CN116064596A - Saccharomyces cerevisiae multidrug resistance transcription factor gene YRR mutant YRR1 T185A And applications thereof - Google Patents
Saccharomyces cerevisiae multidrug resistance transcription factor gene YRR mutant YRR1 T185A And applications thereof Download PDFInfo
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- CN116064596A CN116064596A CN202211290911.3A CN202211290911A CN116064596A CN 116064596 A CN116064596 A CN 116064596A CN 202211290911 A CN202211290911 A CN 202211290911A CN 116064596 A CN116064596 A CN 116064596A
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Abstract
The invention discloses a saccharomyces cerevisiae multi-drug tolerance transcription factor gene YRR1 mutant, which is named YRR1 T185A The nucleotide sequence of the gene is shown as SEQ ID No. 1. The invention also discloses a recombinant plasmid pJFE1-YRR1 containing the mutant T185A The recombinant plasmid and mutant YRR1 T185A Use of mutant YRR1 in constructing Saccharomyces cerevisiae cells with high vanillin resistance T185A Use in improving vanillin resistance of Saccharomyces cerevisiae. Experiments prove that: under the stress of vanillin, transfer into YRR1 T185A Mutant Saccharomyces cerevisiae strain BY4741 (yrr 1.DELTA. YRR 1.1 T185A ) The maximum specific growth rate is 0.125h ‑1 Compared with wild type YRR1 WT Is a strain of BY4741 (yrr 1. Delta., YRR 1) WT ) 34% higher, and the lag phase is 8h shorter than that of the control strain. The mutant provided by the invention is more suitable for the construction of high vanillin tolerance saccharomyces cerevisiae strains for producing vanillin/alcohol or producing second-generation fuel ethanol or other high-value compounds by taking lignocellulose as a raw material.
Description
Technical Field
The Saccharomyces cerevisiae multidrug resistance transcription factor gene YRR mutant YRR1 T185A And a construction method and application thereof, belonging to the field of biotechnology.
Background
Multidrug resistance results from the enhanced extrusion of biologically active molecules, which are present in a variety of organisms ranging from fungi to human cells. One key feature of this adaptation is the activation of expression of multiple drug delivery proteins by sensitive detection of intracellular xenobiotics by transcriptional activators. The proteome of Saccharomyces cerevisiae (Saccharomyces cerevisiae) possesses homologs with other eukaryotic systems, including fungal pathogens such as Candida albicans and Candida albicans, as well as humans, which are ideal systems for exploring how gene regulation affects the function of ATP-dependent transporters (ABC transporters) in eukaryotes. YRR1 is an important multi-drug tolerance-related transcription factor gene in Saccharomyces cerevisiae responsible for activating ATP-dependent transporter expression. Its polymorphism confers a different resistance phenotype to Saccharomyces cerevisiae against 152 different compounds.
Vanillin is one of the most popular fragrances in the world, is widely used in the food, fragrance, and cosmetic industries, and can also be used as a chemical intermediate for producing herbicides, defoamers, polishes, and pharmaceutical industry intermediates for producing pharmaceuticals such as papaverine, levodopa, and trimethoprim (Bezerra et al, 2016; kim et al, 2014), and has extremely high economic value. At present, vanillin is mainly obtained from two sources, namely plant extraction and artificial synthesis. Natural vanillin is expensive, up to $ 1200-4000 per kilogram, 300 times the price of the synthetic product. Chemically synthesized vanillin is not allowed to be used in industries such as food and pharmaceutical industry because other compounds which may have health risks are contained in the product, and the chemical method also causes problems of environmental pollution. The metabolic engineering means is utilized to construct a vanillin synthesis way in saccharomyces cerevisiae, and the biosynthesis of vanillin by taking glucose or ferulic acid as a raw material becomes a current research and development hot spot, wherein vanillin produced by taking ferulic acid as a substrate is considered as natural vanillin which can be used for foods. Vanillin however faces its own toxicity during biosynthesis to inhibit host cells. Vanillin can destroy cell membrane structure, reduce activity of central metabolism related enzyme, break DNA, and inhibit translation process, thereby severely inhibiting microorganism growth and subsequent fermentation process, and limiting vanillin yield improvement. Meanwhile, vanillin is a main phenolic inhibitor in lignocellulose hydrolysate which is a raw material for producing the secondary fuel ethanol, and can be used for synergistic effect with other inhibitors such as furfural, 5-hydroxymethylfurfural, acetic acid and the like, so that the mass production of the secondary fuel ethanol is severely limited. At present, biological detoxification is utilized to eliminate the inhibition effect of the inhibitor, but the link greatly increases the production cost.
