CN112143729B - Promoter for starting RNA expression of phaffia rhodozyma and application thereof - Google Patents

Promoter for starting RNA expression of phaffia rhodozyma and application thereof Download PDF

Info

Publication number
CN112143729B
CN112143729B CN201910576458.4A CN201910576458A CN112143729B CN 112143729 B CN112143729 B CN 112143729B CN 201910576458 A CN201910576458 A CN 201910576458A CN 112143729 B CN112143729 B CN 112143729B
Authority
CN
China
Prior art keywords
promoter
seq
phaffia rhodozyma
polymerase iii
rna polymerase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910576458.4A
Other languages
Chinese (zh)
Other versions
CN112143729A (en
Inventor
王士安
张宁
李福利
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Original Assignee
Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qingdao Institute of Bioenergy and Bioprocess Technology of CAS filed Critical Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority to CN201910576458.4A priority Critical patent/CN112143729B/en
Publication of CN112143729A publication Critical patent/CN112143729A/en
Application granted granted Critical
Publication of CN112143729B publication Critical patent/CN112143729B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Mycology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the field of yeast biotechnology and synthetic biology, in particular to a promoter of RNA polymerase III of phaffia rhodozyma and application thereof. The promoter is a red-hair yeast RNA polymerase III promoter; the promoter is SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or DNA nucleotide sequence having 70% homology with the above sequences and having promoter activity. The promoter disclosed by the invention is applied to a red-hair yeast CIRPR-Cas 9 system to guide the expression of gRNA in the system, and the gene editing efficiency is improved by 4 times.

