U.S. patent application number 13/610015 was filed with the patent office on 2014-03-13 for recombinant thermotolerant yeast with a substitute heat shock protein 104 promoter.
This patent application is currently assigned to NATIONAL CHUNG CHENG UNIVERSITY. The applicant listed for this patent is Hau-Ren Chen, Hsin-Cheng Chen, KUANG-TSE HUANG, Yu-Shiuan Lai, Wen-Chien Lee, Yu-Wei Liang, Meng-Tsu Tsai, Chang-Yu Wu, Yu-Long Wu, Ju-Ping Yeh. Invention is credited to Hau-Ren Chen, Hsin-Cheng Chen, KUANG-TSE HUANG, Yu-Shiuan Lai, Wen-Chien Lee, Yu-Wei Liang, Meng-Tsu Tsai, Chang-Yu Wu, Yu-Long Wu, Ju-Ping Yeh.
Application Number | 20140073054 13/610015 |
Document ID | / |
Family ID | 50158733 |
Filed Date | 2014-03-13 |
United States Patent
Application |
20140073054 |
Kind Code |
A1 |
HUANG; KUANG-TSE ; et
al. |
March 13, 2014 |
RECOMBINANT THERMOTOLERANT YEAST WITH A SUBSTITUTE HEAT SHOCK
PROTEIN 104 PROMOTER
Abstract
The invention provides a yeast strain and a method for making
the same. The method has the step of replacing the regulation
region upstream of the hsp104 gene in the genome of the yeast, so
as to accelerate and prolong the expression span of hsp104 gene and
enhance the capability of the yeast to ferment and produce ethanol
in a high-temperature environment. The yeast is capable of
fermenting glucose at a temperature higher than 42.degree. C. to
produce ethanol, or biomass ethanol, wherein the ethanol production
ratio based on fermentation of glucose is higher than 97%. Being
able to synchronize the degradation/hydrolysis stage and
fermentation stage of biomass ethanol producing process, the yeast
in accordance with the present invention is able to lower the
production cost of biomass ethanol and further raise the
productivity with its high ethanol production ratio.
Inventors: |
HUANG; KUANG-TSE; (Chiayi
County, TW) ; Chen; Hau-Ren; (Chiayi County, TW)
; Lee; Wen-Chien; (Chiayi County, TW) ; Wu;
Chang-Yu; (Chiayi County, TW) ; Chen; Hsin-Cheng;
(Chiayi County, TW) ; Wu; Yu-Long; (Chiayi County,
TW) ; Tsai; Meng-Tsu; (Chiayi County, TW) ;
Yeh; Ju-Ping; (Chiayi County, TW) ; Liang;
Yu-Wei; (Chiayi County, TW) ; Lai; Yu-Shiuan;
(Chiayi County, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HUANG; KUANG-TSE
Chen; Hau-Ren
Lee; Wen-Chien
Wu; Chang-Yu
Chen; Hsin-Cheng
Wu; Yu-Long
Tsai; Meng-Tsu
Yeh; Ju-Ping
Liang; Yu-Wei
Lai; Yu-Shiuan |
Chiayi County
Chiayi County
Chiayi County
Chiayi County
Chiayi County
Chiayi County
Chiayi County
Chiayi County
Chiayi County
Chiayi County |
|
TW
TW
TW
TW
TW
TW
TW
TW
TW
TW |
|
|
Assignee: |
NATIONAL CHUNG CHENG
UNIVERSITY
Chiayi County
TW
|
Family ID: |
50158733 |
Appl. No.: |
13/610015 |
Filed: |
September 11, 2012 |
Current U.S.
Class: |
435/483 ;
435/254.21; 435/320.1; 536/23.2 |
Current CPC
Class: |
C12P 7/06 20130101; Y02E
50/10 20130101; C07K 14/395 20130101; C12N 15/81 20130101; Y02E
50/17 20130101 |
Class at
Publication: |
435/483 ;
435/254.21; 435/320.1; 536/23.2 |
International
Class: |
C12N 15/81 20060101
C12N015/81 |
Claims
1. A yeast substrain deposited as deposit CCTCC M 2012254 in
International Depository Authority China Center for Type Culture
Collection (CCTCC), wherein said yeast substrain is a Saccharomyces
cerevisiae substrain having an upstream regulation region of hsp104
gene which has been replaced with a nucleic acid fragment
comprising: a first portion comprising two Flp/FRT flippase
reaction sites, and a sequence of an antibiotic resistance gene
between the two Flp/FRT flippase reaction sites; and a second
portion downstream of the first portion and comprising a regulation
region of hsp26 gene.
2. A method for making a Saccharomyces cerevisiae substrain capable
of fermenting at a temperature higher than 42.degree. C. to produce
ethanol comprising: obtaining a plasmid comprising: a nucleic acid
fragment comprising: a first portion comprising two Flp/FRT
flippase reaction sites and a sequence of a antibiotic resistance
gene between the two Flp/FRT flippase reaction sites, and a second
portion downstream of the first portion and comprising a regulation
region of hsp26 gene; and two homologous sequences flanking
upstream and downstream of the nucleic acid fragment corresponding
to hsp104 gene and a upstream regulation region of the hsp104 gene;
transforming a Saccharomyces cerevisiae strain with the plasmid;
and screening a Saccharomyces cerevisiae substrain the upstream
regulation region of the hsp104 gene of which has been replaced
with the nucleic acid fragment.
3. The method as claimed in claim 2, wherein the antibiotic
resistance gene is the sequence of KanMX6 for resistance against
kanamycin.
4. The method as claimed in claim 2, wherein each of the Flp/FRT
flippase reaction sites is selected from a group consisting of: a
FRT sequence and a FRT sequence comprising a core region having
less than 4 mutated nucleotides.
5. The method as claimed in claim 2, wherein each of the two
homologous sequences has a length of 40 base pairs.
6. The method as claimed in claim 2, wherein the Saccharomyces
cerevisiae strain belongs to Saccharomyces cerevisiae Kyokai strain
series.
7. A plasmid for making a Saccharomyces cerevisiae substrain
capable of fermenting at a temperature higher than 42.degree. C. to
produce ethanol comprising: a nucleic acid fragment comprising: a
first portion comprising two Flp/FRT flippase reaction sites and a
sequence of a antibiotic resistance gene between the two Flp/FRT
flippase reaction sites, and a second portion downstream of the
first portion and comprising a regulation region of hsp26 gene; and
two homologous sequences flanking upstream and downstream of the
nucleic acid fragment and each comprising a sequence corresponding
to the genomic region covering hsp104 gene and a upstream
regulation region of the hsp104 gene.
8. A nucleic acid fragment for making a Saccharomyces cerevisiae
substrain capable of fermenting at a temperature higher than
42.degree. C. to produce ethanol comprising: a first portion
comprising two Flp/FRT flippase reaction sites and a sequence of a
antibiotic resistance gene between the two Flp/FRT flippase
reaction sites, and a second portion downstream of the first
portion and comprising a regulation region of hsp26 gene.
9. A yeast substrain, the upstream regulation region of the hsp104
gene of which has been replaced with a nucleic acid, capable of
fermenting at a temperature higher than 42.degree. C. to produce
ethanol at an ethanol production ratio higher than 95%.
10. A yeast substrain, which is made by the method as claimed in
claim 2.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a novel recombinant yeast
substrain, especially to a recombinant yeast substrain suitable for
the production of ethanol by fermentation under 42.degree. C. with
high efficiency. The recombinant yeast substrain was constructed by
replacing the genomic regulatory region for yeast gene hsp104 using
linear DNA transformation, so as to alter the behavior of Hsp104
protein under stress and to facilitate the production of alcohol by
high-temperature fermentation.
[0003] 2. Description of the Prior Art
[0004] Biomass ethanol is a biomass fuel that generates bio-energy
and is obtained by conversion of biomass. Said biomass may be
molasses or plants such as maize, wheat or potatoes that allow
production of biomass ethanol through the processes of fermentation
and distillation. Biomass ethanol is known as a feasible way to
reduce dependence on fossil fuels.
[0005] Materials for making biomass ethanol are roughly classified
into three categories:
[0006] 1) sugar materials derived from monosaccharide-abundant
sugar corps such as sugar cane or sorgo;
[0007] 2) starch materials derived from wheat or corn; and
[0008] 3) cellulosic materials derived from agricultural
wastes.
[0009] Materials of the first and second categories primarily come
from food corps. Using such material leads to the zero-sum
competition between food and energy applications that are both
based on food corps. Materials of the third category, though raise
no competition between food and energy applications, require a high
production cost yet to be overcome.
[0010] The method for making ethanol from the cellulosic materials
of the third category may be largely divided into four stages:
[0011] 1) pretreatment with weak acid, weak base or ammonia-gas
explosions to separate cellulose or hemicellulose from the complex
comprising binding lignin, so as to facilitate the chemical or
biological processing in following stages.
[0012] 2) degeneration or hydrolysis for obtaining free sugars;
[0013] 3) fermentation of mixed hexose and pentose to produce
ethanol; and
[0014] 4) collection and distillation of the product for obtaining
biomass ethanol.
[0015] One of the means for lowering the production cost is to
synchronize the degeneration/hydrolysis stage and the fermentation
stage.
[0016] However, the operation temperature for the cellulose enzyme
employed in the degeneration/hydrolysis stage is approximately
45-60.degree. C., which is higher than the fermentation
temperatures of most industrial brewing yeasts (Saccharomyces
cerevisiae). Should a yeast with high-temperature tolerance capable
of working under the high temperature of the
degeneration/hydrolysis stage be cultivated, such a thermotolerant
yeast would synchronously proceed the tasks of the
degeneration/hydrolysis stage and the fermentation stage, so as to
lower the production cost of biomass ethanol.
[0017] Only a few of antecedent technologies are relevant to
thermotolerant yeast as below listed works:
[0018] Yamada et al. (US patent application publication number: US
2010/0062506), with screening media containing high concentration
of sugar and alcohol thermotolerant, have isolated yeast
Kluyveromyces marxianus capable of producing ethanol by
fermentation of sugar cane juice and peaking best productivity as
high as 1.51 g of ethanol per liter per hour at 40.degree. C.
[0019] Forrester et al. (US patent application publication number:
US 2011/0033907) have isolated Saccharomyces cerevisiae strains
YE1358 and YE1615. At 37.degree. C., fermentation of 250 g/L maize
flour and 2 mM CaCl.sub.2 with YE 1615 gives approximately 130 g/L
ethanol.
[0020] Abbas et al. (US patent application publication number: US
2009/0155872) have constructed a plasmid comprising 1) H.
polymorpha (P. angusta) glyceraldehyde-3 phosphate dehydrogenase
(GADPH) promoter-heat shock protein 104 (hsp104) gene and 2) GADPH
promoter-xylulokinase. Transforming H. polymorpha with the plasmid
suppresses the activity of acid trehalase (ATH1) and raises the
capability of H. polymorpha to ferment 12% xylose for producing
ethanol.
[0021] Edgardo et al. (Edgardo et al. 2008, Enzym. Microb. Tech.
43, 120-123) have screened Saccharomyces cerevisiae at
35-45.degree. C. to isolate strains capable of growing and
fermenting glucose at 42.degree. C. However, the fermentation
efficiency of the strain is 75% lower than the theoretical value in
a solution of 50 g/L glucose concentration at 40.degree. C., and is
25% lower at 52.degree. C.
[0022] Benjaphokee et al. (Benjaphokee et al. 2012, N. Biotechnol.
29,379-386), by crossing the spores of thermotolerat Saccharomyces
cerevisiae HB8-3A and ethanol-productive Saccharomyces cerevisiae
TISTR5606, have obtained a strain, TJ14, capable of fermenting
glucose at 41.degree. C. in a pH 3.5 solution of glucose
concentration 100 g/L with a peak fermentation efficiency as high
as 90%.
[0023] Lindquist and Kim have identified that a molecular chaperone
in yeast, the molecular chaperone Hsp104, is capable of separating
gathered proteins and refold the same, which raises the
thermotolerance of the yeast (Bosl, et al. 2006, J. Struct. Biol.
156, 139-148). Hsp104 and Hsp70/40 extract polypeptide chains out
from an aggregated protein complex and facilitate the refolding of
the same (Lee et al. 2003, Cell 115, 229-240; Waghmare et al. 2003,
Biotechniques 34, 1024-1028; Storici et al. 1999, Yeast 15,
271-283; Weibezahn et al. 2005, Biol. Chem. 386, 739-744). Heat
shock protein and other molecular chaperones are indispensable for
cellular stasis of a cell. In normal circumstances, it is vital
that these chaperones are sufficiently expressed. When under
environmental stress, misfolded proteins accumulate and disrupt
cellular physiological conditions. Misfolded proteins may further
induce generation of chaperones, which help restore and maintain
normal cellular physiological conditions. Comparing with other
chaperones, Hsp104 is insignificant under normal conditions and
thus its low concentration. However, under fatal environmental
stress, the concentration of Hsp104 acutely rises in a short period
of time to restore the activities of the disabled proteins
accumulated in the cell. In Saccharomyces cerevisiae, Hsp104,
though expression level of which raises responsive to a stress, the
expression level lowers in a few hours, which fails to allow
Saccharomyces cerevisiae to survive at high temperature for a
significantly long period of time and thus makes Saccharomyces
cerevisiae unsuitable for synchronized hydrolysis and
fermentation.
[0024] Altered expression of recombinant genes to enhance tolerance
of yeasts to stress is practicable. However, few available
recombination tags tend to be insufficient. In order to reuse the
recombination tags that are few in number, the FLP/FRT
recognition-site-specific recombination system is employed. The
natural flippase recombinase gene, flp, is derived from the 2 .mu.m
plasmid of Saccharomyces cerevisiae; FRT stands for "Flippase
recombinase Recognition Target." The FRT comprises 34 base pairs
(bp/bps): 5'-GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC-3', and is divided
into two regions. The first consecutive 13 bps and last 13
consecutive bps belong to a complementary region, which is the FLP
recognition site. The central 8 bps are named as the core region,
which is asymmetric. FLP recognizes two identical FRT in the same
direction and flip-out the gene flanked by the two FRT to
accomplish specific gene recombination. The possibility of the
occurrence of recombination based on two non-identical FRTs is
extreme low (Storici et al. 1999, Yeast 15, 271-283).
[0025] Due to the lack of thermotolerant Saccharomyces cerevisiae
having glucose fermentation efficiency higher than 95% and method
for making same, it is apparent that there is a present need for
such means to lower the production cost of cellulosic biomass
ethanol.
[0026] To overcome the shortcomings that the prior art fails to
provide a thermotolerant yeast and fails to lower the production
cost for biomass ethanol, the present invention provides a
thermotolerant yeast with a substitute heat shock protein 104
promoter to mitigate or obviate the aforementioned problems.
SUMMARY OF THE INVENTION
[0027] The main objective of the invention is to provide a new
recombinant Saccharomyces cerevisiae having an ethanol production
ratio higher than 95% based on fermentation of glucose at a
temperature higher than 42.degree. C.
[0028] The method for making the yeast in accordance with the
present invention comprises the step of replacing the regulation
region upstream of the hsp104 gene in the genome of the yeast, so
as to accelerate and prolong the expression span of hsp104 gene and
enhance the capability of the yeast to ferment and produce ethanol
in a high-temperature environment.
[0029] The yeast in accordance with the present invention has been
deposited in International Depository Authority (IDA) China Center
for Type Culture Collection (CCTCC) and assigned a deposit
designation "CCTCC M 2012254," which is a recombinant yeast
substrain derived from the Saccharomyces cerevisiae strain sake
yeast Kyokai 6.
[0030] The deposited yeast deposited as deposit CCTCC M 2012254 in
CCTCC is capable of fermenting glucose at a temperature higher than
42.degree. C. to produce ethanol, or biomass ethanol, wherein the
ethanol production ratio based on fermentation of glucose is higher
than 97%. Being able to synchronize the degradation/hydrolysis
stage and fermentation stage of biomass ethanol producing process,
the yeast in accordance with the present invention is able to lower
the production cost of biomass ethanol and further raise the
productivity with its high ethanol production ratio.
[0031] Other objectives, advantages and novel features of the
invention will become more apparent from the following detailed
description when taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a schematic drawing of the construction of plasmid
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R (40 homo) pTZ57R/T for a
yeast genome exchange platform;
[0033] FIG. 2a is an image of an electrophoresis result of a PCR
product FRT-KanMX6-FRT, i.e., a fragment containing KanMX6 flanking
by two FRTs, sizing 1431 bps;
[0034] FIG. 2b is an image of an electrophoresis result of DNA
fragments obtained by cleaving FRT-KanMX6-FRT yT&A with EcoRI
and XhoI, which respectively sizing 2726 bps and 1433 bps;
[0035] FIG. 3a is an image of an electrophoresis result of a PCR
product HSP26.sup.R sizing 985 bps;
[0036] FIG. 3b is an image of an electrophoresis result of DNA
fragments obtained by cleaving FRT-KanMX6-FRT-HSP26.sup.R yT&A
with EcoRI and BamHI, which respectively sizing 2709 bps and 2416
bps;
[0037] FIG. 4 is an image of an electrophoresis result that in
lanes 1 to 5 of which are: 1) plasmid FRT-KanMX6-FRT-HSP26.sup.R
yT&A, 2) plasmid FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R, 3) a
linear DNA fragment obtained from XhoI and BamHI digested plasmid
FRT.sup.B-KanMX6-FRT.sup.B yT&A, 4) a fragment cleaved from
plasmid FRT-KanMX6-FRT-HSP26.sup.R yT&A by XhoI and BamHI
digestion, 5) plasmid FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R
yT&A obtained from ligation of the fragments as in lane 3 and
lane 4;
[0038] FIG. 5 is an image of an electrophoresis result that lanes 1
and 2 are loaded with linear DNA fragments obtained from BamHI
digested plasmid FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R (40 homo)
pTZ57R/T;
[0039] FIG. 6 is an image of an electrophoresis result of colony
PCR products of Kyokai 6-HSP104.sup.R/26.sup.R Kan and s288c
yeast.
[0040] FIG. 7 is an image of a thermotolerance spot assay wherein
"WT" represents Kyokai 6 and "PR" represents Kyokai
6-HSP104.sup.R/26.sup.R Kan;
[0041] FIG. 8 is a chart of the result of a shake flask
fermentation experiment wherein "WT" represents Kyokai 6 and "PR"
represents Kyokai 6-HSP104.sup.R/26.sup.R Kan; and
[0042] FIG. 9 is a chart of the result of a fermentation experiment
using fermenters wherein "WT" represents Kyokai 6 and "PR"
represents Kyokai 6-HSP104.sup.R/26.sup.R Kan.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] An embodying of the present invention first involves in
constructing a specific targeted integration of kanamycin
resistance-associated non-selectable DNA promoter exchange
platform, which employs linear DNA transformation to replace the
upstream regulation region of hsp104 gene in yeast genome, so as to
accelerate and prolong the expression span of hsp104 gene and
enhance the capability of the yeast to ferment and produce ethanol
at a high-temperature.
[0044] As shown in FIG. 1, the construction of plasmid pTZ57R/T for
a yeast genome exchange platform using
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R (40 homo) employs antibiotic
resistance gene KanMX6 with a sequence as shown in SEQ ID NO: 9 and
uses plasmid pYC6ct-KanMX6 and the chromosome of Saccharomyces
cerevisiae strain s288c as PCR templates accompanying SEQ ID NOs:
1-7 as primers.
[0045] SEQ ID NO: 1 is a forward primer for KanMX6 and SEQ ID NO: 2
is a backward primer for KanMX6, while SEQ ID NO: 3 is a forward
primer for hsp26 and SEQ ID NO: 4 is a backward primer for hsp26. A
sequence having KanMX6 for resistance against kanamycin, a fragment
containing KanMX6 and the regulation region of hsp26 flanked by two
FRT sequences is obtained by using the two of the aforementioned
primer pairs. The sequence of KanMX6 is first integrated into
vector yT&A and the regulation region of hsp26 (HSP26.sup.R)
digested with XhoI and BamHI is subsequently integrated to the
yT&A vector. The plasmid so constructed is named as
FRT-KanMX6-FRT-HSP26.sup.R yT&A.
[0046] In order to lower the rate of homologous recombination
between designed plasmid and the FRT sequence of the yeast 2 .mu.m
plasmid, a fragment containing KanMX6 flanked by FRT having mutated
FRT core regions is obtained by using SEQ ID NO: 5 as the forward
primer for FRT.sup.B and SEQ ID NO: 6 as the backward primer for
FRT.sup.B. The fragment containing KanMX6 is first integrated into
vector yT&A and the regulation region of hsp26 (HSP26.sup.R)
digested with XhoI and BamHI is subsequently integrated to the
yT&A vector. The plasmid constructed as aforementioned is named
as FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R yT&A.
[0047] The FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R fragment is
further manipulated as being flanked by two homologous sequences
(40 homo) in order to exchange with the upstream regulation region
of hsp104 gene.
[0048] FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R yT&A is then
cleaved by BamHI to be linear DNA. SEQ ID NO: 7 is a forward primer
for adding a homologous sequence and SEQ ID NO: 8 is a backward
primer for adding another homologous sequence, both homologous
sequences, though of different sequences, are 40 bps in length and
abbreviated "40 homo." PCR with the primer pair, SEQ ID NOs: 7 and
8, gives a DNA fragment that is further integrated into a
TA-cloning vector pTZ57R/T. The plasmid constructed with the
foregoing process is named as
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R (40 homo) pTZ57R/T.
[0049] Before transformating a yeast strain of Saccharomyces
cerevisiae Kyokai strain series with plasmid
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R (40 homo) pTZ57R/T with,
e.g., lithium acetate, the plasmid is first cleaved with CaiI to be
linear DNA. The linear DNA made from the plasmid is used to
transform Saccharomyces cerevisiae strains Kyokai 6 and s288C. The
colonies with successfully replaced DNA, i.e., Saccharomyces
cerevisiae substrains of which upstream regulation region of hsp104
has been replaced with the FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R
fragment, are confirmed by colony PCR. The Saccharomyces cerevisiae
substrain, which is derived from the Kyokai 6 strain, having
sequences successfully replaced with a PCR primer pair designed
based on the peripheral sequence of the replaced DNA fragment,
i.e., SEQ ID NOs: 10 and 11, is named as Kyokai
6-HSP104.sup.R/26.sup.R Kan.
[0050] The thermotolerance of Kyokai 6-HSP104.sup.R/26.sup.R Kan is
tested using spot assay. Being incubated in yeast peptone dextrose
agar plate (YPD agar plate) at 30 to 46.degree. C. for 16 hours,
Kyokai 6-HSP104.sup.R/26.sup.R Kan demonstrates superior
thermotolerance than Kyokai 6 from which it is originated. When
shake flask fermenting at 42.degree. C. in an YPD medium, Kyokai
6-HSP104.sup.R/26.sup.R Kan reaches a saturated cell density in 7
hours, which indicates a faster growth rate than Kyokai 6. In
addition, only at the 8-hour time point does Kyokai
6-HSP104.sup.R/26.sup.R Kan give trace ethanol, which may be
attributed to the leak-in of oxygen to the shake flask when
sampling and the consumption of ethanol by Kyokai
6-HSP104.sup.R/26.sup.R Kan that performs respiration at a higher
rate.
[0051] When fermented with fermenter in 5 L of YPD at 42.degree.
C., Kyokai 6-HSP104.sup.R/26.sup.R Kan grows faster than Kyokai 6.
Kyokai 6-HSP104.sup.R/26.sup.R Kan consumes 80 g/L glucose in 14
hours and produce ethanol based on glucose at an ethanol production
ratio of 97%.
EXAMPLE 1
[0052] The instant example demonstrates the preparation and
purification of plasmids. A feasible embodiment has been carried
out as follows:
[0053] 2 mL of LB medium supplied with 100 .mu.g/mL of ampicillin
containing ECOS.TM. 101 (Yeastern, E. coli DH5.alpha.) transformed
with plasmid pYC6ct-KanMX6 cultivated at 37.degree. C. under 175
rpm shaking for 12 hours was added in 1.75-mL microcentrifuge tubes
to be centrifuged for 1 minutes at 14000 rcf. The supernatant was
removed leaving bacteria pellet, which was then repetitively
pipetted and resuspended with 200 .mu.L of MiniPrep-V.sup.2
solution II. The suspension was stood for 5 minutes and then
repetitively pipetted and resuspended with 300 .mu.L of
MiniPrep-V.sup.2 solution III, after which centrifugation at 14000
rcf for 5 minutes was performed.
[0054] 700 .mu.L of supernatant was collected in spin columns
(collection tubes) and centrifuged for 30 seconds. The liquid in
the spin columns was removed and 700 .mu.L of wash solution was
added. An additional 30-second centrifugation was performed and the
wash solution was removed. The spin columns were centrifuged for 3
minutes and respectively placed in 1.75 mL microcentrifuge tubes
with their lids left open. The open spin columns were dried at
60.degree. C. for 20-30 minutes, after which 55 .mu.L of Elution
solution was added and the spin columns were centrifuged for 2
minutes. The solution in the spin columns now contained the
purified plasmid. 1 .mu.L of the solution were used for measuring
the concentration and purity of the plasmid with Nano Drop.RTM.
1000.
EXAMPLE 2
[0055] The instant example demonstrates purification of chromosome
of Saccharomyces cerevisiae. One feasible embodiment has been
performed as follows:
[0056] 1.5 mL of YPD medium containing Saccharomyces cerevisiae
cultivated at 30.degree. C. under 200 rpm shaking for 16 hours was
added into a 1.75-mL microcentrifuge tube and centrifuged at 12000
rpm for 1 minute. The supernatant was removed and 300 .mu.L of
Master Pure.TM. Lysis solution was used to resuspend the yeast. The
suspension was allowed to react at 65.degree. C. for 15 minutes,
followed by a 5-minute ice bath. The tube was supplied with 150
.mu.L of Master Pure.TM. MPC solution (Protech) and was shaked by a
tube-shaker for 10 seconds, after which a centrifugation at 12000
rpm was performed for 10 minutes. 500 .mu.L of supernatant was
taken to a new 1.75-mL microcentrifuge tube and mixed with 500
.mu.L of isopropanol by repetitive inversion, the mixture of which
was than centrifuged at 12000 rpm for 10 minutes. The supernatant
was removed and a washing with 500 .mu.L of 70% alcohol was
performed prior to a short centrifugation. The washing alcohol was
removed and 35 .mu.L of TE buffer (pH 8) was used to dissolve the
DNA. The DNA solution was than stored at -20.degree. C.
EXAMPLE 3
[0057] The instant example demonstrates the construction of plasmid
FRT-KanMX6-FRT yT&A. One feasible embodiment has been performed
as follows:
[0058] A PCR program setting forth an initial denaturation at
temperature 95.degree. C. for 7 minutes, 30 cycles and a final
extension for 10 minutes was employed in the instant embodiment.
Each of the cycles has a denaturation step at 95.degree. C. for 1
minute, an annealing step at 70.degree. C. for 1 minute and an
extention step at 72.degree. C. for 1.7 minute. Plasmid
pYC6ct-KanMX6 was used as the PCR template and SEQ ID NOs: 1 and 2
were used as forward and backward primers.
[0059] As shown in FIG. 2a, a sequence, 1431-bps in length,
containing a KanMX6 gene for resistance against kanamycin and two
FRT sequences flanking the KanMX6 gene was obtained. The sequence
was confirmed with voltage-fixed electrophoresis using a 1%
argarose gel at 100 V and 50 mA for 50 minutes.
[0060] The DNA fragment of the DNA sequence containing the KanMX6
gene was cutted off and purified from the agarose gel using
QIAquick Gel Extraction Kit (Qiagen). 100 ng of the DNA fragment
was mixed with 2 .mu.L of yT&A vector (Yeastern), 2 .mu.L of
Ligation buffer, 0.2 .mu.L of T4 DNA ligase and DI water filling up
the total volume to 20 .mu.L. The DNA mixture was allowed to react
for 1 hour and deactivated at 70.degree. C. for 5 minutes. 10 .mu.L
of the deactivated DNA mixture was added to a 1/3 filled tube of
ECOS.TM. 101 competent cells, followed by 30 minutes of ice bath,
45-90 seconds of 42.degree. C. water bath and another 5-minute ice
bath. 200 .mu.L of LB broth was added to the competent cells, which
was then spreded on a LB agarose plate containing 100 .mu.g/mL of
ampicillin and incubated at 37.degree. C. overnight. Yeast strains
were so cultivated. As shown in FIG. 2b, the plasmids of the
strains were purified and screened with 1% agarose gel
electrophoresis to select substrains having the plasmid containing
the FRT-KanMX6-FRT yT&A fragment. The plasmids of selected sub
strains were sequenced and further confirmed.
EXAMPLE 4
[0061] The instant example demonstrates the construction of plasmid
FRT-KanMX6-FRT-HSP26.sup.R yT&A. One feasible embodiment has
been performed as follows:
[0062] As shown in FIG. 3a, a PCR program setting forth an initial
denaturation at temperature 95.degree. C. for 7 minutes, 30 cycles
and a final extension for 10 minutes was employed in the instant
embodiment. Each of the cycles has a denaturation step at
95.degree. C. for 1 minute, an annealing step at 52.degree. C. for
1 minute and an extention step at 72.degree. C. for 1.7 minute. The
chromosome of Saccharomyces cerevisiae strain s288c was used as the
PCR template and SEQ ID NOs: 3 and 4 were used as forward and
backward primers. The PCR product was a 985-bp DNA fragment
containing HSP26.sup.R.
[0063] As shown in FIG. 3b, a mixture of 25 .mu.L of the PCR
product, 3 .mu.L of restriction enzyme buffer and 2 .mu.L of XhoI
and BamHI restriction enzymes was allowed to react at 37.degree. C.
for 2 hours followed by deactivation at 80 or 65.degree. C. for 20
minutes and electrophoresis using 1% agarose gel for purifing a
XhoI and BamHI-digested DNA fragment of the sequence containing
HSP26.sup.R as aforementioned, which was then ligated to XhoI and
BamHI-digested plasmid FRT-KanMX6-FRT yT&A, in order to
construct FRT-KanMX6-FRT-HSP26.sup.R yT&A. The resulting
plasmid FRT-KanMX6-FRT-HSP26.sup.R yT&A was submitted to
sequencing for further confirmation.
EXAMPLE 5
[0064] The instant example demonstrates the construction of plasmid
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R yT&A. One feasible
embodiment has been performed as follows:
[0065] As shown in FIG. 4, a PCR program setting forth an initial
denaturation at temperature 95.degree. C. for 7 minutes and 33
cycles was employed in the instant embodiment. Each of the cycles
has a denaturation step at 95.degree. C. for 1 minute, an annealing
step at 70.degree. C. for 1 minute and an extention step at
72.degree. C. for 1.7 minute. A linear DNA made from cleaving
plasmid FRT-KanMX6-FRT-HSP26.sup.R yT&A with BamHI was used as
the PCR template and SEQ ID NOs: 5 and 6 were used as forward and
backward primers. The PCR product was a sequence, 1451-bps in
length, containing a KanMX6 gene for resistance against kanamycin
and two FRT.sup.B sequences flanking the KanMX6 gene was obtained.
A DNA fragment of the aforementioned sequence was isolated on and
purified from a 1% agarose gel using electrophoresis and QlAquick
Gel Extraction Kit (Qiagen).
[0066] 100 ng of the DNA fragment was mixed with 2 .mu.L of
yT&A vector (Yeastern), 2 .mu.L of Ligation buffer, 0.2 .mu.L
of T4 DNA ligase and DI water filling up the total volume to 20
.mu.L, allowed to react at 22.degree. C. for 1 hour and then
deactivated at 70.degree. C. for 5 minutes. 10 .mu.L of the
deactivated DNA mixture was added into a 1/3 filled tube of
competent cell ECOS.TM. 101, followed by 30 minutes of ice bath,
45-90 seconds of 42.degree. C. water bath and another 5-minute ice
bath. 200 .mu.L of LB broth was added to the competent cells, which
was then spreded on a LB agarose plate containing 100 .mu.g/mL of
ampicillin and incubated at 37.degree. C. overnight. Yeast strains
were so cultivated. The plasmids of the strains were purified and
screened with 1% agarose gel electrophoresis to select substrains
having the plasmid containing the FRT.sup.B-KanMX6-FRT.sup.B
yT&A fragment.
[0067] The XhoI-BamHI fragment of plasmid
FRT-KanMX6-FRT-HSP26.sup.R yT&A containing HSP26.sup.R was
cleaved off with the restriction enzymes and integrated into the
plasmid FRT.sup.B-KanMX6-FRT.sup.B yT&A to obtain plasmid
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R yT&A.
EXAMPLE 6
[0068] The instant example demonstrates the construction of plasmid
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R (40 homo) pTZ57R/T. One
feasible embodiment has been performed as follows:
[0069] As shown in FIG. 5, a PCR program setting forth an initial
denaturation at temperature 95.degree. C. for 3 minutes, 30 cycles
and a final extension for 30 minutes was employed in the instant
embodiment. Each of the cycles has a denaturation step at
95.degree. C. for 1 minute, an annealing step at 66.8.degree. C.
for 2 minutes and an extention step at 72.5.degree. C. for 3
minutes. A linear DNA made from plasmid
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R yT&A with BamHI
digestion was used as the PCR template and SEQ ID NOs: 7 and 8 were
used as forward and backward primers, wherein the first 40 bps of
SEQ ID NOs: 7 and 8 are respectively homologous sequences to the
upstream regulation region of hsp104 gene, for synthesizing two
40-bp homologous sequences (40 homo) flanking the
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R fragment, so as to exchange
with the upstream regulation region of hsp104 gene. The resulting
DNA fragment was integrated into a TA-cloning vector pTZ57R/T to
constitute plasmid FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R (40 homo)
pTZ57R/T then used for transforming ECOS.TM. Blue (Yeastern, E.
coli XL-1 blue).
EXAMPLE 7
[0070] The instant example demonstrates the transformation of yeast
with plasmid FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R (40 homo)
pTZ57R/T. One feasible embodiment has been performed by lithium
acetate transformation of yeast as follows:
[0071] Yeasts were pre-incubated for 12 hours and the yeast-density
of the suspension was adjusted that the OD.sub.600 was from 2 to 4,
after which the incubated suspension was diluted that OD.sub.600
was from 0.2 to 0.4. The suspension was further incubated for 3
hours while maintaining its OD.sub.600 to be less than 0.6. The
suspension was then centrifuged at 4.degree. C. and 12000 rpm for 5
minutes. The supernatant was removed and 25 mL of sterilized
deionized water was added to resuspend the yeast pellet. The
suspension was centrifuged at 4.degree. C. for 5 minutes at 12000
rpm. The supernant was removed and the yeast pellet was left. 0.3
mL of TE/LiAc (10 mM Tris; 10 mM EDTA, 0.1 M LiAc, pH 7.5) was
added and yeast competent cells were prepared.
[0072] In one 1.75-mL microcentrifuge tube, more than 0.1 .mu.g of
a linear DNA made from Call digested
FRT.sup.B-KanMX6-FRT.sup.B-HSP26.sup.R (40 homo) pTZ57R/T, and 0.1
mg of carrier DNA (sheared, denatured herring testes DNA) was added
and mixed 0.1 mL of yeast competent cells were further added,
immediate followed by addition of 0.6 mL of PEG/LiAc (10 mM Tris;
10 mM EDTA, 0.1 M LiAc, 40% PEG4000, pH 7.5). The microcentriguge
tube was shaked with a tube shaker for 10 seconds allowing well
mixing of the aforementioned ingredients, and then incubated in a
constant-temperature incubator at 30.degree. C. and being shaked at
200 rpm for 30 minutes. 70 .mu.L DMSO was then added and mixed by
repetitive inversions. A 42.degree. C. heat shock with a duration
of 15 minutes was immediately performed. A 5-minute ice bath
followed. 1 mL of YP medium was added. After a 3-hour incubation at
30.degree. C. shaking at 200 rpm, 200 .mu.L of the incubated
suspension was spreaded on a YPD G418 solid medium for
screening.
EXAMPLE 8
[0073] The instant example demonstrates the screening of yeast
substrains with yeast colony PCR. One feasible embodiment has been
performed as follows:
[0074] Yeast colony PCR was performed using Yeast Protein Kit.TM.
(Zymo Research). 20 .mu.L of Y-Lysis Buffer and 1 .mu.L of
Zymolyase solution were added in a 1-mL microcentrifuge tube. Trace
yeast from a colony on the aforementioned solid medium was
transferred in to the mixture in the microcentrifuge tube and was
incubated at 37.degree. C. for 1 hour. 2 .mu.L of the incubated
suspension was used for PCR.
[0075] As shown in FIG. 6, the substrain derived from Kyokai 6
strain whose DNA was successfully replaced represented a PCR
product being a 2.5 kb DNA fragment. The substrain was named Kyokai
6-HSP104.sup.R/26.sup.R Kan, based on which a deposit, CCTCC M
2012254, has been made in an IDA, i.e., CCTCC.
EXAMPLE 9
[0076] The instant example demonstrates the thermotolerance spot
assay for yeast.
[0077] Yeast substrain Kyokai 6-HSP104.sup.R/26.sup.R Kan was
picked with a toothpick into 1.5 mL of YPD medium and cultivated
submerged. When OD.sub.600 value of the suspension became 2 to 4,
the suspension was 10-fold diluted and re-activated. 3 hours later,
OD.sub.600 value was further measured and adjusted to 0.3 with YP
medium. A suspension so adjusted was defined a 1X suspension. 100
.mu.L of 1X suspension was 10-fold diluted and defined as a
10.sup.-1X suspension. With similar methods, 10.sup.-2X, 10.sup.-3X
and 10.sup.-4X suspensions were made. 10-.mu.L aliquots of the
aforementioned suspensions of different concentrations was serially
dropped on YPD solid mediums and respectively incubated at
30.degree. C., 37.degree. C., 40.degree. C., 42.degree. C.,
44.degree. C. and 46.degree. C. for 16 hours. As shown in FIG. 7,
Kyokai 6-HSP104.sup.R/26.sup.R Kan demonstrated superior
thermotolerance than the Kyokai 6 strain from which it is derived
from.
EXAMPLE 10
[0078] The instant example demonstrates the shake flask
fermentation experiment at 42.degree. C.
[0079] With a toothpick or similar tool, trace Kyokai
6-HSP104.sup.R/26.sup.R Kan was taken to 5 mL of YPD medium to
perform submerged cultivation. When the OD.sub.600 value of the
cultivation medium had been higher than 7, the cultivation medium
was diluted 10-fold diluted and re-activated for 3 hours. The
OD.sub.600 value was measured again and the cultivation medium was
adjusted with YPD medium that the glucose concentration was 80 g/L
and the OD value was 0.3. Shake flask fermentation was performed at
42.degree. C. At every 1-hour interval, two 1-mL samples were taken
from the fermentation suspension. One of the 1-mL sample of each
time point was used for OD values measurement. The other 1-mL
sample of each time point was centrifuged at 13000 rpm, from which
supernatant was collected and filtered through a 0.22 .mu.m filter,
so as to measure the glucose concentration and ethanol
concentration with HPLC.
[0080] As shown in FIG. 8, when performing shake flask fermenting
at 42.degree. C. in an YPD medium, Kyokai 6-HSP104.sup.R/26.sup.R
Kan reached a saturated cell density in 7 hours, which indicated a
faster growth rate than Kyokai 6. In addition, only at the 8-hour
time point did Kyokai 6-HSP104.sup.R/26.sup.R Kan give trace
ethanol, which may be attributed to the leak-in of oxygen into the
shake flask when sampling and the consumption of ethanol by Kyokai
6-HSP104.sup.R/26.sup.R Kan that performs respiration at a higher
rate.
EXAMPLE 11
[0081] The instant example demonstrates the fermentation experiment
using fermenters at 42.degree. C.
[0082] With a toothpick or similar tool, Kyokai
6-HSP104.sup.R/26.sup.R Kan that had grown to a rod or string shape
was submitted to 100 mL of YPD medium to perform submerged
cultivation. When the OD.sub.600 value of the cultivation medium
had been higher than 7, the cultivation medium was diluted that the
OD.sub.600 value returned to 7, wherein the total volume was 1000
mL 3L of glucose solution containing 400 g of glucose and 1 L of
4.times. concentration YP medium were added into the fermenter. The
fermenter was kept in a condition that its agitator were rotating
at 200 rpm, the temperature was 42.degree. C. and the pH value was
5.
[0083] When the condition became stable, 100 mL medium having an OD
of 7 was poured in. After 2 minutes of stirring, 20 mL of
fermentation suspension was taken as a 0-hour sample. 10 mL of the
fermentation suspension sample was centrifuged at 14000 rpm for 5
minutes. The supernatant was taken and filtered with a 0.22 .mu.m
filter and the concentrations of glucose and ethanol were measured
with HPLC. 10 mL of DI water was added to resuspend the pellet. The
resuspended suspension was centrifuged at 14000 rpm for 5 minutes,
after which the supernatant was removed and 10 mL DI water was
added to resuspend the pellet. 1 mL of the suspension was used to
measure OD value and the rest was centrifuged at 14000 rpm for 5
minutes and, having the supernatant removed, stored at -80.degree.
C. Foregoing sampling and measurement was repeated at time points
until the HPLC measured glucose concentration dropped to 0.
[0084] As shown in FIG. 9, Kyokai 6-HSP104.sup.R/26.sup.R Kan grew
faster than Kyokai 6 and consumed 80 g/L glucose in 14 hours. The
ethanol production ratio of Kyokai 6-HSP104.sup.R/26.sup.R Kan
based on glucose was as high as 97%.
Sequence CWU 1
1
11154DNASaccharomyces cerevisiaesource1..54/mol_type="DNA"
/note="forward primer for KanMX6" /organism="Saccharomyces
cerevisiae" 1gaagttccta ttctctagaa agtataggaa cttcgacatg gaggcccaga
atac 54260DNASaccharomyces cerevisiaesource1..60/mol_type="DNA"
/note="backwards for KanMX6" /organism="Saccharomyces cerevisiae"
2ctcgaggaag ttcctatact ttctagagaa taggaacttc cagtatagcg accagcattc
60329DNASaccharomyces cerevisiaesource1..29/mol_type="DNA"
/note="forward for HSP26" /organism="Saccharomyces cerevisiae"
3ccgctcgaga tataacctac cataggaca 29425DNASaccharomyces
cerevisiaesource1..25/mol_type="DNA" /note="backward for HSP26"
/organism="Saccharomyces cerevisiae" 4cgcggatccg ttaatttgtt tagtt
25580DNASaccharomyces cerevisiaesource1..80/mol_type="DNA"
/note="forward for FRTB" /organism="Saccharomyces cerevisiae"
5gaattcgaag ttcctattct ctagttcgta taggaacttc ccatgggcgg ccgccatatg
60gacatggagg cccagaatac 80660DNASaccharomyces
cerevisiaesource1..60/mol_type="DNA" /note="backward for FRTB"
/organism="Saccharomyces cerevisiae" 6ctcgaggaag ttcctatacg
aactagagaa taggaacttc cagtatagcg accagcattc 60774DNASaccharomyces
cerevisiaesource1..74/mol_type="DNA" /note="forward for 40 homo"
/organism="Saccharomyces cerevisiae" 7tttttccaga attttctaga
agggttatta attacaatct gaagttccta ttctctagtt 60cgtataggaa cttc
74869DNASaccharomyces cerevisiaesource1..69/mol_type="DNA"
/note="backward for 40 homo" /organism="Saccharomyces cerevisiae"
8tcgttagagc cctttctgta aattgcgttt ggtcgttcat gttaatttgt ttagtttgtt
60tgtttgctt 6991492DNASaccharomyces
cerevisiaesource1..1492/mol_type="DNA" /note="KanMX6 gene sequence"
/organism="Saccharomyces cerevisiae" 9cggatccccg ggttaattaa
ggcgcgccag atctgtttag cttgcctcgt ccccgccggg 60tcacccggcc agcgacatgg
aggcccagaa taccctcctt gacagtcttg acgtgcgcag 120ctcaggggca
tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc
180atttgcatcc atacattttg atggccgcac ggcgcgaagc aaaaattacg
gctcctcgct 240gcagacctgc gagcagggaa acgctcccct cacagacgcg
ttgaattgtc cccacgccgc 300gcccctgtag agaaatataa aaggttagga
tttgccactg aggttcttct ttcatatact 360tccttttaaa atcttgctag
gatacagttc tcacatcaca tccgaacata aacaaccatg 420ggtaaggaaa
agactcacgt ttcgaggccg cgattaaatt ccaacatgga tgctgattta
480tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
ctatcgattg 540tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
gcaaaggtag cgttgccaat 600gatgttacag atgagatggt cagactaaac
tggctgacgg aatttatgcc tcttccgacc 660atcaagcatt ttatccgtac
tcctgatgat gcatggttac tcaccactgc gatccccggc 720aaaacagcat
tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
780ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
ttttaacagc 840gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
ataacggttt ggttgatgcg 900agtgattttg atgacgagcg taatggctgg
cctgttgaac aagtctggaa agaaatgcat 960aagcttttgc cattctcacc
ggattcagtc gtcactcatg gtgatttctc acttgataac 1020cttatttttg
acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
1080gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
tccttcatta 1140cagaaacggc tttttcaaaa atatggtatt gataatcctg
atatgaataa attgcagttt 1200catttgatgc tcgatgagtt tttctaatca
gtactgacaa taaaaagatt cttgttttca 1260agaacttgtc atttgtatag
tttttttata ttgtagttgt tctattttaa tcaaatgtta 1320gcgtgattta
tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc
1380agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg
ctgtcgattc 1440gatactaacg ccgccatcca gtttaaacga gctcgaattc
atcgatgata tc 14921037DNASaccharomyces
cerevisiaesource1..37/mol_type="DNA" /organism="Saccharomyces
cerevisiae" 10aagggcacat gcggttgtgg cgagtttcat ggtttaa
371143DNASaccharomyces cerevisiaesource1..43/mol_type="DNA"
/organism="Saccharomyces cerevisiae" 11gagcaattta cagaaagggc
tctaacgatt ttgacgttgg ctc 43
* * * * *