For Saccharomyces cerevisiae, 0.6. 0.6g L -1 The vanillin can seriously inhibit the growth and fermentation of the saccharomyces cerevisiae, the delay time is prolonged to about 12 hours from 2 hours, and the fermentation period is greatly prolonged. Applicant has previously studied that YRR1 deletion mutant BY4741 (yrr 1 delta) exhibits good vanillin tolerance. Meanwhile, the liquid fermentation results show that the maximum specific growth rate of BY4741 (yrr 1 delta) and the specific consumption rate of vanillin are respectively improved BY 142% and 50% compared with the original strain BY4741 under the condition of vanillin stress. Wherein the increase in ribosome synthesis-related gene is one of mechanisms by which YRR1 deletion increases the strain's tolerance to vanillin. However, YRR deletion increases expression of alcohol dehydrogenase and ribosome synthesis-related genes, but not target genes, and YRR deletion causes reduced tolerance of the cells to other inhibitors. The search finds that the expression of the Yrr1p target gene is enhanced to construct a mutant or an element of the Saccharomyces cerevisiae multidrug resistance transcription factor gene YRR1The literature to improve the vanillin tolerance of Saccharomyces cerevisiae has not been reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a saccharomyces cerevisiae multi-drug tolerance transcription factor gene YRR gene mutant YRR1 T185A And a construction method and application thereof.
The saccharomyces cerevisiae multidrug resistance transcription factor gene YRR1 mutant is characterized in that: the mutant was designated YRR1 T185A The nucleotide sequence of the gene is shown as SEQ ID No. 1; the gene sequence is Genebank from Saccharomyces cerevisiae S288C: deoxyribonucleotides 553-555 of YRR of Gene ID 854333 were obtained by mutating ACT to GCA.
The construction of the Saccharomyces cerevisiae multidrug resistance transcription factor gene YRR mutant adopts a fusion PCR method, and is characterized in that: genebank: the YRR ORF 541-568 deoxyribonucleic acid shown in Gene ID 8543336 is set as the overlapping region of fusion PCR, and the region contains mutation sites; the mutation sites were designed on the primers 185R1 and 185F1 in the overlap region; during construction, firstly amplifying the 5' end of the ORF to an overlapping region, wherein the sequence from the 5' end of the ORF to the overlapping region is shown as SEQ ID No.2, and the 5' end of the sequence is provided with a BamHI enzyme cutting site; amplifying the sequence from the overlapping region to the 3' end of the ORF, wherein the sequence from the overlapping region to the 3' end of the ORF is shown as SEQ ID No.3, and the 3' end of the sequence comprises a PstI enzyme cutting site; finally, carrying out fusion PCR on SEQ ID No.2 and SEQ ID No.3 to obtain YRR1 containing enzyme cutting sites T185A The mutant sequence SEQ ID No.4.
The invention provides a recombinant plasmid pJFE1-YRR1 containing a Saccharomyces cerevisiae multi-drug tolerance transcription factor gene YRR1 mutant according to claim 1 T185A The method is characterized in that: the recombinant plasmid is obtained by carrying out enzyme digestion and connection on a nucleotide sequence shown in SEQ ID No.4 and a vector pJFE1, and is mutant YRR1 T185A Expressed under the control of the promoter TEF1p and the terminator PGK1 t.
The invention provides a mutant containing the saccharomyces cerevisiae multi-drug tolerance transcription factor gene YRR or a recombinant plasmid pJFE1-YRR1 T185A Brewing of (C)Recombinant strain of Saccharomyces cerevisiae.
Wherein: the recombinant strain of Saccharomyces cerevisiae is designated BY4741 (yrr 1.DELTA., YRR 1.1) T185A ) Is obtained BY using YRR1 deletion strain BY4741 (yrr 1.DELTA.) as an initial strain, and the recombinant plasmid pJFE1-YRR1 T185A Transformation into Saccharomyces cerevisiae strain BY4741 (yrr 1.DELTA.). The specific construction steps are as follows:
(1) Extraction of Saccharomyces cerevisiae BY4741 genome
Saccharomyces cerevisiae BY4741 strain was cultured overnight in 5mL of YEPD liquid medium, the cells were collected, and the BY4741 genome was extracted BY disrupting cells BY bead milling.
(2) Obtaining the 5' -end of YRR ORF shown in SEQ ID No.2 to the fragment of the overlap region sequence:
PCR amplification was performed using the genome of Saccharomyces cerevisiae BY4741 as a template.
The amplification primer is an upstream primer F1F: CGCGGATCCGCGATGAAAAGAAGAAGCGATGC, downstream primer F1r: GGTTCTCATAGATGCGGGGCCGTATAGTG, underlined mutation sites.
(3) Acquisition of sequence fragments from the overlapping region to the 5' end of the YRR ORF shown in SEQ ID No.3
PCR amplification was performed using the genome of Saccharomyces cerevisiae BY4741 as a template. The amplification primer is an upstream primer F2F: CACTATACGGCCCCGCATCTATGAGAACC, underlined mutation site, downstream primer F2r: TGCACTGCAGTTAATTGTCTTTGTAATCATCGAAAAGAGTG.
