U.S. patent application number 10/983951 was filed with the patent office on 2005-06-30 for modified yeast consuming l-arabinose.
Invention is credited to Becker, Jessica, Boles, Eckhard.
Application Number | 20050142648 10/983951 |
Document ID | / |
Family ID | 26655701 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050142648 |
Kind Code |
A1 |
Boles, Eckhard ; et
al. |
June 30, 2005 |
Modified yeast consuming L-arabinose
Abstract
The present invention relates to a method for producing a
L-arabinose utilizing yeast strain for the production of ethanol,
whereby a yeast strain is modified by introducing and expressing
araA gene (L-arabinose isomerase), araB gene (L-ribulokinase
D.sup.121-N) and araD gene (L-ribulose-5-P 4-epimerase) and
carrying additional mutations in its genome or overexpressing a
TAL1 (transaldolase) gene, enabling it to consume L-arabinose, to
use it as the only carbon source, and to produce ethanol, as well
as a method for producing ethanol using such a modified strain.
Inventors: |
Boles, Eckhard; (Dreieich,
DE) ; Becker, Jessica; (Dusseldorf, DE) |
Correspondence
Address: |
Gauthier & Connors LLP
Suite 3300
225 Franklin Street
Boston
MA
02110
US
|
Family ID: |
26655701 |
Appl. No.: |
10/983951 |
Filed: |
November 8, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10983951 |
Nov 8, 2004 |
|
|
|
PCT/SE03/00749 |
May 7, 2003 |
|
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Current U.S.
Class: |
435/161 ;
435/254.21; 435/483 |
Current CPC
Class: |
C12P 7/06 20130101; Y02E
50/17 20130101; Y02E 50/10 20130101; C12N 15/52 20130101 |
Class at
Publication: |
435/161 ;
435/483; 435/254.21 |
International
Class: |
C12P 007/06; C12N
001/18; C12N 015/74 |
Foreign Application Data
Date |
Code |
Application Number |
May 8, 2002 |
SE |
0201428-0 |
Jul 4, 2002 |
SE |
0202090-7 |
Claims
1. A method for producing an L-arabinose utilizing Saccharomvces
cerevisiae east strain for the production of ethanol, wherein a
yeast strain is modified by introducing and expressing an araA gene
(L-arabinose isomerase), an araB gene (L-ribulokinase) and an araD
gene (L-ribulose-5-P 4-epimerase), and carrying additional
mutations in its genome or overexpressing a TAL1 (transaldolase)
gene, enabling it to consume L-arabinose, and to produce ethanol
thereby from a medium comprising L-arabinose, whereby the yeast
strain is further modified by expressing a mutant form of the E.
coli L-ribulokinase enzyme with reduced activity.
2. The method according to claim 1, wherein the araA gene is a B.
subtilis araA gene.
3. The method according to claim 1, wherein the araA gene is a M.
smegmatis araA gene.
4. The method according to claim 1, wherein the araB gene is a E.
coli araB gene.
5. The method according to claim 1, wherein the araD gene is an E.
coli araD gene.
6. The method according to claim 1, wherein the TAL1 gene is an S.
cerevisiae TAL1 gene.
7. (canceled)
8. The method according to claim 1, wherein the Saccharomyces
cerevisiae strain is a CEN.PK strain, preferably a CEN.PK2-1C.
9. The method according to claim 1, wherein the Saccharomyces
cerevisiae strain is a Saccharomyces cerevisiae W303-strain.
10. (canceled)
11. The method according to claim 1, wherein the yeast strain is
further modified by overexpressing the yeast GAL2 gene.
12. The method according to claim 1, wherein the araB gene is
placed behind a weak promoter.
13. The method according to claim 1, wherein the modifications are
made behind the strong HXT7 promoter fragment on multicopy vectors
in S. cerevisiae CEN.PK-strains.
14. The method according to claim 1, wherein the modifications are
made behind the strong HAX7 promoter fragment on multicopy vectors
in Saccharomyces cerevisiae W303-strains.
15. The method according to claim 1, wherein the amount of
L-arabinose of the growth medium is 2 to 200 g/L.
16. The method according to claim 1, wherein the strain is
Saccharomyces cerevisiae strain JBY25-4M with DSM accession number
15560.
17. The method according to claim 1, wherein the strain is
Saccharomyces cerevisiae strain JBY24-3T with a DSM accession
number 15559.
18. A method for producing ethanol by fermenting yeast, wherein a
modified yeast according to claim 1 ferments a growth medium
containing L-arabinose.
19. The method according to claim 18, wherein the amount of
L-arabinose of the growth medium is 2 to 200 g/L.
Description
TECHNICAL FIELD
[0001] The present invention relates to a modified yeast strain,
preferably a Saccharomyces cerevisiae, consuming L-arabinose while
producing ethanol, as well as a method for producing ethanol.
BACKGROUND OF THE INVENTION
[0002] Fuel ethanol is considered as a suitable alternative to
fossil fuels and it can be produced from plant biomass, which is a
low cost and renewable resource available in large amounts. For
this reason cellulose biomass, which includes agricultural
residues, paper wastes, wood chips, etc., is an ideal abundantly
available source of sugars for the fermentation to ethanol. For
example when glucose is produced from cereals,
hemi-cellulose-containing by-products mainly consisting of the
pentose sugars arabinose and xylose (arabinoxylan) are generated.
These are presently used as a low price cattle feed. But this
resource could be utilized in a more profitable way if it would be
integrated into existing starch processing which yields ethanol and
starch derivatives.
[0003] In the context of conversion of hemi-cellulose sugars,
fermentability of L-arabinose becomes important. The approximation
is often made that hydrolysates generated by dilute acid
pretreatment, contain only D-xylose because this is the most
abundant hemi-cellulose sugar. Resulting from this most studies on
conversion of hemi-cellulose hydrolysates focus on the conversion
of D-xylose. However hemi-cellulose as a heteropolysaccharide
contains pentosans and hexosans. Although xylan is the dominant
pentosan and glucomannan is the dominant hexosan the levels of
arabinan are significant in some biomass materials. In particular
arabinan levels are significant in herbaceous species where it
represents up to 10-20% of total non-glucan carbohydrate. Microbial
biocatalysts selected to develop or ferment hydrolysates derived
from materials with high arabinan content must therefore exhibit
the ability to ferment L-arabinose as well as xylose and preferably
also other sugars to ethanol.
[0004] Many types of yeast, especially Saccharomyces cerevisiae and
related species have traditionally been used for fermenting glucose
based feedstocks to ethanol by anaerobic fermentation because they
are the safest and most effective micro organisms for fermenting
sugars to ethanol. But these superior glucose fermenting yeasts are
unable to ferment xylose and L-arabinose and are also unable to use
these pentose sugars for growth. A few other yeast species such as
Pichia sdpitis and Candida shehatae can ferment xylose to ethanol;
however, they are not as effective as Saccharomyces for
fermentation of glucose and have a relatively low ethanol
tolerance. Thus, they are not suitable for large scale industrial
production of ethanol from biomass. Some yeast can utilize
L-arabinose for growth but no yeast can ferment it to commercial
amounts of ethanol. Unlike yeasts and fungi, most bacteria,
including E. coli and Bacillus subtilis, can utilize L-arabinose
for aerobic growth and are also able to ferment it to various
products including ethanol.
[0005] Sedlak & Ho, Enzyme Microb Technol 28, (2001) pp. 16-24
discloses an expression of E. coli araBAD operon encoding enzymes
for metabolizing L-arabinose in Saccharomyces cerevisiae. The
strain hereby expresses araA, araB and araD, but is incapable of
producing any ethanol.
SUMMARY OF THE INVENTION
[0006] It has now been possible to solve this problem, whereby a
new Saccharomyces cerevisiae yeast strain able to consume
L-arabinose, has been created, and to produce ethanol.
DETAILED DESCRIPTION OF THE PATENT INVENTION
[0007] It has now surprisingly been found possible to overcome the
problem of having a yeast consuming L-arabinose by means of the
present invention by obtaining a method for producing a L-arabinose
utilizing yeast strain for the production of ethanol, which method
is characterized in that a yeast strain is modified by introducing
and expressing B. subtilis araA gene (L-arabinose isomerase), E.
coil araB gene (L-ribulokinase) and E. coli araD gene
(L-ribulose-5-P 4-epimerase), and carrying additional mutations in
its genome or overexpressing the S. cerevisiae TAL1 (transaldolase)
gene, enabling it to consume L-arabinose, and to produce
ethanol.
[0008] The invention will be described more in detail in the
following by reference to a number of experiments described
explaining the nature of the invention.
[0009] The application further encompasses the Saccharomyces
cerevisiae strain JBY25-4M (DSM 15560) and Saccharomyces cerevisiae
strain JBY24-3T (DSM 15559) which were deposited at Deutsche
Sammiung von Mikroorganismen und Zelikulturen on Apr. 4, 2003 under
the Budapest Convention.
[0010] First, the E. coli genes araA (L-arabinose isomerase), araB
(L-ribulokinase) and araD (L-ribulose-5-P 4-epimerase) have been
cloned and overexpressed behind the strong HXT7 promoter fragment
on multicopy vectors in S. cerevisiae CEN.PK-strains. Whereas araA
did not produce any L-arabinose isomerase activity in the yeast
transformants, araB overexpression produced up to 0.7 U/mg protein
L-ribulokinase activity and araD produced up to 0.13 U/mg protein
L-ribulose-5-P 4-epimerase activity. Transformation of CEN.PK2-1C
with all three constructs together did not allow the transformants
to grow on L-arabinose medium. It has been shown that the yeast
galactose permease (Gal2) is able to transport L-arabinose [J.
Bacteriol. 103, 671-678 (1970)]. Simultaneous overexpression of
GAL2 behind the ADH1 promoter together with the bacterial
L-arabinose metabolising genes did also not allow the transformants
to grow on L-arabinose medium.
[0011] Second, cloning and overexpression of the Bacillus subtilis
araA gene behind the strong HXT7 promoter fragment on multicopy
vectors in the S. cerevisiae CEN.PK2-1C strain resulted in an
active protein in-yeast, which produced L-arabinose isomerase
activity in the order of at least some mU/mg protein. Similarly,
overexpression of the Mycobacterium smegmatis araA gene behind the
strong HXT7 promoter fragment on a multicopy vector in the S.
cerevisiae CEN.PK2-1C strain produced L-arabinose isomerase
activity.
[0012] Then, transformants expressing the B. subtilis araA gene
together with the E. coli genes araB and araD as well as the yeast
GAL2 gene were incubated in liquid media (synthetic complete or
synthetic complete/0.1% yeast extract/0.2% peptone) with
L-arabinose as the sole carbon source for several weeks. After 4-5
days of incubation the transformants started to grow slowly in
these media, in contrast to a strain containing only four empty
vectors. Whenever the cells reached an OD.sub.600 of 3-4, they were
inoculated in fresh medium at an OD.sub.600 of 0.3, and grown
further. Growth became faster after 10 days. These observations
indicate the occurrence of spontaneous suppressor mutations
enabling the cells to use L-arabinose more efficiently. Otherwise,
the cells might become somehow adapted to the use of
L-arabinose.
[0013] To distinguish between suppressor mutations or an adaptation
process, the mutant transformants were grown on glucose medium and
then shifted again on arabinose medium. They started to grow on
arabinose medium with only a short lag-phase indicating that indeed
they contain specific mutations enabling the cells to grow on
arabinose. The activities of all three heterologous enzymes were
measured in crude extracts of the original and the mutant
transformants. Whereas the activities of L-ribulose-5-P 4-epimerase
and L-arabinose isomerase were similar in both strains, the
L-ribulokinase activity was strongly reduced in the mutant
transformants.
[0014] When the mutant transformants were selected for loss of
their plasmids they were no longer able to grow on arabinose. The
plasmids were re-isolated and amplified in E. coli. The re-isolated
plasmids were transformed into a CEN.PK2-1C wild-type strain. When
growth on arabinose of these new transformants was compared to the
original mutant transformants, the lag-phase on arabinose medium
was significantly prolonged indicating that additional genomic
mutations had occurred in the mutant transformants enabling them to
grow efficiently on arabinose. Different combinations of original
and re-isolated plasmids were transformed into the mutant JBY25
strain. It turned out that replacing the re-isolated GAL2, araD and
araA plasmids by the corresponding original plasmids did only
slightly affect the ability to grow on arabinose. However,
replacing the re-isolated araB (L-ribulokinase) plasmid by the
corresponding original plasmid resulted in strongly reduced growth
on arabinose.
[0015] When the complete re-isolated L-ribulokinase gene was
sequenced it showed one mutation, which leads to an exchange of
amino acid 121 Asp for an Asn in the conserved sugar kinase domain
of the kinase. Determination of the kinetics of the mutant enzyme
revealed that its Km value for L-ribulose was increased and the
Vmax was decreased.
[0016] Growth experiments with the wild-type and mutant kinases
expressed from centromeric plasmids in strain JBY25 together with
the re-isolated isomerase and epimerase plasmids have also been
performed. In case of the mutant kinase this centromeric plasmid
did not confer good growth on L-arabinose to the transformants. But
the transformants carrying the wild-type kinase on a centromeric
plasmid showed better growth than those transformed with the
overexpressed kinase. This is another indication that the reduced
activity of the kinase is important for better growth on
L-arabinose.
[0017] To find out whether all four plasmids carrying the Bacillus
subtilis L-arabinose isomerase, the E. coli L-ribulokinase and
L-ribulose 5-P 4-epimerase and the yeast Gal2galactose permease,
respectively, are necessary for growth on L-arabinose, the mutant
strain was transformed with different combinations of re-isolated
and empty plasmids (without any gene for L-arabinose metabolism).
Transformants lacking the L-arabinose isomerase, the L-ribulokinase
or the L-ribulose 5-P 4-epimerase but transformed with the other
three re-isolated plasmids did not show any growth on L-arabinose
indicating that these genes are absolutely necessary for the
utilization of L-arabinose. Transformants lacking the overexpressed
galactose permease are able to grow on L-arabinose medium, but with
slightly decreased growth rates as compared to the mutant strain
containing all four re-isolated plasmids, indicating that
over-expression of a transporter is not necessary for growth on
L-arabinose but can improve it.
[0018] To test whether only one or more mutations in the genome of
the CEN.PK2-1C wild-type strain enable the transformants to grow on
L-arabinose, and whether these mutation(s) are recessive or
dominant, the mutant strain and also the wild-type strain, each
transformed with the four plasmids for L-arabinose metabolism were
crossed with a haploid wild-type strain. Afterwards, growth on
L-arabinose was investigated. The diploid mutant strain exhibited
faster growth on L-arabinose than the diploid control strain. But
the diploid mutant strain did not grow as well as the haploid
mutant strain transformed with the four plasmids. The diploid
mutant strain was sporulated and tetrade analysis was performed.
The results indicate that there is more than one mutation in the
genome of the strain with at least one being dominant and another
one being recessive.
[0019] Moreover, overexpression of S. cerevisiae TAL1
(transaldolase) together with B. subtilis araA (L-arabinose
isomerase), mutant E. coli araB (L-ribulokinase), and E. coli araD
(L-ribulose-5-P 4-epimerase) resulted in growth on L-arabinose
already in the CEN.PK2-1C wild-type strain.
[0020] Ethanol production was determined with the IBY25 mutant
strain transformed with the four re-isolated plasmids and incubated
in a growth medium with 20 g/L L-arabinose. Under oxygen-limiting
conditions at a culture OD.sub.600nm=15-20, ethanol production
rates reached up to 0.06 g ethanol/g dry weight and hour.
[0021] We have now demonstrated that it is possible to transfer the
method for producing an L-arabinose utilizing yeast strain to other
Saccharomyces cerevisiae strains that are different from the CEN.PK
strains.
[0022] We have used the W303 S. cerevisiae strain that is not
related to the CEN.PK strains, and have transformed this strain
with the plasmids expressing B. subtilis araA gene (L-arabinose
isomerase), the mutant E. coli araB gene with reduced activity
(L-ribulokinase), E. coli araD gene (L-ribulose-5-P 4-epimerase),
and S. cerevisiae TAL1 (transaldolase) gene.
[0023] The transformants could grow on a defined medium with
L-arabinose as the sole carbon source, although very slowly. Then,
cells were incubated in liquid medium (synthetic complete/0.1%
yeast extract/0.2% peptone) with L-arabinose as the sole carbon
source for several days. After 4-5 days of incubation the
transformants started to grow faster in this medium, in contrast to
a W303 strain containing only four empty vectors. Whenever the
cells reached an OD.sub.600 of 3-4, they were inoculated in fresh
medium at an OD.sub.600 of 0.3, and grown further. Finally, after
20 days this resulted in a strain able to grow on L-arabinose
medium much more faster, and able to ferment L-arabinose to
ethanol.
[0024] The invention is a modified yeast strain expressing the
bacterial B. subtilis araA gene (L-arabinose isomerase), E. coli
mutant araB gene (L-ribulokinase D.sup.121-N) and E. coli araD gene
(L-ribulose-5-P 4-epimerase), and carrying additional mutations in
its genome or overexpressing the S. cerevisiae TAL1 (transaldolase)
gene, enabling it to consume L-arabinose, to use it as the only
carbon source, and to produce ethanol.
[0025] Normally the growth medium will contain about 20 g of
L-arabinose/L. However, growth and production of ethanol will occur
between 2 and 200 g/L. There is no need for further sugars, and
thus L-arabinose can be used alone. It is possible that
co-consumption of xylose and arabinose could work, but this has not
been determined so far.
* * * * *