Description

Promoter for starting RNA expression of phaffia rhodozyma and application thereof
Technical Field
The invention relates to the field of yeast biotechnology and synthetic biology, in particular to a promoter of RNA polymerase III of phaffia rhodozyma and application thereof.
Background
The yeast has important value for scientific research and human life. Since the twenty-first century, research efforts in the model of saccharomyces cerevisiae have won nobel physiological or medical prizes in 2001, 2013, and 2016, respectively. On the industrial side, the global market for Saccharomyces cerevisiae and related products reaches $ 71 billion in 2016, and is projected to $ 100.7 billion in 2020, as counted by BCC Research. In addition to saccharomyces cerevisiae, there are a large number of yeasts in nature, and these non-model yeasts are rich in species diversity, genetic diversity, and metabolic diversity, but their development of applications is weak.
With the development of genome sequencing and modern biotechnology, the feasibility and significance of non-model biological research is gradually highlighted (Russell JJ, Theriot JA, sound P, et al bmc Biology,2017,15: 55). The non-model ascomycete yeast has characteristics in the aspects of protein expression, bioenergy, bio-based chemical synthesis and the like, such as: kluyveromyces yeast synthesizes beta-galactosidase, Scheffersomyces stipitis metabolizes xylose, Yarrowia lipolytica synthesizes citric acid, fats and oils, etc. (Johnson EA. appl Microbiol Biotechnol,2013,97: 503-. Modeless basidiomycete yeasts are distinguished in protein drug synthesis, secondary metabolite synthesis, contaminant degradation, etc., for example: phaffia rhodozyma (Phaffia rhodozyma) synthesizes astaxanthin, Pseudozyma antarctica synthesizes ribavirin, Trichosporon yeast degrades aromatic pollutants, Sporobolomyces yeast synthesizes special fatty acids, etc. (Johnson EA. appl Microbiol Biotechnol,2013,97: 7563-7577).
Among basidiomycetous yeasts, phaffia rhodozyma has attracted attention for the synthesis of astaxanthin. Astaxanthin has super-strong antioxidant capacity, and has the functions of resisting tumor, resisting inflammation and preventing cardiovascular and cerebrovascular diseases (Ambati R, Phang SM, Ravi S, et al. Mar Drugs,2014,12: 128-. The market for astaxanthin will reach $ 15 billion by the year 2020 as assessed by BCC research. China already enters an aging society, and carotenoid products such as astaxanthin and the like have important prevention and improvement effects on degenerative diseases, hypertension, hyperglycemia, hyperlipidemia and the like in the aging society. The astaxanthin-synthesizing ratio of phaffia rhodozyma can be 50-85% of the cell carotenoid, and has outstanding advantages in growth speed, cell density, pollution resistance and the like, and is considered as a potential cell factory for astaxanthin production (Bellor N, Molina M, David-Palma M, et al, BMC Genomics,2016,17: 901). However, the phaffia rhodozyma is a non-model yeast, the regulation mechanism of astaxanthin anabolism is not clear, and a synthesis technology platform is lacked, so that the application and development of the phaffia rhodozyma are severely limited. The genomic sequence of Phaffia rhodozyma was published in 2015, which also reflects the weakness of the relevant basic research (Sharma R, Gassel S, Steiger S, et al. BMC Genomics 2015,16: 233). The publication of genome sequences promotes the research of multiomics, and recently, comparative genomics, P450 omics and metabonomics data are published in succession, which provides important reference for the research of astaxanthin synthesis by phaffia rhodozyma. Basidiomycetes yeasts have found more than 700 types, but the model biotechnology strains are lacked so far, and the Phaffia rhodozyma is developed into a model, so that the development and the application of the economically important yeasts are very facilitated.
In recent years, crispr (clustered regulated intercurrent short palindromic repeats) gene editing technology has been rapidly developed, and the technology has been developed in various yeasts including saccharomyces cerevisiae, yarrowia lipolytica, pichia pastoris, kluyveromyces lactis, schizosaccharomyces pombe, candida albicans, cryptococcus neoformans, and the like (stopicek V, Holkenbrink C, Borodina i. fems yeast research,2017,17: 1-16). However, this technique has not been reported for an economically important basidiomycete yeast. The system established in the phaffia rhodozyma can provide reference for the construction of technical platforms of other non-model yeasts. The key to establishing the technology is to obtain a gRNA (guide RNA) promoter with high expression. Aiming at the problem, the invention identifies, clones and verifies the effective red-hair yeast gRNA and explains the application thereof.
Disclosure of Invention
The invention aims to provide a red-hair yeast RNA polymerase III promoter and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a promoter for promoting the RNA expression of phaffia rhodozyma, which is a phaffia RNA polymerase III promoter; the promoter is SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or DNA nucleotide sequence having 70% homology with the above sequences and having promoter activity.
The nucleotide shown as SEQ ID NO. 1 or the DNA sequence with 70 percent of homology and promoter activity is a promoter P of RNA polymerase III of phaffia rhodozymaSCR1。
The nucleotide shown as SEQ ID NO. 2 or the DNA sequence which has 70 percent of homology and has promoter activity is a promoter P of RNA polymerase III of phaffia rhodozymaU6A。
The nucleotide shown as SEQ ID NO. 3 or the DNA sequence with 70 percent of homology and promoter activity is a promoter P of RNA polymerase III of phaffia rhodozymaU6B。
The promoter is RNA polymerase III promoter and tRNA of phaffiaAlaArtificial hybrid initiation of gene fusion; the artificial hybrid promoter is SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 or DNA nucleotide sequence which has 70% homology with the above sequences and has promoter activity.
The tRNAAlaThe gene is phaffia rhodozyma tRNA shown by SEQ ID NO. 4 nucleotideAlaA gene.