(4) Saccharomyces cerevisiae YRR1 with enzyme cutting site shown in SEQ ID No.4 T185A Acquisition of nucleotide sequence fragments of mutant genes
Fusion PCR is carried out by taking nucleotide sequence fragments of SEQ ID No.2 and SEQ ID No.3 as templates, and Saccharomyces cerevisiae YRR1 with enzyme cutting sites shown in SEQ ID No.4 is obtained through PCR amplification T185A Mutant genes. Wherein: the primer is
Upstream primer F1F: CGCGGATCCGCGATGAAAAGAAGAAGCGATGC the number of the individual pieces of the plastic,
downstream primer F2r: TGCACTGCAGTTAATTGTCTTTGTAATCATCGAAAAGAGTG.
(5) Recombinant plasmid pJFE1-YRR1 T185A Is obtained by (a)
Saccharomyces cerevisiae YRR1 with enzyme cutting site shown in SEQ ID No.4 T185A The mutant gene and the vector pJFE1 were digested with BamHI and PstI, ligated, and then transformed with E.coli. In the presence of 100 mug mL -1 Screening was performed on LB plates for ampicillin. Picking up the transformant to extract the plasmid to obtain recombinant plasmid pJFE1-YRR1 T185A 。
(6) Recombinant plasmid pJFE1-YRR1 T185A The strain BY4741 (yrr 1.DELTA.) was transformed into Saccharomyces cerevisiae YRR, a deletion strain.
The recombinant plasmid pJFE1-YRR1 thus obtained was used T185A The cells were transferred into Saccharomyces cerevisiae BY4741 (yrr. Delta.) BY the lithium acetate method and screened on plates containing SC-URA. The mutant transformant obtained by screening is Saccharomyces cerevisiae YRR1 T185A Mutant strain designated BY4741 (yrr 1.DELTA., YRR 1.1) T185A )。
(7) PCR sequencing verification of mutants:
single colonies were picked from the screening plates and incubated in SC-URA broth at 30℃for 24h at 200rpm. Plasmid BY4741 of the recombinant strain was then extracted (yrr 1.DELTA., YRR 1.1 T185A ) To obtain recombinant plasmid pJFE1-YRR1 T185A Sequencing was carried out by company. Sequencing of the upstream primer was F1F, the downstream primer was F2r, at YRR1 T185A Two ends.
As a result, it was confirmed that the 553-555 deoxyribonucleotides of YRR (Genebank: gene ID: 854333) were mutated from ACT to GCA.
The invention relates to application of a saccharomyces cerevisiae multidrug resistance transcription factor gene YRR1 mutant in improving the vanillin resistance of saccharomyces cerevisiae.
The invention relates to an application of a saccharomyces cerevisiae multidrug resistance transcription factor gene YRR1 mutant in construction of a saccharomyces cerevisiae cell with high vanillin resistance.
Mutant YRR1 constructed according to the invention T185A Is always positioned in the cell nucleus, and can promote the regulation and control of the expression of target genes SNQ2 and YOR1 under the condition of no pressure stress. The mutant of the invention can be used for improving vanillin resistance of Saccharomyces cerevisiae, and can not damage the resistance of Saccharomyces cerevisiae to other inhibitors such as 4-Nitroquinone N-oxide (4-NQO).
The recombinant plasmid pJFE1-YRR1 T185A Use in the construction of a s.cerevisiae cell with high vanillin resistance.
The Saccharomyces cerevisiae recombinant strain BY4741 (yrr delta, YRR 1) T185A ) Use in verifying increased vanillin tolerance.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a mutant YRR1 engineered by the design T185A And expression plasmids thereof, which enable high level expression of the downstream target gene to be activated even in the absence of inhibitors, YRR 1. The expression constructed by the invention is YRR1 T185A The mutant strain was isolated in SC-URA medium containing 6mM vanillin stress, compared with the control strain BY4741 transformed into wild type YRR1 (yrr Δ, YRR 1) WT ) The maximum specific growth rate of the mutant is 0.125h -1 Compared with wild type YRR1 WT Is a strain of BY4741 (yrr 1. Delta., YRR 1) WT ) 34% higher. Compared with a control strain, the delay period is shortened by 8 hours, the fermentation period is greatly shortened, and a foundation is provided for constructing the saccharomyces cerevisiae cells with high vanillin resistance. The mutant strain provided by the invention is more suitable for the construction of the high vanillin tolerance saccharomyces cerevisiae strain for producing vanillin/alcohol or producing second-generation fuel ethanol or other high-value compounds by taking lignocellulose as a raw material, and has great application value. And the mutant recombinant strain provided BY the invention has more advantages compared with YRR deletion strain BY4741 (yrr 1 delta): YRR1 has an important role in the tolerance of many inhibitors such as 4-NQO and salicylic acid, and the YRR1 deletion causes the Saccharomyces cerevisiae to have a sensitive phenotype on both inhibitors as described above (Cui et al, 1998; kodo et al, 2013). The recombinant strain containing the mutant of the invention does not reduce the resistance of Saccharomyces cerevisiae to other toxic substances due to YRR1 deletion.
