一组用于酿酒酵母多拷贝整合的质粒工具包

Plasmid kit for saccharomyces cerevisiae multi-copy integration

文档序号：3336 发布日期：2021-09-17 浏览：64次中文

1. The saccharomyces cerevisiae multicopy integration gene expression frame is characterized in that the expression frame consists of a weak promoter sequence and a screening gene sequence with a degradation label deg; the weak promoter comprises P_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1The screening gene comprises ScTRP1, KlLEU2, KlURA3, ScMET15, SpHIS5, natMX, hpHMX, kanMX, patMX and bleMX.

2. The gene expression cassette of claim 1, wherein the weak promoter P is a promoter for expression of a gene_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1The nucleotide is shown in SEQ ID NO. 1-10 respectively; the screening gene is provided with a degradation label, and a nucleotide sequence formed by the screening gene and the degradation label is shown in SEQ ID NO. 11-20.

3. A saccharomyces cerevisiae multicopy integration plasmid consisting of a gene expression cassette and a pre-integration expression cassette according to claim 1 or 2; the pre-integration expression frame consists of an upstream and downstream homologous arm sequence of a Ty transposon, a terminator sequence and a green fluorescent protein expression frame.

4. The Saccharomyces cerevisiae multicopy integration plasmid of claim 3, wherein the plasmid is a plasmid that integrates multiple copies of Saccharomyces cerevisiae gene into a single geneTy transposon sequences include Ty1Cons, Ty2Cons, Ty3Cons, Ty4Cons and Ty5 Cons; the terminator comprises T_RFC5-T_POL30、T_SEC13-T_PNP1、T_MTD1-T_RPF2、T_LEU2-T_NFS1、T_DSF1-T_HXT13、T_TIM21-T_GSC2、T_RRP12-T_TAF3、T_RNA14-T_BUB2、T_ADH1And T_CYC1。

5. The Saccharomyces cerevisiae multicopy integration plasmid of claim 3, wherein the upstream homology arm, the green fluorescent protein expression cassette, the terminator sequence, the gene expression cassette, the downstream homology arm are ligated in that order.

6. The Saccharomyces cerevisiae multicopy integration plasmid of claim 3, wherein the green fluorescent protein expression cassette comprises a promoter P_GAL7Promoter P_GAL7Upstream of (2) comprises a terminator sequence T_GAL10。

7. A strain for producing taxifolin, which expresses relevant genes for producing taxifolin by the saccharomyces cerevisiae multicopy integration plasmid of any one of claims 3-6, and the genes are expressed by the following three modules:

a first module: overexpression of the relevant genes from tyrosine or phenylalanine to p-coumaric acid synthesis:

from tyrosine to p-coumaric acid genes include ARO4^fbr、ARO7^fbr、FjTAL、EcaroL；

The genes from phenylalanine to p-coumaric acid include ARO4^fbr、ARO7^fbr、FjTAL、EcaroL、SmPAL、SmC4H；

And a second module: overexpresses genes related to p-coumaric acid to naringenin production, including Pc4CL, PhCHS, and MsCHI;

and a third module: naringenin-to-taxifolin synthesis related genes including SmF 3' H, SmCPR and SmF 3H.

8. The strain of claim 7, wherein the strain is a starting strain of Saccharomyces cerevisiae C800.

9. A method for synthesizing taxifolin, which is characterized in that the strain of claim 7 or 8 is used for de novo synthesis of taxifolin by using glucose or ethanol as a substrate.

10. Use of the saccharomyces cerevisiae multicopy integration gene expression cassette of claim 1 or 2, or the saccharomyces cerevisiae multicopy integration plasmid of any one of claims 3 to 6, or the strain of claim 7 or 8 for the production of a protein of interest or taxifolin and derivatives thereof.

Background

Saccharomyces cerevisiae, a model strain of eukaryotes, is commonly used for introducing heterologous pathways to synthesize high value-added products. These approaches typically involve several to tens of genes, and multiple co-expression of multiple genes in s.cerevisiae requires multiple integrations, which is inefficient, and the integration sites and selection tags do not satisfy the subsequent integration procedures. Therefore, there is a need for a highly efficient, high copy and stable insertion site for integrating genes requiring overexpression into these sites to achieve efficient expression of heterologous genes.