Use of a promoter for promoting RNA expression in saccharomyces cerevisiae to promote expression of grnas, to promote expression of snornas, or to promote expression of RNA polymerase III transcripts in the CIRSPR gene editing system.
A recombinant vector containing the promoter.
The recombinant vector is an episomal vector or an integrating vector; wherein the free vector or the integrated vector contains any one promoter of the red-hair yeast RNA polymerase III promoter or the fusion promoter.
An expression cassette containing the promoter.
A recombinant bacterium containing the recombinant vector.
The invention has the advantages that:
the promoter takes a DNA conserved sequence identified by a yeast RNA polymerase III transcription factor TFIIIC as a reference, and the RNA polymerase III promoter is identified from a phaffia rhodozyma (Xanthophyllomyces dendrorhous) genome through sequence comparison and cloning analysis; meanwhile, taking a yeast transfer RNA (tRNA) sequence as a reference, identifying tRNA from the phaffia rhodozyma genome through sequence alignment and clone analysisAlaGenes, promoters and tRNAs with RNA polymerase IIIAlaThe artificial hybrid promoter formed by gene fusion can effectively promote the RNA expression of the phaffia rhodozyma. The promoter obtained by the invention can be applied to a CRISPR-Cas9 gene editing system of phaffia rhodozyma to guide the expression of gRNA in the system, and the CRISPR-Cas9 system is applied to the gene editing of the phaffia rhodozyma, so that compared with self homologous recombination, the gene editing efficiency is improved by 4 times, and the RNA promoter provided by the invention has strong starting capability and high gRNA expression level, and can be used for guiding Cas9 protein to cut a genome target sequence at a fixed point. Except for the patent, a promoter capable of guiding the expression of gRNA in phaffia rhodozyma has not been reported.
Drawings
FIG. 1 shows the conserved sequence of promoter of RNA polymerase III, which is exemplified by Saccharomyces cerevisiae, according to an embodiment of the present invention; wherein, the recognition region of the transcription factor TFIIIC is BoxA and BoxB.
FIG. 2 shows the Vienna RNA Package 2.0 software-predicted tRNA from Phaffia rhodozyma according to the embodiment of the present inventionAlaThe secondary structure of the gene.
FIG. 3 is a diagram illustrating the examination of the nuclear localization sequence of Phaffia rhodozyma using eGFP as the tracer molecule according to an embodiment of the present invention; in the right panel, the nuclear localization sequence NLS used is indicated to correctly direct eGFP into the nucleus.
FIG. 4 is a schematic diagram of a CRISPR-Cas9 integration plasmid of phaffia rhodozyma provided by the embodiment of the invention.
FIG. 5 is an editing phenotype diagram of the genes of Phaffia rhodozyma provided by the embodiment of the invention, wherein the left diagram is ET1 and ET2 target sequence sites, the right diagram is ET3 target sequence sites, white colonies are correctly edited clones, and red colonies are unedited clones.
FIG. 6 is a diagram of the repair types of phaffia rhodozyma gene editing provided in the embodiment of the present invention, in which ET3-3, ET3-4 and ET3-6 are base substitutions, and ET3-5 is a base deletion.
Detailed Description
The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.
The invention takes the DNA conserved sequence identified by RNA polymerase III transcription factor TFIIIC of saccharomyces cerevisiae and candida albicans as reference, and identifies the RNA polymerase III promoter from the genome of the phaffia rhodozyma by sequence comparison. And identifying tRNA genes from the genome of the phaffia rhodozyma by taking a saccharomyces cerevisiae transfer RNA (tRNA) sequence as a reference through sequence alignment. A fusion promoter of RNA polymerase III promoter and tRNA gene was constructed using Gibson DNA assembly. The function of the promoter was verified in the CIRSPR-Cas9 system.
The method specifically comprises the following steps:
a: the promoter of the RNA polymerase III of the phaffia has all DNA sequences shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, and DNA sequences which have 70 percent of homology with the shown sequences and have promoter activity.
B: the phaffia RNA fusion promoter has all DNA sequences shown as SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, and DNA sequences which have 70 percent of homology with the shown sequences and have promoter activity.
C: a vector carrying any one of the RNA polymerase III promoters described in (A) or (B), which includes an episomal vector or an integrating vector.
D: and (C) recombinant bacteria of the phaffia rhodozyma transformed with any one of the vectors.
Examples
1. Materials and methods
1.1 strains and culture conditions
Phaffia rhodozyma strain CBS 6938 (purchased from the Netherlands CBS culture Collection) is used as a material. YPD liquid medium (glucose 20g/L, peptone 20g/L, yeast extract 10g/L) was used for the conventional culture under 22 ℃ and 250 rpm.
1.2 prediction of RNA polymerase III promoter
By using the promoter sequences of SCR1 and U6 genes of Saccharomyces cerevisiae and Candida albicans as reference and applying the optize for Somewhat similar sequences program in NCBI Blastn program package, the homologous sequence of the genome of the Phaffia rhodozyma is obtained from the genome data of the Xanthophyllomyces dendrorhous (tall: 5421). Obtaining a homologous sequence of a tRNA gene of the phaffia rhodozyma by using the same method and taking a Saccharomyces cerevisiae transfer RNA (tRNA) sequence as a reference through NCBI Blastn sequence comparison; the sequence was obtained as follows:
taking a promoter of a Saccharomyces cerevisiae SCR1 gene as a reference sequence to obtain a promoter P of a RNA polymerase III of the phaffia rhodozyma with nucleotide shown in SEQ ID NO. 1SCR1。
The promoter P of the RNA polymerase III of the phaffia rhodozyma with the nucleotide shown in SEQ ID NO. 2 and two homologous sequences obtained by taking the promoter of the candida albicans U6 gene as a reference sequenceU6A(ii) a Phaffia rhodozyma RNA polymerase III promoter P of nucleotide shown in SEQ ID NO. 3U6B。
