acids, and from
free acetate in the presence of the enzyme acetyl-CoA synthase.
There are several key rate limiting steps in the biosynthesis of
A-CoA. The overexpression of the enzymes catalyzing these rate
limiting steps increases the intracellular levels of A-CoA. The
A-CoA node serves as a connecting point at which several metabolic
pathways intersect. Enhancing the A-CoA flux, i.e., the amount of
A-CoA generated in a given time, through the A-CoA node is a useful
strategy for increasing the production of compounds that require
A-CoA for their biosynthesis.
[0006] CoA and A-CoA are precursors to many industrially useful
compounds. A-CoA is also a substrates in alcohol acetyl transferase
reactions that produce various acetate esters. In addition, A-CoA
and its condensation product acetoacetyl-CoA are involved in the
biological production of various polyhydroxybutyrates (PHBs). A-CoA
can be carboxylated to malonyl-CoA and subsequently enter pathways
to isoprenoid and terpenoid compounds through mevalonate. In sum,
enhancing the intracellular pools/flux of A-CoA has implications in
improving the production of the useful compounds derived from
A-CoA.
[0007] Existing methodologies focus on the engineering of metabolic
pathways by overexpressing enzymes that are directly involved in
the production of a target compound. The invention claimed and
described herein differs from existing methodologies in that in the
present invention, cellular metabolism is altered to increase
glycolytic flux and to direct this increased flux towards the
production of precursor molecules such as A-CoA. The increased
production of A-CoA in turn increases the production of target
compounds such as esters, PHBs and polyketides.
[0008] Metabolic engineering has the potential to considerably
improve process productivity by manipulating the throughput of
metabolic pathways. Most current metabolic engineering studies
focus on manipulating enzyme levels through the amplification,
addition, or deletion of a particular pathway. However, cofactors
play an essential role in a large number of biochemical reactions
and their manipulation has the potential to be used, as an
additional tool to achieve desired metabolic engineering goals. In
addition, cofactor manipulation may also provide an additional
means to study cellular metabolism, in particular the interplay
between cofactor levels/fluxes and metabolic fluxes.
SUMMARY OF THE INVENTION
[0009] An aspect of the invention provides a method for increasing
the levels of CoA or A-CoA in an E. coli strain through the genetic
manipulation of the strain. Another aspect of the invention
provides a microorganism with increased intracellular levels of CoA
or A-CoA.
[0010] An aspect of the invention provides a method for
manipulating the metabolism of a cell, comprising expression at
elevated levels of one or more enzymes involved in A-CoA
metabolism, wherein the cell displays increased flux through the
A-CoA node.
[0011] A further aspect of the invention provides a microorganism
which expresses one or more enzymes involved in A-CoA metabolism at
elevated levels, wherein said microorganism displays increased flux
through the A-CoA node.
[0012] An aspect of the invention provides a method of producing
isoamyl acetate in a cell comprising expression at elevated levels
of one or more enzymes involved in A-CoA metabolism, wherein the
cell displays increased flux through the A-CoA node
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The accompanying drawings which are incorporated in and
constitute a part of this specification exemplify the invention and
together with the description, serve to explain the principles of
the invention:
[0014] FIG. 1 illustrates the metabolic pathway at the A-CoA
node;
[0015] FIG. 2 illustrates the involvement of pantothenate kinase in
the biosynthesis of CoA;
[0016] FIG. 3 illustrates a plasmid construct used for the
overexpression of pantothenate kinase;
[0017] FIG. 4 illustrates metabolite concentrations of acetate,
glucose and succinate in bacterial strains overexpressing
pantothenate kinase;
[0018] FIG. 5 illustrates intracellular CoA and A-CoA levels of
steady state chemostat cultures;
[0019] FIG. 6 illustrates intracellular CoA and A-CoA levels in
bacterial strains overexpressing pantothenate kinase;
[0020] FIG. 7 illustrates levels of CoA, A-CoA and isoamyl acetate
in bacterial strains overexpressing pantothenate kinase in the
presence of pantothenate supplement;
[0021] FIG. 8 illustrates the glucose uptake rate of steady state
chemostat cultures;
[0022] FIG. 9 illustrates the acetate production rate of steady
state chemostat cultures;
[0023] FIG. 10 illustrates isoamyl acetate concentrations in the
strains tested; and
[0024] FIG. 11 illustrates pyruvic acid concentrations in the
strains tested.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0025] Reference will now be made in detail to embodiments of the
invention, examples of which are illustrated in the accompanying
drawings.
