CN117701409A - Acid-resistant saccharomyces cerevisiae engineering strain, construction method and application thereof - Google Patents
Acid-resistant saccharomyces cerevisiae engineering strain, construction method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention discloses an acid-resistant saccharomyces cerevisiae engineering strain, a construction method and application thereof. The acid-resistant saccharomyces cerevisiae engineering strain is obtained by performing the following operations on a saccharomyces cerevisiae original strain: at least one of overexpressing SSK2 gene, knocking out NUP145 gene, and knocking out KTR6 gene. The acid-resistant saccharomyces cerevisiae engineering strain provided by the invention can be effectively fermented in an acidic environment with the pH value below 3.0, so that the fermentation performance of the strain at low pH value is improved, the treatment process is simplified, and the cost is effectively reduced. The yeast engineering bacteria have huge fermentation production potential in low pH acid environment and have better application prospect in industrial application.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an acid-resistant saccharomyces cerevisiae engineering strain, a construction method and application thereof.
Background
Succinic acid, which was originally found in amber, is also known as Succinic Acid (SA). It is a tetracarboxylic acid, an intermediate metabolite of tricarboxylic acid cycle, widely found in humans, animals, plants and microorganisms. Its molecular formula is C 4 H 6 O 4 The molecular weight is 118.09. Succinic acid is widely used in the industries of medicine, agriculture, food and the like, and particularly is used as a surfactant, a thickener, an electroplated ion chelating agent, a food acidulant and a key raw material of medicines. More critical, bio-based succinic acid has an important role in sustainable biochemistry as a platform chemical. It is the basis for synthesizing important chemicals such as 1, 4-Butanediol (BDO), gamma-butyrolactone and tetrahydrofuran. Currently, succinic acid is synthesized by chemical synthesis and biosynthesis. While chemically synthesized succinic acid is dominant in the market place, bio-based succinic acid is exhibiting a rapidly growing trend. This growth is due in part to the rising cost of conventional petroleum-based production; global concerns about climate and environmental problems have increased, and society and industry have prompted the search for more optimal solutions. The cell factory is used for producing the bio-based succinic acid, so that the traditional petrochemical succinic acid is supplemented or replaced, and the method is a sustainable development mode.
Saccharomyces cerevisiae may be used for succinic acid biosynthesis. However, when yeast synthesizes succinic acid, continuous production and discharge of succinic acid to the outside of cells causes a decrease in pH of the medium, and acid stress is applied to cells, affecting their performance. Therefore, key gene targets are mined, and the saccharomyces cerevisiae is subjected to metabolic modification by adopting a genetic engineering means, so that the tolerance of the saccharomyces cerevisiae to low-pH environment is enhanced, and the saccharomyces cerevisiae is beneficial to the application of the saccharomyces cerevisiae in fermentation synthesis of acid substances such as succinic acid. Therefore, the neutralizing agent and the acid added after the fermentation are avoided to recycle the product, and the method has important significance in reducing the production cost and improving the economic benefit.
Disclosure of Invention
In this study we aimed to construct a strain of Saccharomyces cerevisiae that is tolerant to low pH, when fermented in an acidic environment (e.g. to produce succinic acid), without the need for pH adjustment by addition of neutralizing agents, simplifying the production process, reducing costs and improving overall production efficiency. For this reason, we have studied the acid tolerance mechanism of Saccharomyces cerevisiae and determined three genes SSK2, NUP145 and KTR6 as key genes. Through over-expression or knockout of these three genes, the tolerance of Saccharomyces cerevisiae to low pH environments (high concentration succinic acid) was successfully enhanced. This finding provides an optimization strategy for succinic acid production. Specifically, by over-expressing SSK2 genes on saccharomyces cerevisiae chromosome or knocking out NUP145 and KTR6 genes, the low pH (pH < 3.0) acid stress caused by high concentration succinic acid tolerance of the yeast is successfully improved, and a larger application potential is created for producing succinic acid by the yeast.
The primary aim of the invention is to provide an acid-resistant saccharomyces cerevisiae engineering strain.
The invention also aims to provide a construction method of the acid-resistant saccharomyces cerevisiae engineering strain.
Still another object of the present invention is to provide the use of the above-mentioned acid-resistant Saccharomyces cerevisiae engineering strain.
The aim of the invention is achieved by the following technical scheme:
an acid-resistant saccharomyces cerevisiae engineering strain is obtained by performing the following operations on a saccharomyces cerevisiae starting strain: at least one of overexpressing SSK2 gene, knocking out NUP145 gene, and knocking out KTR6 gene.
The saccharomyces cerevisiae starting strain is preferably a strain with CEN.PK background, is a commonly used yeast strain, and has good representativeness and universality in the wide application of metabolic engineering and systematic biological research in industry and academia (Microb Cell face, 2012,11,36); more preferably Saccharomyces cerevisiae IMX581.
The manipulation is preferably over-expression of SSK2 gene, knockout of NUP145 gene, knockout of KTR6 gene or knockout of NUP145 gene and KTR6 gene.
The gene overexpression is preferably performed by homologous recombination.
The knockout is preferably a traceless knockout by a method of homologous recombination or a CRISPR/Cas9 gene editing technique.
The acids include, but are not limited to, succinic acid.
The acid resistance refers to the environment with the pH value less than 3; more preferably, an environment with ph=2.65 can be tolerated.
The construction method of the acid-resistant saccharomyces cerevisiae engineering strain preferably comprises the following steps:
(1) Constructing pROS10-X3 knockout plasmid, and amplifying to obtain SSK2 repair fragments with pGPD promoters;
(2) Constructing pROS10-NUP145 knockout plasmid and amplifying to obtain NUP145 repair fragment;
(3) Constructing pROS10-KTR6 knockout plasmid and amplifying to obtain KTR6 repair fragments;
(4) Constructing pROS10-N20_1-NUP145-N20_2-KTR6 double-knock plasmid, and amplifying to obtain NUP145 repair fragment and KTR6 repair fragment;
(5) Transforming the pROS10-X3 knockout plasmid obtained in the step (1) and the SSK2 repair fragment with the pGPD promoter into a saccharomyces cerevisiae starting strain, and constructing to obtain a strain S1;
(6) Transforming the pROS10-NUP145 knockout plasmid and the NUP145 repair fragment obtained in the step (2) into a saccharomyces cerevisiae starting strain, and constructing to obtain a strain S2;
(7) Transforming the pROS10-KTR6 knockout plasmid and the KTR6 repair fragment obtained in the step (3) into a saccharomyces cerevisiae starting strain, and constructing to obtain a strain S3;
(8) And (3) transforming the pROS10-N20_1-NUP145-N20_2-KTR6 double-knocked-down plasmid obtained in the step (4) into a Saccharomyces cerevisiae starting strain by using NUP145 and KTR6 repair fragments, and constructing a strain S4.
The pROS10-X3 knockout plasmid described in step (1) is preferably obtained by: using plasmid pROS10 as a template, and amplifying pROS10SK-K-F/pROS10SK-K-R by using a primer pair to obtain a plasmid frame with a screening mark URA 3; amplifying pROS10-Gibson/X3-N20 with primer pair pROS10 as template to obtain gRNA with 20bp target X3; the gRNA with 20bp target X3 and the plasmid frame with the screening mark URA3 are spliced together by using Gibson assembly technology to obtain pROS10-X3 knockout plasmid.
The SSK2 repair fragment with pGPD promoter described in step (1) is preferably obtained by the following steps: splicing an SSK2 gene fragment with a nucleic acid sequence shown as SEQ ID No.1 and a plasmid frame with a pGPD promoter to obtain a plasmid p416-GPD-SSK2; then using plasmid p416-GPD-SSK2 as a template, and amplifying by OE-SSK2-F/OE-SSK2-R to obtain an SSK2 repair fragment with pGPD promoter; wherein the plasmid framework with pGPD promoter is obtained by digestion of plasmid p416GPD using restriction enzymes BamHI, xhoI.
The pROS10-NUP145 knockout plasmid described in step (2) is preferably obtained by: using plasmid pROS10 as a template, and amplifying pROS10SK-K-F/pROS10SK-K-R by using a primer pair to obtain a plasmid frame with a screening mark URA 3; amplifying pROS10-Gibson/NUP145-N20 by using plasmid pROS10 as a template to obtain gRNA with 20bp targeted NUP 145; the plasmid frame with the screening mark URA3 and the gRNA with the 20bp targeted NUP145 are spliced by using the Gibson assembly technology to obtain pROS10-NUP145 knockout plasmid.
The NUP145 repair fragment obtained by amplification described in step (2) is preferably obtained by the following steps: and carrying out PCR amplification on the template obtained by self annealing through a primer pair NUP145-RF-F/NUP145-RF-R to obtain a NUP145 repair fragment.
The pROS10-KTR6 knockout plasmid described in the step (3) is preferably obtained by: using plasmid pROS10 as a template, and amplifying pROS10SK-K-F/pROS10SK-K-R by using a primer pair to obtain a plasmid frame with a screening mark URA 3; amplifying pROS10-Gibson/KTR6-N20 by using plasmid pROS10 as a template to obtain gRNA with 20bp targeted KTR 6; and splicing the plasmid frame with the screening mark URA3 and the gRNA with the 20bp targeted KTR6 by using a Gibson assembly technology to obtain the pROS10-KTR6 knockout plasmid.
The KTR6 repair fragment in the step (3) is preferably obtained by the following steps: and carrying out PCR amplification on the template obtained by self annealing through a primer pair KTR6-RF-F/KTR6-RF-R to obtain a KTR6 repair fragment.
The pROS10-N20_1-NUP145-N20_2-KTR6 double-knocked-down plasmid described in step (4) is preferably obtained by: using plasmid pROS10 as a template, and amplifying by a primer pROS10DK-Frame to obtain a plasmid Frame with a screening mark URA 3; using plasmid pROS10 as a template, amplifying pROS10-Gibson/NUP145-N20 to obtain gRNA with 20bp targeted NUP145, and amplifying pROS10-Gibson/KTR6-N20 to obtain gRNA with 20bp targeted KTR 6; the plasmid frame with the screening mark URA3, the gRNA with the 20bp targeting NUP145 and the gRNA with the 20bp targeting KTR6 are spliced by using the Gibson assembly technology to obtain the pROS10-N20_1-NUP145-N20_2-KTR6 double-knocked plasmid.
The NUP145 repair fragment obtained by amplification described in step (4) is preferably obtained by the following steps: and carrying out PCR amplification on the template obtained by self annealing through a primer pair NUP145-RF-F/NUP145-RF-R to obtain a NUP145 repair fragment.
The KTR6 repair fragment in the step (4) is preferably obtained by the following steps: and carrying out PCR amplification on the template obtained by self annealing through a primer pair KTR6-RF-F/KTR6-RF-R to obtain a KTR6 repair fragment.
The saccharomyces cerevisiae starting strain in the step (5), the step (6), the step (7) and the step (8) is preferably a strain with CEN.PK background; more preferably IMX581.
The application of the acid-resistant saccharomyces cerevisiae engineering strain in fermentation production in a low-pH acidic environment.
The low pH acidic environment refers to an environment with pH less than 3; preferably an environment as low as ph 2.65.
The acid is preferably succinic acid.
Compared with the prior art, the invention has the following advantages and effects:
the invention successfully constructs the yeast engineering bacteria capable of tolerating acid stress (high-concentration succinic acid), and improves the growth capacity of the strain in an acid environment with the pH value lower than 3.0. The invention focuses on three key targets related to succinic acid tolerance, and breaks through fermentation limitation of saccharomyces cerevisiae in a very acidic environment by means of over-expressing SSK2 genes or knocking out NUP145 and KTR6 genes. The strain acid resistance is improved, so that a neutralizing agent is not required to be added in the fermentation process to maintain the pH, the production flow is simplified, and the production cost is reduced.
The yeast engineering strains can be grown and fermented in an acidic environment (such as fermentation production of succinic acid), and have good industrial application potential.
Drawings
FIG. 1 is a schematic diagram of a plasmid according to example 1; wherein, (A) is a schematic diagram of a plasmid carrying a URA3 screening marker with p416-GPD inserted into an overexpressed gene SSK2; (B) Schematic representation of pROS10 single-knock plasmid carrying URA3 selectable marker and corresponding N20 fragment of NUP145 gene; (C) Schematic diagram of pROS10 single-knock plasmid carrying URA3 selection marker and corresponding N20 fragment of KTR6 gene; (D) Schematic representation of pROS10 double-knocked-down plasmid carrying the corresponding N20 fragment of URA3 selection marker, NUP145 gene and KTR6 gene.
FIG. 2 is a schematic diagram showing the sequencing results of two N20 sites in pROS10-N20_1-NUP145-N20_2-KTR 6.
FIG. 3 is a diagram of agarose gel electrophoresis after cleavage of p416-GPD using restriction enzymes BamHI, xhoI; wherein lane M is a DNA molecular weight marker.
FIG. 4 is a graph showing the results of plasmid identification obtained by the construction of example 1; wherein, (A) is a PCR identification agarose gel electrophoresis chart of the successful integration of the strain and the over-expression of SSK2 gene; (B) Is a PCR identification agarose gel electrophoresis chart of the successful single-knock NUP145 and KTR6 genes of the strain; (C) Is a PCR identification agarose gel electrophoresis chart of the strain IMX581 which is used for knocking out NUP145 and KTR6 genes simultaneously; lane M is a DNA molecular weight marker.
FIG. 5 is a graph showing the results of acid resistance tests of yeast engineering strains S1, S2, S3 and S4 cultured in a medium containing high concentration succinic acid; wherein S0 (i.e., IMX 581) served as a control.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified;
the experimental methods in the following examples, in which specific conditions are not specified, are generally carried out according to conventional conditions, such as those described in "molecular cloning Experimental guidelines (Beijing: scientific Press, 2017)", and "Yeast genetic methods Experimental guidelines (Beijing: scientific Press, 2016)".
CRISPR techniques applied in the examples described below are described in the prior art (FEMS Yeast Research,2015,15 (2): fov 004.). Furthermore, the plasmid p416GPD used in the following examples is disclosed in document "Yeast vectors for the controlled expression of heterologous proteins in different genetic backgroups. Gene 1995, 156:119-122"; plasmid pROS10 and strain IMX581 are disclosed in the literature "CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae. FEMS Yeast Research,2015,15, fov004" and are available from EUROSCARF.
For a better understanding of the present invention, a further description will be given of a Saccharomyces cerevisiae (Saccharomyces cerevisiae) CEN.PK background strain as the starting strain, and IMX581 as the specific example.
The media referred to in the following examples are as follows:
LB liquid medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl; the solvent is deionized water; 2% agar powder is added into the solid culture medium.
Ampicillin-resistant LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl; the solid medium was sterilized by adding 2% agar powder, cooled to about 40℃and 100. Mu.g/mL ampicillin (sterilized by filtration).
YPD medium: 20g/L peptone, 10g/L yeast extract, 20g/L glucose (injection: sterilized alone before addition); the solvent is deionized water; 2% agar powder is added into the solid culture medium.
SC+30g/L SA medium: 0.77g/L CSM-Ura,1.7g/L YNB w/o aa and (NH) 4 ) 2 SO 4 ，5.0g/L(NH 4 ) 2 SO 4 0.02g/L Uracil (Uracil), 30g/L Succinic Acid (SA), 20g/L glucose (note: added after sterilization alone); the solvent is deionized water.
SC-Ura auxotroph Medium: 0.77g/L CSM-Ura,1.7g/L YNB w/o aa and (NH) 4 ) 2 SO 4 ，5.0g/L(NH 4 ) 2 SO 4 20g/L glucose (added after independent sterilization), regulating the pH value to 5.5-6.0, and using deionized water as a solvent; 2% agar powder is added into the solid culture medium.
5-FOA solid screening Medium: 0.77g/L CSM-Ura,1.7g/L YNB w/o aa and (NH) 4 ) 2 SO 4 ，5.0g/L(NH 4 ) 2 SO 4 0.05g/L Uracil (Uracil), 0.75 g/L5-FOA, 20g/L glucose (filter sterilized with 22 μm filter), and deionized water as solvent; 2% agar (injection: added with the above liquid after sterilization alone).
The methods involved in the following examples are as follows:
1. constructing a plasmid:
(1) Amplifying the corresponding fragments and frames required by Gibson assembly by a PCR means;
(2) The Gibson assembly method, specific operations were performed according to the NEB Gibson Assembly Cloning kit (cat No. E2611, NEB) instructions;
(3) 5. Mu.L of the assembly system was transformed into 50. Mu.L of E.coli DH 5. Alpha. Competent cells, plated onto ampicillin-resistant LB solid medium, and cultured overnight;
(4) Positive clones were obtained by screening, plasmids were extracted after amplification culture, and the specific extraction procedure was performed according to the instructions of HiPure Plasmid Micro Kit (cat. No. P1001-03, magen).
2. Yeast strain transformation:
unless otherwise indicated, all methods of lithium acetate conversion are used, and specific operations are described in relevant standard specifications.
3. Engineering Saccharomyces cerevisiae Strain OD 600nm Is characterized by comprising the following steps:
inoculating Saccharomyces cerevisiae single colony in 3mL fermentation medium, and shake culturing at 30deg.C at 200 rpm. Diluting fermentation broth at a proper ratio, and using VioletExternal spectrophotometer measuring OD 600nm 。
The primer sequences involved in the following examples are shown in Table 1:
table 1: primer sequence (5 '-3')
EXAMPLE 1 construction of an engineering strain of Saccharomyces cerevisiae tolerant to high concentration succinic acid
1.1 overexpression of SSK2 to improve the succinic acid tolerance of Saccharomyces cerevisiae
The process of overexpressing SSK2 is as follows:
(1) Construction of plasmid pROS10-X3
A. Using plasmid pROS10 as a template, and amplifying pROS10SK-K-F/pROS10SK-K-R by using a primer pair to obtain a plasmid frame with a screening mark URA 3;
PCR reaction system: 2 XMaster Mix 20. Mu. L, pROS10SK-K-F (10. Mu.M) 1.6. Mu. L, pROS10SK-K-R (10. Mu.M) 1.6. Mu. L, pROS10 10.4. Mu. L, ddH 2 O16.4. Mu.L, total volume 40. Mu.L.
PCR reaction conditions: 3min at 95 ℃; 15Sec at 95 ℃,15 Sec at 56 ℃, 3min at 72 ℃,34 cycles; and at 72℃for 5min.
B. Amplifying pROS10-Gibson/X3-N20 with primer pair pROS10 as template to obtain gRNA with 20bp target X3; the PCR reaction system is basically the same as A, and the difference is that: use of the primers herein;
PCR reaction conditions: 3min at 95 ℃;95 ℃ 15Sec, 56 ℃ 15Sec, 72 ℃ 30Sec,34 cycles; and at 72℃for 5min.
C. The fragment was spliced with the plasmid frame carrying the selection marker URA3 using Gibson assembly techniques, and the resulting plasmid was constructed and designated pROS10-X3.
(2) Construction of plasmid p416-GPD-SSK2 and repair fragment pGPD-SSK2
The plasmid p416GPD is taken as a template, and restriction enzymes BamHI and XhoI are used for enzyme digestion of the plasmid, so that a plasmid frame with pGPD promoter is obtained, and the enzyme digestion electrophoresis result is shown in FIG. 3;
the genome of the yeast strain is taken as a template, and a 4804bp SSK2 gene fragment is obtained by amplification of a primer P416-SSK2-F/P416-SSK 2-R; wherein, the coding nucleic acid sequence of SSK2 is shown in SEQ ID No.1; the PCR reaction system is basically the same as (1), except that: using the primers herein, the template yeast genome was 0.1. Mu.L, ddH 2 O is 16.7 mu L; the PCR reaction conditions are the same as those of (1) A;
the fragment was spliced with the plasmid framework carrying pGPD promoter by Gibson assembly technique, and the resulting plasmid was constructed and designated as p416-GPD-SSK2, the structure of which is shown in FIG. 1 (A).
The 5747bp repair fragment pGPD-SSK2 is obtained by amplification of OE-SSK2-F/OE-SSK2-R by taking the constructed plasmid p416-GPD-SSK2 as a template. The PCR reaction system is basically the same as (1), except that: use of the primers herein and templates; the PCR reaction conditions were the same as in (1) A.
(3) Plasmid pROS10-X3 and repair fragment pGPD-SSK2 were transformed together into strain IMX581 by lithium acetate transformation at a molar ratio of 1:1, spread onto SC-Ura solid culture and cultured at 30℃for 3-4 days, and single colony PCR was performed using primers X3-P1/GPD-P2 and CYC1-P1/X3-P2 (results are shown in FIG. 4 (A)), positive transformants were selected and streaked onto 5-FOA solid medium to remove plasmids.
(4) The monoclonal transfer plates obtained on the 5-FOA solid medium were selected and verified on SC-Ura and YPD solid media for successful plasmid removal at 30℃for 2-3 days.
(5) The strain grown on YPD solid medium to give the plasmid-free strain was designated Saccharomyces cerevisiae S1.
1.2 knockout of NUP145 to improve acid resistance of Saccharomyces cerevisiae
NUP145 gene knockout process is as follows:
the coding nucleic acid sequence of NUP145 is shown in SEQ ID No.2.
(1) Construction of plasmid pROS10-N20-NUP145
Using plasmid pROS10 as a template, and amplifying pROS10SK-K-F/pROS10SK-K-R by using a primer pair to obtain a plasmid frame with a screening mark URA 3; the PCR reaction system is 1.1 (1), and the PCR reaction conditions are 1.1 (1) A.
Amplifying pROS10-Gibson/NUP145-N20 by using plasmid pROS10 as a template to obtain gRNA with 20bp targeted NUP 145; the PCR reaction system is 1.1 (1), and the PCR reaction conditions are 1.1 (1) B.
The above fragment was spliced to a plasmid frame carrying the selectable marker URA3 using Gibson assembly techniques, and the resulting plasmid was constructed and designated pROS10-NUP145.
(2) PCR amplified NUP145 repair fragment
And synthesizing a 59bp primer pair NUP145-RF-F/NUP145-RF-R which is completely matched with the upstream and downstream of the NUP145, and obtaining the targeted repair fragment with 50bp homology arms with the upstream and downstream of the NUP145 through PCR amplification and gel recovery.
PCR reaction system: 2 XMaster Mix 20. Mu. L, NUP145-RF-F (10. Mu.M) 2. Mu. L, NUP145-RF-R (10. Mu.M) 2. Mu. L, ddH 2 O16. Mu.L, total volume 40. Mu.L.
PCR reaction conditions: 3min at 95 ℃; 15Sec at 95 ℃,15 Sec at 56 ℃,15 Sec at 72 ℃ and 34 cycles; and at 72℃for 5min.
(3) Plasmid pROS10-NUP145 was transformed into strain IMX581 together with the repair fragment at a molar ratio of 1:1 by a lithium acetate transformation method, spread on SC-Ura solid culture, cultured at 30℃for 3-4 days, single colony PCR was performed using the primer pair NUP145-VER-F/NUP145-VER-R, and positive transformants were selected (the result is shown in FIG. 4 (B)), streaked on 5-FOA solid culture, and cultured at 30℃for 2-3 days.
(4) The monoclonal transfer plates obtained on the 5-FOA solid medium were selected on SC-Ura and YPD solid medium to verify that they were successfully deplasmidized.
(5) The strain grown on YPD solid medium to give the plasmid-free strain was designated Saccharomyces cerevisiae S2.
1.3 knockout of KTR6 to improve acid resistance of Saccharomyces cerevisiae
The KTR6 gene knockout process is as follows:
the coding nucleic acid sequence of KTR6 is shown in SEQ ID No.3.
(1) Construction of plasmid pROS10-N20-KTR6
Using plasmid pROS10 as a template, and amplifying pROS10SK-K-F/pROS10SK-K-R by using a primer pair to obtain a plasmid frame with a screening mark URA 3; the PCR reaction system is 1.1 (1), and the PCR reaction conditions are 1.1 (1) A.
Amplifying pROS10-Gibson/KTR6-N20 by using plasmid pROS10 as a template to obtain gRNA with 20bp targeted KTR 6; the PCR reaction system is 1.1 (1), and the PCR reaction conditions are 1.1 (1) B.
The fragment was spliced with the plasmid frame carrying the selection marker URA3 using Gibson assembly technique, and the resulting plasmid was constructed and designated pROS10-KTR6.
(2) PCR amplified KTR6 repair fragment
And synthesizing a 59bp primer pair KTR6-RF-F/KTR6-RF-R which is completely matched with the upstream and downstream of KTR6, and obtaining the targeted repair fragment with 50bp homology arms with the upstream and downstream of KTR6 through PCR amplification and gel recovery. The PCR reaction system and the PCR reaction conditions are 1.2 (2).
(3) Plasmid pROS10-KTR6 together with the repair fragment was transformed into strain IMX581 by lithium acetate transformation at a molar ratio of 1:1, spread onto SC-Ura solid culture and cultured at 30℃for 3-4 days, single colony PCR was performed using primer KTR6-VER-F/KTR6-VER-R pairs for verification, and positive transformants were selected (the results are shown in FIG. 4 (B)), streaked on 5-FOA solid culture and cultured at 30℃for 2-3 days.
(4) The monoclonal transfer plates obtained on the 5-FOA solid medium were selected on SC-Ura and YPD solid medium to verify that they were successfully deplasmidized.
(5) The strain grown on YPD solid medium to give the plasmid-free strain was designated Saccharomyces cerevisiae S3.
1.4 overlapping and knocking out NUP145+KTR6 to improve acid resistance of Saccharomyces cerevisiae
Nup145+ktr6 stack knockout process is as follows:
(1) Construction of plasmid pROS10-N20_1-NUP145-N20_2-KTR6
Using plasmid pROS10 as template, amplifying the pROS10DK-Frame primer (the primer is used as upstream primer and downstream primer) to obtain a plasmid Frame with a screening mark URA 3; the PCR reaction system is 1.1 (1), and the PCR reaction conditions are 1.1 (1) A.
Amplifying pROS10-2N20-1/NUP145-N20-1 to obtain gRNA with 20bp targeted NUP145 by using the plasmid pROS10 as a template; the PCR reaction system is the same as that of 1.1 (1); the PCR reaction conditions were substantially the same as those of 1.1 (1) B, except that the extension time after annealing was 1min at 72 ℃.
The plasmid pROS10 is used as a template, and the primer pair pROS10-2N20-2/KTR6-N20-2 is used for amplification to obtain the gRNA with 20bp targeted KTR6. The PCR reaction system is the same as that of 1.1 (1); the PCR reaction conditions were substantially the same as those of 1.1 (1) B, except that the extension time after annealing was 1min at 72 ℃.
The above fragments were spliced together with the plasmid frame carrying the selection marker URA3 using Gibson assembly techniques to construct a plasmid designated pROS10-N20_1-NUP145-N20_2-KTR6, and the sequencing results of the two N20 s obtained by sequencing the plasmids are shown in FIG. 2.
(2) PCR amplification of NUP145 and KTR6 repair fragments
Synthesizing a 59bp primer pair NUP145-RF-F/NUP145-RF-R which is completely matched with the upstream and downstream of the NUP145, and obtaining a targeted repair fragment with a 50bp homology arm with the upstream and downstream of the NUP145 through PCR amplification and gel recovery; the PCR reaction system and the PCR reaction conditions are 1.2 (2).
And synthesizing a 59bp primer pair KTR6-VF-F/KTR6-VF-R which is completely matched with the upstream and downstream of KTR6, and obtaining the targeted repair fragment with 50bp homology arms with the upstream and downstream of KTR6 through PCR amplification and gel recovery. The PCR reaction system and the PCR reaction conditions are 1.2 (2).
(3) Plasmid pROS10-N20_1-NUP145-N20_2-KTR6 was transformed together with two repair fragments in a molar ratio of 2:1:1 into strain IMX581 by lithium acetate transformation, spread onto SC-Ura solid culture and cultured at 30℃for 3-4 days, single colony PCR was performed using primer pairs NUP145-VER-F/NUP145-VER and KTR6-VER-F/KTR6-VER pairs (the results are shown in FIG. 4 (C)), positive transformants were obtained by screening, and positive transformants were streaked on 5-FOA solid culture and cultured at 30℃for 2-3 days.
(4) The monoclonal transfer plates obtained on the 5-FOA solid medium were selected on SC-Ura and YPD solid medium to verify that they were successfully deplasmidized.
(5) The strain grown on YPD solid medium to give the plasmid-free strain was designated Saccharomyces cerevisiae S4.
Example 2 engineering strains tolerant to Low pH acid stress
(1) Inoculating the above Saccharomyces cerevisiae strains IMX581, S1, S2, S3, and S4 from plate to 3mL YPD fermentation medium, culturing at 30deg.C and 200rpm for 12 hr, and culturing at OD 600nm =0.1 inoculated into sc+30g/L SA medium for growth, where IMX581 is a control bacterium, shown as S0 in the figure.
(2) After 96h of growth, the bacterial liquid is diluted by different times according to the concentration, and then the OD is measured by an ultraviolet spectrophotometer 600nm 。
From the results (fig. 5), S1, S2, S3, S4 have good acid resistance. In 30g/L succinic acid SC medium (pH=2.65), the acid tolerance (expressed as cell growth density) of S1, S2, S3, S4 was increased by 54%, 148%, 300%, 359% respectively compared with that of S0 (IMX 581). These engineered yeast strains show great potential for industrial application for succinic acid fermentative production in acidic environments with pH below 3.0.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. An acid-resistant saccharomyces cerevisiae engineering strain is characterized in that: the acid-resistant saccharomyces cerevisiae engineering strain is obtained by performing the following operations on a saccharomyces cerevisiae starting strain: at least one of overexpressing SSK2 gene, knocking out NUP145 gene, and knocking out KTR6 gene.
2. The acid-resistant saccharomyces cerevisiae engineering strain according to claim 1, wherein the strain is characterized in that:
the gene overexpression is chromosome integration overexpression genes by a homologous recombination method;
the knockout is a traceless knockout by a homologous recombination method or a CRISPR/Cas9 gene editing technology;
the acid is succinic acid.
3. The acid-resistant saccharomyces cerevisiae engineering strain according to claim 1, wherein the strain is characterized in that:
the saccharomyces cerevisiae starting strain is a strain with a CEN.PK background;
the operation is to over express SSK2 gene, knock out NUP145 gene, knock out KTR6 gene or knock out NUP145 gene and KTR6 gene;
the acid is succinic acid.
4. A method for constructing an acid-resistant saccharomyces cerevisiae engineering strain according to any one of claims 1 to 3, characterized by comprising the following steps:
(1) Constructing pROS10-X3 knockout plasmid, and amplifying to obtain SSK2 repair fragments with pGPD promoters;
(2) Constructing pROS10-NUP145 knockout plasmid and amplifying to obtain NUP145 repair fragment;
(3) Constructing pROS10-KTR6 knockout plasmid and amplifying to obtain KTR6 repair fragments;
(4) Constructing pROS10-N20_1-NUP145-N20_2-KTR6 double-knock plasmid, and amplifying to obtain NUP145 repair fragment and KTR6 repair fragment;
(5) Transforming the pROS10-X3 knockout plasmid obtained in the step (1) and the SSK2 repair fragment with the pGPD promoter into a saccharomyces cerevisiae starting strain, and constructing to obtain a strain S1;
(6) Transforming the pROS10-NUP145 knockout plasmid and the NUP145 repair fragment obtained in the step (2) into a saccharomyces cerevisiae starting strain, and constructing to obtain a strain S2;
(7) Transforming a saccharomyces cerevisiae original strain with pROS10-KTR6 knockout plasmid and a KTR6 repair fragment, and constructing to obtain a strain S3;
(8) The pROS10-N20_1-NUP145-N20_2-KTR6 double knocked-down plasmid and NUP145 and KTR6 repair fragments are transformed into a Saccharomyces cerevisiae starting strain, and a strain S4 is constructed.
5. The method for constructing an acid-resistant saccharomyces cerevisiae engineering strain according to claim 4, wherein the method is characterized in that:
the pROS10-X3 knockout plasmid described in step (1) was obtained by the following steps: using plasmid pROS10 as a template, and amplifying pROS10SK-K-F/pROS10SK-K-R by using a primer pair to obtain a plasmid frame with a screening mark URA 3; amplifying pROS10-Gibson/X3-N20 with primer pair pROS10 as template to obtain gRNA with 20bp target X3; splicing gRNA with 20bp target X3 and a plasmid frame with a screening mark URA3 by using Gibson assembly technology to obtain pROS10-X3 knockout plasmid;
the SSK2 repair fragment with pGPD promoter in the step (1) is obtained by the following steps: splicing an SSK2 gene fragment with a nucleic acid sequence shown as SEQ ID No.1 and a plasmid frame with a pGPD promoter to obtain a plasmid p416-GPD-SSK2; then using plasmid p416-GPD-SSK2 as a template, and amplifying by OE-SSK2-F/OE-SSK2-R to obtain an SSK2 repair fragment with pGPD promoter; wherein, the plasmid frame with pGPD promoter is obtained by using restriction enzyme BamHI and XhoI to digest plasmid p416 GPD;
the pROS10-NUP145 knockout quality described in step (2) was obtained by: using plasmid pROS10 as a template, and amplifying pROS10SK-K-F/pROS10SK-K-R by using a primer pair to obtain a plasmid frame with a screening mark URA 3; amplifying pROS10-Gibson/NUP145-N20 by using plasmid pROS10 as a template to obtain gRNA with 20bp targeted NUP 145; splicing the plasmid frame with the screening mark URA3 and the gRNA with the 20bp targeted NUP145 by using a Gibson assembly technology to obtain pROS10-NUP145 knockout plasmid;
the NUP145 repair fragment obtained by amplification described in step (2) is obtained by the following steps: carrying out PCR amplification on a template obtained by self annealing through a primer pair NUP145-RF-F/NUP145-RF-R to obtain a NUP145 repair fragment;
the pROS10-KTR6 knockout plasmid described in the step (3) is obtained by the steps of: using plasmid pROS10 as a template, and amplifying pROS10SK-K-F/pROS10SK-K-R by using a primer pair to obtain a plasmid frame with a screening mark URA 3; amplifying pROS10-Gibson/KTR6-N20 by using plasmid pROS10 as a template to obtain gRNA with 20bp targeted KTR 6; splicing a plasmid frame with a screening mark URA3 and gRNA with 20bp targeted KTR6 by using a Gibson assembly technology to obtain pROS10-KTR6 knockout plasmid;
the KTR6 repair fragment in the step (3) is obtained by the following steps: carrying out PCR amplification on a template obtained by self annealing through a primer pair KTR6-RF-F/KTR6-RF-R to obtain a KTR6 repair fragment;
the pROS10-N20_1-NUP145-N20_2-KTR6 double-knocked-down plasmid described in step (4) was obtained by: using plasmid pROS10 as a template, and amplifying by a primer pROS10DK-Frame to obtain a plasmid Frame with a screening mark URA 3; using plasmid pROS10 as a template, amplifying pROS10-Gibson/NUP145-N20 to obtain gRNA with 20bp targeted NUP145, and amplifying pROS10-Gibson/KTR6-N20 to obtain gRNA with 20bp targeted KTR 6; splicing the plasmid frame with the screening mark URA3 with the gRNA with the 20bp targeting NUP145 and the gRNA with the 20bp targeting KTR6 by using a Gibson assembly technology to obtain pROS10-N20_1-NUP145-N20_2-KTR6 double-knock plasmid;
the NUP145 repair fragment obtained by amplification described in step (4) is obtained by the following steps: carrying out PCR amplification on a template obtained by self annealing through a primer pair NUP145-RF-F/NUP145-RF-R to obtain a NUP145 repair fragment;
the KTR6 repair fragment in the step (4) is obtained by the following steps: and carrying out PCR amplification on the template obtained by self annealing through a primer pair KTR6-RF-F/KTR6-RF-R to obtain a KTR6 repair fragment.
6. Use of the acid-resistant saccharomyces cerevisiae engineering strain according to any one of claims 1-3 in fermentation production in a low pH acidic environment.
7. The use of the acid-tolerant saccharomyces cerevisiae engineering strain according to claim 6 in fermentation production in a low pH acidic environment, wherein the acid-tolerant saccharomyces cerevisiae engineering strain is characterized in that:
the low pH acidic environment refers to an environment with pH less than 3.
8. The use of the acid-tolerant saccharomyces cerevisiae engineering strain according to claim 7 in fermentation production in a low pH acidic environment, wherein the acid-tolerant saccharomyces cerevisiae engineering strain is characterized in that:
the acid is succinic acid.
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