With Saccharomyces cerevisiae tRNAAlaThe gene is a reference sequence to obtain the Phaffia rhodozyma tRNA of the nucleotide shown in SEQ ID NO. 4AlaA gene.
The obtained sequences SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3 are fused with the sequence SEQ ID NO 4 respectively to obtain the sequences SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7.
1.3 design of Gene editing target sequences
The gRNA target sequence was designed using CRISPY-web. Searching for Scaffold _79 (ID: LN483167.1) of the X.dendrorhous genome of Saccharomyces cerevisiae, using the CrtE gene (1539235-1541616) as a target; the primer sequences were obtained as described in Table 1.
1.4 vector construction
1) Vector construction was performed by the Gibson assembly method using the primers shown in Table 1. The CRISPR/Cas9 vector includes a Cas9 expression element, a gRNA transcription element, and a screening marker. The expression of Cas9 required a nuclear localization signal, optimized the Nuclear Localization Sequence (NLS) SV40 according to the x.dendrorhous codon bias and placed at the 5' end of eGFP amplification primers to examine whether exogenous SV40 nuclear localization signal was suitable for phaffia rhodozyma. The eGFP gene fragment with the SV40 sequence and the amplification primers of the framework p61hte are NeGFP-F/NeGFP-R and p61hte-F/p61hte-R respectively. The SV40-eGFP and p61hte fragments were PCR amplified with KAPA HiFi high fidelity DNA polymerase, respectively, in 25ul amplification systems (2 XKAPA Mix,12.5 ul; 0.75ul each for 10uM primers; 1ul template; water to 25ul) to obtain each DNA sequence. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 98 deg.C for 20 s, annealing at 60-72 deg.C for 15 s, and extension at 72 deg.C for 15 s/kb with cycle number of 29-35; extension at 72 ℃ for 6 minutes. The two DNA fragments were assembled by Gibson Assembly method, the concentration of the DNA fragments in the reaction system was controlled at 100 and 200ng per reaction, the total volume was 10ul, the Assembly conditions were 50 ℃ for 1 hour. After the reaction, 2ul of the transformed DH5 alpha competent cells were obtained, the positive clones were obtained by colony PCR and DNA sequencing, verification and screening, and the constructed plasmid was named pCYP-eGFP.
2) When constructing a Cas9 expression vector, the Cas9 gene of a pCRCT plasmid (purchased from Addgene) is codon optimized according to the codon preference of X.dendrorhous, synthesized by Wuxi Qinglan biology company, fused with an SV40 nuclear localization sequence at both the N-terminal and the C-terminal of Cas9, and an assembly fragment is amplified by using primers C9-F/R. Cas9 genes are all placed in PADH4Behind the promoter. The extension of the backbone fragment was performed using plasmid pCYP-eGFP as template and primers pC9-F/pC 9-R. And (3) respectively carrying out PCR amplification by using KAPA HiFi high-fidelity DNA polymerase, wherein the amplification systems are 25ul, and the amplification conditions and the assembly conditions are consistent with the records in the step 1.4 and the step 1). The resulting plasmid was designated pCYP-Cas 9.
3) The resulting gRNA promoter was assembled with gRNA sequences by Gibson method. The ribosome rDNA sequence is set at two ends of the expression cassette for homologous recombination and integration, and the screening label is geneticin. GGPP synthetase gene CrtE is used as a verification target for CRISPR editing, and a target gene sequence of 20bp is introduced into a vector through PCR amplification. When a plasmid for verifying transformation efficiency is constructed, a CrtE gene knockout plasmid is constructed as a control, the upstream and downstream homology arms of a CrtE gene are assembled with a zeocin resistance gene expression cassette, and on the basis, CRISPR/Cas9 elements with ET2 and ET3 are assembled with a vector with a CrtE homology arm. The construction process is as follows:
amplifying a plasmid skeleton by using a primer PgRNA-F/PgRNA-R by taking the plasmid pUG6-rDNALONG-CrtE as a template; the plasmid pCYP-Cas9 is used as a template, and a primer gCAS-F/gCAS-R is used for amplifying a Cas9 protein; plasmid pUG6-CRISPR was obtained by Gibson assembly. Plasmid pUG6-CRISPR is used as a template, primers pCopg1-F/pCopg1-R and ET1-F/ET1-R are respectively used for amplifying a plasmid skeleton and a DNA fragment with a target sequence ET1, and the plasmid pUG6-CRISPR-ET1 is obtained through Gibson assembly. Plasmid pUG6-CRISPR-ET2 is obtained by using plasmid pUG6-CRISPR as a template and primers pCopg2-F/pCopg1-R and ET1-F/ET2-R to amplify a plasmid skeleton and a DNA fragment with a target sequence ET2 respectively through Gibson assembly. Primers pCopg3-F/pCopg1-R and ET1-F/ET3-R are respectively used for amplifying a plasmid skeleton and a DNA fragment with a target sequence ET3, and the plasmid pUG6-CRISPR-ET3 is obtained by Gibson assembly. Using plasmid pUG6 as a template, and applying a primer pUG6-F/pUG6-R to amplify a skeleton; amplifying a CrtE homologous fragment by using a primer Edir-F/Edir-R by using a phaffia rhodozyma genome as a template; plasmid pUG6D1 was obtained by Gibson assembly. Amplifying a skeleton by using a primer pEdisr-F/pEdisr-R by taking the plasmid pUG6D1 as a template; the plasmid pUG6-Zeocin is used as a template, Zeocin genes are amplified by using a primer zeo-F/zeo-R, and the plasmid pUG6D2 is obtained through Gibson assembly. Amplifying a plasmid skeleton by using a primer pEZin-F/pEZin-R by taking the plasmid pUG6D2 as a template; amplifying a CrtE homologous fragment by using a primer EZ-F/EZ-R by taking a phaffia rhodozyma genome as a template; the plasmid pEZin is obtained by Gibson assembly. Amplifying a Cas9-gRNA fragment by using a primer GT2-F/GT2-R by taking the plasmid pUG6-CRISPR-ET2 as a template; amplifying a plasmid skeleton by using a primer pUEZ-F/R by taking a plasmid pEZin as a template; gibson assembles to obtain plasmid pEgET 2. All PCR amplifications used KAPA HiFi high fidelity DNA polymerase, the amplification system was 25ul, the amplification conditions and assembly conditions were consistent with those described in step 1.4, 1).