[0026] An application of this invention can be to increase the
production of esters, PHBs and polyketides. Coenzyme A (CoA) and
A-CoA are precursors to fatty acid biosynthesis. Hence with the
manipulation of CoA and A-CoA, fatty acid biosynthesis can
potentially be altered.
[0027] Esters are an important class of chemical compounds used in
food and flavor industries. Certain of the useful compounds derived
from an increase in the levels of CoA and A-CoA include, but are
not limited to, succinate, isoamyl alcohol and isoamyl acetate.
Esters such as isoamyl acetate may be used in nail polish, lacquer
coatings, plasticizers, food flavoring compounds and other
industrial applications. The increased production of A-CoA is also
useful in the production of target compounds such as esters, PHBs
and polyketides.
[0028] FIG. 1 shows the intersection of metabolic pathways at the
A-CoA node. Pyruvate is oxidatively decarboxylated to A-CoA by
pyruvate dehydrogenase (PDH), which subsequently enters the
tricarboxylic acid (TCA) cycle. In the presence of an alcohol,
A-CoA may be converted to an ester using an alcohol
acetyltransferase (AAT). In the presence of inorganic phosphate
(Pi), A-CoA may be converted to acetyl phosphate by
phosphotransacetylase (PTA), which in turn may be converted to
acetate using acetate kinase (ACK).
[0029] FIG. 2 shows the involvement of pantothenate kinase (PanK)
in the CoA biosynthetic pathway. Also shown is the negative
regulation of PanK by CoA and acetyl CoA.
[0030] In general, the invention relies on the introduction of one
or more genes into a microorganism, which in turn result in
increased intracellular levels of CoA and/or A-CoA. In an
embodiment of the invention, an isolated recombinant construct
comprising the gene encoding PanK is introduced into an E. coli
strain.
[0031] In an alternate embodiment of the invention, an isolated
recombinant construct comprising the gene encoding pyruvate
dehydrogenase (PDH) is introduced into an E. coli strain together
with an isolated recombinant construct comprising the gene encoding
PanK.
[0032] In an embodiment of the invention, an E. coli strain is
transformed with an isolated recombinant construct comprising the
gene encoding PanK, where the panK gene is under the control of the
lac promoter and additionally comprising the ATF2 (Alcohol
Acetyltransferase 2) gene under the control of the ptb
(Phosphotransbutyrylase) promoter.
[0033] In general, the invention relies on the introduction of one
or more genes into a microorganism, where the genes encode enzymes
that catalyze one or more rate limiting steps of A-CoA
biosynthesis. An example of an enzyme involved in a rate limiting
step of A-CoA synthesis is pantothenate kinase. Overexpression of
the gene encoding pantothenate kinase along with simultaneous
supplementation of precursor pantothenic acid, significantly
increases intracellular CoA levels (FIG. 1).
[0034] Another example of an enzyme involved in a rate limiting
step of A-CoA synthesis is pyruvate dehydrogenase. Overexpression
of pyruvate dehydrogenase in the presence of elevated levels of
pantothenate kinase along with simultaneous supplementation of
precursor pantothenic acid, leads to the increased carbon flux from
pyruvate to A-CoA.
[0035] A third example of an enzyme involved in a rate limiting
step of A-CoA synthesis is pyruvate oxidoreductase. Overexpression
of pyruvate oxidoreductase in the presence of elevated levels of
pantothenate kinase along with simultaneous supplementation of
precursor pantothenic acid, leads to the increased carbon flux from
pyruvate to A-CoA.
