WO2022178741A1 - 一种具有启动、终止双功能调控元件的构建方法和双功能元件库 - Google Patents

一种具有启动、终止双功能调控元件的构建方法和双功能元件库 Download PDF

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WO2022178741A1
WO2022178741A1 PCT/CN2021/077808 CN2021077808W WO2022178741A1 WO 2022178741 A1 WO2022178741 A1 WO 2022178741A1 CN 2021077808 W CN2021077808 W CN 2021077808W WO 2022178741 A1 WO2022178741 A1 WO 2022178741A1
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sequence
termination
bifunctional
nucleotide sequence
seq
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张根林
刘政洋
倪晓霞
张燕
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石河子大学
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • 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
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  • the invention belongs to the technical field of synthetic biology, and in particular relates to a construction method and a library of bifunctional elements with a dual function regulation element of initiation and termination.
  • gene circuits require a complete gene expression cassette - including promoters, genes, and terminators, while the construction of metabolic pathways requires the connection of different expression cassettes.
  • chemical methods can synthesize DNA fragments of a certain length, in order to achieve larger
  • the assembly of expression modules still requires DNA assembly technology, including GoldenGate method, In-fusion technology, Gibson assembly, DNA assembler, etc.
  • Promoters CYC1p, TYS1p, etc., terminators ADH1t, PDALt, etc. are often used in the prior art, but separate promoters and terminators are often time-consuming when used for gene circuit or pathway assembly, and when the assembly pathway is long, assembly Efficiency is also limited.
  • the purpose of the present invention is to provide a construction method and a library of bifunctional elements with activation and termination dual-function regulatory elements.
  • the pathway construction process can be simplified and assembly enhanced.
  • the invention provides a construction method with a dual function regulation element of initiation and termination, and the initiation sequence and the termination sequence are assembled according to the principle of first termination and then initiation;
  • the promoter sequence includes an upstream active sequence and a core promoter sequence
  • the upstream active sequence includes a Mig1/Mig2 binding site, a Rap1 binding site, a Gcr1 binding site, a weakMig1 binding site, a GC box, a CAAT box and an octamer A body frame
  • the core promoter sequence includes a TATA box, a BRE element, an MTE element and an Inr element;
  • the termination sequence includes an efficiency element, a positional element and polyA.
  • nucleotide sequence of the Mig1/Mig2 binding site is shown in SEQ ID No.1;
  • the nucleotide sequence of the Rap1 binding site is shown in SEQ ID No.2;
  • the nucleotide sequence of the Gcr1 binding site is shown in SEQ ID No.3;
  • the nucleotide sequence of the weakMig1 binding site is shown in SEQ ID No.4;
  • the nucleotide sequence of the GC box is GGGCGG;
  • the nucleotide sequence of the CAAT box is CCAATCT;
  • the nucleotide sequence of the octamer box is ATGCAAAT.
  • the nucleotide sequence of the TATA box is TATATAAA;
  • the nucleotide sequence of the BRE element is GGACGCC
  • the nucleotide sequence of the MTE element is shown in SEQ ID No.5;
  • the nucleotide sequence of the Inr element is TTAATAT.
  • the nucleotide sequence of the efficiency element is TATATATA
  • the nucleotide sequence of the position element is AATAAA.
  • the core promoter sequence is linked before the upstream active sequence.
  • the termination sequence is linked before the core promoter sequence.
  • the termination sequence is inserted between the core promoter sequence and the upstream active sequence.
  • the present invention also provides a regulatory element with activation and termination dual functions constructed based on the construction method described in the above technical solution, and the nucleotide sequence of the dual-functional regulatory element with activation and termination is as shown in SEQ ID No. 6 Show.
  • the present invention also provides a bifunctional element library obtained based on the construction method described in the above technical solution, the initiation sequence and the termination sequence are assembled according to the principle of first termination and then initiation, and various kinds of the initiation sequence and the termination sequence are adjusted. Alignment of sequences and spacers, resulting in a library of bifunctional elements.
  • the present invention provides a construction method and a library of bifunctional elements with dual functions of initiation and termination.
  • the initiation sequence and the termination sequence are assembled according to the principle of first termination and then initiation; the initiation sequence includes an upstream active sequence and a core initiation sequence.
  • the upstream active sequence includes Mig1/Mig2 binding site, Rap1 binding site, Gcr1 binding site, weakMig1 binding site, GC box, CAAT box and octamer box;
  • the core promoter sequence includes TATA Blocks, BRE elements, MTE elements and Inr elements;
  • the termination sequences include efficiency elements, positional elements and polyA.
  • the regulatory elements with start and stop functions constructed by the construction method provided by the present invention are used in pathway construction. Since the number of fragments is reduced in the construction process, the construction process of the metabolic pathway is simplified, and the assembly efficiency is improved by about approx. 18%.
  • Fig. 1 is the functional verification result of core promoter
  • Fig. 2 is the functional verification result of promoter
  • Fig. 3 is the functional verification result of terminator
  • Fig. 4 is the functional verification result of the dual function element
  • Figure 5 is a library of bifunctional elements of different strengths.
  • the present invention provides a method for constructing a control element with dual functions of initiation and termination.
  • the initiation sequence and the termination sequence are assembled according to the principle of termination first and initiation later;
  • the initiation sequence includes an upstream active sequence and a core promoter sequence;
  • the upstream active sequence includes Mig1/Mig2 binding site, Rap1 binding site, Gcr1 binding site, weakMig1 binding site, GC box, CAAT box and octamer box;
  • the core promoter sequence includes TATA box, BRE element, MTE elements and Inr elements;
  • the termination sequences include efficiency elements, positional elements and polyA.
  • the nucleotide sequence of the Mig1/Mig2 binding site is shown in SEQ ID No.1; the nucleotide sequence of the Rap1 binding site is shown in SEQ ID No.2; the Gcr1
  • the nucleotide sequence of the binding site is shown in SEQ ID No.3; the nucleotide sequence of the weak Mig1 binding site is shown in SEQ ID No.4; the nucleotide sequence of the GC frame is GGGCGG;
  • the nucleotide sequence of the CAAT box is CCAATCT; the nucleotide sequence of the octamer box is ATGCAAAT.
  • SEQ ID No. 1 GATCGTAGCCCCGGATTTAC.
  • SEQ ID No. 2 GATCGTACACCTGGACATAC.
  • SEQ ID No. 3 GATCGTAGAGCTTCCACTAC.
  • SEQ ID No. 4 GATCGTAGCCCCACAAATAC.
  • the nucleotide sequence of the TATA box is TATATAAA; the nucleotide sequence of the BRE element is GGACGCC; the nucleotide sequence of the MTE element is shown in SEQ ID No. 5; the Inr The nucleotide sequence of the element is TTAATAT.
  • SEQ ID No. 5 CGAGCCGAGCA.
  • the nucleotide sequence of the efficiency element is TATATATA
  • the nucleotide sequence of the position element is AATAAA
  • the present invention preferably links the core promoter sequence before the upstream active sequence.
  • the present invention preferably links the termination sequence before the core promoter sequence, or inserts the termination sequence between the core promoter sequence and the upstream active sequence. After the above ligation or insertion, the transcriptional level makes its initiation and termination functions complete and independent of each other.
  • the present invention also provides a regulatory element with activation and termination dual functions constructed based on the construction method described in the above technical solution, and the nucleotide sequence of the dual-functional regulatory element with activation and termination is as shown in SEQ ID No. 6 shown, as follows:
  • the present invention also provides a bifunctional element library obtained based on the construction method described in the above technical solution, the initiation sequence and the termination sequence are assembled according to the principle of first termination and then initiation, and various kinds of the initiation sequence and the termination sequence are adjusted. Alignment of sequences and spacers, resulting in a library of bifunctional elements.
  • yeast constitutive promoter consists of an upstream active sequence and a core promoter sequence.
  • the upstream active sequence contains a large number of transcription factor binding sites, and the yeast transcription factor binding site Mig1 is selected.