TABLE 1 PCR amplification primers used in the present invention
Figure BDA0002112161180000061
Figure BDA0002112161180000071
1.5 Phaffia rhodozyma transformation
A single colony of strain CBS 6938 was picked from YPD plate medium, inoculated into a 250ml shake flask containing 30ml YPD (YPD components of glucose 20g/L, peptone 20g/L, yeast extract 10g/L), and cultured at 22 ℃ for 48 hours. Inoculating the above cultured bacterial liquid into 500ml shake flask containing 50ml YPD to the final concentration of OD6000.02, and then cultured at 22 ℃ to OD600About 1.2, taking about 14-20 hours. The culture was centrifuged, resuspended in potassium phosphate buffer (50mM, pH 7.0), DTT was added to a final concentration of 25mM, and treated for 15 min. Then, centrifuged at 4 ℃ and treated with ice-cold sucrose buffer STM (270mM sucrose,10mM Tris-HCl, pH 7.5, and 1mM MgCl2) Washed twice, and the washed cells were resuspended in 0.1ml of STM buffer for use. 60. mu.l of the above-obtained competent cells were taken, 10. mu.g of the gene expression cassette DNA was added, and electrotransformation was carried out under conditions of 1000. omega., 800V and 25. mu.F. The transformation solution was resuspended in 600. mu.l of ice-cold (ca. 0 ℃) YPD, incubated at 22 ℃ for 2.5h, and 100. mu.l was plated on the selection plate. The resistance concentrations of the selected plates were G418200. mu.g/ml, respectively.
1.6 functional analysis of RNA polymerase III promoter
The CIRPR-Cas 9 gene editing clone is screened through red and white colony difference, and the white colony is the correct editing clone. The correct editing clone is selected, the DNA sequence is determined, and the function of the RNA polymerase III promoter is tested through sequence comparison. And the function of the RNA polymerase III promoter is tested by analyzing the gene editing efficiency of the red-hair-like yeast CIRPR-Cas 9.
2. Results and analysis
2.1 prediction of RNA polymerase III promoter
By taking the promoter sequences of SCR1 and U6 genes of Saccharomyces cerevisiae and Candida albicans as reference (figure 1), the homologous promoter of the Phaffia rhodozyma is identified through NCBI Blastn sequence alignment, and the searched sequences are shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. The Saccharomyces cerevisiae transfer RNA (tRNA) sequence is used as a reference, the NCBI Blastn sequence alignment is carried out to identify the tRNA gene of the phaffia rhodozyma (figure 2), and the searched gene sequence is shown as SEQ ID NO: 4. The tRNA was fused with the predicted SCR1 and U6, respectively, and the resulting fusion promoters are shown in SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7.
2.2 verification of the Nuclear localization sequence
Since the Cas9 gene needs to be localized in the nucleus to function, validation of the nuclear localization sequence was first performed using fluorescent proteins. Exogenous SV40 sequence cctaagaagaagagaaaggtc was selected as the validation target, and eGFP fluorescent protein was used as the tracer. The localization of the fluorescent protein eGFP in the cell is observed by a fluorescence microscope, and the result shows that the fluorescent protein eGFP presents obvious green fluorescence at the position of the cell nucleus, which indicates that the eGFP protein can successfully enter the cell nucleus, and indicates that SV40NLS can be used for guiding the Cas9 protein to enter the cell nucleus of phaffia rhodozyma (fig. 3).
2.3 effectiveness of RNA polymerase III promoter
The function of the predicted RNA polymerase III promoter was verified by transformation of the CRISPR-Cas9 plasmid in phaffia rhodozyma (fig. 4). As a result of studies with three different target sequences in the CrtE gene, it was found that transformants targeting ET2 and ET3 exhibited white colonies as correctly edited strains without addition of donor DNA (FIG. 5). This indicates that the predicted RNA polymerase III promoter PSCR1、PU6AAnd PU6BAnd each with tRNAAlaThe fusion promoter of (3) can be successfully used for expression of gRNA. DNA sequencing is carried out on the CrtE locus of the transformant, and the result shows that 1308bp bases of the CrtE 3-2 strain genome are deleted, 6bp bases of the CrtE3-5 strain genome are deleted, and bases are replaced by other clones, so that the correct function of the promoter is proved (figure 6).
2.4 editing efficiency of CRISPR/Cas9 System
Through the experiment, the CRISPR/Cas9 system of phaffia rhodozyma can be determined to successfully edit target DNA sequences of ET2 and ET 3. For gene knockout, the knockout efficiency of the gene editing method and the homologous recombination method is compared. Results show that by adopting a CRISPR/Cas9 system and taking ET2 and ET3 as double-strand break and gene knockout targets, the knockout efficiency of the CrtE gene is obviously higher than that of a homologous recombination method, and is respectively improved by 4.1 times and 2.9 times, so that the promoter P is supported on the other handSCR1、PU6AAnd PU6BAnd each with tRNAAlaThe fusion promoter can guide the expression of gRNA, and the constructed CRISPR/Cas9 system can effectively improve the gene editing efficiency of phaffia rhodozyma.
>SEQ ID NO:1
SCR1 promoter
tgtcgttagtgtcatcggattgatctctatcccaggcctgatgacaggagcgatcatcggtggttcttccgtcgaacaag
ccgcgaaattacaaagtacatctcgaaatttcgatactttgaactttgcctttaaaacttttagatcgactgatatactc
ttcgtttcctttaccaaaagtgatcttgatgtttatgatcagcgcttcttctgcactatccggttagtccttcttcctcc
tatcgatcgcctagtcccaatcagaactcctttgaattgactcctgatcctttgttttccatcttcaactctcgccaata
gtgctggcagccatgatattcacattctcggtcgtattcgaccctcaacatcgaatcagagacgacaggatatattcgtc
cgagagcgtgctttctggtgggatcagaagtgttgggaagatcggcgtgttgggttggaaatcgatcaaaaatctaggag
gaaaggagcgaaggccatgagagtaagaac
>SEQ ID NO:2
U6A promoter
agttgcacctccatactgtgcagtcttaaagctcttttcgtcctgggcaaccaggacatcaataatatcgaatacaatac
agagaagattagcattgcccctgcataaggatgacacgattcgatcagagtggagaccttcgggtcaaattattttttta
ctcatgtatctacatattctgaattccacatgggcatataatctcaggaaagcaagaggttgctatatatgtatcagaac
gtaataacttatgacagatggaggtaggcaacacagtccgcttcct