[0036] The inventive system and methods described herein may be
used to manipulate the production of A-CoA through the
overexpression of any active enzyme that is capable of increasing
the carbon flux through the A-CoA node.
[0037] An embodiment of the invention provides a method of
increasing the intracellular pool of A-CoA by elevated expression
of at least one gene which encodes an enzyme involved in A-CoA
biosynthesis.
[0038] As used herein, the enzymes involved in A-CoA metabolism
includes all enzymes whose elevated expression results in an
increase in the carbon flux through the A-CoA node. These enzymes
include enzymes that mediate the conversion of pyruvate to A-CoA,
as well as enzymes that catalyze one or more rate-limiting steps of
the A-CoA biosynthesis pathway. These enzymes include, but are not
limited to, pyruvate dehydrogenase, pyruvate formate lyase,
pyruvate oxidoreductase, pantothenate kinase, and mixtures
thereof.
[0039] Another important enzyme that plays a role in the
biosynthesis of CoA is phosphopantetheine adenylyltransferase
(CoAD). In an embodiment of the invention, overexpression of CoAD
leads to the increased carbon flux through the A-CoA node.
[0040] In other embodiments of the inventions, the A-CoA level is
enhanced through the deletion of an A-CoA utilizing pathway. An
alternate embodiment of the invention shows an enhancement of A-CoA
levels through the reduction of A-CoA flux through one or more
A-CoA utilizing pathways. Examples of such A-CoA utilizing pathways
include, but are not limited to, acetate formation pathway of
acetate kinase and phosphotransacetylase, the TCA cycle entry of
citrate synthase (citrate synthase formation), the fatty acid
biosynthesis pathway, the formation of malonyl-CoA (malonate
formation), and the condensation of acetyl-CoA via a thiolase
(acetoacetate or acetoacetyl CoA formation). These strategies for
reduction of utilization of A-CoA can be used in combination with
the strategies to increase acetyl-CoA to yield additional
incremental increases that are useful in directing metabolism in
particular types of cells. Additional ways to increase the level of
A-CoA directly through the enzymes that uptake acetic acid such as
A-CoA synthetase or other acyl-CoA synthetases that uptake other
acids (e.g., propionic acid or butyric acid) could be used in
combination with the above-listed strategies.
EXAMPLE 1
Plasmid Construction
[0041] Plasmid pGS367 (Pyruvate dehydrogenase expression plasmid)
was obtained from Dr J. R. Guest of Dept of Molecular Biology and
Biotechnology, University of Sheffield, Sheffield, UK. Plasmid
pSJ380 bearing the panK (Pantothenate Kinase) gene cloned in
pET-15b (NOVAGEN.TM.) vector under the control of T7 promoter was
obtained from Dr. Suzanne Jackowski of Biochemistry Dept, St Jude
Children's Research Hospital, Memphis, Tenn. A 1.5 kb XbaI-BamHI
fragment containing the panK gene was cloned into the high copy
number plasmid pUC19 to yield the construct pRV380, following which
it was cloned into the plasmid pDHK29 using the same restriction
sites to yield the construct pRV480. The construct, pRV480, bearing
the panK gene is compatible with pGS367. The ATF2 (Alcohol
Acetyltransferase 2) gene along with the ptb
(Phosphotransbutyrylase) promoter was amplified by PCR the
construct pTAAT (which carries the ATF2 gene of yeast) as template
DNA. The forward and reverse primers used were as follows:
1 5'-CCCAAGCTTTGTGGATGGAGTTAAGTCAGTAGAAAG-3' (forward primer); [SEQ
ID NO: 1] and 5'-CCATCGATTTAAAGCGACGCAAATTCGCC-3' (reverse primer)
[SEQ ID NO: 2]
[0042] The forward and reverse primers contain HindIII and ClaI
restriction sites respectively, which allowed the amplified PCR
fragment to be cloned into the corresponding restriction sites of
the plasmid pRV480 to yield pATCA (FIG. 3). The newly created pATCA
construct contains panK gene under the control of the lac promoter
and ATF2 gene under the control of the ptb promoter. This newly
constructed plasmid pATCA, bearing the genes panK and ATF2 is
compatible with pGS367.