Although YRR1 deletion increased vanillin tolerance, it was compared to mutant YRR1 provided by the invention T185A The mechanism for improving vanillin tolerance is different, and YRR1 T185A The reason for the mutant to increase vanillin tolerance is to enhance the expression of the Yrr1p target gene, while YRR1 deletion increases the expression of alcohol dehydrogenaseNon-target gene expression.
Drawings
FIG. 1 shows BY4741 (yrr 1.DELTA. YRR 1.1 in the absence of inhibitors T185A ) And BY4741 (yrr 1 delta, YRR1 WT ) Expression levels of the Yrr1p target genes SNQ2 and YOR1 in the strain.
FIG. 2 is BY4741 (yrr 1. Delta., YRR 1) T185A ) And BY4741 (yrr 1 delta, YRR1 WT ) Growth profile under 6mM vanillin stress.
FIG. 3 is BY4741 (yrr 1. Delta., YRR 1) T185A ) And BY4741 (yrr 1. Delta.) gradient growth experiments at 4-NQO pressure.
Detailed Description
The present invention will be described in detail with reference to the following drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limiting in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
Summary of the media and experimental materials involved in the following examples:
(1) Culture medium
Saccharomyces cerevisiae was cultured with SC-URA medium: yeast complete synthesis medium (SC): yeast base Nitrogen Source 1.77g L -1 Ammonium sulfate 5g L -1 ，CSM-URA 0.77g L -1 Sterilizing at 115deg.C for 30min, and adding sterilized 400g L -1 Glucose mother liquor to final concentration of 20g L -1 。
LB medium: 5g L -1 Yeast powder, 10g L -1 Peptone, 10g L -1 Sodium chloride. Adding agar powder to the solid culture medium to 2%, and screening and adding AMP to 150 mug mL to the solid culture medium -1 Liquid medium was screened supplemented with AMP to 100. Mu.g mL -1 . YEPD medium: 20g L -1 Peptone, 10g L -1 Yeast powder, 20g L -1 Glucose; sterilizing at 115 deg.C for 30min.
(2) Enzymes and reagents
The DNA polymerase used for PCR was Phata Max Super Fidelity DNA Polymerase P505-d2, available from Nanjinouzan Biotechnology Co., ltd (Vazyme). Restriction enzymes and T4 DNA Ligase (ThermoFisher) were purchased from Thermo Inc. SYBR Green Master Mix Kit the kit was purchased from the Wohan Aibotac Biotechnology Co. ABScript II RT Master Mix reverse transcription kit PK20403 was purchased from Bottak Biotechnology Inc. of Wuhan.
(3) Strains and plasmids
Saccharomyces cerevisiae BY4741 is purchased from vast Proteus plasmid platformhttp://www.miaolingbio.com。
Saccharomyces cerevisiae S288C was purchased from Tex (Wuhan) Biotechnology Co., ltd or Feng Shou (Shanghai) Biotechnology Co., ltd in Yu Zhili.
The construction method of the Saccharomyces cerevisiae YRR deleted strain BY4741 (yrr 1 delta) is shown in the doctor's thesis of the inventor Wang Xinning (analysis of the inhibitor vanillin tolerance mechanism in lignocellulose hydrolysate BY Saccharomyces cerevisiae), and in 2017, the doctor's thesis of Shandong university.
Coli trans5 a was purchased from beijing, all gold.
pUG6 plasmid construction is described in A new efficient gene disruption cassette for repeated use in budding yeast [ J ]. Nucleic Acids Res,1996,24 (13): 2519-2524.
The sequence information and construction method of pJFE1 are disclosed in reference (doi: 10.1016/j. Jbiosc.2015.05.014.Epub 2015Jul 7.PMID:26160406.).
Other materials, reagents, and the like, unless otherwise specified, are commercially available.
Example 1: recombinant plasmid pJFE1-YRR1 T185A Construction of (3)
(1) Extraction of Saccharomyces cerevisiae BY4741 genome
Saccharomyces cerevisiae BY4741 is inoculated into 2mL of YEPD medium, cultured at 30 ℃ for 200rp overnight, the thalli are collected BY centrifugation, the thalli are washed once BY 1mL of sterile water, 200 mu L of lysis buffer solution is added for suspending cells, the cells are transferred into a screw cell tube containing 0.4g of glass beads, 200 mu L of phenol/chloroform/isoamyl alcohol (25:24:1) is added, after vortex oscillation for 3min, 200 mu L of TE solution is added for uniform mixing, and 13000r/min centrifugation is carried out for 10min at 4 ℃, and the transfer is carried outAdding 1ml of absolute ethanol at-20 ℃ into a new Eppendorf tube to precipitate DNA, centrifuging for 10min at 13000r/min at 4 ℃, and discarding the supernatant. After drying in vacuo, 400. Mu.L of TE solution was added to dissolve DNA, and 10. Mu.L of 10mg mL was added -1 After incubation at 37℃for 15min, 10. Mu.L of 4mol L was added -1 After mixing with 1mL of absolute ethanol, centrifuging for 10min at 13000r/min, removing supernatant, drying, and adding 50 μl of ddH 2 O is dissolved.
Wherein: preparing a lysis buffer solution: 2% Triton X-100,1% SDS,100mmol L -1 NaCl，10mmol L -1 Tris-Cl，1mmol L -1 EDTA, pH adjusted to 8.0.
(2) Acquisition of sequence fragment from 5' end to overlapping region of YRR1 Gene shown in SEQ ID No.2
PCR amplification was performed using the genome of Saccharomyces cerevisiae BY4741 as a template.
The amplification primer is an upstream primer F1F: CGCGGATCCGCGATGAAAAGAAGAAGCGATGC, downstream primer F1r: GGTTCTCATAGATGCGGGGCCGTATAGTG, underlined mutation sites.
The PCR system is as follows:
the PCR procedure was as follows:
(3) Acquisition of sequence fragments from the overlapping region to the 5' end of the YRR ORF shown in SEQ ID No.3
PCR amplification was performed using the genome of Saccharomyces cerevisiae BY4741 as a template. The amplification primer is an upstream primer F2F: CACTATACGGCCCCGCATCTATGAGAACC, underlined mutation site, downstream primer F2r: TGCACTGCAGTTAATTGTCTTTGTAATCATCGAAAAGAGTG.
The PCR system is as follows:
the PCR procedure was as follows:
(4) Acquisition of the fragment with cleavage site shown in SEQ ID No.4
Fusion PCR amplification was performed using SEQ ID No.2 and SEQ ID No.3 as templates. The primer is an upstream primer F1F: CGCGGATCCGCGATGAAAAGAAGAAGCGATGC, downstream primer F2r: TGCACTGCAGTTAATTGTCTTTGTAATCATCGAAAAGAGTG.
The PCR system is as follows:
the PCR procedure was as follows:
(5) YRR1 of Saccharomyces cerevisiae with enzyme cutting site shown in SEQ ID No.4 T185A Cleavage of the mutant Gene and cleavage of pJFE1
The PCR product was purified by agarose gel electrophoresis experiments. The PCR product was subjected to agarose gel electrophoresis, and then placed under an ultraviolet lamp, and the gel containing the DNA to be recovered was carefully cut off, placed in an Eppendorf tube, and weighed.
The recovery and purification of DNA fragments in agarose Gel was performed using the E.Z.N.A Gel Extraction kit from Omega Bio-tek company (USA) according to the kit instructions. YRR1 after recovery T185A The fragment was digested with the restriction enzymes BamHI and PstI in the following system:
plasmid pJFE1 was digested with restriction enzymes BamHI and PstI in the following system:
YRR1 T185A and pJFE1 was purified after cleavage and operated according to the instructions of the OMEGA PCR product purification kit (D6492-01).
(6)YRR1 T185A Connection to pJFE1
Will YRR1 T185A The fragment was ligated to linearized pJFE 1. The connection is carried out for 40min at 25 ℃, and the connection system is as follows:
(7) Coli transformation
10 mu L of the ligation product obtained in (6) was added to the E.coli trans 5. Alpha. Competence, ice-bathed for 30min, heat-shocked for 90s at 42℃and ice-bathed again for 3min. 200. Mu.L of LB medium was added and incubated at 37℃for 1h. After incubation, the supernatant was removed by centrifugation, diluted with water and applied to a medium containing 150. Mu.g mL -1 Ampicillin was cultured on LB plates at 37℃for 12 hours.
(8) Coli plasmid extraction and validation
Single colony E.coli on plates was inoculated onto 5mL containing 100. Mu.g mL -1 Ampicillin was cultured in LB liquid medium for 12 hours, and plasmids were extracted according to the OMEGA plasmid miniprep kit (D9643-01). Then, the promoter and terminator fragments on the vector plasmid are used as primers to be sent to the biological company for sequencing.
Sequencing and confirming to obtain recombinant plasmid pJFE1-YRR1 T185A Wherein the mutant YRR1 T185A The nucleotide sequence of the gene is shown as SEQ ID No. 1. YRR1 T185A Expressed under the control of the promoter TEF1p and the terminator PGK1 t.
Example 2: recombinant plasmid pJFE1-YRR1 T185A Transformation of Saccharomyces cerevisiae BY4741 (yrr 1. Delta.)