>SEQ ID NO:3
U6B promoter
gcttcctttcgtcctgggcaaccaggacatcaataatatcgaatacaatacagagaagattagcattgcccctgcataag
gatgacacgattcgatcagagtggagaccttcgggtcaaattatttttttaactcacatatcctcatatgtgcctatgag
gatgcttgtgtactgccattctttgatgcttttcctttcttcccttcttccctttctgttttgatcgcaaagcaggatct
cctgctcaattgactagccttcaataaggattgacacaagtgatgacgatcacgtagcgcaagaaagggtagaaggcaag
aaaggcgaggaggtgaggaaaaccgccgcagtgaaatgcgagacgacagaccatctcacttttctaagatccttcactcc
ttcctctcatctttcatcccactttcttcttctctcttggactgtcttataagttgattccatcacca
>SEQ ID NO:4
tRNAAlaSequence of
GTGGGGACCTGGCGTAGTGGTAGCGCCCAGCCTTTGCGTGGCTGTAGCCCCGGTTCGATTCCGGGGGTCTCCAACC
>SEQ ID NO:5
SCR1-tRNAAlaHybrid promoters
tgtcgttagtgtcatcggattgatctctatcccaggcctgatgacaggagcgatcatcggtggttcttccgtcgaacaag
ccgcgaaattacaaagtacatctcgaaatttcgatactttgaactttgcctttaaaacttttagatcgactgatatactc
ttcgtttcctttaccaaaagtgatcttgatgtttatgatcagcgcttcttctgcactatccggttagtccttcttcctcc
tatcgatcgcctagtcccaatcagaactcctttgaattgactcctgatcctttgttttccatcttcaactctcgccaata
gtgctggcagccatgatattcacattctcggtcgtattcgaccctcaacatcgaatcagagacgacaggatatattcgtc
cgagagcgtgctttctggtgggatcagaagtgttgggaagatcggcgtgttgggttggaaatcgatcaaaaatctaggag
gaaaggagcgaaggccatgagagtaagaacGTGGGGACCTGGCGTAGTGGTAGCGCCCAGCCTTTGCGTGGCTGTAGCCCCGGTTCGATTCCGGGGGTCTCCAACC
>SEQ ID NO:6
U6A-tRNAAlaHybrid promoters
agttgcacctccatactgtgcagtcttaaagctcttttcgtcctgggcaaccaggacatcaataatatcgaatacaatac
agagaagattagcattgcccctgcataaggatgacacgattcgatcagagtggagaccttcgggtcaaattattttttta
ctcatgtatctacatattctgaattccacatgggcatataatctcaggaaagcaagaggttgctatatatgtatcagaac
gtaataacttatgacagatggaggtaggcaacacagtccgcttcctTGGGGACCTGGCGTAGTGGTAGCGCCCAGCCTT
TGCGTGGCTGTAGCCCCGGTTCGATTCCGGGGGTCTCCAACC
>SEQ ID NO:7
U6B-tRNAAlaHybrid promoters
gcttcctttcgtcctgggcaaccaggacatcaataatatcgaatacaatacagagaagattagcattgcccctgcataag
gatgacacgattcgatcagagtggagaccttcgggtcaaattatttttttaactcacatatcctcatatgtgcctatgag
gatgcttgtgtactgccattctttgatgcttttcctttcttcccttcttccctttctgttttgatcgcaaagcaggatct
cctgctcaattgactagccttcaataaggattgacacaagtgatgacgatcacgtagcgcaagaaagggtagaaggcaag
aaaggcgaggaggtgaggaaaaccgccgcagtgaaatgcgagacgacagaccatctcacttttctaagatccttcactcc
ttcctctcatctttcatcccactttcttcttctctcttggactgtcttataagttgattccatcacca
TGGGGACCTGGCGTAGTGGTAGCGCCCAGCCTTTGCGTGGCTGTAGCCCCGGTTCGATTCCGGGGGTCTCCAACC
Sequence listing
<110> institute of bioenergy and Process in Qingdao, China academy of sciences
<120> promoter for promoting RNA expression of phaffia rhodozyma and application thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 510
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgtcgttagt gtcatcggat tgatctctat cccaggcctg atgacaggag cgatcatcgg 60
tggttcttcc gtcgaacaag ccgcgaaatt acaaagtaca tctcgaaatt tcgatacttt 120
gaactttgcc tttaaaactt ttagatcgac tgatatactc ttcgtttcct ttaccaaaag 180
tgatcttgat gtttatgatc agcgcttctt ctgcactatc cggttagtcc ttcttcctcc 240
tatcgatcgc ctagtcccaa tcagaactcc tttgaattga ctcctgatcc tttgttttcc 300
atcttcaact ctcgccaata gtgctggcag ccatgatatt cacattctcg gtcgtattcg 360
accctcaaca tcgaatcaga gacgacagga tatattcgtc cgagagcgtg ctttctggtg 420
ggatcagaag tgttgggaag atcggcgtgt tgggttggaa atcgatcaaa aatctaggag 480
gaaaggagcg aaggccatga gagtaagaac 510
<210> 2
<211> 286
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agttgcacct ccatactgtg cagtcttaaa gctcttttcg tcctgggcaa ccaggacatc 60
aataatatcg aatacaatac agagaagatt agcattgccc ctgcataagg atgacacgat 120
tcgatcagag tggagacctt cgggtcaaat tattttttta ctcatgtatc tacatattct 180
gaattccaca tgggcatata atctcaggaa agcaagaggt tgctatatat gtatcagaac 240
gtaataactt atgacagatg gaggtaggca acacagtccg cttcct 286
<210> 3
<211> 468
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcttcctttc gtcctgggca accaggacat caataatatc gaatacaata cagagaagat 60
tagcattgcc cctgcataag gatgacacga ttcgatcaga gtggagacct tcgggtcaaa 120
ttattttttt aactcacata tcctcatatg tgcctatgag gatgcttgtg tactgccatt 180
ctttgatgct tttcctttct tcccttcttc cctttctgtt ttgatcgcaa agcaggatct 240
cctgctcaat tgactagcct tcaataagga ttgacacaag tgatgacgat cacgtagcgc 300
aagaaagggt agaaggcaag aaaggcgagg aggtgaggaa aaccgccgca gtgaaatgcg 360
agacgacaga ccatctcact tttctaagat ccttcactcc ttcctctcat ctttcatccc 420
actttcttct tctctcttgg actgtcttat aagttgattc catcacca 468
<210> 4
<211> 76
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gtggggacct ggcgtagtgg tagcgcccag cctttgcgtg gctgtagccc cggttcgatt 60
ccgggggtct ccaacc 76
<210> 5
<211> 586
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgtcgttagt gtcatcggat tgatctctat cccaggcctg atgacaggag cgatcatcgg 60
tggttcttcc gtcgaacaag ccgcgaaatt acaaagtaca tctcgaaatt tcgatacttt 120
gaactttgcc tttaaaactt ttagatcgac tgatatactc ttcgtttcct ttaccaaaag 180
tgatcttgat gtttatgatc agcgcttctt ctgcactatc cggttagtcc ttcttcctcc 240
tatcgatcgc ctagtcccaa tcagaactcc tttgaattga ctcctgatcc tttgttttcc 300
atcttcaact ctcgccaata gtgctggcag ccatgatatt cacattctcg gtcgtattcg 360
accctcaaca tcgaatcaga gacgacagga tatattcgtc cgagagcgtg ctttctggtg 420
ggatcagaag tgttgggaag atcggcgtgt tgggttggaa atcgatcaaa aatctaggag 480
gaaaggagcg aaggccatga gagtaagaac gtggggacct ggcgtagtgg tagcgcccag 540
cctttgcgtg gctgtagccc cggttcgatt ccgggggtct ccaacc 586
<210> 6
<211> 361
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
agttgcacct ccatactgtg cagtcttaaa gctcttttcg tcctgggcaa ccaggacatc 60
aataatatcg aatacaatac agagaagatt agcattgccc ctgcataagg atgacacgat 120
tcgatcagag tggagacctt cgggtcaaat tattttttta ctcatgtatc tacatattct 180
gaattccaca tgggcatata atctcaggaa agcaagaggt tgctatatat gtatcagaac 240
gtaataactt atgacagatg gaggtaggca acacagtccg cttccttggg gacctggcgt 300
agtggtagcg cccagccttt gcgtggctgt agccccggtt cgattccggg ggtctccaac 360
c 361
<210> 7
<211> 543
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gcttcctttc gtcctgggca accaggacat caataatatc gaatacaata cagagaagat 60
tagcattgcc cctgcataag gatgacacga ttcgatcaga gtggagacct tcgggtcaaa 120
ttattttttt aactcacata tcctcatatg tgcctatgag gatgcttgtg tactgccatt 180
ctttgatgct tttcctttct tcccttcttc cctttctgtt ttgatcgcaa agcaggatct 240
cctgctcaat tgactagcct tcaataagga ttgacacaag tgatgacgat cacgtagcgc 300
aagaaagggt agaaggcaag aaaggcgagg aggtgaggaa aaccgccgca gtgaaatgcg 360
agacgacaga ccatctcact tttctaagat ccttcactcc ttcctctcat ctttcatccc 420
actttcttct tctctcttgg actgtcttat aagttgattc catcaccatg gggacctggc 480
gtagtggtag cgcccagcct ttgcgtggct gtagccccgg ttcgattccg ggggtctcca 540
acc 543