[0043] Relevant plasmid constructs were transformed into DH10B or
YBS121 bacterial strain to carry out certain exemplary embodiments
of the invention.
[0044] The plasmids used in certain embodiments of the invention
are set forth in Table 1 below. The transformed bacterial strains
used in certain embodiments of the invention are set forth in Table
2 below.
2 TABLE 1 Plasmid Properties pGS367 Pyruvate dehydrogenase
expression plasmid pRV480 Pantothenate kinase expression plasmid
pATCA Pantothenate kinase expression plasmid where the panK gene is
under the control of the lac promoter and additionally containing
the ATF2 gene under the control of the ptb promoter ptac-85
IPTG-inducible bacterial expression vector
[0045]
3TABLE 2 ATCC Recombinant Deposit Strain No. Properties
DH10B(ptac-85, Overexpresses pantothenate kinase pRV480)
DH10B(pGS367, Overexpresses pantothenate kinase and pRV480)
pyruvate dehydrogenase DH10B(ptac-85, Overexpresses pantothenate
kinase pATCA) expression plasmid where the panK gene is under the
control of the lac promoter and additionally containing the ATF2
gene under the control of the ptb promoter DH10B(pGS367,
Overexpresses pantothenate kinase pATCA) expression plasmid where
the panK gene is under the control of the lac promoter and
additionally containing the ATF2 gene under the control of the ptb
promoter, and pyruvate dehydrogenase YBS121 Overexpresses
pantothenate kinase (pATCA, expression plasmid where the panK gene
ptac-85) is under the control of the lac promoter and additionally
containing the ATF2 gene under the control of the ptb promoter
YBS121 Overexpresses pantothenate kinase (pATCA, expression plasmid
where the panK gene pGS367) is under the control of the lac
promoter and additionally containing the ATF2 gene under the
control of the ptb promoter, and pyruvate dehydrogenase
DH10B(pUC19) Control DH10B(pRV380) Overexpresses panK DH10B(pKmAT,
Control pUC19) DH10B(pKmAT, Overexpresses panK pRV380)
EXAMPLE 2
Bioreactor Experiments
[0046] Bioreactor studies were performed in a 1 liter (l) BIOFLO
110.TM. fermentor with 0.5 l working volume to provide a controlled
environment with 0.5 liter working volume. The dilution rate was
maintained at either 0.15/hr or 0.35/hr until it reached a steady
state after 4 to 6 residence times. The temperature was controlled
at 37.degree. C. The pH was measured using a glass electrode
(METTLER-TOLEDO.TM.) and controlled at a set point of 7.0 by adding
3N HNO.sub.3 or 3N NaOH. Dissolved oxygen (DO) was monitored using
a polarographic oxygen electrode (METTLER-TOLEDO.TM.) and the DO
was maintained above 80% saturation by an automated controller
which adjusts the agitation appropriately using a feed back control
loop. The air was filtered through a 0.22-.mu.m inline filter and
delivered to the culture at a flow rate of 2.5 liters/min. The
initial agitation speed was set at 500 rpm. The effluent gases were
bubbled through a 1 M CuSO.sub.4 solution to prevent release of
bacteria. Samples were taken during the steady state phase after 4,
5 and 6 residence times.
EXAMPLE 3
Aerobic Shake Flask Experiments
[0047] Since isoamyl alcohol and isoamyl acetate are volatile
compounds, aerobic shake flask experiments were carried out in
flasks capped with rubber stoppers. The rubber stopper facilitates
headspace gas sampling for analysis of volatile compounds (isoamyl
acetate and isoamyl alcohol) and also prevents their escape from
the flask. For aerobic cultures, 10 ml culture medium was used in a
250 ml Erlenmeyer flask and preliminary experiments have shown that
the high headspace to culture medium ratio (240:10 air-to-liquid
ratio) provided sufficient aeration over the course of the
experiment. The cultures were grown in an orbital shaker at the
required temperature. At the end of the experiment (24 hrs), the
cultures were analyzed for isoamyl acetate production.
EXAMPLE 4
Quantification of Isoamyl Compounds
[0048] Isoamyl alcohol and isoamyl acetate content was determined
by headspace gas chromatography. The flask or the tube, as the case
may be, was heated at 50.degree. C. for 30 minutes and 1 ml of head
space gas was injected into HEWLETT-PACKARD.TM. 6000 series gas
chromatograph equipped with an ALLTECH.TM. 6'.times.1/4".times.2 mm
POROPAK.TM. QS 80/100 column. A 6% ethyl acetate solution was used
as internal standard.
EXAMPLE 5
Acetate Formation in an Aerobic Chemostat
[0049] The specific acetate production rate for the two strains
DH10B(pUC19) and DH10B(pRV480) is shown in FIG. 4. The results show
that the overexpression of PanK leads to an increase in acetate
levels and suggests that higher carbon flux through the A-CoA node
was achieved by expressing PanK. This result was confirmed by the
decreased levels of succinate in the strain expressing PanK (FIG.
4).
EXAMPLE 6
Overexpression of Pantothenate Kinase
[0050] The variation in CoA/A-CoA levels was studied in a batch
reactor to study the overexpression of pantothenate kinase.
[0051] The intracellular CoA/A-CoA levels were studied using the
recombinant strains DH10B(pUC19) and DH10B(pRV480) in a batch
reactor using M9 medium. The results show that the overexpression
of PanK leads to an increase in CoA/A-CoA levels (FIG. 5).
Additionally, the increase in CoA levels is greater than the
observed increase in A-CoA levels.
[0052] The intracellular CoA/A-CoA levels were studied in the same
two strains above in the presence of 5 mM pantothenic acid (FIG.
6a). The strain overexpressing PanK showed higher levels of
intracellular A-CoA in the presence of pantothenic acid relative to
the non-supplemented control experiments.
EXAMPLE 7
Isoamyl Acetate Production
[0053] Two recombinant strains were constructed, DH10B(pKmAT,
pUC19) and DH10B(pKmAT, pRV380). The latter strain overexpresses
PanK and displays higher isoamyl acetate production relative to the
control strain (FIG. 6).
EXAMPLE 8
Coa/A-Coa Levels
[0054] The variation in CoA/A-CoA levels was studied in an aerobic
chemostat to study the coexpression of pyruvate dehydrogenase and
pantothenate kinase, and the results are shown in FIG. 7. The
precursor compound pantothenic acid (5 mM) was supplemented in all
these experiments as a substrate for the overexpressed pantothenate
kinase to increase intracellular CoA/A-CoA levels.
[0055] The intracellular CoA/A-CoA levels were studied using the
recombinant strains DH10B(ptac-85, pRV480) and DH10B(pGS367,
pRV480) in an aerobic chemostat using Luria Broth medium at two
different dilution rates (0.15/hr and 0.35/hr). Both strains
overexpress pantothenate kinase and are supplemented with
pantothenate in the culture medium, which enables them to have an
elevated levels of intracellular CoA/A-CoA. However, only the
strain DH10B(pGS367, pRV480) overexpresses pyruvate dehydrogenase
whereas the strain DH10B(ptac-85, pRV480) carries a control
plasmid. The intracellular levels of CoA/A-CoA are below the
detection limit of HPLC (.about.0.04 nmol) for both the strains at
a dilution rate of 0.15/hr. At such a low dilution rate the E. coli
culture at steady state corresponds more to the stationary phase of
cell growth. This observation is consistent with the observation
that the CoA/A-CoA levels were negligible in the stationary growth
phase.
[0056] At a dilution rate of 0.35/hr, the intracellular CoA/A-CoA
levels were within the detectable range of HPLC. At this higher
dilution rate, the cell culture at steady state corresponds to
exponential growth phase and the intracellular levels of CoA and
A-CoA are significant and detectable. This is again consistent with
earlier studies where high levels of CoA and A-CoA levels were
observed during the exponential growth phase. However, there was no
significant change in the intracellular A-CoA level with the
overexpression of pyruvate dehydrogenase in addition to
pantothenate kinase (FIG. 7).