  • /Mig2 binding site sequence is: GATCGTAGCCCCGGATTTAC
  • Rap1 binding site sequence is: GATCGTACACCTGGACATAC
  • Gcr1 binding site sequence is: GATCGTAGAGCTTCCACTAC
  • weak Mig1 binding site sequence is: GATCGTAGCCCCACAAATAC
  • transcription factor binding site can effectively improve promoter transcription level .
  • a transcription factor binding site that assists RNAII polymerase to bind to DNA GC box: GGGCGG, CAAT box: CCAATCT, octamer box: ATGCAAAT; core promoter sequence comprises: TATA box TATATAAA is located at -25 upstream of the initiation site ⁇ -30bp, BRE element GGACGCC is located at -37 ⁇ -32bp upstream of the initiation site, MTE element CGAGCCGAGCA is located at +11 ⁇ +18bp downstream of the initiation site, and Inr element: TTAATAT.
  • the promoter sequence designed in this example includes the upstream active sequence UAS and the core promoter sequence; wherein the change of the connecting sequence, the selection of the transcription factor binding site and the different arrangement sequence will cause the promoter strength to change.
  • UAS upstream active sequence
  • core promoter sequence wherein the change of the connecting sequence, the selection of the transcription factor binding site and the different arrangement sequence will cause the promoter strength to change.
  • the upstream active sequence UAS (SEQ ID No. 7): AGTGATAATC GATCGTAGCCCCGGATTTAC GTAAGTTCTG GATCGTACACCTGGACATAC AGATTGTGAT GGCAATCT CGATTACACT GATCGTAGAGCTTCCACTAC TAAATCGGT GATCGTAGC CCCACAAATAC CGTGATACTG, the underlined sequences are the above-mentioned yeast transcription factor binding sites Mig1/Mig2, Rap1, Gcr1, weakMig1 in sequence. The remaining sequences are randomly connected sequences to ensure that the upstream active sequence has the lowest homology to yeast, so as to prevent the sequence from homologous recombination in yeast, so that it cannot function normally.
  • a TAT TCTTTT CGAGCCGAGCA CTACGGACCG the underlined sequence is BRE element, TATA box, Inr element (+1 is the transcription start site) and MTE element.
  • the rest of the sequences are T-rich random connection sequences, and the selection of T-rich sequences can make RNA II polymerase better combine with DNA sequences.
  • the above-mentioned upstream active sequence is directly connected with the core promoter sequence to form an artificial promoter sequence, and after chemical synthesis, a promoter sequence fragment (obtained by amplification with primer 1 and primer 2) is obtained by amplifying.
  • the primer sequences are:
  • Primer 1 (SEQ ID No. 9): CGGGATCC AGTGATAATCGATCG, the underlined sequence is the overlapping sequence of the BamH I restriction site.
  • Primer 2 (SEQ ID No. 10):
  • the underlined sequence is the overlapping sequence of the Not I restriction site.
  • the reporter gene eGFP fragment was obtained by querying from the Genebank gene bank, and the eGFP gene fragment was obtained by amplification after chemical synthesis (amplified with primer 3 and primer 4).
  • the primer sequences are:
  • the underlined sequence is the overlapping sequence of the Not I restriction site.
  • Primer 4 (SEQ ID No. 12): GAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGA TTATTTGTATAGTTCATCCA, the underlined sequence is the overlapping sequence of CYC1t.
  • the terminator CYC1t fragment was obtained by querying the Genebank gene library, and the CYC1t gene fragment was obtained by amplification after chemical synthesis (amplified with primer 5 and primer 6).
  • the primer sequences are:
  • Primer 5 (SEQ ID No. 13): CTGGGATTACACATGGCATGGATGAACTATACAATAA TCATGTAATTAGTTATGTCAC, the underlined sequence is the overlapping sequence of eGFP.
  • Primer 6 (SEQ ID No. 14): CGAGCTC GCAAATTAAAGCCTTCGAGCG, the underlined sequence is the overlapping sequence of the Sac I restriction site.
  • eGFP-CYC1t was constructed by overlapping extension PCR method
  • the fragment eGFP-CYC1t and the single-copy plasmid pRS41H were double digested with BamH I and Not I with Not I and Sac I restriction enzymes, respectively. After column recovery, eGFP-CYC1t and the linearized plasmid pRS41H were separated by T7 ligase. Connected to form the start function verification vector plasmid 1: pRS41H-eGFP-CYC1t.
  • the promoter sequence was obtained by amplification and the verification vector 1 was double digested with BamHI and Not I.
  • the digested vector and the promoter sequence column were recovered and connected with T7 to form a complete expression vector: pRS41H-Promoter-eGFP-CYC1t.
  • the function and strength of the promoter were determined by the expression level of the fluorescent protein.
  • the core promoter validation results are shown in Figure 1.
  • DCP1, DCP2, respectively are core promoter sequences with different random sequences.
  • the TATA box type in SCP is different from DCP. It can be seen from Figure 1 that different random sequences and different TATA box types can change the strength of the core promoter.
  • mutaDCP2 is a combination of the TATA box elements in the DCP2 core promoter sequence. The fluorescence intensity was consistent with the blank value, indicating that the TATA box is an important core promoter element.
  • shotDCP1 is a truncated DCP1 core initiation sequence after random sequence, and its intensity is lower than that of DCP1
  • the genome sequence of Saccharomyces cerevisiae was queried in the NCBI database, and the sequence information of the 3'-UTR terminator downstream of the structural gene of Saccharomyces cerevisiae was obtained by bioinformatics analysis. : TTTCAAA composition.
  • the sequence of elements described above is linked into an artificial terminator regulatory element.
  • the specific sequence is as follows (SEQ ID No.15):
  • the underlined sequence is the sequential connection of the above elements, and the remaining sequences are random connection sequences. The difference in the random sequence will also cause a change in the termination efficiency, and this embodiment only takes this sequence for explanation.
  • the termination sequence fragment (obtained by primer 7 and primer 8) was obtained by amplification.
  • the amplification primers are:
  • Primer 7 (SEQ ID No. 16): CGGGATCC AAGACAGTTTATATATAACGATAG, the underlined sequence is the overlapping sequence of the BamH I restriction site.
  • Primer 8 (SEQ ID No. 17): TTTCCTTTTGCGGCCGC TAGGCACGTCTGC, the underlined sequence is the overlapping sequence of the Not I restriction site.
  • the yeast constitutive promoter TYS1p gene sequence was obtained by querying Genebank, the genome of Saccharomyces cerevisiae was extracted, and the TYS1p gene fragment (obtained by primer 9 and primer 10) was obtained by amplification.
  • the primer sequences are:
  • Primer 9 (SEQ ID No. 18): ACGCGTCGAC TCCTTGCGCTTACTCGAATAG ⁇ the overlapping sequence of the underlined sequence and the Sal I restriction site.
  • Primer 10 (SEQ ID No. 19): 5'> GACAACTCCAGTGAAAAGTTCTTCTCCCTTGCTCACCAT TGTTATCGTCAATTAGAGTA overlapping sequence of underlined sequence and eGFP.
  • the reporter gene eGFP fragment was obtained by querying from the Genebank gene bank, and the eGFP gene fragment was obtained by amplification after chemical synthesis (amplified with primer 11 and primer 12).
  • the primer sequences are:
  • Primer 11 (SEQ ID No. 20): CGGGATCC CGTATATATATATAACTG, the underlined sequence is the overlapping sequence of the BamH I restriction site.
  • Primer 12 (SEQ ID No. 21): ATTAGCATCCATAACCGCATACTCTAATTGACGATAACA ATGGTGAGCAAGGGAGAAGA, the underlined sequence is the overlapping sequence with the promoter TYS1p.
  • TYS1p and eGFP gene fragments were used as common templates, and TYS1p-eGFP was constructed by overlapping extension PCR method;
  • TYS1p-eGFP and single-copy plasmid pRS41H were double digested with Sal I and BamHI restriction enzymes, respectively. After column recovery, TYS1p-eGFP and linearized pRS41H were ligated with T7 ligase to form a termination function verification vector plasmid 2: pRS41H-TYS1p-eGFP.
  • the different termination sequences obtained by amplification and verification vector 2 were double digested with BamH I and Not I.
  • the digested vector and the termination sequence column were recovered and then connected with T7 to form a complete expression vector: pRS41H-YTS1p-eGFP-Terminator.
  • the function and strength of the terminator were determined by the expression of fluorescent protein. The results of the functional verification of the terminator are shown in Figure 3.
  • control is the negative control for deletion of terminator
  • T1, T2, T3 are the termination sequences after adding different Linker sequences
  • T4 is the termination sequence with too long Linker sequence
  • the other three are mutation efficiency element, position element and Ploy(A) after the termination sequence.
  • Sequence analysis was performed on the start and stop sequences with moderate expression in the above examples. Since it is not appropriate to insert too long fragments between the terminator elements, the termination sequence is inserted into the initiation sequence. After the core promoter sequence reaching the basal transcription level is placed in the upstream active sequence, the termination sequence is placed before the promoter sequence and between the upstream active sequence and the core promoter sequence, respectively. Detection of its transcriptional level makes its initiation and termination functions complete and independent of each other. A regulatory element with dual functions of initiation and termination is formed. The specific sequence is as follows:
  • the bifunctional element sequence obtained by primer 13 and primer 14 was obtained by amplification.
  • the primer sequences are:
  • Primer 13 (SEQ ID No. 22): CGGGATCC AAGACAGTTTATATATAACG, the underlined sequence is the overlapping sequence of the BamH I restriction site.
  • Primer 14 (SEQ ID No. 23): TTTTCCTTTTGCGGCCGC CGGTCCGTAGTGCTCGGCTC, the underlined sequence is the overlapping sequence of the Not I restriction site.
  • Primer 15 (SEQ ID No. 24): ATTGTGATCTTCCTTCAAAGTTAGGCCATAAACTTAAC TCATGTAATTAGTTATGTCAC, the underlined sequence is the overlapping sequence of mTagBFP2.
  • Primer 16 (SEQ ID No. 25): CGAGCTC GCAAATTAAAGCCTTCGAGC, the underlined sequence is the overlapping sequence of the Sac I restriction site.
  • the blue fluorescent protein mTagBFP2 fragment was obtained by querying from the Genebank gene library, and the mTagBFP2 gene fragment (obtained by primer 17 and primer 18) was obtained by amplification after chemical synthesis.
  • the primer sequences are:
  • Primer 17 (SEQ ID No. 26): ATAAGAATGCGGCCGC ATGGTTTCAAAGGGTGAGGAAC, the underlined sequence is the overlapping sequence of the Not I restriction site.
  • Primer 18 (SEQ ID No. 27): GGCGTGAATGTAAGCGTGACATAACTAATTACATGA GTTAAGTTTATGGCCTAACTTTG, the underlined sequence is the overlapping sequence of CYC1t.
  • mTagBFP3 and CYC1t gene fragments were used as common templates, and mTagBFP3-CYC1t was constructed by overlapping extension PCR method;
  • the mTagBFP2-CYC1t and the verification vector 1pRS41H-TYS1p-eGFP were double digested with Not I and Sac I restriction enzymes, respectively. After the column was recovered, the mTagBFP2-CYC1t and the linear verification vector 2 were connected with T7 ligase to form Dual function verification vector 3: pRS41H-TYS1P-eGFP-BamH I-Not I-mTagBFP2-CYC1t.
  • the amplified bifunctional element and verification vector 3 were double digested with BamHI and Not I.
  • the digested vector and the bifunctional element column were recovered and connected with T7 to form a complete expression vector: pRS41H-TYS1P-eGFP-bifunctional element-mTagBFP2-CYC1t.
  • the functional integrity and strength of the bifunctional element were determined by detecting the expression levels of the two fluorescent proteins.
  • Figure 4 shows the results of the functional verification of the dual function element.
  • the bifunctional element sequence mentioned in this example is T+2.0UAS+DCP2, and T is the termination sequence; the other upstream active sequences UAS, core promoter DCP1 and SCP are the promoter elements mentioned in Example 1, 2.0 UAS represents the upstream active sequence with the addition of two transcription factors, and Only T+UAS represents the bifunctional element lacking the core promoter sequence. It can be seen from Figure 4 that the function of the bifunctional element is complete, it can normally terminate the green fluorescent protein in the previous transcription unit, and can also normally start the blue fluorescent protein in the second transcription unit. By deleting the core promoter sequence, the expression level of the second transcription unit was significantly decreased, indicating that the core promoter sequence had a great influence on the promoter function.
  • the three functional verification vectors 1pRS41H-eGFP-CYC1t, 2pRS41H-TYS1p-eGFP, and 3pRS41H-TYS1P-eGFP-BamH I-Not I-mTagBFP2-CYC1t formed by connection in the above embodiment were transformed into Escherichia coli competent state respectively.
  • the cells were smeared on the screening plate containing Amp resistance, and after colony PCR and sequencing verification, three E. coli engineering bacteria containing the verification vector were obtained.
  • the digested vector and the bifunctional element column were recovered and connected to T7 to form three complete expression vectors: TYS1P-eGFP-bifunctional element, bifunctional element-eGFP-CYC1t, TYS1p-eGFP-bifunctional element-mTagBFP2- CYC1t.
  • the three expression vectors were transformed into E. coli competent cells, and then spread on Amp-resistant plates. After overnight culture, single colonies were picked for colony PCR verification. In a liquid medium containing % peptone and 1% yeast powder, the cells were cultured overnight at 37° C. and shaken at 150 rpm, and plasmids were extracted. The constructed three expression plasmids were transformed into Saccharomyces cerevisiae CEN.PK2-1C cells by lithium acetate transformation method to obtain Saccharomyces cerevisiae transformants.
  • Saccharomyces cerevisiae transformants selected from different bifunctional element expression vectors in the bifunctional element library were inoculated in a liquid medium containing 2% glucose, 2% peptone and 1% yeast powder, and incubated at 30°C and 220rpm for 36 hours with shaking.
  • the fluorescence intensity of intracellular eGFP and mTagBFP2 was detected by cytometer, the fluorescence intensity of Saccharomyces cerevisiae CEN.PK2-1C and empty verification vector transformants were used as blank, and the fluorescence intensity of expression cassette TYS1p-eGFP-CYC1t was used as a reference.
  • the bifunctional elements can normally perform the functions of initiation and termination, and a bifunctional element library is formed after verifying the bifunctional elements of different strengths.
  • Example 5 Application of bifunctional elements in lycopene pathway construction.
  • the heterologous synthesis pathway of lycopene was selected as the verification object, and two key synthases in the lycopene synthesis pathway were selected: Erwinia phytoene synthase CrtB (GenBank: KC954270.1) and phytoene synthase CrtB (GenBank: KC954270.1) from red lycopene Phytophthora phytoene desaturase CrtI (GenBank: AY177424.1), constructs a plasmid expression cassette in vitro.
  • the natural promoters and terminators were used to compare with bifunctional elements.
  • the natural promoters were CYC1p and TYS1p; the terminators were ADH1t and PDALt.
  • a complete plasmid containing pRS41H-(CYC1p-CrtB-ADH1t)-(TYS1p-CrtI-ADALt) was assembled simultaneously by OE-PCR and Gibson assembly.
  • the yeast promoter CYC1p gene sequence was obtained by querying from Genebank, and after chemical synthesis, the CYC1p gene fragment (obtained by primer 19 and primer 20) was obtained by amplification.
  • Primer 19 (SEQ ID No. 28): CCGGGCCCCCCCTCGAGG GCATGCATGTGCTCTGTATGTATATAAAAC, the underlined sequence is the overlapping sequence of the vector plasmid pRS41H.
  • Primer 20 (SEQ ID No. 29): GTTCAACAAAGATGGGTTGTTCAT TATTAATTTAGTGTGTGTATTTGTGTTTGTGTC, the underlined sequence is the overlapping sequence of CrtB.
  • the phytoene synthase CrtB (GenBank: KC954270.1) gene sequence of Erwinia was obtained by querying Genebank, and after chemical synthesis, the CrtB gene fragment (obtained by amplification with primer 21 and primer 22) was obtained by amplification.
  • Primer 21 (SEQ ID No. 30): GACACACAAACACAAATACACACACTAAATTAATA ATGAACAACCCATCTTTGTTGAAC, the underlined sequence is the overlapping sequence of CYC1p.
  • Primer 22 (SEQ ID No. 31): CTTTAAAATTTGTATACACTTATTTTTTTTATAACT TTACAATGGTCTTTGCCACAAGT, the underlined sequence is the overlapping sequence of ADH1t.
  • yeast terminator ADH1t gene sequence was obtained by querying Genebank, the genome of Saccharomyces cerevisiae was extracted, and the ADH1t gene fragment was obtained by amplification (amplified with primer 23 and primer 24)
  • Primer 23 (SEQ ID No. 32): ACTTGTGGCAAAGACCATTGTAA AGTTATAAAAAAAATAAGTGTATACAAATTTTAAAG, the underlined sequence is the overlapping sequence of CrtB.
  • Primer 24 (SEQ ID No. 33): CCTATTCGAGTAAGCGCAAGGA TCGGCATGCCGGTAGAGG, the underlined sequence is the overlapping sequence of TYS1p.
  • the yeast terminator TYS1p gene sequence was obtained by querying Genebank, the genome of Saccharomyces cerevisiae was extracted, and the TYS1p gene fragment (obtained by primer 25 and primer 26) was obtained by amplification.
  • Primer 25 (SEQ ID No. 34): CCTCTACCGGCATGCCGA TCCTTGCGCTTACTCGAATAGG, the underlined sequence is the overlapping sequence of ADH1t.
  • Primer 26 (SEQ ID No. 35): GTCTTGGTCTTGTTCCTTACCCAT TGTTATCGTCAATTAGAGTATGCGG, the underlined sequence is the overlapping sequence of CrtI.
  • the phytoene desaturase CrtI (GenBank: AY177424.1) gene sequence of Phaffia rhodozyma was obtained by querying from Genebank. After chemical synthesis, the CrtI gene fragment was obtained by amplification (amplified with primer 27 and primer 28)
  • Primer 27 (SEQ ID No. 36): CCGCATACTCTAATTGACGATAACA ATGGGTAAGGAACAAGACCAAGAC, the underlined sequence is the overlapping sequence of TYS1p.
  • Primer 28 (SEQ ID No. 37): CCTACGATTTAATTAATCCCT TTAGAAAGCCAAAACACCAACAGATC, the underlined sequence is the overlapping sequence of ADALt.
  • the yeast-terminated ADALt gene sequence was obtained by querying Genebank, the genome of Saccharomyces cerevisiae was extracted, and the ADALt gene fragment was obtained by amplification (amplified with primer 29 and primer 30).
  • Primer 29 (SEQ ID No. 38): GATCTGTTGGTGTTTTGGCTTTCTAA AGGGATTAATTAAATCGTAAGG, the underlined sequence is the overlapping sequence of CrtI.
  • Primer 30 (SEQ ID No. 39: GGCGGCCGCTCTAGAACTAGTG CCCACCAGACTTCAATTTTTG, the underlined sequence is the overlapping sequence of pRS41H.
  • pRS41H empty vector As a template, a linearized pRS41H gene fragment (amplified with primer 31 and primer 32) was obtained by amplification.
  • Primer 31 (SEQ ID No. 40: CAAAAATTGAAGTCTGGTGGGG CACTAGTTCTAGAGCGGCCGCC, the underlined sequence is the overlapping sequence of ADALt.
  • Primer 32 (SEQ ID No. 41: GTTTTATACATACAGAGCACATGCATGC CCTCGAGGGGGGGCCCGG, the underlined sequence is the overlapping sequence of CYC1p.
  • the gene fragments obtained with the above primers were simultaneously constructed into a complete plasmid containing pRS41H-CYC1p-CrtB-ADH1t-TYS1p-CrtI-ADALt by one-step ligation.
  • the promoters CYC1p and TYS1p and the terminators ADH1t and PDALt were used to replace U7, U8 and U9 in the bifunctional element library.

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Abstract

一种具有启动、终止双功能调控元件的构建方法和双功能元件库，涉及合成生物学技术领域。所述构建方法包括：将启动序列和终止序列按照先终止后启动的原则进行组装；所述启动序列包括上游活性序列和核心启动子序列，所述上游活性序列包括Mig1/Mig2结合位点、Rap1结合位点、Gcr1结合位点、weak Mig1结合位点、GC框、CAAT框和八聚体框；所述核心启动子序列包括TATA框、BRE元件、MTE元件和Inr元件；所述终止序列包括效率元件、位置元件和polyA。采用所述具有启动、终止双功能调控元件应用到途径构建中，简化了构建过程，途径组装效率相比于现有技术提高了约18％。

Description

一种具有启动、终止双功能调控元件的构建方法和双功能元件库 技术领域
本发明属于合成生物学技术领域，具体涉及一种具有启动、终止双功能调控元件的构建方法和双功能元件库。
背景技术
复杂的化合物经常被应用于溶剂、燃料、聚合物、纺织品、营养品、香料和药品。然而，由于工业化对环境的污染、温室气体影响造成的气候变化以及原油短缺的担忧，促使人们寻找新的、可持续的方式来供应合成化学品的供应。代谢工程与合成生物学的出现为研究和发展生物技术提供了崭新思路，代谢途径和遗传电路通常被引入微生物中，以产生化学物质或实现新的功能。酵母作为传统工业微生物能够以安全可靠的方式生产高值天然产物、化工产品和材料等。这类实验通常需要将异源基因电路或代谢途径在酵母体内表达，实现目标产物的合成。
基因电路的构建需要一个完整的基因表达盒-包括启动子、基因、终止子，而代谢途径的构建需要将不同表达盒连接到一起，虽然化学方法可以合成一定长度的DNA片段，但为了更大表达模块的组装仍然需要DNA组装技术，包括GoldenGate方法、In-fusion技术、Gibson组装、DNA assembler等。现有技术中常使用启动子CYC1p、TYS1p等，终止子ADH1t、PDALt等，但单独的启动子和终止子用于基因电路或途径组装时常常耗时较长，且当组装途径较长时，组装效率也有限。
发明内容
有鉴于此，本发明的目的在于提供一种具有启动、终止双功能调控元件的构建方法和双功能元件库，采用本发明提供的构建方法构建得到的具有启动、终止双功能调控元件应用到途径构建中，可以简化途径构建过程，增强组装。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种具有启动、终止双功能调控元件的构建方法，将启动序列和终止序列按照先终止后启动的原则进行组装；
所述启动序列包括上游活性序列和核心启动子序列，所述上游活性序列包括Mig1/Mig2结合位点、Rap1结合位点、Gcr1结合位点、weakMig1结合位点、GC框、CAAT框和八聚体框；所述核心启动子序列包括TATA框、BRE元件、MTE元件和Inr元件；
所述终止序列包括效率元件、位置元件和polyA。
优选的，所述Mig1/Mig2结合位点的核苷酸序列如SEQ ID No.1所示；
所述Rap1结合位点的核苷酸序列如SEQ ID No.2所示；
所述Gcr1结合位点的核苷酸序列如SEQ ID No.3所示；
所述weakMig1结合位点的核苷酸序列如SEQ ID No.4所示；
所述GC框的核苷酸序列为GGGCGG；
所述CAAT框的核苷酸序列为CCAATCT；
所述八聚体框的核苷酸序列为ATGCAAAT。
优选的，所述TATA框的核苷酸序列为TATATAAA；
所述BRE元件的核苷酸序列为GGACGCC；
所述MTE元件的核苷酸序列如SEQ ID No.5所示；
所述Inr元件的核苷酸序列为TTAATAT。
优选的，所述效率元件的核苷酸序列为TATATATA，所述位置元件的核苷酸序列为AATAAA。
优选的，将所述核心启动子序列连接在上游活性序列前。
优选的，将所述终止序列连接在核心启动子序列前。
优选的，将所述终止序列插入在核心启动子序列和上游活性序列之间。
本发明还提供了一种基于上述技术方案所述的构建方法构建得到的具有启动、终止双功能调控元件，所述具有启动、终止双功能调控元件的核苷酸序列如SEQ ID No.6所示。
本发明还提供了一种基于上述技术方案所述的构建方法得到的双功能元件库，将启动序列和终止序列按照先终止后启动的原则进行组装，调节所述启动序列和终止序列中各种序列的排列顺序和间隔区，得到双功能元件库。
本发明提供了一种具有启动、终止双功能调控元件的构建方法和双功能元件库，将启动序列和终止序列按照先终止后启动的原则进行组装；所述启动序列包括上游活性序列和核心启动子序列，所述上游活性序列包括Mig1/Mig2结合位点、Rap1结合位点、Gcr1结合位点、weakMig1结合位点、GC框、CAAT框和八聚体框；所述核心启动子序列包括TATA框、BRE元件、MTE元件和Inr元件；所述终止序列包括效率元件、位置元件和polyA。采用本发明提供的构建方法构建得到的具有启动、终止双功能调控元件应用到途径构建中，由于构建过程中减少了片段数量，简化代谢途径构建过程，组装效率相比于现有技术提高了约18％。
附图说明
图1为核心启动子的功能验证结果；
图2为启动子的功能验证结果；
图3为终止子的功能验证结果；
图4为双功能元件的功能验证结果；
图5为不同强度的双功能元件库。
具体实施方式
本发明提供了一种具有启动、终止双功能调控元件的构建方法，将启动序列和终止序列按照先终止后启动的原则进行组装；所述启动序列包括上游活性序列和核心启动子序列，所述上游活性序列包括Mig1/Mig2结合位点、Rap1结合位点、Gcr1结合位点、weakMig1结合位点、GC框、CAAT框和八聚体框；所述核心启动子序列包括TATA框、BRE元件、MTE元件和Inr元件；所述终止序列包括效率元件、位置元件和polyA。
在本发明中，所述Mig1/Mig2结合位点的核苷酸序列如SEQ ID No.1所示；所述Rap1结合位点的核苷酸序列如SEQ ID No.2所示；所述Gcr1结合位点的核苷酸序列如SEQ ID No.3所示；所述weak Mig1结合位点的核苷酸序列如SEQ ID No.4所示；所述GC框的核苷酸序列为GGGCGG；所述CAAT框的核苷酸序列为CCAATCT；所述八聚体框的核苷酸序列为ATGCAAAT。
SEQ ID No.1：GATCGTAGCCCCGGATTTAC。
SEQ ID No.2：GATCGTACACCTGGACATAC。
SEQ ID No.3：GATCGTAGAGCTTCCACTAC。
SEQ ID No.4：GATCGTAGCCCCACAAATAC。
在本发明中，所述TATA框的核苷酸序列为TATATAAA；所述BRE元件的核苷酸序列为GGACGCC；所述MTE元件的核苷酸序列如SEQ ID No.5所示；所述Inr元件的核苷酸序列为TTAATAT。
SEQ ID No.5：CGAGCCGAGCA。
在本发明中，所述效率元件的核苷酸序列为TATATATA，所述位置元件的核苷酸序列为AATAAA。
本发明优选将所述核心启动子序列连接在上游活性序列前。本发明优选将所述终止序列连接在核心启动子序列前，或者将所述终止序列插入在核心启动子序列和上游活性序列之间。上述连接或插入后，转录水平使其启动和终止功能完整且互不影响。
本发明还提供了一种基于上述技术方案所述的构建方法构建得到的具有启动、终止双功能调控元件，所述具有启动、终止双功能调控元件的核苷酸序列如SEQ ID No.6所示，具体如下：
SEQ ID No.6：
Figure PCTCN2021077808-appb-000001
本发明还提供了一种基于上述技术方案所述的构建方法得到的双功能元件库，将启动序列和终止序列按照先终止后启动的原则进行组装，调节所述启动序列和终止序列中各种序列的排列顺序和间隔区，得到双功能元件库。
为了进一步说明本发明，下面结合实例对本发明进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
启动功能的设计与验证。
通过NCBI数据库分析大量酿酒酵母启动子序列信息得知：酵母组成型启动子由上游活性序列和核心启动子序列组成，其中上游活性序列包含大量转录因子结合位点，选用酵母转录因子结合位点Mig1/Mig2结合位点序列为：GATCGTAGCCCCGGATTTAC、Rap1结合位点序列为：GATCGTACACCTGGACATAC、Gcr1结合位点序列为：GATCGTAGAGCTTCCACTAC、weak Mig1结合位点序列为：GATCGTAGCCCCACAAATAC，转录因子结合位点可有效提高启动子转录水平。及优选辅助RNAII聚合酶与DNA结合的转录因子结合位点：GC框：GGGCGG、CAAT框：CCAATCT、八聚体框：ATGCAAAT；核心启动子序列包含：TATA框TATATAAA位于起始位点上游-25～-30bp处、BRE元件GGACGCC位于起始位点上游-37～-32bp处、MTE元件CGAGCCGAGCA位于起始位点下游+11～+18bp处和Inr元件：TTAATAT组成。
本实施例所设计的启动序列包含上游活性序列UAS及核心启动子序列；其中连接序列的变化、转录因子结合位点的选择不同及排列顺序不同均会造成启动子强度发生变化。本实施例仅取一例作为讲解对象。
上游活性序列UAS(SEQ ID No.7)：AGTGATAATC GATCGTAGCCCCGGATTTACGTAAGTTCTG GATCGTACACCTGGACATACAGATTGTGAT GGCAATCTCGATTACACT GATCGTAGAGCTTCCACTACTAAATCGGT GATCGTAGC CCCACAAATACCGTGATACTG，下划线序列依次为上述酵母转录因子结合位点Mig1/Mig2、Rap1、Gcr1、weakMig1。其余序列为随机连接序列，以确保上游活性序列与酵母同源性最低，以防止该序列在酵母体内发生同源重组，从而不能正常行使其功能。
核心启动子序列(SEQ ID No.8)：
TATTGTATTTTCACA GGACGCC
Figure PCTCN2021077808-appb-000002
CTCTTGTTTTCTTCTTTTCTC TTA +1A TATTCTTTT CGAGCCGAGCACTACGGACCG，下划线序列依次为BRE元件、TATA框、Inr元件(+1处为转录起始位点)及MTE元件。其余序列为T-rich随机连接序列，选用T-rich序列可使RNAⅡ聚合酶更好的与DNA序列结合。
将上述上游活性序列与核心启动子序列直接连接形成人工启动子序列，化学合成后扩增获得启动子序列片段(以引物1、引物2扩增获得)。
引物序列为：
引物1(SEQ ID No.9)： CGGGATCCAGTGATAATCGATCG，下划线序列为BamH I酶切位点的重叠序列。
引物2(SEQ ID No.10)：
TTTTCCTTTTGCGGCCGCCGGTCCGTAGTGCTCGGC，下划线序列为Not I酶切位点的重叠序列。
验证载体质粒1的构建：
从Genebank基因库中查询获得报告基因eGFP片段，化学合成后扩增获得eGFP基因片段(以 引物3、引物4扩增获得)。
引物序列为：
引物3(SEQ ID No.11)：
ATAAGAATGCGGCCGCATGGTGAGCAAGGGAGAAGAAC，下划线序列为Not I酶切位点的重叠序列。
引物4(SEQ ID No.12)： GAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATTATTTGTATAGTTCATCCA，下划线序列为CYC1t的重叠序列。
从Genebank基因库中查询获得终止子CYC1t片段，化学合成后扩增获得CYC1t基因片段(以引物5、引物6扩增获得)。
引物序列为：
引物5(SEQ ID No.13)： CTGGGATTACACATGGCATGGATGAACTATACAAATAATCATGTAATTAGTTATGTCAC，下划线序列为eGFP的重叠序列。
引物6(SEQ ID No.14)： CGAGCTCGCAAATTAAAGCCTTCGAGCG，下划线序列为Sac I酶切位点的重叠序列。
设计缺失启动子的基因表达盒，通过将双功能元件构建至表达盒前即可验证其终止功能是否完整。
利用引物3和引物6，以eGFP和CYC1t基因片段为共同模板，通过重叠延伸PCR方法连接构建成eGFP-CYC1t；
用Not I和Sac I限制性内切酶对片段eGFP-CYC1t和单拷贝质粒pRS41H分别进行BamH I与Not I双酶切，柱回收后，用T7连接酶将eGFP-CYC1t和线性化的质粒pRS41H连接，形成启动功能验证载体质粒1：pRS41H-eGFP-CYC1t。
将扩增获得启动子序列和验证载体1使用BamH I和Not I双酶切。将酶切完成的载体和启动子序列柱回收后T7连接，形成一个完整的表达载体：pRS41H-Promoter-eGFP-CYC1t。通过荧光蛋白的表达量确定启动子的功能及强度。核心启动子验证结果见图1。
以未做改动的空酵母CEN.PK2-1C和未添加启动子序列的验证载体1酵母转化子emp-B1，作为阴性对照。DCP1、DCP2、分别为取不同随机序列的核心启动子序列。SCP中的TATA框类型与DCP不同，从图1可以看出不同的随机序列及不用的TATA框类型可使核心启动子的强度发生变化，mutaDCP2是将DCP2核心启动子序列中的TATA框元件进行了突变，其荧光强度与空白值一致，说明TATA框为重要的核心启动子元件。shotDCP1为随机序列截短后的DCP1核心启动序列，其强度较DCP1有所降低
上游活性序列与核心启动子序列结合后的完整启动子强度验证结果见图2。
由图2结果可以看出，在添加由两种转录因子结合位点组成的上游活性序列UAS后，启动子转录水平有了明显提升，2.0UAS为添加了4种转录因子结合位点的上游活性序列，启动子强度有了进一步的提升。在UAS与突变核心启动子DCP2中TATA序列结合后。
实施例2
终止功能的设计与验证。
在NCBI数据库中查询酿酒酵母基因组序列，通过生物信息学分析获得酿酒酵母结构基因下游的3’-UTR终止子的序列信息，其中终止序列由效率元件：TATATATA，定位元件：AATAAA及Ploy(A)：TTTCAAA组成。将上述元件顺序列连接成人工终止子调控元件。具体序列如下(SEQ ID No.15)：
AAGACAGTT TATATATAACGATAGT AATAAATTATTCAGTTGATAATCTCG TTTCAAAGCAGACGTGCCTA。下划线序列为上述元件依次顺序连接，其余序列为随机连接序列。其中随机序列的不同也会造成终止效率的改变，本实施例仅取这一序列做讲解。
化学合成后扩增获得终止序列片段(以引物7、引物8扩增获得)。
扩增引物为：
引物7(SEQ ID No.16)： CGGGATCCAAGACAGTTTATATATAACGATAG，下划线序列为BamH I酶切位点的重叠序列。
引物8(SEQ ID No.17)： TTTTCCTTTTGCGGCCGCTAGGCACGTCTGC，下划线序列为Not I酶切位点的重叠序列。
验证载体质粒2的构建：
通过从Genebank查询获得酵母组成型启动子TYS1p基因序列，提取酿酒酵母基因组，扩增获得TYS1p基因片段(以引物9、引物10扩增获得)。
引物序列为：
引物9(SEQ ID No.18): ACGCGTCGACTCCTTGCGCTTACTCGAATAG<下划线序列与Sal I酶切位点的重叠序列。
引物10(SEQ ID No.19):5’> GACAACTCCAGTGAAAAGTTCTTCTCCCTTGCTCACCATTGTTATCGTCAATTAGAGTA下划线序列与eGFP的重叠序列。
从Genebank基因库中查询获得报告基因eGFP片段，化学合成后扩增获得eGFP基因片段(以引物11、引物12扩增获得)。
引物序列为：
引物11(SEQ ID No.20)： CGGGATCCCGTATATATATATAACTG，下划线序列为BamH I酶切位点的重叠序列。
引物12(SEQ ID No.21): ATTAGCATCCATAACCGCATACTCTAATTGACGATAACAATGGTGAGCAAGGGAGAAGA，下划线序列为与启动子TYS1p重叠序列。
设计缺失终止子的基因表达盒，通过将双功能元件构建至表达盒后即可验证其终止功能是否完整。
利用引物9和引物12，以TYS1p和eGFP基因片段为共同模板，通过重叠延伸PCR方法连接构建成TYS1p-eGFP；
用Sal I和BamH I限制性内切酶对TYS1p-eGFP和单拷贝质粒pRS41H分别进行双酶切，柱回收后，用T7连接酶将TYS1p-eGFP和线性化pRS41H连接，形成终止功能验证载体质粒2：pRS41H-TYS1p-eGFP。
将扩增获得的不同终止序列和验证载体2使用BamH I和Not I双酶切。将酶切完成的载体和终止序列柱回收后T7连接，形成一个完整的表达载体：pRS41H-YTS1p-eGFP-Terminator。通过荧光蛋白的表达量确定终止子的功能及强度。终止子功能验证结果见图3。
control为缺失终止子的阴性对照，T1、T2、T3为添加不同Linker序列后的终止序列，T4为Linker序列过长的终止序列，其余三个分别为突变效率元件、位置元件及Ploy(A)后的终止序列。由图3可知，在添加不同的随机序列后对终止子的强度影响不大，但是在突变效率元件、定位元件、及Ploy(A)时，可大幅降低终止子强度。当随机序列过长时，也会降低终止子强度，所以终止序列中的三个元件不宜变化且距离不能过长。
实施例3
启动终止双功能元件的构建及验证
将上述实施例中表达强度适中的启动与终止序列进行序列分析。由于终止子元件间不宜插入过长片段，所以将终止序列插入启动序列中。将达到基础转录水平的核心启动子序列置于上游活性序列后，分别将终止序列放置于启动子序列之前、上游活性序列与核心启动子序列之间。检测其转录水平使其启动和终止功能完整且互不影响。形成一种具有启动、终止双功能调控元件。具体序列如下：
AAGACAGTT TATATATAACGATAGT AATAAATTATTCAGTTGATAATCTCG TTTCAAAGCAGACGTGCCTAAGTGATAATC GATCGTAGCCCCGGATTTACGTAAGTTCTG GATCGTACACCTG GACATACAGATTGTGAT GGCAATCTCGATTACACT GATCGTAGAGCTTCCACTACTAAATCGGT GATCGTAGCCCCACAAATACCGTGATACTGTATTGTATTTTCACA GGACGCC
Figure PCTCN2021077808-appb-000003
CTCTTGTTTTCTTCTTTTCTC TTAATATTCTTTT CGAGCCGAGCACTACGGACCG。该序列为实施例1及实施例2中提及的终止序列和启动序列顺序连接而成，本实施例仅取将终止序列置于启动序列之前的序列做讲解，其余理性结合方式在结果中展示其验证结果。
化学合成后扩增获得双功能元件序列(以引物13、引物14扩增获得)。
引物序列为：
引物13(SEQ ID No.22)： CGGGATCCAAGACAGTTTATATATAACG，下划线序列为BamH I酶切位点的重叠序列。
引物14(SEQ ID No.23)： TTTTCCTTTTGCGGCCGCCGGTCCGTAGTGCTCGGCTC，下划线序列为Not I酶切位点的重叠序列。
验证载体质粒3的构建：
与实施例1相同的方法，不同的酶切位点扩增CYC1t(以引物15、引物16扩增获得)
引物15(SEQ ID No.24)： ATTGTGATCTTCCTTCAAAGTTAGGCCATAAACTTAACTCATGTAATTAGTTATGTCAC，下划线序列为mTagBFP2的重叠序列。
引物16(SEQ ID No.25)： CGAGCTCGCAAATTAAAGCCTTCGAGC，下划线序列为Sac I酶切位点的重叠序列。
从Genebank基因库中查询获得蓝色荧光蛋白mTagBFP2片段，化学合成后扩增获得mTagBFP2基因片段(以引物17、引物18扩增获得)。
引物序列为：
引物17(SEQ ID No.26)： ATAAGAATGCGGCCGCATGGTTTCAAAGGGTGAGGAAC，下划线序列为Not I酶切位点的重叠序列。
引物18(SEQ ID No.27)： GGCGTGAATGTAAGCGTGACATAACTAATTACATGAGTTAAGTTTATGGCCTAACTTTG，下划线序列为CYC1t的重叠序列。
利用引物15和引物18，以mTagBFP3和CYC1t基因片段为共同模板，通过重叠延伸PCR方法连接构建成mTagBFP3-CYC1t；
用Not I和Sac I限制性内切酶对mTagBFP2-CYC1t和验证载体1pRS41H-TYS1p-eGFP分别进行双酶切，柱回收后，用T7连接酶将mTagBFP2-CYC1t和线性化验证载体2连接，形成双功能验证载体3：pRS41H-TYS1P-eGFP-BamH I-Not I-mTagBFP2-CYC1t。
将扩增获得的双功能元件和验证载体3使用BamH I和Not I双酶切。将酶切完成的载体和双功能元件柱回收后T7连接，形成一个完整的表达载体：pRS41H-TYS1P-eGFP-双功能元件-mTagBFP2-CYC1t。通过检测两种荧光蛋白的表达量确定双功能元件的功能完整性及强度。双功能元件功能验证结果见图4。
本实施例中提及的双功能元件序列为T+2.0UAS+DCP2，T为终止序列；其余上游活性序列UAS、核心启动子DCP1及SCP为实施例1中提及的启动子元件内容，2.0UAS代表添加了两种转录因子的上游活性序列，Only T+UAS代表缺失核心启动序列的双功能元件。由图4可以看出双功能元件的功能完整，可以正常终止前一个转录单元中的绿色荧光蛋白，也可以正常启动第二个转录单元中的蓝色荧光蛋白。通过缺失核心启动序列，第二转录单元表达量明显下降，说明核心启动序列对启动功能影响很大。通过改变双功能序列中的元件顺序可以导致两个转录单元的表达量发生变化。但是当终止序列置于启动子序列之后对启动功能有很大影响，所以仅理性结合方式仅选用第一种：终止序列置于启动序列之前，以及第二种：终止序列置于上游活性序列与核心启动子序列之间，这两种结合方式。
将上述实施例中连接形成的三种功能验证载体1pRS41H-eGFP-CYC1t、验证载体2pRS41H-TYS1p-eGFP、验证载体3pRS41H-TYS1P-eGFP-BamH I-Not I-mTagBFP2-CYC1t分别转 化大肠杆菌感受态细胞，涂在含有Amp抗性的筛选平板上，经过菌落PCR和测序验证获得含有验证载体的三种大肠杆菌工程菌。
实施例4
双功能元件的构建及双功能元件库的建立及应用
分别挑取含有实施例3的验证载体1、2、3的大肠杆菌单菌落，接种于含有2％氯化钠、2％蛋白胨和1％酵母粉的液体培养基中，37℃、150rpm震荡过夜培养，提取质粒，三个验证载体均使用BamH I和Not I双酶切，使其线性化，用于双功能元件连接。将扩增获得的双功能元件序列使用BamH I和Not I双酶切。将酶切完成的载体和双功能元件柱回收后T7连接，形成三个完整的表达载体：TYS1P-eGFP-双功能元件、双功能元件-eGFP-CYC1t、TYS1p-eGFP-双功能元件-mTagBFP2-CYC1t。
将三种表达载体转化至大肠杆菌感受态细胞中，后涂布于Amp抗性平板，过夜培养后挑取单菌落进行菌落PCR验证，取阳性结果的菌株接种于含有2％氯化钠、2％蛋白胨和1％酵母粉的液体培养基中，37℃、150rpm震荡过夜培养，提取质粒。将构建完成的三个表达质粒用醋酸锂转化法转化到酿酒酵母CEN.PK2-1C细胞中，获得酿酒酵母转化子。
选择不同结构的双功能元件进行扩增，按照上述步骤，可获得含有不同双功能元件表达载体和酿酒酵母转化子。
选择双功能元件库中的不同双功能元件表达载体的酿酒酵母转化子接种于含有2％葡萄糖、2％蛋白胨和1％酵母粉的液体培养基中，30℃、220rpm震荡培养36小时，利用流式细胞仪检测细胞内eGFP和mTagBFP2的荧光强度，以酿酒酵母CEN.PK2-1C及空验证载体转化子的荧光强度为空白，表达盒TYS1p-eGFP-CYC1t的荧光强度为参照，发现本发明构建的双功能元件可以正常行使启动和终止的功能，验证不同强度的双功能元件后形成双功能元件库。
在通过改变元件的排列、不同元件间的连接序列及不同元件的选用后，可以得到一系列不同强度的双功能元件库
元件库中代号的具体结合方式见表1。
表1 元件库中代号的具体结合方式
代号 具体结合方式
U1 T+UAS+DCP1
U2 T+UAS+DCP2
U3 T+UAS+SCP1
U4 T+2.0UAS+DCP1
U5 T+2.0UAS+DCP2
U6 T+2.0UAS+SCP1
U7 2.0UAS+T+DCP2
U8 3.0UAS+T+DCP1
U9 4.0UAS+T+DCP2
U10 5.0UAS+T+DCP2
U11 2.0UAS+2.0T+DCP2
U12 2.0UAS+3.0T+DCP2
U13 2.0UAS+4.0T+DCP2
不同强度的双功能元件表达情况见图5。
实施例5：双功能元件在番茄红素途径构建中的应用。
本案例选取番茄红素异源合成途径为验证对象，选择番茄红素合成途径中的2个关键合成酶：欧文氏菌的八氢番茄红素合成酶CrtB(GenBank:KC954270.1)和来自红发夫酵母的八氢番茄红素去饱和酶CrtI(GenBank:AY177424.1)，在体外构建质粒表达盒。分别使用天然启动子和终止子与双功能元件做对比，天然启动子选择CYC1p，TYS1p；终止子选择ADH1t，PDALt。以OE-PCR和Gibson assembly两种组装方法同时组装成含有pRS41H-(CYC1p-CrtB-ADH1t)-(TYS1p-CrtI-ADALt)的完整质粒。
通过从Genebank查询获得酵母启动子CYC1p基因序列，化学合成后，扩增获得CYC1p基因片段(以引物19、引物20扩增获得)。
引物19(SEQ ID No.28)： CCGGGCCCCCCCTCGAGGGCATGCATGTGCTCTGTATGTATATAAAAC，下划线序列为载体质粒pRS41H的重叠序列。
引物20(SEQ ID No.29)： GTTCAACAAAGATGGGTTGTTCATTATTAATTTAGTGTGTGTATTTGTGTTTGTGTGTC，下划线序列为CrtB的重叠序列。
通过从Genebank查询获得欧文氏菌的八氢番茄红素合成酶CrtB(GenBank:KC954270.1)基因序列，化学合成后，扩增获得CrtB基因片段(以引物21、引物22扩增获得)。
引物21(SEQ ID No.30)： GACACACAAACACAAATACACACACTAAATTAATAATGAACAACCCATCTTTGTTGAAC，下划线序列为CYC1p的重叠序列。
引物22(SEQ ID No.31)： CTTTAAAATTTGTATACACTTATTTTTTTTATAACTTTACAATGGTCTTTGCCACAAGT，下划线序列为ADH1t的重叠序列。
通过从Genebank查询获得酵母终止子ADH1t基因序列，提取酿酒酵母基因组，扩增获得ADH1t基因片段(以引物23、引物24扩增获得)
引物23(SEQ ID No.32)： ACTTGTGGCAAAGACCATTGTAAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAG，下划线序列为CrtB的重叠序列。
引物24(SEQ ID No.33)： CCTATTCGAGTAAGCGCAAGGATCGGCATGCCGGTAGAGG，下划线序列为TYS1p的重叠序列。
通过从Genebank查询获得酵母终止子TYS1p基因序列，提取酿酒酵母基因组，扩增获得TYS1p基因片段(以引物25、引物26扩增获得)。
引物25(SEQ ID No.34)： CCTCTACCGGCATGCCGATCCTTGCGCTTACTCGAATAGG，下划线序列为ADH1t的重叠序列。
引物26(SEQ ID No.35)： GTCTTGGTCTTGTTCCTTACCCATTGTTATCGTCAATTAGAGTATGCGG，下划线序列为CrtI的重叠序列。
通过从Genebank查询获得红发夫酵母的八氢番茄红素去饱和酶CrtI(GenBank:AY177424.1)基因序列，化学合成后，扩增获得CrtI基因片段(以引物27、引物28扩增获得)
引物27(SEQ ID No.36)： CCGCATACTCTAATTGACGATAACAATGGGTAAGGAACAAGACCAAGAC，下划线序列为TYS1p的重叠序列。
引物28(SEQ ID No.37)： CCTTACGATTTAATTAATCCCTTTAGAAAGCCAAAACACCAACAGATC，下划线序列为ADALt的重叠序列。
通过从Genebank查询获得酵母终止ADALt基因序列，提取酿酒酵母基因组，扩增获得ADALt基因片段(以引物29、引物30扩增获得)。
引物29(SEQ ID No.38)： GATCTGTTGGTGTTTTGGCTTTCTAAAGGGATTAATTAAATCGTAAGG，下划线序列为CrtI的重叠序列。
引物30(SEQ ID No.39： GGCGGCCGCTCTAGAACTAGTGCCCACCAGACTTCAATTTTTG，下划线序列为的pRS41H重叠序列。
以pRS41H空载体为模板，扩增获得线性化的pRS41H基因片段(以引物31、引物32扩增获得)。
引物31(SEQ ID No.40： CAAAAATTGAAGTCTGGTGGGCACTAGTTCTAGAGCGGCCGCC，下划线序列为ADALt的重叠序列。
引物32(SEQ ID No.41： GTTTTATATACATACAGAGCACATGCATGCCCTCGAGGGGGGGCCCGG，下划线序列为CYC1p的重叠序列。
以上述引物获得的基因片段，同时使用一步连接，构建成含有pRS41H-CYC1p-CrtB-ADH1t-TYS1p-CrtI-ADALt的完整质粒。
以双功能元件做对照，选用双功能元件库中的U7、U8、U9替代所用的启动子CYC1p、TYS1p，终止子ADH1t、PDALt。
以相同的实验条件，操作手法，不同的组装方法，并作3组平行实验。做出以下对比(表2)对比后发现使用双功能元件简化构建步骤后，使用不同DNA组装方法其连接效率约有18％的提升。
表2 双功能元件用于途径组装时的效果
Figure PCTCN2021077808-appb-000004
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (9)

  1. 一种具有启动、终止双功能调控元件的构建方法，其特征在于，将启动序列和终止序列按照先终止后启动的原则进行组装；
    所述启动序列包括上游活性序列和核心启动子序列，所述上游活性序列包括Mig1/Mig2结合位点、Rap1结合位点、Gcr1结合位点、weak Mig1结合位点、GC框、CAAT框和八聚体框；所述核心启动子序列包括TATA框、BRE元件、MTE元件和Inr元件；
    所述终止序列包括效率元件、位置元件和polyA。
  2. 根据权利要求1所述的构建方法，其特征在于，所述Mig1/Mig2结合位点的核苷酸序列如SEQ ID No.1所示；
    所述Rap1结合位点的核苷酸序列如SEQ ID No.2所示；
    所述Gcr1结合位点的核苷酸序列如SEQ ID No.3所示；
    所述weakMig1结合位点的核苷酸序列如SEQ ID No.4所示；
    所述GC框的核苷酸序列为GGGCGG；
    所述CAAT框的核苷酸序列为CCAATCT；
    所述八聚体框的核苷酸序列为ATGCAAAT。
  3. 根据权利要求1所述的构建方法，其特征在于，所述TATA框的核苷酸序列为TATATAAA；
    所述BRE元件的核苷酸序列为GGACGCC；
    所述MTE元件的核苷酸序列如SEQ ID No.5所示；
    所述Inr元件的核苷酸序列为TTAATAT。
  4. 根据权利要求1所述的核苷酸序列，其特征在于，所述效率元件的核苷酸序列为TATATATA，所述位置元件的核苷酸序列为AATAAA。
  5. 根据权利要求1所述的构建方法，其特征在于，将所述核心启动子序列连接在上游活性序列前。
  6. 根据权利要求1所述的构建方法，其特征在于，将所述终止序列连接在核心启动子序列前。
  7. 根据权利要求1所述的构建方法，其特征在于，将所述终止序列插入在核心启动子序列和上游活性序列之间。
  8. 一种基于权利要求1所述的构建方法构建得到的具有启动、终止双功能调控元件，其特征在于，所述具有启动、终止双功能调控元件的核苷酸序列如SEQ ID No.6所示。
  9. 一种基于权利要求1～7任一项所述的构建方法得到的双功能元件库，其特征在于，将启动序列和终止序列按照先终止后启动的原则进行组装，调节所述启动序列和终止序列中各种序列的排列顺序和间隔区，得到双功能元件库。
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JIANG HUIHUI: "Research Progress of Promoter Elements in Pichia Pastoris Expression System", JOURNAL OF CHAOHU COLLEGE, vol. 19, no. 3, 31 December 2017 (2017-12-31), pages 67 - 72, XP055961888, ISSN: 1672-2868 *
KEMLER IRIS, BUCHER ETIENNE, SEIPEL KATJA, MÜLLER-LMMERGLÜCK MICHAEL M., SCHAFFNER WALTER: "Promoters with the octamer DNA motif (ATGCAAAT) can be ubiquitous or cell type-specific depending on binding affinity of the octamer site and Oct-factor concentration", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 19, no. 2, 1 January 1991 (1991-01-01), GB , pages 237 - 242, XP055961882, ISSN: 0305-1048, DOI: 10.1093/nar/19.2.237 *
SANTANGELO GEORGE M.: "Glucose Signaling in Saccharomyces cerevisiae", MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 70, no. 1, 1 March 2006 (2006-03-01), US , pages 253 - 282, XP055961881, ISSN: 1092-2172, DOI: 10.1128/MMBR.70.1.253-282.2006 *
SARMA NAYAN J., KRISTINE WILLIS: "The new nucleoporin - Regulator of transcriptional repression and beyond ", NUCLEUS, vol. 3, no. 6, 9 October 2012 (2012-10-09), XP055961886, DOI: 10.4161/nucleus.22427 *
TANG HONGTING, WU YANLING, DENG JILIANG, CHEN NANZHU, ZHENG ZHAOHUI, WEI YONGJUN, LUO XIAOZHOU, KEASLING JAY D.: "Promoter Architecture and Promoter Engineering in Saccharomyces cerevisiae", METABOLITES, vol. 10, no. 8, pages 320, XP055918931, DOI: 10.3390/metabo10080320 *
WANG ZHAOXIA: "Design of Terminator of Saccharomyces Cerevisiae and Application in Pathway Engineering", BASIC SCIENCES, CHINA MASTER’S THESES FULL-TEXT DATABASE, 15 January 2020 (2020-01-15), XP055961892 *
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