To avoid Saccharomyces cerevisiae chromosome in situ YRR1 WT We used YRR deletion strain BY4741 (yrr 1.DELTA.) as starting strain. The recombinant plasmid pJFE1-YRR1 was subjected to T185A The transformation into Saccharomyces cerevisiae strain BY4741 (yrr. Delta.) gives a recombinant strain of Saccharomyces cerevisiae, designated BY4741 (yrr. Delta., YRR 1) T185A )。
Sequencing the recombinant plasmid pJFE1-YRR1 T185A Transformation was performed BY the lithium acetate method into Saccharomyces cerevisiae YRR1 deleted strain BY4741 (yrr 1.DELTA.). The specific operation is as follows:
BY4741 (yrr 1.DELTA.) single colonies were picked and cultured overnight in 1-2mL of YEPD medium, and the overnight cultured colonies were transferred to 40mL of YEPD to give an initial cell concentration OD 600 Shaking culture at 30℃to OD at 0.2 600 Between 0.6 and 1.0, collecting the cells by centrifugation, washing with sterile water, and washing with 1mL of 0.1mol L -1 LiAc resuspended cells were transferred to a new sterile Eppendorf tube, the supernatant was centrifuged off and the cells were resuspended in 400. Mu.L of 0.1mol L -1 LiAc; the resuspended bacteria solution was divided into 50. Mu.L portions and placed in a sterile Eppendorf tube, centrifuged, and the supernatant was discarded, followed by the addition of 240. Mu.L of 50% (w/v) PEG-3350, 36. Mu.L of 1mol L -1 LiAc，10μL 10mg mL -1 Single-stranded fish essence DNA (boiled in boiling water for 5min before use, rapidly placed on ice for half an hour), sterilized redistilled water and plasmid (or DNA fragment) 70 μl, and shake-mixed. Heat-preserving for 30min at 30 ℃ and heat-shocking for 25min in a 42 ℃ water bath. Supernatant was removed by centrifugation, resuspended cells were incubated in YEPD for 2-4h, centrifuged and resuspended in sterile water and plated on plates containing SC-URA for selection. The mutant transformant obtained by screening is Saccharomyces cerevisiae YRR1 T185A Mutant strain designated BY4741 (yrr 1.DELTA., YRR 1.1) T185A )。
Example 3: BY4741 (yrr 1. Delta., YRR 1) T185A ) In improving vanillin resistanceApplication in (Vanillin tolerance)
(1)BY4741(yrr1Δ,YRR1 T185A ) Effect on the expression levels of the YRR1 target genes SNQ2 and YOR1 in the absence of inhibitors
BY4741 (yrr 1 delta, YRR 1) T185A ) Single colonies were grown overnight in SC-URA medium at 30℃for about 24 hours at 200 r/min. At an initial OD 600 0.2, transfer to 20mL fresh SC-URA medium for secondary activation, 30 ℃ 200r/min overnight culture to mid-log. At an initial OD 600 The overnight cultures were transferred to 40mL of SC-URA medium at 30℃and 200rpm. Culturing the strain to OD 600 1.0, cells were collected, the cells were washed with ultrapure water at 4℃and the supernatant was removed by centrifugation. mu.L of TES buffer (10 mM Tris-HCl, pH 8.0;10mM NaCl;1mM EDTA) was added to resuspend the cells, and an equal volume of phenol was added. Vortex mix for 5min, incubate in 65 ℃ water bath for 20min, reverse every 5min. The ice bath was kept for 5min and centrifuged at 4 ℃. The aqueous phase was transferred to an RNase-free EP tube and 400. Mu.L of phenol was added. Mix the samples and centrifuge at 4 ℃. The aqueous phase was transferred to an RNase-free EP tube and 400. Mu.L of chloroform was added. Mix the samples and centrifuge at 4 ℃. The aqueous phase was transferred to an EP tube for RNase and three volumes of 95% ethanol were added. Mix the samples, centrifuge at 4 ℃, discard the supernatant. The precipitate was dissolved by adding 500. Mu.L of 75% ethanol, and the supernatant was removed by centrifugation. The solution was washed twice with 75% ethanol. The RNA was dried and 30. Mu.L of DEPC (diethyl pyrocarbonate) was dissolved in water.
The total RNA after treatment was reverse transcribed into cDNA using the ABScript II RT Master Mix reverse transcription kit (PK 20403/Wuhan Aiboltag Biotechnology Co., ltd.). In the reverse transcription process, genomic DNA was removed by first treating with 4 XgDNA reverse Mix. In the reverse transcription reaction system, no RT Control (No reverse transcriptase) was used as a negative Control reaction to examine whether genomic DNA remained in the RNA template. The reverse transcription procedure is: 25 ℃/5min, 42 ℃/15min, 85 ℃/5sec, 4 ℃/Hold.