EXAMPLE 9
Glucose Uptake and Acetate Formation
[0057] The specific glucose uptake rate for the two strains
DH10B(ptac-85, pRV480) and DH10B(pGS367, pRV480) at two different
dilution rates is shown in FIG. 8. Both strains showed higher
glucose uptake rate at the higher dilution rates. At a dilution
rate of 0.35/hr, the control strain DH10B(ptac-85, pRV480),
exhibited a significantly higher uptake rate than DH10B(pGS367,
pRV480), which overexpresses both PanK and PDH.
[0058] The specific acetate production rate for DH10B(pGS367,
pRV480) is significantly higher than the control strain at both
dilution rates (FIG. 9). At the dilution rate of 0.15/hour,
DH10B(pGS367, pRV480) displays a 103% increase in acetate
production. At a dilution rate of 0.35/hour, DH10B(pGS367, pRV480)
displays a 53% increase in acetate production. These results
suggested that higher carbon flux through the A-CoA node was
achieved by co-expressing both PanK and PDH.
EXAMPLE 10
Coexpression of PDH and Pank
[0059] Two recombinant strains were constructed, DH10B(ptac-85,
pATCA) and DH10B(pGS367, pATCA). Both strains overexpress
pantothenate kinase due to which both strains have elevated
CoA/A-CoA levels when the cell culture medium is supplemented with
pantothenate. Similarly both the strains overexpress alcohol
acetyltransferase and therefore can produce isoamyl acetate when
isoamyl alcohol is added externally to the cell culture medium.
However, only the strain DH10B(pGS367, pATCA) overexpresses PDH
thereby enhancing the carbon flux from pyruvate to A-CoA in this
strain. The production of isoamyl acetate was studied in both
strains to elucidate the effect of this coexpression on isoamyl
acetate production. No increase in isoamyl acetate production was
observed upon overexpression of pyruvate dehydrogenase in addition
to pantothenate kinase (data not shown).
[0060] The results of isoamyl acetate production can be explained
if the competition of acetate production pathway at the A-CoA node
is taken into consideration. The enzyme alcohol acetyltransferase
(AAT), which condenses isoamyl alcohol and A-CoA to form isoamyl
acetate, might be competing less effectively with
phosphotransacetylase for the common substrate A-CoA.
Phosphotransacetylase (PTA) catalyses the formation of acetyl
phosphate from A-CoA, the first step in the formation of acetate.
The PTA enzyme has greater affinity towards A-CoA when compared to
AAT. This observation suggests that the acetate production pathway
might be stronger than the ester production pathway and possibly
drains the enhanced carbon flux.
EXAMPLE: 11
Channeling Enhanced Carbon Flux to Isoamyl Acetate Production
[0061] Since the acetate production pathway is more competitive
than the isoamyl acetate production pathway at the A-CoA node, it
was hypothesized that with the inactivation of acetate production
pathway, the carbon flux could be more efficiently channeled to
ester production. Under such conditions the enhanced carbon flux
through the A-CoA node can have a beneficial effect on ester
production. To test this hypothesis, a ackA-pta deletion mutant (a
strain containing mutant copies acetate kinase (ackA) and
phosphoacetyltransferase (pta)) YBS121 was used to construct two
recombinant strains, YBS121(ptac-85, pATCA) and YBS121(pGS367,
pATCA).
[0062] The supplementation of pantothenic acid is necessary in
addition to overexpression of pantothenate kinase to increase
intracellular CoA/A-CoA levels. This
supplementation/non-supplementation of pantothenic acid to the
culture medium was used as control parameter to maintain
intracellular CoA/A-CoA levels at elevated/basal levels. A series
of triplicate experiments were performed to study the effect of
CoA/A-CoA manipulation and PDH overexpression on isoamyl acetate
production both individually and in combination. Even though the
plasmid pATCA, overexpresses PanK, the supplementation of the
precursor pantothenic acid is required to increase CoA/A-CoA
levels. The results of these experiments are shown in FIG. 10.