Claims (8)

1. A promoter for promoting RNA expression in Phaffia rhodozyma, comprising: the promoter is a red-hair yeast RNA polymerase III promoter; the promoter is DNA nucleotide sequence shown in SEQ ID NO. 2 and SEQ ID NO. 3.
2. The promoter for promoting RNA expression of Phaffia rhodozyma according to claim 1, wherein: the nucleotide shown as SEQ ID NO. 2 is a promoter P of RNA polymerase III of phaffia rhodozymaU6A。
3. The promoter for promoting RNA expression of Phaffia rhodozyma according to claim 1, wherein: the nucleotide shown as SEQ ID NO. 3 is a promoter P of RNA polymerase III of phaffia rhodozymaU6B。
4. Use of the promoter for promoting RNA expression of Phaffia rhodozyma according to any one of claims 1 to 3, wherein: use of a promoter according to any one of claims 1 to 3 to promote expression of grnas, to promote expression of snornas or to promote expression of RNA polymerase III transcripts in the CIRSPR gene editing system.
5. A recombinant vector comprising the promoter of any one of claims 1-3.
6. The recombinant vector of claim 5, wherein: the recombinant vector is an episomal vector or an integrating vector; wherein the free vector or the integrated vector contains any one promoter of the RNA polymerase III promoters of the phaffia rhodozyma.
7. An expression cassette comprising the promoter of any one of claims 1-3.
8. A recombinant bacterium comprising the recombinant vector of claim 5.
CN201910576458.4A 2019-06-28 2019-06-28 Promoter for starting RNA expression of phaffia rhodozyma and application thereof Active CN112143729B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910576458.4A CN112143729B (en) 2019-06-28 2019-06-28 Promoter for starting RNA expression of phaffia rhodozyma and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910576458.4A CN112143729B (en) 2019-06-28 2019-06-28 Promoter for starting RNA expression of phaffia rhodozyma and application thereof

