U.S. patent application number 11/108870 was filed with the patent office on 2005-08-18 for ethanol production.
This patent application is currently assigned to Elsworth Biotechnology Limited. Invention is credited to Cusdin, Fiona, Green, Edward, Javed, Muhammad, Milner, Paul.
Application Number | 20050181492 11/108870 |
Document ID | / |
Family ID | 9900827 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181492 |
Kind Code |
A1 |
Javed, Muhammad ; et
al. |
August 18, 2005 |
Ethanol production
Abstract
The present invention relates to the production of ethanol as a
product of bacterial fermentation. In particular this invention
relates to a novel method of gene inactivation and gene expression
based upon homologous recombination.
Inventors: |
Javed, Muhammad; (Dagenham,
GB) ; Cusdin, Fiona; (Horley, GB) ; Milner,
Paul; (Ickenham, GB) ; Green, Edward;
(Guildford, GB) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
Elsworth Biotechnology
Limited
Guildford
GB
|
Family ID: |
9900827 |
Appl. No.: |
11/108870 |
Filed: |
April 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11108870 |
Apr 19, 2005 |
|
|
|
09971361 |
Oct 5, 2001 |
|
|
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60247017 |
Nov 13, 2000 |
|
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Current U.S.
Class: |
435/161 ;
435/252.31 |
Current CPC
Class: |
C12N 9/00 20130101; C12P
7/065 20130101; Y02E 50/17 20130101; C12N 9/0004 20130101; Y02E
50/10 20130101 |
Class at
Publication: |
435/161 ;
435/252.31 |
International
Class: |
C12P 007/06; C12N
001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 6, 2000 |
GB |
0024554.8 |
Claims
1-12. (canceled)
13. A method of inactivating a native gene encoding lactate
dehydrogenase and inserting one or more expressible genes
comprising homologous recombination.
14. The method according to claim 13 wherein the one or more
expressible genes are a gene encoding pyruvate decarboxylase and a
gene encoding alcohol dehydrogenase.
15. The method according to claim 14 wherein the gene encoding
pyruvate decarboxylase and the gene encoding alcohol dehydrogenase
form part of a pyruvate decarboxylase (PDC) operon operatively
linked to an insertion element (IE) sequence comprising nucleotides
651-3800 of SEQ ID NO:1.
16. The method according to claim 13 in which the gene encoding
pyruvate decarboxylase is from a Zymomonas sp.
17. The method according to claim 16 in which the gene encoding
pyruvate decarboxylase is from Zymomonas mobilis.
18. The method according to claim 15 wherein the gene encoding
alcohol dehydrogenase is from Bacillus strain LN.
19. The method according to any one of claims 13 to 18 wherein an
insertion sequence and a PDC operon, or portions thereof, are
stably integrated into a chromosome of a recombinant bacterium.
20. The method according to any one of claims 13 to 14 comprising
using a shuttle vector which is able to replicate in E. coli and
Bacillus strains.
21-46. (canceled)
47. The method of claim 20 wherein the shuttle vector is pUBUC-IE.
Description
[0001] This application claims benefit of U.S. Application Ser. No.
60/247,017, filed Nov. 13, 2000, and U.K. Application No.
0024554.8, filed Oct. 6, 2000, both incorporated herein by
reference.
[0002] This invention relates to the production of ethanol as a
product of bacterial fermentation. In particular this invention
relates to a novel method of gene inactivation and gene expression
based upon homologous recombination.
[0003] 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.
[0004] 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.
[0005] 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 the
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.
[0006] Ethanologenic microorganisms, such as Zymomonas mobilis and
yeast, are capable of a second type of anaerobic fermentation
commonly referred to as 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. DNA which encodes both of these enzymes in Z. mobilis
has 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 Escherichia 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. 5,554,520 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.
[0007] U.S. Pat. No. 5,482,846 discloses the simultaneous
transformation of mesophilic 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 bacteria
transformed with the pdc gene alone will produce ethanol.
[0008] EP-A-0761815 describes a method of homologous recombination
whereby a sporulation gene is inserted into Bacillus
thurengiensis.
[0009] EP-A-0603416 describes a method of homologous recombination
whereby an arbitary gene is inserted into Lactobacillus
delbrueckii.
[0010] EP-A-0415297 describes a method of producing Bacillus
strains expressing a mutant protease.
[0011] Biwas et al., (J. Bacteriol., 175, 3628-3635, 1993)
describes a method of homologous recombination whereby Lactococcus
lactis has a chromosomal gene replaced by a plasmid carried
modified copy. The method uses a thermosensitive plasmid and cannot
be used to transform a thermophilic bacterium.
[0012] A key improvement in the production of ethanol using
biocatalysts can be achieved with thermophilic microorganisms that
operate at high temperature. The conversion rate of carbohydrates
into ethanol is much faster. For example, ethanol productivity in a
thermophilic Bacillus is up to ten-fold faster than a conventional
yeast fermentation process which operates at 30.degree. C.
Consequently, a smaller production plant is required for a given
volumetric productivity, thereby reducing plant construction costs.
At high temperature, there is a reduced risk of contamination in
the fermenter from other microorganisms, resulting in less
downtime, increased plant productivity and a lower energy
requirement for feedstock sterilisation. Moreover, fermentation
cooling is not required, reducing operating costs further. The heat
of fermentation helps to evaporate ethanol, which reduces the
likelihood of growth inhibition from high ethanol concentrations, a
common problem with most bacterial fermentations. Ethanol
evaporation in the fermenter head space also facilitates product
recovery.
[0013] The inventors' strain originates from a wild-type isolate
that is a natural composting organism and far more suited for the
conversion of sugars found in agricultural feedstocks to ethanol
than traditional mesophilic microorganisms. The base strain
possesses all the genetic machinery for the conversion of hexose
and pentose sugars, and cellobiose to ethanol; the inventors have
simply blocked the LDH pathway to increase ethanol yields. This
process is called self-cloning and does not involve expression of
foreign DNA. Consequently, the resulting organism does not fall
under the safety regulations imposed on the use of genetically
modified organisms (GMOs).
[0014] In contrast, conventional biocatalysts are either good
ethanol producers unable to utilise pentose sugars or poor ethanol
producers that can utilise pentose sugars. These organisms have
been genetically modified using complex genetic techniques so that
they can convert both hexose and pentose sugars to ethanol.
However, there are doubts about the stability of these recombinant
organisms and concerns over safety since such organisms fall under
the GMO safety regulations. Moreover, recombinant mesophiles have
expensive nutrient requirements and are sensitive to high salt
concentrations and feedstock inhibitors.
[0015] The metabolic reactions leading to lactic acid formation
(LDH pathway) have been blocked by chemical mutagenesis and the
resulting strain TN is lactate negative and produces ethanol in
high yield. However, the mutant strain is unstable and
spontaneously reverts to the lactacte-producing wild-type.
Revertants grow faster than the mutant at low pH and in high sugar
concentrations, and rapidly `take-over` in continuous culture.
During `take-over`, the main fermentation product changes from
ethanol to lactate.
[0016] The inventors initiated a molecular biology program to
tackle the stability problem and gain a better insight into the
genetic systems involved in ethanol formation. The inventors first
developed genetic techniques to specifically manipulate the
organism and a sporulation deficient mutant amenable to genetic
manipulation was then selected in continuous culture. The inventors
then sequenced several key metabolic genes; lactate dehydrogenase
(ldh), lactase permease (lp), alcohol dehydrogenase (adh) and a
novel insertion sequence located within the ldh gene. DNA sequence
analysis of the ldh gene from the chemically mutated strain
revealed that the gene had been inactivated by the insertion of a
naturally occurring insertion sequence element (IE) (also referred
to as an IS element) in the coding region of the gene.
Transposition into (and out of) the ldh gene and subsequent gene
inactivation is itself unstable, resulting in reversion.
[0017] The inventors determined that the IE sequence within the ldh
gene provides a large area for homologous recombination. It was
therefore proposed that the stability of the ldh mutation could be
improved by integration of plasmid DNA into the IE sequence already
present within the ldh gene of strain TN.
[0018] The stability of the ldh gene mutation was improved by
specific homologous recombination between a plasmid and the
insertion sequence within the ldh gene. The resulting strain is a
sporulation deficient, facultatively anaerobic, Gram-positive
Bacillus which exhibits improved ethanol production characteristics
in continuous culture. Results show that this new type of mutant is
completely stable and has superior growth characteristics and
ethanol productivity than the first mutants generated by chemical
mutagenesis.
[0019] Strain improvement has been achieved through a novel method
of gene integration based on homologous recombination. The site of
integration and plasmid for recombination can also be used to
integrate and overexpress native or heterologous genes.
[0020] Southern hybridisation studies indicated that 3 copies of a
transposable insertion sequence element (IE) are present on the
chromosome of Bacillus strain LLD-R. The insertion sequence is 3.2
kb long and comprises three DNA open reading frame sequences
(ORF's) that are potentially translatable into proteins. ORF1
exhibits no homology to any protein in the National Center for
Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov/)
whereas istA and istB display significant homology to a family of
known transposase enzymes. Bacillus strain TN was developed from
LLD-R following chemical mutagenesis (FIGS. 9A and 9B), and one
copy of the insertion sequence was found within the structural ldh
gene resulting in inactivation of the ldh gene and a lactate
negative phenotype, the main metabolic product of fermentation
thereby changing from lactate to ethanol. The DNA sequence of the
ldh gene and the IE sequence (underlined) from Bacillus strain TN
are shown in FIG. 1. The amino acid sequence of L-LDH is shown in
FIG. 11.
[0021] However, this insertion proved to be relatively unstable and
the mutant strain TN spontaneously reverts back to strain TN-R with
a functional ldh gene. The main metabolic product of fermentation
changes from ethanol to lactate as shown in FIG. 2 which shows the
genetic instability of Bacillus mutant strain TN.
[0022] The IE sequence was amplified from TN chromosomal DNA by
PCR. Primers were chosen from the ldh gene sequence that flanked
the insertion sequence and a HindIII restriction site was
introduced into the upstream primer and a XbaI restriction site was
introduced into the downstream primer to create convenient
restriction sites for subsequent cloning into plasmid pUBUC. A 3.2
kb PCR fragment containing the insertion sequence was trimmed using
HindIII and XbaI restriction endonucleases and subsequently cloned
into plasmid pUBUC resulting in plasmid pUBUC-IE (FIG. 5).
[0023] In vivo methylation of plasmid DNA to prevent its
restriction after transformation of Bacillus sp. was achieved after
transformation, propagation in and purification from E. coli TOP10
cells harbouring plasmid pMETH. Methylated pUBUC-IE was then used
to transform Bacillus strain TN. Transformants were first isolated
on TGP agar plates (kanamycin) at 52.degree. C. Transformants were
then screened using PCR amplification of the ldh gene. Failure to
amplify a PCR product (greater than 10 kb using set PCR conditions)
using LDH primers suggested that at least one copy of the plasmid
had become integrated into the chromosome.
[0024] The new strain, TN-T9, was grown under pH controlled
conditions in continuous culture without kanamycin selection to
check for strain stability. Stability of strain TN-T9 was confirmed
using sub-optimal fermentation conditions such that residual sugar
was present within the fermentation medium; conditions which favour
reversion. The fermentation ran continuously for 750 hours without
any trace of lactate production despite the presence of residual
sugar within the fermentation medium, pyruvate excretion and
numerous deviations from the set conditions. Ethanol was produced
in relatively large amounts throughout the fermentation FIG. 4,
indicating that the ldh gene mutation in strain TN-T9 is stable in
continuous culture under the experimental conditions provided.
[0025] The inventors have also optimised the fermentation
conditions for cell growth and ethanol production for Bacillus
strain TN-T9.
[0026] In summary the inventors have developed a dual system for
improving the stability of the ldh mutant whilst expressing pdc and
adh genes optionally using a pdc/adh operon. The inventors have
also isolated and sequenced a novel ldh gene and insertion sequence
element, as well as novel lactate permease and alcohol
dehydrogenase genes. Furthermore, the inventors have developed a
technique for the integration of plasmid DNA into the chromosome
and selection of recombinant Bacillus sp and have developed a set
of optimised conditions for the production of ethanol by bacterial
fermentation.
[0027] Accordingly, a first aspect of the present invention relates
to a recombinant thermophilic, Gram-positive bacterium which has
been transformed using a method of homologous recombination for
stabilising a gene mutation and for inserting an expressible
gene.
[0028] The invention also provides a recombinant thermophilic,
Gram-positive bacterium in which the stability of the ldh mutation
has been enhanced by homologous recombination between a plasmid and
the chromosomal DNA of the bacterium resulting in a strain for the
production of ethanol as a product of bacterial fermentation.
[0029] Preferably, the Gram-positive bacterium is a strain of B.
thermoglucosidasius.
[0030] Preferably, the Gram-positive bacterium has been transformed
with a plasmid harbouring an IE sequence as set forth in FIG. 1, or
a functional portion or variant thereof. Advantageously, the IE
sequence of FIG. 1, or functional variant or portion thereof, is
stably incorporated into the chromosome of the recombinant
bacterium by homologous recombination.
[0031] Preferably, integration of the IE sequence into the
chromosome of the recombinant bacterium will result in the
inactivation of the native ldh gene.
[0032] Preferably, the Gram-positive bacterium is Bacillus strain
TN-T9 (NCIMB Accession no. NCIMB 41075 deposited on 8 Sep. 2000 in
accordance with the terms of the Budapest Treaty).
[0033] Alternatively, it is preferred that the Gram-positive
bacterium is Bacillus strain TN-TK (NCIMB Accession no. NCIMB 41115
deposited on 27 Sep. 2001 in accordance with the terms of the
Budapest Treaty).
[0034] The present invention also relates to a Gram-positive
bacterium obtained by selecting mutants of TN-T9 which are
kanamycin sensitive. A suitable method for obtaining such strains
is described in the appended examples.
[0035] Preferably, the Gram-positive bacterium is sporulation
deficient.
[0036] According to a second aspect of the present invention there
is provided a Gram-positive bacterium wherein a native ldh gene has
been inactivated by homologous recombination and one or more
expressible genes have been inserted into the chromosomal DNA of
the bacterium. Furthermore, gene expression may be increased by
increased gene copy number following multiple insertions of the
plasmid into the insertion sequence either as a result of one round
or repeated rounds of integration.
[0037] The one or more expressible genes may be inserted into one
or more IE sequences present in the chromosomal DNA of the
bacterium. For example, there are 3 IE sequences on the chromosome
of strains TN-T9 and TN-TK.
[0038] The gene to be expressed may be native to Bacillus such as
alcohol dehydrogenase or foreign (i.e. heterlogous such as pyruvate
decarboxylase from Z. mobilis and .alpha.-amylase from B.
stearothermophilus. The genes may also be arranged in an operon
under the same transcriptional control. Gene expression may be
regulated by manipulating the copy number of the gene and by using
different transcriptional promoter sequences.
[0039] Preferably, the one or more genes are pdc and/or adh.
[0040] The amino acid sequence of adh is shown in FIG. 12.
[0041] According to a third aspect of the invention there is
provided a method of inactivating a native ldh gene and inserting
one or more expressible genes into the chromosome of a bacterium by
homologous recombination.
[0042] Preferably the bacterium is a thermophilic Gram-positive
bacterium.
[0043] Preferably, the gene to be inactivated is a native ldh gene
and the one or more expressible genes are a pdc gene and a adh
gene.
[0044] Preferably, the pdc gene and the adh gene form part of a PDC
operon operatively linked to the IE sequence of FIG. 1 on the same
plasmid.
[0045] Preferably the pdc gene is heterologous to the cell.
[0046] Preferably, both the IE sequence of FIG. 1 and the PDC
operon, or portions thereof, are stably integrated into the
chromosome of the bacterium.
[0047] Advantageously, the method of gene inactivation and
expression comprises the use of a shuttle vector, as set forth in
FIG. 5, which is able to replicate in E. coli and Bacillus strains
at temperatures up to 54.degree. C.
[0048] According to a fourth aspect of the present invention there
is provided a shuttle vector which is able to replicate in both E.
coli and Bacillus sp at temperatures up to 54.degree. C., which
confers resistance to ampicillin and kanamycin and which harbours
the IE sequence, or a portion thereof as set forth in FIG. 1, from
Bacillus strain TN.
[0049] Preferably, the shuttle vector is pUBUC-IE as set forth in
FIG. 5.
[0050] Preferably, the shuttle vector will contain a PDC operon
comprising a pdc gene and a adh gene under the control of the ldh
promoter and operably linked to the IE sequence of FIG. 1.
[0051] According to a fifth aspect of the present invention there
is provided a method of selecting for recombinant Bacillus sp at
high temperature wherein plasmid DNA has been stably integrated
into the ldh gene of the recombinant bacterium by homologous
recombination, comprising use of PCR to select for recombinants
that do not contain the native ldh gene and IE sequence.
[0052] Preferably, successful integration of the insertion sequence
into the ldh gene will be indicated by failure to amplify a PCR
product from the ldh gene of the recombinant bacterium.
[0053] The present invention also provides one or more polypeptides
encoded by the sequence shown in FIG. 1 from nucleotide 652 to
nucleotide 3800, or a functional variant or portion thereof,
wherein the one or more polypeptides have the biological activity
of a transposase.
[0054] The one or more polypeptides may have the biological
activity of a transposase taken alone or when combined with other
polypeptides.
[0055] Preferably, the one or more polypeptides has the amino acid
sequence shown in FIG. 13, FIG. 14 or FIG. 15 or a functional
portion or variant thereof.
[0056] The functional portions or variants retain at least part of
the transposase function of the polypeptide shown in FIG. 13, FIG.
14 or FIG. 15. Preferably the portions are at least 20, more
preferably at least 50 amino acids in length. Furthermore, it is
preferred that the variants have at least 80%, more preferably at
least 90% and most preferably at least 95% sequence homology with
the polypeptide shown in FIG. 13, FIG. 14 or FIG. 15. Homology is
preferably measured using the BLAST program.
[0057] According to a sixth aspect of the invention there is
provided a DNA sequence as set forth in FIG. 6, or a functional
variant thereof, which codes for a polypeptide having the
biological activity of the enzyme lactate dehydrogenase.
[0058] According to a seventh aspect of the present invention there
is provided a DNA sequence as set forth in FIG. 7B, or a functional
variant thereof, which codes for a polypeptide having the
biological activity of the enzyme lactate permease.
[0059] According to an eigth aspect of the present invention there
is provided a DNA sequence as set forth in FIG. 8, or a functional
variant thereof, which codes for a polypeptide having the
biological activity of the enzyme alcohol dehydrogenase.
[0060] In this specification, functional variants include DNA
sequences which as a result of sequence additions, deletions or
substitutions, or which by virtue of the degeneracy of the genetic
code, hybridise to and/or encode a polypeptide having a lactate
dehydrogenase lactate permease or alcohol deydrongenase activity.
Preferably, the variants have at least 80%, more preferably 90% and
most preferably 95% sequence homology to the sequence shown in the
Figures. Homology is preferably measured using the BLAST
program.
[0061] A ninth aspect of the invention also provides a method for
improving the stability of the ldh mutant comprising expressing
genes using a pdc/adh operon.
[0062] A tenth aspect of the present invention relates to a
technique for the integration and selection of recombinant Bacillus
sp in accordance with the invention.
[0063] According to the final eleventh aspect of the present
invention there is provided a process for the production of ethanol
by bacterial fermentation of the Gram-positive bacterium of the
present invention comprising optimised fermentation conditions of
pH, temperature, redox values and specific dilution rates for cell
growth and ethanol production. Preferably, the fermentation
conditions will comprise a pH range of between 5.5-7.5 and a
temperature range of 40-75.degree. C. with redox values being
between -360-400 mV and dilution rates between 0.3 and 0.8
h.sup.-1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] The production of recombinant bacteria in accordance with
the present invention will now be described, by way of example
only, with reference to the drawings in which:
[0065] FIG. 1 shows the nucleotide sequence of a DNA sequence
comprising an insertion element (IE), wherein the IE sequence is
underlined;
[0066] FIG. 2 is a schematic representation of the genetic
instability of strain TN;
[0067] FIG. 3 is a schematic representation of the method for LDH
gene inactivation by single-crossover recombination in Bacillus
mutant strain TN;
[0068] FIG. 4 is a graphical representation showing the stability
of Bacillus mutant strain TN-T9 in continuous culture for over 750
hours;
[0069] FIG. 5 is a schematic representation of shuttle vector
pUBUC-IE;
[0070] FIG. 6 shows the DNA sequence of a novel lactate
dehydrogenase gene and translated amino acid sequence from Bacillus
strain LN;
[0071] FIG. 7A shows the partial DNA sequence of a novel lactate
permease gene and the translated amino acid sequence from Bacillus
strain LN;
[0072] FIG. 7B shows the full DNA sequence of a novel lactate
permease gene and the translated ammo acid sequence from Bacillus
strain LN;
[0073] FIG. 8 shows the DNA sequence of a novel alcohol
dehydrogenase gene (underlined) from Bacillus strain LN;
[0074] FIG. 9 is a schematic representation showing (A) the
development of Bacillus strain TN-T9 and (B) the development of
Bacillus strains TN-T9 and TN-TK;
[0075] FIG. 10 shows the construction of an artificial PDC
operon;
[0076] FIG. 11 shows the amino acid sequence of L-lactate
dehydrogenase (ldh) from the TN strain;
[0077] FIG. 12 shows the amino acid sequence of alcohol
dehydrogenase (adh) from the TN strain;
[0078] FIG. 13 shows the amino acid sequence of a transposase
encoded by the IE sequence.
[0079] FIG. 14 shows the amino acid sequence of a transposase
encoded by the IE sequence.
[0080] FIG. 15 shows the amino acid sequence of a transposase
encoded by the IE sequence.
EXAMPLES
[0081] Materials and Methods
[0082] Construction of Plasmid pUBUC
[0083] A shuttle vector for the transfer of DNA between E. coli and
the inventor's thermophilic Bacillus strains was developed by
fusing plasmids pUC18 and pUB110. Plasmid pUB110 is a widely used
vector that was isolated from Staphyloccocus aureus which confers
resistance to kanamycin and which can replicate in B.
stearothermophilus at temperatures up to 54.degree. C. Narumi et
al., 1992 Biotechnology Techniques 6, No. 1. Plasmids pUB110 and
pUC18 were linearised with EcoR1 and BamH1, and then ligated
together to form pUBUC (6.4 kb). Plasmid pUBUC has a temperature
sensitive replicon, and cannot replicate above 54.degree. C. making
it an ideal host for gene integration, via homologous recombination
at elevated temperatures.
[0084] Construction of Plasmid pMETH
[0085] A 1.1 kb fragment containing the met gene was amplified from
Haemophilus aeygptius chromosomal DNA by PCR. The gene was verified
by DNA sequencing. The met gene was trimmed with BamHI and XbaI,
and then subcloned into the expression plasmid pCL1920, previously
linearised with BamH1 and XbaI. The resultant plasmid pMETH was
transformed into E. coli TOP 10. E. coli TOP 10 cells harbouring
pMETH were propagated and the culture was harvested for subsequent
transformation and in vivo methylation using a method described by
Tang et al (1994) Nuc. Acid Res. 22 (14). Competent cells were
stored in convenient aliquots at -70.degree. C. prior to
transformation.
[0086] PCR Amplification
[0087] The IE sequence was amplified from TN chromosomal DNA by PCR
using primers LDH7 and LDH8. The concentration of reactants and the
PCR procedure used were those recommended in the Expand.TM. High
Fidelity PCR System (Roche Diagnostics). PCR amplification from
lyophilised cells was achieved after 30 cycles in a Genius
thermocycler (Techne, Ltd., Cambridge). The sequence of the
upstream primer, LDH7, was 5'-AAGCTT GAT GAA ATC COG ATT TGA TGG-3'
and the sequence of the downstream primer, LDH8 was 5'-TCTAGA GCT
AAA TTT CCA AGT AGC-3'. These primers were chosen from the ldh gene
sequence that flanked the insertion sequence. A HindIII restriction
site was introduced into the upstream primer and a XbaI restriction
site was introduced into the downstream primer to create convenient
restriction sites for subsequent cloning (introduced sites are
underlined).
[0088] Construction of Plasmid pUBUC-IE
[0089] The manipulation, transformation and isolation of plasmid
DNA in E. coli was performed using standard procedures (Maniatis).
A 3.2 kb PCR fragment containing the insertion sequence was trimmed
with HindIII and XbaI and then cloned into plasmid pUBUC. The
resulting shuttle plasmid, referred to as pUBUC-IE (FIG. 5) can
replicate in E. coli and Bacillus strains at temperatures up to
54.degree. C., confers resistance to ampicillin and kanamycin, and
harbours the IE sequence from Bacillus strain TN.
[0090] Construction of PDC Operon
[0091] Bacillus strain TN converts the intracellular metabolite
pyruvate to acetyl-CoA via the PFL or PDH pathway. Acetyl-CoA is
then reduced to acetaldehyde and then to ethanol in reactions
catalysed by AcDH and ADH, respectively. The introduction of a
foreign PDC enzyme provides the cells with an alternative pathway
for ethanol production that involves decarboxylation of pyruvate by
PDC to form acetaldehyde which is then reduced to ethanol by the
native ADH enzyme. Both PDC and ADH are involved in the conversion
of pyruvate to ethanol.
[0092] We have shown that expression of Z. mobilis pdc from plasmid
pZP-1 improves cell growth and stability of the mutant strain TN.
However, we did not see any significant increase in ethanol
formation. Therefore, we decided to increase pdc gene expression
and co-express the native adh gene from Bacillus TN.
[0093] In plasmid pZP-1, the pdc gene was placed under the control
of the ldh promoter sequence from B. stearothermophilus NCA1503. We
decided to change the promoter with the ldh promoter from Bacillus
LN (construct 1). We then placed the adh gene from Bacillus strain
LN under the control of the ldh promoter (construct 2). Finally,
both pdc (from Z. mobilis and adh (from Bacillus LN) were placed
under the control of the ldh promoter sequence (construct 3). All
the genes have been amplified by PCR from Z. mobilis (pdc) and
Bacillus strain LN (ldh promoter and pdc), trimmed with the
appropriate restriction enzymes, ligated together and cloned into
an E. coli plasmid vector. The 3 constructs were cloned into the
replicative shuttle vectors pUBUC, pFC1 or the integrative shuttle
vector pUBUC-IE for chromosomal integration.
Example 1
[0094] Transformation of TN
[0095] Plasmid pUBUC-IE was methylated in vivo after
transformation, propagation in and purification from E. coli TOP10
cells harbouring plasmid pMETH. Methylated pUBUC-IE was then used
to transform Bacillus strain TN. Bacillus strain TN cells were
grown at 65.degree. C. in 50 ml of TGP medium until the absorbance
at 600 nm (A.sub.600) reached 0.5-0.6. The culture was chilled on
ice for 15-30 min. The cells were harvested by centrifugation and
washed once in 10 ml and twice in 5 ml of cold TH buffer (272 mM
trehalose and 8 mM HEPES; pH 7.5 with KOH). The cell pellet was
re-suspended in 400 .mu.l of TH buffer and stored at 4.degree. C.
prior to electroporation. Methylated plasmid DNA was used to
transform strain TN by electroporation based on a method previously
described by Narumi et al (1992) Biotechnology Techniques 6(1). The
competent cells were dispensed into 90 .mu.l aliquots and mixed
with 2 .mu.l of methylated plasmid DNA (250 ng/.mu.l). The mixture
was transferred to cold electroporation cuvettes (0.2 cm electrode
gap) and incubated on ice for 5 minutes. The suspensions were then
subjected to a 2.5 kV discharge from a 25 .mu.F capacitor and the
pulse control was set at 201 ohms (time constant, .tau.=5 ms) using
a EquiBio Easyject electroporator. The cells were immediately
transferred to 5 ml of pre-warmed TGP, incubated at 52.degree. C.
for 1 hr, and plated on TGP agar (10 .mu.g/ml kanamycin). The
plates were incubated for 24-48 hours at 52.degree. C.
[0096] Selection of Recombinants
[0097] The following method was used to select for chromosomal
integration of the temperature sensitive plasmid pUBUC-IE by
homologous recombination.
[0098] 1. Transformants were grown in 5 ml of TGP (kanamycin)
medium at 52.degree. C. for 24 hours.
[0099] 2. 50 ml of fresh TGP (kanamycin) medium was inoculated with
1 ml from O/N culture and incubated in a shaking water bath at
52.degree. C. until a OD.sub.600 was reached .about.0.5.
[0100] 3. 15 ml of the above culture was centrifuged at 4100 rpm
for 5 min at 5.degree. C. and the pellet was resuspended in 150
.mu.l of TGP (10:g/ml kanamycin) medium and spread on TGP
(kanamycin) plates.
[0101] 4. The plates were incubated at 68.degree. C. for 16
hours.
[0102] 5. The isolated colonies were picked and analysed for
plasmid integration into the insertion sequence site by PCR.
[0103] Screening of TN Integrants
[0104] TN integrants were isolated at 68.degree. C. Failure to
amplify a PCR product using LDH primers in TN integrants indicated
that at least one copy of plasmid pUBUC-IE had become integrated
into the chromosome. As a result of integration the new strain
TN-T9 was found to be more stable with regard to ldh reversion and
"take over" than the parental strain TN.
[0105] Stability of Strain TN-T9
[0106] The fermentation was run under sub-optimal conditions such
that residual sugar was present in the medium; conditions which
favour reversion. The fermentation ran continuously for over 750
hours without any trace of lactate production despite residual
sugar, pyruvate excretion and numerous deviations from the set
conditions. Ethanol was produced in relatively large amounts
throughout the fermentation. Kanamycin was not used to select for
integratnts throughout the entire fermentation. These results
indicate that the ldh gene mutation in TN-T9 is stable in
continuous culture under the experimental conditions (pH 7.0,
65.degree. C. with a 2% sugar feed).
[0107] Ethanol Yields and Productivity
[0108] The fermentation conditions have been optimised for ethanol
production from glucose, xylose and glucose/xylose based
feedstocks.
1 Culture type: continuous Temperature: 65.degree. C. pH: 6.8 Sugar
concentration in feed: 2-10% Sparge gas: air Redox: >-350 mV
(controlled through air flow rate) Dilution rate: 0.36-0.6
h.sup.-1
[0109] Under these conditions the ethanol yields obtained were
between 0.4-0.5 g/g sugar. Ethanol productivities, using a dilution
rate of 0.5 h.sup.-1, were approximately 4 and 8 g
ethanol/litre/hour on 2 and 4% sugar feeds, respectively.
Example 2
[0110] Selection of the Kanamycin Sensitive Strain--TN-TK
[0111] Bacillus TN-TK is a kanamycin sensitive derivative of TN-T9.
This strain is completely stable with regard to the ldh mutation
and an ideal host for plasmid borne expression involving kanamycin
as a selectable marker.
[0112] TN-T9 was first grown at 68.degree. C. for 24 hours in 5 ml
of TGP supplemented with kanamycin (10 .mu.g/ml). Approximately 100
ml of culture was spread on two TGP (Km) agar plates and incubated
overnight at 68.degree. C. Several hundred colonies were obtained
and 100 were transferred to fresh TGP (Km) plates using a sterile
toothpick. After overnight growth at 68.degree. C., the colonies
were transferred (by replica plating) to fresh TGP plates and TGP
(Km) plates and grown overnight at 68 C.
[0113] Two kanamycin sensitive colonies were obtained on TGP but
not on the corresponding TGP (Km) plate. The ldh gene regions from
these colonies were amplified by PCR and found to be comparable in
size to the disrupted ldh gene from TN-T9 (parental strain). PCR
was used to demonstrate that the strains had lost the gene
conferring resistance to kanamycin. One derivative referred to as
TN-TK was chosen for further growth experiments. These experiments
confirmed that the kanamycin sensitivity and ldh mutation were
completely stable.
* * * * *