RT-qPCR analysis was performed using a SYBR Green Master Mix Kit kit (Wohan Ebolac Biotechnology Co., ltd.). The reaction system: 2X Universal SYBR Green Fast qPCR Mix/10. Mu.L; cDNA template/Variable (1-10 ng); forward primer (10. Mu.M)) 0.4. Mu.L; reverse primer (10. Mu.M)/0.4. Mu.L; ddH 2 O/to 20. Mu.L. The procedure for fluorescent quantitative PCR instrument (Roche Molecular Biochemicals, germany) was: a dissolution profile is automatically generated at 95 ℃/10sec, 60 ℃/10sec, 72 ℃/30 sec. The YRR target genes SNQ2 (qCPR primers: SNQ2-F and SNQ 2-R) and YOR1 (qCPR primers: YOR1-F and YOR 1-R) which have been reported to be verified in the literature were selected as subjects. The real-time quantitative PCR was performed to verify that ACT1 of Saccharomyces cerevisiae was selected as an internal reference marker Gene (Genebank: gene ID: 850504) (qCPR primers: actin-F and Actin-R).
Wherein, the primer sequence for fluorescent quantitative PCR is as follows:
RT-qPCR calculation method: ct= -k lgX 0 +b (linear equation) with which the initial concentration slope of the sample can be calculated from the Ct value = -1/lg (1+e), when the amplification efficiency e=1, the slope = -3.32. When the amplification efficiency is in the range of 90% -110%, the slope is in the range of-3.1 to-3.59. When the amplification efficiencies of two genes of interest are the same, a mathematical calculation model based on fluorescence quantification can be simplified to ΔΔct for comparison of differences or change ratios between different samples. For example, if the difference in the expression levels of the a gene and the b gene in a cell line is compared, ACT1 is selected as the reference gene, and the Ct value of the reference gene is subtracted from the Ct value of the a gene to obtain ΔCt 1 Subtracting the Ct value of the reference gene from the Ct value of the b gene to obtain a delta Ct 2 Then use DeltaCt 1 -ΔCt 2 =ΔΔct, whereas the difference in expression level of the a, b genes is 2 -ΔΔCt 。
Control strain (wild-type YRR1 anaplerotic strain) BY4741 (yrr 1.DELTA., YRR 1) WT ) Also with reference to the above method.
The results show that BY4741 (yrr 1. Delta., YRR 1) T185A ) The expression level of the target gene was higher than that of the control strain BY4741 (yrr 1.DELTA., YRR 1.1) WT ) Respectively increased by 5.4 times (SNQ 2) and 4.5 times (YOR 1) (see FIG. 1).
(2)BY4741(yrr1Δ,YRR1 T185A ) Vanillin tolerance test
BY4741 (yrr 1 delta, YRR 1) T185A ) Single colonies were grown in 1-2mL of SC-URA medium at 30℃for about 24 hours at 200 r/min. Starting with OD 600 0.2, transfer to 20mL fresh medium for secondary activation, 30 ℃ 200r/min overnight culture to mid-log phase. At an initial OD 600 At 0.2, the overnight cultures were transferred to 40mL containing 6mmol L -1 Shaking culture was performed in SC-URA medium containing vanillin at 30℃and 200rpm. Sample OD is measured by periodic sampling 600 。
The maximum specific growth rate calculation method comprises the following steps: logarithmic growth phase ln (OD) 600 ) Linear regression slope over time.
BY4741(yrr1Δ,YRR1 T185A ) The maximum specific growth rate under the stress of vanillin is 0.125h -1 (see FIG. 2), the delay period is 10h.
Wild-type YRR anaplerotic strain BY4741 (yrr Δ, YRR 1) WT ) Shake flask fermentation under vanillin stress was also performed as described above, and the calculation showed that the maximum specific growth rate was 0.082h -1 The delay time is 18h.
From the fermentation results, BY4741 (yrr 1.DELTA., YRR 1.1) T185A ) The maximum specific growth rate of (a) was higher than that of the control strain BY4741 (yrr 1.DELTA., YRR 1.1 WT ) The delay time is shortened by 8h and is 34% higher than that of the traditional Chinese medicine.
(3) Mutant strain BY4741 (yrr 1. Delta., YRR 1) T185A ) Comparison with YRR1 deletion Strain BY4741 (yrr 1.DELTA.) in 4-NQO resistance
BY4741 (yrr 1. Delta., YRR 1) T185A ) In the presence of 6mmol L -1 Gradient growth experiments were performed on SC-URA plates of vanillin. The specific operation is as follows:
inoculation of BY4741 (yrr 1. Delta., YRR 1) T185A ) Culturing in 1-2mL SC-URA medium at 30deg.C overnight to logarithmic phase, and maintaining the culture product at initial OD 600 Transferring to 5mL of fresh culture medium for 0.2, culturing overnight, centrifuging to collect thalli, washing the suspended thalli with 1mL of sterile water for 3 times, centrifuging at 8000r/min, suspending thalli with sterile water again, and standing at 30 ℃ for 9h to prepare resting cells. Preparing a culture medium, pouring the culture medium into a plate, and naturally airing. Adjusting the concentration of the bacterial suspension to OD 600 Sequentially performing 10-time gradient dilution to 1.0, sequentially taking 4 mu L of bacterial suspension, dripping the bacterial suspension on a test plate, placing the test plate in a 30 ℃ incubator for culturing for 1-3 days, observing colony growth conditions, and photographing.
Control strain (YRR 1 deleted strain) BY4741 (yrr 1 Δ) was also performed as described above.
The results are shown in FIG. 3, from which it is evident that at 0.2ug mL -1 BY4741 (yrr 1.DELTA., YRR 1.1) T185A ) Growth conditions were superior to control strains.
Claims (9)
1. A saccharomyces cerevisiae multidrug resistance transcription factor gene YRR mutant, characterized in that: the mutant was designated YRR1 T185A The nucleotide sequence of the gene is shown as SEQ ID No. 1; the gene sequence is Genebank from Saccharomyces cerevisiae S288C: deoxyribonucleotides 553-555 of YRR of Gene ID 854333 were obtained by mutating ACT to GCA.
2. The saccharomyces cerevisiae multi-drug resistant transcription factor gene YRR1 mutant is constructed by a fusion PCR method, and is characterized in that: genebank: YRR1 ORF Nos. 1546 to 1569 deoxyribonucleic acid shown in Gene ID 854333 are arranged as an overlapping region of fusion PCR, and the region contains a mutation site; the mutation sites were designed on the primers 185R1 and 185F1 in the overlap region; during construction, firstly amplifying the 5' end of the ORF to an overlapping region, wherein the sequence from the 5' end of the ORF to the overlapping region is shown as SEQ ID No.2, and the 5' end of the sequence is provided with a BamHI enzyme cutting site; amplifying the sequence from the overlapping region to the 3' end of the ORF, wherein the sequence from the overlapping region to the 3' end of the ORF is shown as SEQ ID No.3, and the 3' end of the sequence comprises a PstI enzyme cutting site; finally, carrying out fusion PCR on SEQ ID No.2 and SEQ ID No.3 to obtain YRR1 containing enzyme cutting sites T185A The mutant sequence SEQ ID No.4.
3. Recombinant plasmid pJFE1-YRR1 containing Saccharomyces cerevisiae multidrug resistance transcription factor gene YRR mutant according to claim 1 T185A The method is characterized in that: the recombinant plasmid is prepared by carrying out enzyme digestion and connection between a nucleotide sequence shown in SEQ ID No.4 and a vector pJFE1Obtained mutant YRR1 T185A Expressed under the control of the promoter TEF1p and the terminator PGK1 t.
4. A mutant containing the Saccharomyces cerevisiae multidrug resistance transcription factor gene YRR of claim 1 or a recombinant plasmid pJFE1-YRR1 of claim 2 T185A Is a recombinant strain of Saccharomyces cerevisiae.
5. The recombinant strain of Saccharomyces cerevisiae according to claim 4, wherein: the recombinant strain of Saccharomyces cerevisiae is designated BY4741 (yrr 1.DELTA., YRR 1.1) T185A ) Is obtained BY using YRR1 deletion strain BY4741 (yrr 1.DELTA.) as an initial strain, and the recombinant plasmid pJFE1-YRR1 T185A Transformation into Saccharomyces cerevisiae strain BY4741 (yrr 1.DELTA.).
6. Use of the saccharomyces cerevisiae multidrug resistance transcription factor gene YRR1 mutant of claim 1 for increasing saccharomyces cerevisiae vanillin resistance.
7. Use of the saccharomyces cerevisiae multi-drug resistant transcription factor gene YRR1 mutant of claim 1 for constructing saccharomyces cerevisiae cells with high vanillin resistance.
8. The recombinant plasmid pJFE1-YRR1 of claim 3 T185A Use in the construction of a s.cerevisiae cell with high vanillin resistance.
9. A recombinant strain of Saccharomyces cerevisiae BY4741 as defined in claim 5 (yrr 1.DELTA. YRR 1.1 T185A ) Use in verifying increased vanillin tolerance.
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| CN110042067A (en) * | 2019-04-16 | 2019-07-23 | 山东大学 | A kind of method and its mutant strain improving recombinant Saccharomyces cerevisiae bacterial strain xylose utilization ability |
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| CN110042067A (en) * | 2019-04-16 | 2019-07-23 | 山东大学 | A kind of method and its mutant strain improving recombinant Saccharomyces cerevisiae bacterial strain xylose utilization ability |
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