Publications (2)

Publication Number Publication Date
CN112143729A CN112143729A (en) 2020-12-29
CN112143729B true CN112143729B (en) 2022-05-17

Family

ID=73869468

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910576458.4A Active CN112143729B (en) 2019-06-28 2019-06-28 Promoter for starting RNA expression of phaffia rhodozyma and application thereof

Country Status (1)

Country Link
CN (1) CN112143729B (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108064287A (en) * 2015-01-06 2018-05-22 帝斯曼知识产权资产管理有限公司 For solving the CRISPR-CAS systems of fat yeast host cell
WO2017105991A1 (en) * 2015-12-18 2017-06-22 Danisco Us Inc. Methods and compositions for t-rna based guide rna expression

Also Published As

Publication number Publication date
CN112143729A (en) 2020-12-29

Similar Documents

Publication Publication Date Title
Cernak et al. Engineering Kluyveromyces marxianus as a robust synthetic biology platform host
Wang et al. Molecular tools for gene manipulation in filamentous fungi
US20170088845A1 (en) Vectors and methods for fungal genome engineering by crispr-cas9
CN104245938B (en) Genetic manipulation and expression system for handle rest fungus subphylum and smut subphylum
CN111073902B (en) CRISPR/dCas9 vector for improving expression level of gliotoxin biosynthesis gene and construction method and application thereof
CN111057713A (en) CRISPR/Cas9 vector applicable to erwinia bacterium FS110 and construction method and application thereof
US8198089B2 (en) Flocculent yeast and method for production thereof
Woodcraft et al. The expanding CRISPR toolbox for natural product discovery and engineering in filamentous fungi
EP3412765B1 (en) Method for producing mutant filamentous fungi
CN112553238B (en) CRISPR/Cas9 vector applicable to coniothyrium minitans FS482 as well as construction method and application thereof
US11332751B2 (en) Methods and compositions for production of aromatic and other compounds in yeast
CN112143729B (en) Promoter for starting RNA expression of phaffia rhodozyma and application thereof
CN112760338B (en) CRISPR/Cpf1 vector suitable for deep-sea fungi FS140 and construction method and application thereof
CN112359043B (en) CRISPR/Cas9 vector applicable to phomopsis FS508 and construction method and application thereof
Cernak et al. Engineering Kluyveromyces marxianus as a robust synthetic biology platform host. mBio 9: e01410-18
CN114085784A (en) Recombinant yeast with high cytochrome P450 expression and application thereof
US20150147774A1 (en) Expression construct for yeast and a method of using the construct
CN116555269B (en) Bumblebee candida utilis inducible promoter and application thereof
CN111394349B (en) Rhodosporidium toruloides RNA polymerase III type promoter and application thereof
Yuan et al. A CUP1-tag mediated approach for multi-copy gene integration in budding yeast
EP4130254A1 (en) Eukaryotic cell lysates comprising exogenous enzymes and methods for preparing the same
CN111394350B (en) Rhodosporidium toruloides RNA polymerase III type promoter and application thereof
CN115786385A (en) CRISPR/Cas9 vector suitable for deep-sea fungus FS441 and construction method and application thereof
Juergens et al. Genome editing in Kluyveromyces and Ogataea yeasts using a
Lund Nielsen et al. Genes Linked to Production of Secondary Metabolites in Talaromyces atroroseus Revealed Using CRISPR-Cas9

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant