U.S. patent application number 09/754083 was filed with the patent office on 2002-03-21 for ethanol production.
Invention is credited to Baghaei-Yazdi, Namdar, Green, Edward, Javed, Muhammad.
Application Number | 20020034816 09/754083 |
Document ID | / |
Family ID | 9883239 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020034816 |
Kind Code |
A1 |
Green, Edward ; et
al. |
March 21, 2002 |
Ethanol production
Abstract
This invention relates to ethanol production as a product of
bacterial fermentation.
Inventors: |
Green, Edward; (Surrey,
GB) ; Javed, Muhammad; (Essex, GB) ;
Baghaei-Yazdi, Namdar; (London, GB) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
9883239 |
Appl. No.: |
09/754083 |
Filed: |
January 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60177199 |
Jan 21, 2000 |
|
|
|
Current U.S.
Class: |
435/252.31 ;
435/485 |
Current CPC
Class: |
C12P 7/065 20130101;
C12N 9/0006 20130101; C12R 2001/07 20210501; C12P 7/56 20130101;
C12N 1/205 20210501; Y02E 50/10 20130101 |
Class at
Publication: |
435/252.31 ;
435/485 |
International
Class: |
C12N 001/21; C12N
015/75 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 6, 2000 |
GB |
0000185.9 |
Claims
1. A Gram-positive bacterium which has been transformed with a
heterologous gene encoding pyruvate decarboxylase or a functional
equivalent thereof, but has solely native alcohol dehydrogenase
function.
2. A Gram-positive bacterium according to claim 1 wherein the
bacterium is a Bacillus sp.
3. A Gram-positive bacterium according to claim 1 or 2 wherein the
bacterium is a thermophile.
4. A Gram-positive bacterium according to claim 2 or 3 wherein the
Bacillus is selected from B. stearothermophilus; B. calvodax; B.
caldotenax, B. thermoglucosidasius, B. coagulans, B. licheniformis,
B. thermodenitrificans, and B. caldolyticus.
5. A Gram-positive bacterium according to claim 1, 2, 3 or 4
wherein the gene encoding lactate dehydrogenase expression has been
inactivated.
6. A Gram-positive bacterium according to claim 5 in which the
lactate dehydrogenase gene has been inactivated by homologous
recombination.
7. A Gram-positive bacterium according to any preceding claim in
which the heterologous gene is from Zymomonas sp or from
Saccharomyces cerevisiae.
8. A Gram-positive bacterium according to claim 7 in which the
heterologous gene is from Z. mobilis.
9. A Gram-positive bacteria according to claim 7 in which the
heterologous gene is pdc 5 from S. cerevisiae.
10. A Gram-positive bacterium according to the preceding claim
wherein the heterologous gene is incorporated into the chromosome
of the bacterium.
11. A Gram-positive bacterium according to any one of claims 1 to 9
in which the bacterium has been transformed with a plasmid
comprising the heterologous gene.
12. A Gram-positive bacterium according to claim 11, wherein the
plasmid is pFC1.
13. A Gram-positive bacteria according to any one of claims 1 to 9,
wherein the heterologous gene is operatively linked to the lactate
dehydrogenase promoter from Bacillus strain LN (NCIMB accession
number 41038).
14. Strains LN (NCIMB accession number 41038); LN-T (E31, E32); TN
NCIMB accession number 41039); TN-P1; TN-P3; LN-S (J8) (NCIMB
accession number 41040); LN-D (NCIMB accession number 41041);
LN-D11 and LN-P1.
15. A recombinant, sporulation deficient, thermophilic Bacillus
which grows at greater than 50.degree. C.
16. A recombinant sporulation deficient, thermophilic Bacillus
which grows at greater than 50.degree. C. and which is not B.
licheniformis.
17. A method of producing ethanol comprising culturing a bacterium
or strain according to any one of claims 1 to 13 under suitable
conditions.
18. A method according to claim 17 in which the method is operated
at a temperature between 40-75.degree. C.
19. A method according to claim 18 operated at a temperature of
52-65.degree. C.
20. A method according to claim 18 operated at a temperature of
60-65.degree. C.
21. A method of producing L-lactic acid comprising culturing strain
LN under suitable conditions.
22. A nucleic acid molecule comprising the lactate dehydrogenase
promoter region of strain LN (NCIMB accession number 41038).
23. The nucleic acid molecule of claim 22, wherein the nucleic acid
molecule comprises the nucleic acid sequence shown in FIG. 8.
24. Plasmid pFC1.
25. Plasmid pFC1-PDC1.
Description
[0001] This invention relates to the production of ethanol as a
product of bacterial fermentation. In particular, the invention
relates to ethanol production by thermophilic strains of Bacillus
sp.
[0002] Many bacteria have the natural ability to metabolise simple
sugars into a mixture of acidic and neutral fermentation products
via the process of glycolysis. Glycolysis is the series of
enzymatic steps whereby the six carbon glucose molecule is broken
down, via multiple intermediates, into two molecules of the three
carbon compound pyruvate. The glycolytic pathways of many bacteria
produce pyruvate as a common intermediate. Subsequent metabolism of
pyruvate results in a net production of NADH and ATP as well as
waste products commonly known as fermentation products. Under
aerobic conditions, approximately 95% of the pyruvate produced from
glycolysis is consumed in a number of short metabolic pathways
which act to regenerate NAD.sup.+via oxidative metabolism, where
NADH is typically oxidised by donating hydrogen equivalents via a
series of steps to oxygen, thereby forming water, an obligate
requirement for continued glycolysis and ATP production.
[0003] Under anaerobic conditions, most ATP is generated via
glycolysis. Additional ATP can also be regenerated during the
production of organic acids such as acetate. NAD.sup.+is
regenerated from NADH during the reduction of organic substrates
such as pyruvate or acetyl CoA. Therefore, the fermentation
products of glycolysis and pyruvate metabolism include organic
acids, such as lactate, formate and acetate as well as neutral
products such as ethanol.
[0004] The majority of facultatively anaerobic bacteria do not
produce high yields of ethanol either under aerobic or anaerobic
conditions. Most facultative anaerobes metabolise pyruvate
aerobically via pyruvate dehydrogenase (PDH) and the tricarboxylic
acid cycle (TCA). Under anaerobic conditions, the main energy
pathway for the metabolism of pyruvate is via
pyruvate-formate-lyase (PFL) pathway to give formate and
acetyl-CoA. Acetyl-CoA is then converted to acetate, via
phosphotransacetylase (PTA) and acetate kinase (AK) with the
co-production of ATP, or reduced to ethanol via acetaldehyde
dehydrogenase (AcDH) and alcohol dehydrogenase (ADH). In order to
maintain a balance of reducing equivalents, excess NADH produced
from glycolysis is re-oxidised to NAD.sup.+by lactate dehydrogenase
(LDH) during the reduction of pyruvate to lactate. NADH can also be
re-oxidised by AcDH and ADH during the reduction of acetyl-CoA to
ethanol but this is a minor reaction in cells with a functional
LDH. Theoretical yields of ethanol are therefore not achieved since
most acetyl CoA is converted to acetate to regenerate ATP and
excess NADH produced during glycolysis is oxidised by LDH.
[0005] Ethanologenic organisms, such as Zymomonas mobilis and
yeast, are capable of a second type of anaerobic fermentation
commonly referred to as an alcoholic fermentation in which pyruvate
is metabolised to acetaldehyde and CO.sub.2 by pyruvate
decarboxylase (PDC). Acetaldehyde is then reduced to ethanol by ADH
regenerating NAD.sup.+Alcoholic fermentation results in the
metabolism of 1 molecule of glucose to two molecules of ethanol and
two molecules of CO.sub.2. The genes which encodes both of these
enzymes in Z. mobilis have been isolated, cloned and expressed
recombinantly in hosts capable of producing high yields of ethanol
via the synthetic route described above. For example; U.S. Pat. No.
5,000,000 and Ingram et al (1997) Biotechnology and Bioengineering
58, Nos. 2 and 3 have shown that the genes encoding both PDC (pdc)
and ADH (adh) from Z. mobilis can be incorporated into a "pet"
operon which can be used to transform Eseherichia coli strains
resulting in the production of recombinant E. coli capable of
co-expressing the Z. mobilis pdc and adh. This results in the
production of a synthetic pathway re-directing E. coli central
metabolism from pyruvate to ethanol during growth under both
aerobic and anaerobic conditions. Similarly, U.S. Pat. No. 5,554520
discloses that pdc and adh from Z. mobilis can both be integrated
via the use of a pet operon to produce Gram negative recombinant
hosts, including Erwina, Klebsiella and Xanthomonas species, each
of which expresses the heterologous genes of Z. mobilis resulting
in high yield production of ethanol via a synthetic pathway from
pyruvate to ethanol.
[0006] U.S. Pat. No. 5,482,846 discloses the simultaneous
transformation of Gram positive Bacillus sp with heterologous genes
which encode both the PDC and ADH enzymes so that the transformed
bacteria produce ethanol as a primary fermentation product. There
is no suggestion that the bacteria may be transformed with the pdc
gene alone.
[0007] A key improvement in the production of ethanol using
biocatalysts can be achieved if operating temperatures are
increased to levels at which the ethanol is conveniently removed in
a vapourised form from the fermentation medium. However, at the
temperatures envisioned, traditional mesophilic microorganisms,
such as yeasts and Z. mobilis, are incapable of growth. This has
led researchers to consider the use of thermophilic, ethanologenic
bacteria as a functional alternative to traditional mesophilic
organisms. See EP-A-0370023.
[0008] The use of thermophilic bacteria for ethanol production
offers many advantages over traditional processes based upon
mesophilic ethanol producers. Such advantages include the ability
to ferment a wide range of substrates, utilising both cellobiose
and pentose sugars found within the dilute acid hydrolysate of
lignocellulose, as well as the reduction of ethanol inhibition by
continuous removal of ethanol from the reaction medium using either
a mild vacuum or gas sparging. In this way, the majority of the
ethanol produced may be automatically removed in the vapour phase
at temperatures above 50.degree. C. allowing the production phase
to be fed with high sugar concentrations without exceeding the
ethanol tolerance of the organism, thereby making the reaction more
efficient. The use of thermophilic organisms also provides
significant economic savings over traditional process methods based
upon lower ethanol separation costs.
[0009] The use of facultative anaerobes also provides a number of
advantages in allowing a mixed aerobic and anaerobic process. This
facilitates the use of by-products of the anaerobic phase to
generate further catalytic biomass in the aerobic phase which can
then be returned to the anaerobic production phase.
[0010] The inventors have produced sporulation deficient variants
of a thermophilic, facultatively anaerobic, Gram-positive bacterium
which exhibit improved ethanol production-related characteristics.
This approach has a number of important advantages over
conventional processes using both traditional and recombinant
mesophilic bacteria, including simplification of the transformation
process by using only the pdc gene of Z. mobilis in strains that
already produce ethanol and have a `native` adh gene. Expression of
pdc has resulted in a significant increase in ethanol production by
the recombinant organism and has unexpectedly improved the
organism's growth characteristics. Recombinant microorganisms,
which prior to transformation with the pdc gene were highly
unstable and difficult to culture, show significant increases in
growth and survival rates both aerobically and anaerobically as
well as an increase in the rate of ethanol production near to
theoretical yields.
[0011] Accordingly, a first aspect of the invention relates to a
Gram-positive bacterium which has been transformed with a
heterologous pdc gene, but which has solely a native alcohol
dehydrogenase function. The gene may encode a functional equivalent
of pyruvate decarboxylase Functional equivalents of pyruvate
decarboxylase include expression products of insertion and deletion
mutants of natural pdc gene sequences.
[0012] It is possible that organisms which carry out glycolysis or
a variant thereof can be engineered, in accordance with the present
invention, to convert as much as 67% of the carbon in a sugar
molecule via glycolysis and a synthetic metabolic pathway
comprising enzymes which are encoded by heterologous pdc and native
adh genes. The result is an engineered organism which produces
ethanol as its primary fermentation product.
[0013] The Gram-positive bacterium is preferably a Bacillus, The
bacterium may be a thermophile. Where the Gram-positive bacterium
is a Bacillus it is preferably selected from B. stearothermophilus;
B. calvodex; B. caldotenax; B. thermoglucosidasius, B. coagulans,
B. licheniformis, B. thermodenitrificans, and B. caldolytics.
[0014] A gene encoding lactate dehydrogenase may be inactivated in
the Gram-positive bacterium of the invention. For example, the
lactate dehydrogenase gene may be inactivated by homologous
recombination. The heterologous pdc gene may be from Zymomonas sp,
preferably Z. mobilis or may be from yeast e.g the S. cerevisae pdc
5 gene.
[0015] The heterologous gene may be incorporated into the
chromosome of the bacterium. Alternatively, the bacterium may be
transformed with a plasmid comprising the heterologous gene.
Preferably the bacterium is transformed using plasmid is pFC1, more
preferably with pFC1-PDC1 The invention includes Gram-positive
bacteria, preferably a Bacillus sp including the operon of the
invention. The Bacillus sp may be selected from B.
stearothermophilus; B. calvodex; B. caldotenax, B.
thermoglucosidasius, B. coagulans, B. licheniformis, B.
thermodenitrificans, and B. caldolyrticus. The operon may be
incorporated into the genome of the Bacillus. Multiple copies of
the PDC operon may be incorporated into the genome.
[0016] In a preferred embodiment of the present invention the
Gram-positive bacteria has the heterologous gene operatively linked
to the lactate dehydrogenase promoter from Bacillus strain LN
(NCIMB accession number 41038) so that the heterologous gone is
under the control of the promoter. The sequence of the promoter
region from strain LN is shown in FIG. 8.
[0017] The invention also provides strains LN (NCIMB accession
number 41038); LN-T (E31, E32); TN (NCIMB accession number 41039);
TN-P1; TN-P3; LN-S (J8) (NCIMB accession number 41040); LN-D (NCIMB
accession number 4104 1); LN-D11 and LN-P1.
[0018] According to another aspect of file invention, there is
provided a recombinant, sporulation deficient, thermophilic
Bacillus which grows at greater than 50.degree. C. The Bacillus is
preferably not B. licheniformis.
[0019] A second aspect of the present invention relates to a method
of producing ethanol using bacteria of the invention maintained
under suitable conditions.
[0020] The method may be operated at a temperature between
40-75.degree. C.; preferably at a temperature of 52-65.degree. C.;
most preferably at a temperature of 60-65.degree. C.
[0021] The present invention also relates to a method of producing
L-lactic acid using strain LN.
[0022] The present invention also provides a nucleic acid molecule
comprising the lactate dehydrogenase promoter of strain LN (NCIMB
accession number 41038). The sequence of a nucleic acid molecule
comprising the lactate dehydrogenase promoter of strain LN is shown
in FIG. 7, Preferably the nucleic acid molecule comprises a
functional fragment of the nucleic acid sequence shown in FIG. 7. A
functional fragment is defined as a fragment that function as a
promoter and enables the expression of an operably linked gene.
[0023] The present invention also provides plasmid pFC1. The
structure of this plasmid is shown schematically in FIG. 8.
[0024] The present invention also provides plasmid pFC1-PDC1. The
structure of this plasmid is shown schematically in FIG. 9.
[0025] The production of recombinant bacteria in accordance with
the invention will now be described, by way of example only, with
reference to the accompanying drawings, FIGS. 1 to 10 in which:
[0026] FIG. 1 is a schematic representation showing the production
of bacterial strains of the invention;
[0027] FIG. 2 illustrates the metabolic pathway whereby sugars are
metabolised to produce ethanol by bacterial strains of the
invention;
[0028] FIG. 3 is a schematic representation illustrating the method
of LDH gene inactivation by single crossover recombination;
[0029] FIG. 4 is a schematic representation illustrating the method
of LDH gene inactivation by double crossover recombination;
[0030] FIG. 5 is a schematic representation illustrating the method
of LDH gene inactivation and heterologous gene expression by double
crossover recombination;
[0031] FIG. 6 provides details about the PDC gene and promoter
construct;
[0032] FIG. 7 provides sequence data about the LDH promoter from
Bacillus LN;
[0033] FIG. 8 is a schematic diagram of the replicative plasmid
pFC1;
[0034] FIG. 9 is a schematic diagram of the PDC construct 2 cloned
into the pFC1 plasmid; and
[0035] FIG. 10 shows the construction of an artificial PDC
operon.
[0036] The inventors initiated a strain development program to
overcome inherent strain limitations in respect of ethanol
production, such as instability and sporulation under adverse
conditions. Physiological manipulation and selection for strains
with superior growth characteristics has been achieved in
continuous culture, whereas a more targeted genetic approach has
been used to engineer strains with greater stability and superior
production characteristics.
[0037] In accordance with FIG. 1, all strains were ultimately
derived from a previously isolated Bacillus isolate PSII, a novel,
thermophilic, Gram-positive, spore-forming, facaltative anaerobe.
PSII ferments a wide range of organic compounds from sugars,
including hernicelluloses, to organic acids such as acetate,
formate and lactate and small amounts of ethanol at temperatures
between 50.degree. C. and 70.degree. C.
[0038] Bacillus strains LLD-15, LLD-R, LLD-16 and T13 have been
described in EP-A-0370023 and by Amartey et a, (1999) Process
Biochemistry 34 No. 3 pp.289-294. Strain LLD-15 (NCIMB12428) arose
during attempts to obtain mutants of Bacillus stearothermophilus
strain NCA 1503 lacking L-LDH activity by selecting for suicide
substrate resistance (Payton and Hartley (1984) Trends in
Biotechnology, 2 No. 6). Strain LLD-15 was assumed to be a variant
of B. stearothermophilus NCA1503, but is, in fact, derived from
PS11. Strain LLD-R arises spontaneously and reproducibly from
strain LLD-15 and is selected on plates or during continuous
cultures under which it grows more rapidly (i.e. at low pH in media
containing sugars, acetate and formate). LLD-R produces L-lactate
anaerobically and contains high levels of L-LDH, so is therefore,
clearly a wild type revertant of the non-lactate-producing LLD-15
lesion.
[0039] Bacillus strain T13 is an L-lactate deficient mutant of
strain LLD-R. Isolation and characterisation of this mutant strain
has been described previously by, Javed. M. PhD) Thesis, Imperial
College, London. Thus, Bacillus strains LLD-15, LLD-16 and T13 are
mutants of the original isolate, PSII in which the ldh gene has
been inactivated either via spontaneous mutation or by chemical
mutagenesis and the major fermentation product, unlike PSII, is
ethanol. The lactate dehydrogenase gene mutation in LLD-15, LLD-16
and T13 results from the insertion of a transposon into the coding
region of the lactate dehydrogenase gene resulting in
ldhTgeno-type, All three strains are inherently unstable in high
sugar concentrations (>2%) and revert back to lactate producing
strains. Strains T13 and LLD-15 revert to T13-R and LLD-R,
respectively. Strains LLD-15, LLD-R, LLD-16 and T13 also tend to
sporulate under adverse growth conditions such as changes in pH,
temperature and medium compositions, and during periods of nutrient
starvation. Since sporulation often leads to culture washout in a
continuous system, these strains are not suitable for large scale
industrial use in which process parameters fluctuate.
EXAMPLE 1
[0040] Production of LN
[0041] Bacillus strain LN was produced from LLD-R as a spontaneous
sporulation mutant which arose during nitrogen adaptation in
continuous culture. Strain LN is more robust than the parental
strains LLD-15 and LLD-R under vigorous industrial conditions such
as low pH, high sugar concentrations and in crude hydrolysate feed
stocks and is more amenable to plasmid transformation. Strain LN is
sporulation deficient (spo) and is particularly suitable for the
production of high purity L-lactic acid, producing up to 0.4 g of
L-lactate/g of glucose at 65.degree. C. This strain is sensitive to
kanamycin concentrations in excess of 5 .mu.g/ml and an ideal host
for genetic manipulation (ldh inactivation and heterologous gene
expression). Strain LN has been deposited under the terms of the
Budapest Treaty under accession No. NCIMB 41038.
EXAMPLE 2
[0042] Production of Strain LN-S(J8)
[0043] Strains E31 and E32 are spontaneous transposon mutants from
strain LN. Both strains are lactate deficient and produce up to 0.5
g of ethanol/g of glucose at 65.degree. C. They are non-sporulating
and as such more amenable to genetic manipulation than T13.
EXAMPLE 3
[0044] Production of Strain LN-S (J8)
[0045] Strain LN-S (J8) was produced by a single crossover
homologous recombination event between pUBUC-LDH a temperature
sensitive, non-replicative plasmid harbouring an intel region of
the ldh gene and the ldh gene on the chromosome (see FIG. 3). This
resulted in inactivation of the ldh gene and a lactate negative
phenote. This strain is also sporulation deficient and resistant to
kanamycin. It is stable in relatively high sugar concentrations in
continuous culture (with 10 g/L of residual sugar), it has good
growth characteristics and produces relatively high yields of
ethanol. For example, in continuous culture at pH 7.0, 65.degree.
C., dilution rate of 0.1 h.sup.-1, and 50 g/L glucose feed, the
cells produce up to 20 g/L of ethanol (i.e. 0.4 g of ethanol/g of
glucose utilised) for 200 hours or more without any drop in ethanol
yield.
[0046] Strain LN-S (J8) has been deposited with the NCIMB under the
terms of the Budapest Treaty under accession number NCIMB
41040.
EXAMPLE 4
[0047] Production of Strain LN-D
[0048] This strain was produced by a double crossover homologous
recombination event between a linear insertion cassette and the ldh
gene from strain LN (see FIG. 4). The insertion cassette (pUC-IC)
is a non-replicating pUC18 plasmid harbouring a kanamycin
resistance gene flanked by the ldh and lactase pernease (lp) gene
sequences. Recombination inactivated both the ldh and lp genes
resulting in a lactate negative phenotype. This strain is also
sporulation deficient and resistant to kanamycin. Strain LN-D can
tolerate high sugar concentrations (with up to 10 g/L of residual
sugar), it has good growth characteristics and produces relatively
high yields of ethanol. For example, in continuous culture at pH
7.0, 52.degree. C., dilution rate of 0.1 h.sup.-3 and 50 g/L
glucose feed, the cells produce tip to 20 g/L of ethanol (i.e. 0.4
g of ethanol/g of glucose utilised) for 200 hours or more without
any drop in ethanol yield or cell viability. Furthermore, kanamycin
selection was not required to maintain the ldh gene
inactivation.
[0049] Strain LN-D has been deposited with the NCIMB under the
terms of the Budapest Treaty under accession number NCIMB
41041.
EXAMPLE 5
[0050] Production of Strain LN-D11
[0051] The resistance of strain LN-D to kanamycin was cured after
repeated subculture to produce strain LN-D11. This strain is
lactate negative, sporulation deficient and sensitive to kanamycin
concentrations in excess of 5 .mu.g/ml. Strain LN-D11 can tolerate
high sugar concentrations (with up to 10 g/L of residual sugar), it
has good growth characteristics and produces relatively high yields
of ethanol. For example, in continuous culture at pH 7.0,
52.degree. C., dilution rate of 0.1 h.sup.-1, and 50 g/L glucose
feed, the cells produce up to 20 g/L of ethanol (i.e. 0.4 g of
ethanol/g of glucose utilised for 200 hours or more without any
drop in ethanol yield or cell viability. This strain is an ideal
host for heterologous gene expression.
EXAMPLE 6
[0052] Production of Strain LN-DP1
[0053] Strain LN-DP1 was produced from strain LN-D11 after
transformation with the replicative plasmid pBST22-zym (also
referred to as pZP1). The backbone of this vector is pBST22
(originally developed by Liao et al (1986) PNAS (USA) 83: 576-580)
with the entire pdc gene from Z. mobilis under the control of the
ldh promoter sequence from B. stearothermophilus NCA 1503. Strain
LN-DP1 is sporulation deficient; kanamycin resistant, lactate
negative and contains the heterologous pdc gene from Z. mobilis.
Strain LN-DP1 can utilise high sugar concentrations, it has good
growth characteristics and produces relatively high yields of
ethanol. For example, in continuous culture at pH 7.0, 60.degree.
C., dilution rate of 0.1 h.sup.-1, and 50 g/L glucose feed, the
cells produce up to 25 g/L ethanol (i.e. 0.5 g of ethanol/g of
glucose utilised) for 200 hours or more without any drop in ethanol
yield.
EXAMPLE 7
[0054] Production of Strain TN
[0055] Strain TN arose spontaneously from T13 during nitrogen
adaptation in continuous culture. This strain is more robust than
the parental strain under vigorous industrial conditions (i.e. low
pH, high sugar concentrations, and in crude hydrolysate feed
stocks) and is more amenable to plasmid transformation, making it
an ideal host for genetic engineering. Strain TN is sporulation
deficient and lactate negative (the ldh gene has been inactivated
by transposon mutagenesis into the coding region of the ldh gene).
Strain TN is a good ethanol producer on dilute sugar feeds. For
example, in continuous culture at pH 7.0, 70.degree. C., dilution
rate of 0.1 h.sup.-1, and 20 g glucose, the cells produce up to 8
g/L ethanol (i.e. 0.4 g of ethanol/g of glucose utilised) for 100
hours or more without any drop in ethanol yield. This strain is
sensitive to kanamycin concentrations in excess of 5 .mu.g/ml and
an ideal host for genetic manipulation.
[0056] Strain TN has been deposited at the NCIMB under the terms of
the Budapest Treaty under accession number NCIMB 41039.
EXAMPLE 8
[0057] Production of Strain TN-P1
[0058] Strain TN-P1 was produced from stain TN after transformation
with the replicative plasmid pBST22-zym. As previously described,
pBST22-zym was produced by the incorporation of a pdc gene in
vector pBST22. The transformation efficiency of TN was 10-fold
higher with pBST22-zym than pBST22 and the colony site of the
transformants were significantly larger indicating that pdc
expression conferred a growth advantage to the cells.
[0059] TN-1 is also more stable and a better ethanol producer than
TN, especially in sugar concentrations greater than 20 g/L. For
example, in continuous culture controlled at pH 7.0, 52.degree. C.
with a dilution rate of 0.1 h.sup.-1, and a sugar feed containing
20-50 g/L, strain TN-P1 produced up to 0.5 g ethanol/g sugar
utilised for 600 hours with no significant drop in yield or cell
viability. In addition, in continuous culture controlled at pH 7.0,
52.degree. C. with a dilution rate of 0.1 h.sup.-1, and a wheat
crude hydrolysate feed, TN-P1 produced 0.4 g ethanol/g sugar
utilised in for 400 hours with no significant drop in ethanol yield
or culture viability.
[0060] Plasmid selection in strain TN-P1 was first maintained with
kanamycin but the plasmid was found to be relatively stable without
selection and no significant plasmid loss was detected after 300
hours of continuous culture. The plasmid and PDC enzyme were found
to be relatively stable at high temperatures. In continuous culture
controlled at pH 7.0, with a dilution rate of 0.1 h.sup.-1, and a
glucose feed of 30 g/L, there was no significant drop in ethanol
yield or culture viability when the growth temperature was
increased from 52 to 60.degree. C.
EXAMPLE 9
[0061] Production of Strain TN-P3
[0062] Strain TN-P3 was produced from strain TN after
transformation with the replicative plasmid pFC1-PDC1. The backbone
of this vector is pFC1 (FIG. 8) which was formed from a fusion of
pAB124 (Bingharn et al., Gen. Microbiol., 114, 401-408, 1979) and
pUC18. The pdc gene was amplified from Z. mobilis chromosomal DNA
by PCR using the following primers. The restriction sites BamHI and
SacI (underlined) were introduced into the amplified gene.
[0063] 5'-GAGCTCGCAATGAGTTATACTGTC-3'
[0064] 5'-GGATCCCTAGAGGAGCTTGTTA-3'
[0065] The ldh promoter was amplified by PCR from Bacillus LN using
the following primers. The restriction sites SacI and BamHI
(underlined) were introduced into the amplified sequence.
[0066] 5'-GGATCCGGCAATCTGAAAGGAAG-3'
[0067] 5'-GAGCTCTCATCCTTTCCAAAA-3'
[0068] The ldh promoter sequence and pdc gene were digested with
SacI and BamHI and then ligated together (FIG. 6). The construct
was then digested with BamHI and ligated into BamHI digested pFC1
to form plasmid pFC1-PDC1 (FIG. 9).
[0069] Strain TN-P3 is a good ethanol producer and produces yields
in excess of 0.45 g ethanol/g sugar at temperatures between 50 and
60.degree. C.
[0070] Gene Inactivation
[0071] Single-Crossover Recombination (SCO)(FIG. 3)
[0072] SCO or Campbell-type integration was used for directed ldh
gene inactivation. An internal fragment (700 bp) of the target gene
(ldh) was first cloned into pUBUC to form pUBUC-LDH.
[0073] Plasmid PUBUC is a shuttle vector for DNA transfer between
Escherichia coli and Bacillus strains LN and TN and was formed from
the fusion of pUB110 and pUC18. This vector contains a selectable
marker that confers resistance to kanamycin and a Gram-positive and
Gram-negative replicon. The plasmid is temperature sensitive in
Bacillus and cannot replicate above 54.degree. C.
[0074] Plasmid pUBUC-LDH was first methylated (in vivo) and then
transformed into the host strain (LN) at the permissible
temperature (50.degree. C.) for plasmid propagation. The growth
temperature was then increased to 65.degree. C., preventing plasmid
replication and integrants were selected using kanamycin.
Integration of the plasmid DNA into the ldh gene resulted in gene
inactivation and a lactate negative phenotype.
[0075] Double-Crossover Recombination (DCQ (FIGS. 4 & 5)
[0076] DCO or replacement recombination differs from SCO in that it
results in integration of only one copy of the target DNA and
typically, a region of chromosomal DNA is replaced by another
region, either foreign DNA or mutationally altered homologous DNA.
The target DNA (kanamycin marker) was flanked on either side by
mutationally altered fragments of the ldh gene in plasmid pUC-IC (a
non-replicative vector based on pUC18). The vector was first
methylated (in vivo) and then linearised at a site outside the
flanking region (this prevents SCO). The methylated, linearised
plasmid DNA was then transformed into strain LN and integrants were
selected using kanamycin.
[0077] This technique has also be used to inactivate ldh and
integrate a copy of the pdc gene into the chromosome simultaneously
(FIG. 5) and can be applied to other genes of interest.
EXAMPLE 10
[0078] PDC Expression
[0079] In the expression plasmid pBST22-zym, the pdc gene from Z
mobilis is under the control of the ldh promoter sequence from B.
stearothermophilus NCA1503 (FIG. 6). This plasmid was transformed
into strain TN to form TN-P1. Although PDC improved cell growth,
pdc expression and subsequent enzyme activity was relatively weak
and there was only a small increase in ethanol yield. In addition,
PDC activity is sensitive to temperature and rapidly declined at
growth temperatures greater than 60.degree. C.
[0080] Therefore, we increased expression of pc by firstly
replacing the ldh promoter from B. stearothermophilus NCA 1503 with
the ldh promoter sequence from Bacillus sp. LN (FIG. 6). The pdc
gene under the control of ldh promoter from strain LN (construct 2)
was subcloned into pFC1 to form plasmid pFC1-PDC1 (FIG. 9). Plasmid
pFC1 is a shuttle vector that contains a Gram-positive and
Gram-negative replicon and confers resistance to ampicillin and
tetracycline. This plasmid was transformed into strain TN to form
TN-P3.
[0081] Strain Performance
[0082] TN-P3 produces significantly more ethanol than the
untransformed control strain TN.
1 Strain Ethanol Concentration TN 21.5 mM TN-P1 24.0 mM TN-P3 39.5
mM
[0083] The strains were cultured in a culture medium containing JSD
supplemented with 50 mM PIPES buffer and 2% glucose at 54.degree.
C. for 24 hours. TN-P1 and TN-P3 cultures were supplemented with
kanamycin (12 .mu.g/ml) and tetracycline (10 .mu.g/ml),
respectively.
[0084] The thermostability of pdc can be improved by cloning an
alternative pdc gene, encoding a more thermostable PDC enzyme, from
Saccharomyces cerevislae. This gene, referred to as pdc5 will also
be under the control of the ldh promoter from strain LN (see FIG.
9, construct 3). The construct will be cloned into pFC1 and the
resulting plasmid pFC1-PDC5 will be transformed into strain TN.
[0085] Development of an Artificial PDC Operon
[0086] An artificial PDC operon can be constructed using
interchangeble gene sequences from the ldh promoter, pdc genes from
Z. mobilis and the yeast Saccharomyces cerevisiae, and and from LN
(see FIG. 10).
[0087] 1) pdc expression should be increased when the ldh promoter
from B stearothermophilus NCA1503 is replaced by the ldh promoter
from LN and will result in higher ethanol yields.
[0088] 2) the ethanol yields should be increased further if adA
from LN is co-expressed with pdc from Z mobilis in a PDC operon
Ethanol yields should be close to theorehcal values of 0.5 g
ethanol/g sugar.
[0089] 3) the thermostability of pdc may be increased above
64.degree. C. if the Z mobilis pdc gene is replaced by the pdc5
gene from S. cerevisiae. This will increase the growth temperature
from 64 to70.degree. C.
[0090] Integration of the PDC Operon into Strain LN-D11
[0091] By fusing the PDC operon to the insertion element sequence
(first identified in the ldh gene from TN) several copies of the
PDC operon can be introduced into multiple sites on the chromosome
increasing both the stability and gene dosage.
[0092] This expression strategy may also be used for other genes of
interest.
* * * * *