[0063] The strain YBS121(ptac-85, pATCA) produced 0.07 mM isoamyl
acetate without supplementation of pantothenic acid. Upon
supplementation of pantothenic acid, the isoamyl acetate production
in the same strain increased to 0.16 mM, a 225% increase. These
results indicate that the CoA/A-CoA manipulation leads to a 124%
increase in isoamyl acetate production. However, the strain
YBS121(pGS367, pATCA) produced 0.23 mM isoamyl acetate without
supplementation of pantothenic acid, which is a 223% increase
compared to the control strain YBS121(ptac-85, pATCA) (no
pantothenic acid addition). This result shows that overexpression
of pyruvate dehydrogenase is more efficient in increasing isoamyl
acetate production compared to CoA/A-CoA manipulation. However the
same strain (YBS121(pGS367, pATCA)) produced 0.44 mM of isoamyl
acetate upon supplementation of pantothenic acid. The increase in
isoamyl acetate production is about 5-fold, upon simultaneous
manipulation of CoA/A-CoA levels and enhancing carbon flux from
pyruvate node. This significant increase in isoamyl acetate
production illustrate that the strategies of cofactor manipulation
and carbon flux enhancement are synergistic and much more effective
in increasing isoamyl acetate production, than using either of the
strategies alone.
EXAMPLE 12
PDH and Pank Coexpression
[0064] When the above experiments are repeated without any
supplementation of pantothenic acid, notable differences were
observed in the accumulation of pyruvate and the results are as
shown in FIG. 11. The ackA-pta mutation relieves the highly
competitive phosphotransacetylase enzymatic step of the acetate
formation pathway and makes A-CoA more accessible to alcohol
acetyltransferase. However, the inactivation of the acetate
formation pathway leads to metabolic imbalance at the pyruvate
node. The carbon flux is bottled up at the pyruvate node leading to
excretion of pyruvate to the extracellular medium. The recombinant
strain, YBS121(ptac-85, pATCA), an acetate pathway deletion mutant
strain, produced 13.69 mM of pyruvate as expected. Increasing
intracellular CoA/A-CoA levels increases this excretion slightly.
When the intracellular CoA/A-CoA levels were increased in the
strain YBS121(ptac-85, pATCA) upon pantothenic acid
supplementation, it produced 13.81 mM of pyruvate. Overexpression
of pyruvate dehydrogenase could convert some of this excess
pyruvate to A-CoA leading to a decrease in pyruvate excretion. The
strain YBS121(pGS367, pATCA), which overexpresses pyruvate
dehydrogenase produced only 10.97 mM of pyruvate. This
overexpression of pyruvate dehydrogenase lead to a 21% decrease in
pyruvate accumulation. However, a significant amount of pyruvate is
still excreted even in this case. The same strain YBS121(pGS367,
pATCA) when supplemented with pantothenic acid, produced only 1.1
mM of pyruvate, which is a significant drop in pyruvate excretion,
when compared to the control strain YBS121(ptac-85, pATCA). When
the overexpression of pyruvate dehydrogenase is accompanied by an
increase in availability of CoA, most of the excess pyruvate could
be efficiently converted to A-CoA. The coexpression of pyruvate
dehydrogenase and pantothenate kinase relieved the metabolic
imbalance at pyruvate node and the pyruvate excretion dropped to
negligible levels. This metabolic engineering strategy efficiently
channels the excess carbon flux from pyruvate node to A-CoA node in
an acetate pathway deletion mutant. The drop in pyruvate excretion
leads to a more efficient utilization of the carbon source without
any loss at the pyruvate node.
Sequence CWU 1
1
2 1 36 DNA Artificial primer 1 cccaagcttt gtggatggag ttaagtcagt
agaaag 36 2 29 DNA Artificial primer 2 ccatcgattt aaagcgacgc
aaattcgcc 29
* * * * *