There are a number of Long Terminal Repeats (LTRs) in the Saccharomyces cerevisiae genome, one type of LTR being transposons, which in yeast are designated as Ty transposons. There are five types of the Ty transposon sequence matching, including Ty1Cons, Ty2Cons, Ty3Cons, Ty4Cons and Ty5Cons, and the sequence matching between transposons is also low, the copy number of the five Ty transposons on the genome is about 20-50, and the transposons are distributed uniformly on the genome, and homologous recombination rarely occurs, and the structure is stable, so these Ty sites are ideal foreign gene insertion sites (Maury, J.; Germann, S.M.; Ballalobsen, S.A., et al., EasyClonemulti: A Set of Vectors for simple and Multiple genetic Integrations in Saccharomyces cerevisiae [ J ]. PloS One, 2016 (3), e 0194.). However, the current integrated expression based on the Ty locus has various defects, such as insufficient screening tag and low copy number of the obtained transformant; the integrative expression frame uses the same promoter, the same terminator and other expression elements, so that the homologous recombination probability is increased; the application of such integration sites is limited due to the unknown ability of the different Ty sites to coordinate integration.

To further refine the Ty integration protocol, a high copy integration plasmid kit was constructed in this study. In this high copy integration package, the screening tag classes were first expanded, including 5 auxotrophic tags: uracil-deficient tags (KlURA3), leucine-deficient tags (KlLEU2), histidine-deficient tags (SpHIS5), tryptophan-deficient tags (sctp 1), and methionine-deficient tags (ScMET 15); 5 antibiotic screening tags: nolsemicin resistance tag (Nourethericin resistance, natMX), Hygromycin resistance tag (Hygromycin resistance, hpHMX), Bialaphos resistance tag (Bialaphos resistance, patMX) (Goldstein A L, McCusker J H.three and new dominant drug resistance cassettes for gene delivery in Saccharomyces cerevisiae [ J ]. Yeast (Chichester, England)1999,15(14),1541-53.), Geneticin resistance tag (genetic in resistance, KanmX) (Wach A, Brachat A, Pohlmann R, Nuet al, New hematology modules for PCR-base gene delivery [ J ] (PCR-nucleotide sequences J.) (nucleotide J.) (PCR-nucleotide sequences J.) (PCR-nucleotide sequences J.) (nucleotide J.) (III J.) (nucleotide J.),93, nucleotide J.), 30(6), e 23.). Meanwhile, when a screening gene expression frame is constructed, a weak promoter is used for expressing a related screening gene, and a Degradation tag (deg) is added to the C end of the screening gene (Gilon T, Chomsky O, Kulka R G. grading signals for ubiquitin system protein [ J ]. EMBO J1998, 17(10),2759-66.), the expression level of the gene is reduced at the transcription level and the protein level, and the screening pressure is strengthened, so that the integrated copy number of the foreign gene is increased. Finally, all elements such as promoters and terminators are not identical in order to reduce the probability of homologous recombination of foreign genes. Connecting the constructed expression package with a reporter gene green fluorescent protein EGFP, respectively transforming the expression package into a saccharomyces cerevisiae strain, and determining the integration difficulty and the distribution condition of the integrated copy number of the corresponding labels and sites by detecting the number of the obtained transformants and the intensity distribution of the green fluorescent protein of the transformants.

After obtaining the high-copy integration tool and verifying the integration performance of the high-copy integration tool, the taxifolin synthetic pathway genes are integrated on different Ty sites of a saccharomyces cerevisiae genome by using different screening labels and are used for verifying the multi-copy integration performance.

Disclosure of Invention

The Saccharomyces cerevisiae includes model strain S288c and its derivative strains. The plasmids can realize multi-copy integration in a saccharomyces cerevisiae genome through one-time transformation, and the integration sites are mutually independent and have good stability, so that the plasmids can be used for over-expression of genes.

The invention provides a saccharomyces cerevisiae multicopy integrated gene expression cassette, which consists of a weak promoter sequence, a screening gene sequence with a degradation label deg and a terminator; the weak promoter comprises P_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1The screening gene comprises ScTRP1, KlLEU2, KlURA3, ScMET15, SpHIS5, natMX, hpHMX, kanMX, patMX and bleMX.

In one embodiment, the terminator is T_TDH3The nucleotide sequence is SEQ ID NO. 43.

In one embodiment, the weak promoter P_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1The nucleotide is shown in SEQ ID NO. 1-10 respectively; the screening gene is provided with a degradation label which is deg, and the nucleotide sequence is shown in SEQ ID NO. 46; the nucleotide sequences formed by the screened gene and the degradation label are respectively shown in SEQ ID NO. 11-20.

In one embodiment, the weak Promoter is disclosed in the literature Promoter-library-based pathway optimization for efficacy (2S) -naringenin production from p-homologous acid in Saccharomyces cerevisiae (published in 2020).

The invention provides a saccharomyces cerevisiae multicopy integration plasmid, which consists of a gene expression frame and a pre-integration expression frame; the pre-integration expression frame consists of an upstream and downstream homologous arm sequence of a Ty transposon, a terminator sequence and a green fluorescent protein expression frame.

In one embodiment, the Ty transposon sequences comprise Ty1Cons, Ty2Cons, Ty3Cons, Ty4Cons, and Ty5 Cons; the terminator comprises T_RFC5-T_POL30、T_SEC13-T_PNP1、T_MTD1-T_RPF2、T_LEU2-T_NFS1、T_DSF1-T_HXT13、T_TIM21-T_GSC2、T_RRP12-T_TAF3、T_RNA14-T_BUB2、T_ADH1And T_CYC1。

In one embodiment, the saccharomyces cerevisiae multicopy integration plasmid is ligated in the order of upstream homology arm, green fluorescent protein expression cassette, terminator sequence, gene expression cassette, downstream homology arm.

In one embodiment, the upstream and downstream homology arms of Ty1Con1 are shown in SEQ ID No.21 and SEQ ID No.22, respectively; the upstream and downstream homology arms of Ty1Cons2 are shown in SEQ ID NO.23 and SEQ ID NO.24 respectively; the upstream and downstream homology arms of Ty2 are shown in SEQ ID NO.25 and SEQ ID NO.26 respectively; the upstream and downstream homology arms of Ty3 are shown in SEQ ID NO.27 and SEQ ID NO.28, respectively; the upstream and downstream homology arms of Ty4 are shown in SEQ ID NO.29 and SEQ ID NO.30 respectively; the upstream and downstream sequences of all Ty elements have been disclosed in Maury, j; germann, s.m.; baallil Jacobsen, S.A., et al, easy CloneMulti A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae [ J ]. Plous One 2016,11(3), e0150394 (published in 2016)

In one embodiment, the terminator T is_RFC5-T_POL30、T_SEC13-T_PNP1、T_MTD1-T_RPF2、T_LEU2-T_NFS1、T_DSF1-T_HXT13、T_TIM21-T_GSC2、T_RRP12-T_TAF3、T_RNA14-T_BUB2、T_ADH1And T_CYC1The sequences of (A) are respectively shown in SEQ ID NO. 31-40; the terminator sequences are disclosed in the literature, Promoter-library-based optimization for efficacy (2S) -naringenin production from p-homologous acid in Saccharomyces cerevisiae (published in 2020).

In one embodiment, the green fluorescent protein expression cassette comprises a promoter P_GAL7Promoter P_GAL7Upstream of (2) comprises a terminator sequence T_GAL10With a terminator T_GAL10Promoter P of (1)_GAL7Nucleotide sequence ofIs SEQ ID NO.41, and the nucleotide sequence of the green fluorescent protein is SEQ ID NO. 42.

In one embodiment, the selection gene is preferably KlLEU2, KlURA3, SpHIS5, hpmx, natMX.

The invention provides a strain for expressing a target protein, wherein the strain expresses the target protein through a saccharomyces cerevisiae multicopy integration plasmid.

In one embodiment, the protein of interest is inserted upstream of the green fluorescent expression cassette.

The invention provides a strain for producing taxifolin, which expresses relevant genes for producing taxifolin through a saccharomyces cerevisiae multicopy integration plasmid, wherein the genes are expressed through the following three modules:

a first module: overexpression of the relevant genes from tyrosine or phenylalanine to p-coumaric acid synthesis:

from tyrosine to p-coumaric acid genes include ARO4^fbr、ARO7^fbr、FjTAL、EcaroL；

The genes from phenylalanine to p-coumaric acid include ARO4^fbr、ARO7^fbr、FjTAL、EcaroL、SmPAL、SmC4H；

And a second module: overexpresses genes related to p-coumaric acid to naringenin production, including Pc4CL, PhCHS, and MsCHI;

and a third module: naringenin-to-taxifolin synthesis related genes including SmF 3' H, SmCPR and SmF 3H.

In one embodiment, the strain further expresses a malonyl-coa pathway-associated gene ScACC1^S659A ^,S1157A、SeACS2^S641PAnd SpHIS 5.

In one embodiment, C800 is used as the starting strain, which is disclosed in patent document No. CN 111424020A.

The invention provides a method for producing taxifolin, which takes the bacterial strain for producing taxifolin and takes glucose or ethanol as a substrate to synthesize taxifolin from the beginning.

In one embodiment, the strain is culturedTo OD₆₀₀The method comprises the steps of inoculating 20-30 seed liquid into YPD culture medium according to the proportion of 1-5 mL/100mL, culturing at 30-35 ℃ and 200-250 rpm, fermenting for 72 hours, and adding 95% ethanol or glucose at 12h, 24h, 36h and 48h respectively.

In one embodiment, the 95% ethanol is added at a concentration of 0.5% (0.5mL/100mL), the glucose concentration is 500g/L, and the amount added is 0.5% (0.5mL/100 mL).

In one embodiment, the temperature is controlled to 30 ± 0.1 ℃.

In one embodiment, the strain is cultured to obtain the OD₆₀₀The method comprises the steps of inoculating 20-30 seed liquid into YPD culture medium according to the proportion of 1-5 mL/100mL, culturing at 30-35 ℃, 500-600 rpm and the aeration rate of 1-3 vvn, supplementing fed-batch culture medium at the flow rate of 5.0mL/L when the glucose concentration in a reaction system is reduced to 0g/L, controlling the pH to be 5.5 +/-0.1, and fermenting for 72 hours.

The invention provides an application of the saccharomyces cerevisiae multicopy integration gene expression cassette or the saccharomyces cerevisiae multicopy integration plasmid in production of target protein.

The invention provides application of the strain in producing taxifolin.

In one embodiment of the invention, the microbial cell is Saccharomyces cerevisiae strain C800(CEN. PK2-1D, MAT. alpha.; ura 3-52; leu2-3,112; trp 1-289; his 3. delta.1; MAL 2-8)^C(ii) a SUC 2; gal80: KanMX), said strain C800 being disclosed in the patent document with publication number CN 111424020A. .

The invention provides a recombinant bacterium for producing taxifolin and a production method thereof, wherein the method comprises the steps of selecting the multi-copy carrier, integrating a plurality of copies of taxifolin synthetic genes on 3 different Ty sites, and fermenting by using the recombinant bacterium to produce taxifolin.

The invention has the beneficial effects that: the multicopy integrative plasmid toolkit constructed and verified by the invention can realize multicopy, stable and integrative expression of a plurality of exogenous genes on a saccharomyces cerevisiae genome through one-time transformation. And the exogenous gene can be stably and integrally expressed in multiple batches and multiple genes by selecting different screening labels at different Ty sites, so that the high-efficiency expression of the exogenous gene is realized.

Drawings

FIG. 1 is a diagram showing the genotypes of a multicopy plasmid kit (A: 10 combinations of screening gene expression cassettes for multicopy integration; B: 5 pre-integration expression cassettes; C-G: 50 genotypes of a plasmid kit for multicopy integration of Saccharomyces cerevisiae, where the positions and the order of all elements are the positions and the order in an actual plasmid map).

FIG. 2 is a graph showing the distribution of the number of transformants obtained after one transformation (the abscissa is the selection tag and the ordinate is the number of transformants obtained after one transformation.

FIG. 3 shows the fluorescence intensity distribution of multicopy recombinant strains (transformants were obtained by integrating 50 combination expression cassettes of the expression package into the genome of Saccharomyces cerevisiae, 10-30 transformants were randomly selected, the fluorescence intensity distribution was examined, and a box graph was drawn alongside which the fluorescence intensity distribution of each transformant was simultaneously drawn, Panel A: Blank is the fluorescence intensity distribution of strain C800 in YNB medium, and only one copy of EGFP is present on the genome of C887. Panel B-Panel H is the fluorescence intensity distribution of transformants obtained when the selection tags are, in the order of ScTRP1deg, KlURA3deg, ScMET15deg, hphMXdeg, KanMXdeg, patMXdeg, and bleg, when the selection tags are integrated in the order of Ty1Cons1, Ty1Cons2, Ty2Cons, Ty3Cons and Ty4Cons, when the selection tags are integrated in the order of transformants Ty1 Ty 2deg, Ty3Con 3 Ty and Ty4Con, and when the fluorescence intensity distribution of transformants are not obtained when the selection tags are integrated in the order of KlLUE2 deg.C.C.C.S selection tag, when the selection tag is replaced by KlMX 2, and the selection tags are not obtained when the selection tags are replaced by the combination of KlMX 3, and the selection tag is replaced by the selection tag 2.

FIG. 4 is a schematic diagram of the pathway for the synthesis of taxifolin in Saccharomyces cerevisiae (the taxifolin synthesis pathway is divided into three modules; module one: p-coumaric acid synthesis pathway (over-expression of ARO4/ARO7/TAL or ARO4/ARO 7/PAL/C4H); module two: naringenin synthesis pathway (over-expression of 4 CL/CHS/CHI); and module three: taxifolin synthesis pathway (over-expression of F3' H/CPR/F3H)).

FIG. 5 shows the effect of expression of genes at single Ty site and double Ty sites on the integration pathway.

FIG. 6 is a graph showing the effect of expression of a triple Ty site integration pathway gene; in Panel A, the number in parentheses is the fluorescence intensity.

FIG. 7 shows the copy number of the integrated gene at different sites.

FIG. 8 is a graph showing the yield of the de novo synthesized taxin in the 5-L fermenter.

Detailed Description

YNB medium: 0.72g/L yeast nitrogen source basic culture medium and 20g/L glucose.

YPD medium: 10g/L yeast powder, 20g/L peptone and 20g/L glucose.

Leucine, tryptophan, histidine or uracil were added to YNB medium to a final concentration of 50mg/L, or 50mg/L, as required.

Adding various antibiotics into a non-scalding culture medium in a mother liquor form, wherein the concentration of the nourseothricin mother liquor is 100mg/L, and the working concentration is 100 mu g/L; the concentration of the hygromycin mother liquor is 300mg/L, and the working concentration is 300 mug/L; the concentration of the glufosinate-ammonium mother liquor is 800mg/L, and the working concentration is 800 mug/L (if bialaphos is used, the concentration of the mother liquor is 200mg/L, and the working concentration is 200 mug/L); the concentration of the geneticin mother liquor is 200mg/L, and the working concentration is 200 mug/L; the concentration of the mother liquor of bleomycin is 100mg/L, and the working concentration is 100 mug/L. Transformants using the amino acid-deficient selection tag and the glufosinate antibiotic selection tag were plated on YNB plates, and transformants using the selection tags of the other four antibiotics were plated on YPD plates.

20g/L agar powder is added into the solid culture medium.

Feed medium composition: 400g/L glucose, KH₂PO₄ 18g/L，MgSO₄·7H₂O 10.24g/L，K₂SO₄7g/L，Na₂SO₄0.56g/L, 20 mL. L of mother liquor of metal salt^-124 mL. L vitamin mother liquor^-1If necessary, 1g/L of each amino acid was added. Metal salt mother liquor: ZnSO₄·7H₂O 5.75g/L，MnCl₂·4H₂O 0.32g/L，CoCl₂·6H₂O 0.47g/L，NaMoO₄·2H₂O 0.48g/L，CaCl₂·2H₂O 2.9g/L，FeSO₄·7H₂O2.8 g/L, 80mL of 0.5M EDTA (pH 8.0). Vitamin mother liquor: 0.05g/L Biotin (Biotin), 1g/L Calcium pantothenate (Calcium panthenate), 1g/L Nicotinic acid (Nicotinic acid), 25g/L myo-Inositol (myo-Inositol), 1g/L Thiamine hydrochloride (Thiamine HCl), 1g/L Pyridoxal hydrochloride (Pyridoxal HCl), 0.02g/L p-Aminobenzoic acid (p-Aminobenzoic acid)

Saccharomyces cerevisiae CEN. PK2-1D (MAT. alpha.; ura 3-52; leu2-3,112; trp 1-289; his 3. delta.1; MAL2-8^C(ii) a SUC2) for gene expression.

Coli JM109 was used for molecular cloning.

Plasmids pcfB2989, pcfB2988, pcfB2797, pcfB2990, pcfB2796 and pcfB2803(Addgene plasma #63636, #63638, #63639, #63645, #63641, #63646) were given by Irina Borodina & Jeromeo Maury (Maury J, Germann S M, Baallial Jacobsen S A, et al.

Plasmids pMDT-SmPAL and pMDT-SmC4H are Silybum marianum-derived Phenylalanine Ammonia Lyase (PAL) and cinnamate hydroxylase (C4H) obtained by tBLASTN from Silybum marianum transcriptome identification and reverse transcription corresponding to Contig 5930 and Contig265(Lv Y, Gao S, Xu S, et al spatial organization of simple biosyntheses in mile host [ Silybum marianum (L.) Gaertn ] [ J ]. Plant J2017, 92(6), 995) 1004 ], respectively, and the corresponding gene DNA sequences are in Table 1, with the nucleotide sequences being SEQ ID NO.44 and SEQ ID NO.45 in that order.

P-coumaric acid, naringenin, eriodictyol, and taxifolin were purchased from Sigma-Aldrich (st.

Nolsethricin (CAS: 96736-11-7) was purchased from Solobio (N9210).

Hygromycin (Hygromycin B Solution, CAS: 3128-04-9) was purchased from Biotechnology engineering (Shanghai) Inc. (B540725).

Glufosinate Ammonium (Ammonium glufosinate, CAS:77182-82-2) was purchased from Ark Pharm (AGZ 938).

Geneticin (G418 Sulfate, CAS:108321-42-2) was purchased from Biotechnology engineering (Shanghai) Inc. (A100859).

Bleomycin (Bleomycin, CAS:11006-33-0) was purchased from Biotechnology engineering (Shanghai) Inc. (A620212).

The detection method comprises the following steps: taking 100 mu L of fermentation liquor, mixing with 900 mu L of methanol, whirling, shaking, uniformly mixing for 30s, centrifuging at 13500rpm for 5min, and filtering supernatant for later use. Shimadzu high performance liquid detection was used. A chromatographic column: ZORBAX SB-C18(4.6 mm. times.150 mm, Shimadzu). Mobile phase A: 100% acetonitrile, mobile phase B: 100 percent of water, adding 1 per mill (V/V) of trifluoroacetic acid into all mobile phases, filtering, and removing bubbles by ultrasonic waves for later use. Flow rate of mobile phase: 1mL/min, column temperature: 30 ℃, sample introduction: 10 μ L, detection wavelength: 290 nm. Liquid phase procedure: 0-10 min, 10-40% of mobile phase A; 10-15 min, 40-60% of mobile phase A; and (3) distinguishing various substances according to different peak emergence times of different substances by using 60-10% of the mobile phase A for 15-18 min.

Example 1: construction of multicopy plasmid expression kits

In the present invention, Gibson assembly technology (Gibson assembly) is used as an assembly means, and the assembly of all sequences is seamless cloning. Corresponding nucleotide sequences are amplified through PCR, homologous arms of about 20-30bp are contained among the sequences, and all plasmid expression kits can be obtained through Gibbson assembly.

(1) Construction of multiple copy integrated screening Gene expression cassette combinations

Selecting 10 combinations of screening gene expression cassettes (Marker genes, shown as pT 0-pT 9 in Table 2 and FIG. 1) for multi-copy integration, including 10 weak promoter sequences, 10 screening gene sequences with degradation tag deg, and terminator T_TDH3The nucleotide sequence is SEQ ID NO. 43.

10 weak promoters are respectively P_ADE6、P_LEU2、P_URA3、P_PMA1、P_ZWF1、P_ARO7、P_PYC1、P_ADE3、P_YEF3、P_ERG1Corresponding coreThe nucleotide sequence is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10(Gao, S.; Zhou, H.; Zhou, J., et al, Promoter-library-based optimization for efficacy (2S) -naringenin production from p-cortex acid in Saccharomyces cerevisiae [ J].J Agric Food Chem 2020,68(25),6884-6891.)。

10 screening genes with degradation label deg are ScTRP1-deg, KlLEU2-deg, KlURA3-deg, ScMET15-deg, SpHIS5-deg, natMX-deg, hpHphMX-deg, kanMX-deg, patMX-deg and bleMX-deg, and the corresponding nucleotide sequences are SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQ ID No. 20.

(2) Construction of Pre-integration expression cassette

According to the integration site of the saccharomyces cerevisiae, 5 pre-integration expression frames are constructed, including upstream and downstream homologous arm sequences of 5 sets of Ty sequences, 8 bidirectional terminator sequences, 2 unidirectional terminator sequences and a green fluorescent protein expression frame.

The 5 groups of Ty sequences are Ty1Cons1, Ty1Cons2, Ty2, Ty3 and Ty4 in sequence, the upstream/downstream homology arm of Ty1Con1, the upstream/downstream homology arm of Ty1Con2, the upstream/downstream homology arm of Ty2, the upstream/downstream homology arm of Ty3 and the upstream/downstream homology arm of Ty4, and the nucleotide sequences corresponding to the upstream and downstream homology arms of the 5 groups are SEQ ID NO.21/SEQ ID NO.22, SEQ ID NO.23/SEQ ID NO.24, SEQ ID NO.25/SEQ ID NO.26, SEQ ID NO.27/SEQ ID NO.28 and SEQ ID NO.29/SEQ ID NO.30 in sequence.

8 bidirectional terminators are T in sequence_RFC5-T_POL30、T_SEC13-T_PNP1、T_MTD1-T_RPF2、T_LEU2-T_NFS1、T_DSF1-T_HXT13、T_TIM21-T_GSC2、T_RRP12-T_TAF3、T_RNA14-T_BUB22 unidirectional terminators are in turn T_ADH1And T_CYC1The nucleotide sequence corresponding to the bidirectional terminator is SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33 and SEQ ID NO.32 in sequenceThe sequence corresponding to the unidirectional terminator is SEQ ID NO.39 and SEQ ID NO.40 in sequence.

The expression frame of the green fluorescent protein comprises a promoter P_GAL7Promoter P_GAL7Upstream of (2) comprises a terminator sequence T_GAL10The green fluorescent protein is EGFP with terminator T_GAL10Promoter P of (1)_GAL7The nucleotide sequence of the EGFP gene is SEQ ID NO.41, and the nucleotide sequence of the green fluorescent protein is SEQ ID NO. 42. A terminator sequence T is also used in the present invention_TDH3The nucleotide sequence is SEQ ID NO. 43. The invention uses two silybum marianum-derived genes SmPAL and SmC4H, and the nucleotide sequences are SEQ ID NO.44 and SEQ ID NO.45 in sequence.

Construction of 50 plasmid kits for multiple copy integration of s.cerevisiae: the 10 screening gene expression cassettes (Marker genes) are sequentially and respectively inserted into the middle of two double terminators in 5 pre-integration expression cassettes (Gibson assembly technology is used, no redundant base insertion exists, and both insertion are traceless insertion), and then a group of saccharomyces cerevisiae multi-copy integration kit containing 50 plasmids is obtained. Wherein the Ty1Cons1 pre-integrated expression cassette is combined with 10 screening genes to obtain 10 plasmids of pcT111, pcT112, pcT113, pcT114, pcT115, pcT116, pcT117, pcT118, pcT119 and pcT110 in total in sequence. Wherein the Ty1Cons2 pre-integration expression frame is combined with 10 screening genes, and a total of 10 plasmids of pcT121, pcT122, pcT123, pcT124, pcT125, pcT126, pcT127, pcT128, pcT129 and pcT120 are obtained in sequence. The Ty2 pre-integrated expression frame is combined with 10 screening genes, and pcT21, pcT22, pcT23, pcT24, pcT25, pcT26, pcT27, pcT28, pcT29 and pcT20 total 10 plasmids are obtained in sequence. The Ty3 pre-integrated expression frame is combined with 10 screening genes, and pcT31, pcT32, pcT33, pcT34, pcT35, pcT36, pcT37, pcT38, pcT39 and pcT30 total 10 plasmids are obtained in sequence. Wherein the Ty4 pre-integrated expression frame is combined with 10 screening genes to obtain 10 plasmids of pcT41, pcT42, pcT43, pcT44, pcT45, pcT46, pcT47, pcT48, pcT49 and pcT40 in total.

The actual positions of all the genes, promoters, terminators, etc. in the 10 combinations of the screening gene expression cassettes for multicopy integration, 5 pre-integration expression cassettes, and 50 plasmid kits for multicopy integration of s.cerevisiae are shown in FIG. 1.

Gene ScACC1^S659A,S1157A、SeACS^S641P、SmF3′H^D284N、SmCPR^I453V、ARO4^fbr、ARO7^fbrFjTAL, EcaroL and SmF3H, and the sequentially corresponding nucleotide sequences are SEQ ID NO. 47-SEQ ID NO. 55. Other promoters P mentioned_GAL1And P_GAL10The corresponding nucleotide sequences are SEQ ID NO.56 and SEQ ID NO.57 in sequence.

All nucleotide sequences of the present invention are shown in Table 1. The genotypes of the plasmids and strains are shown in table 2. All key primer sequences are shown in table 3.

Table 1 all nucleotide sequences

TABLE 2 genotypes of plasmids and strains

TABLE 3 Key primer sequences

Example 2: validation of integration Capacity of multicopy integrating plasmid tool

Plasmids pcT110 to pcT119 were amplified respectively using primers Ty11-inte-up/down, plasmids pcT120 to pcT129 were amplified respectively using primers Ty12-inte-up/down, plasmids pcT20 to pcT29 were amplified respectively using primers Ty2-inte-up/down, plasmids pcT30 to pcT39 were amplified respectively using primers Ty3-inte-up/down, plasmids pcT40 to pcT49 were amplified respectively using primers Ty4-inte-up/down, and integrated expression frame portions of the vectors were obtained respectively. PCR products are recovered and purified, and then integrated into a Saccharomyces cerevisiae strain by a Saccharomyces cerevisiae efficient transformation method (see Gietz, R.D.; Schiestl, R.H., High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method [ J ]. Nat Protoc 2007,2(1), 31-4.).

When the screening tags are TRP1, LEU2, URA3, HIS5, natMX, hphMX, patMX and bleMX, the strain is transformed into the strain C800. When the screening tag was MET15, strain C815 was selected.

When the selection tag was KanMX, strain C850 was selected. The transformed cells were plated on corresponding screening plates (when the screening gene was TRP1, they were plated on YNB-containing TRP)^-Plate coated with screening gene LEU2 containing YNB-LEU^-Plate coated with YNB-URA when the selection gene is URA3^-Plate, screening gene is applied to YNB-HIS containing HIS5^-Plate coated with YNB-MET when the screening gene is MET15^-Plates were plated on YPD plates containing nourseothricin when the selection gene was natMX, and plated on hphMXYPD plates containing hygromycin, YPD plates containing geneticin coated with the selection gene KanMX, YPD plates containing glufosinate-ammonium coated with the selection gene patMX, and YPD plates containing bleomycin coated with the selection gene bleMX) were cultured at 30 ℃ for 3-5 days until single colonies were obtained. And counting the obtained single colonies, inoculating the single colonies for culture, and detecting the fluorescence intensity. The integration efficiency of each combination in the kit was determined from the number of transformants obtained and the distribution of fluorescence intensity of the transformants. The number distribution of transformants obtained for each combination after one transformation is shown in FIG. 2. The fluorescence intensity distribution range of the multicopy recombinant strains obtained for each combination is shown in FIG. 3.

As can be seen from FIG. 2, about 100-500 transformants can be obtained after a large part of plasmids in the Ty expression package are transformed once, but pcT122, pcT22, pcT32 and pcT 42; pcT25, pcT35, pcT45 and pcT116, pcT26, pcT46 did not obtain transformants after multiple transformations, whereas pcT114, pcT124, pcT24, pcT34 and pcT44 using ScMET15deg as a selection tag obtained more than 1 ten thousand transformants, however, the transformants had almost no fluorescence (fig. 3), and it was found that ScMET15deg was not suitable for high copy integration here.

As can be seen from FIG. 3, the fluorescence intensity values of C887, a blank strain and a strain having only one copy of EGFP, were about 8000 and 25000 (FIG. 3A). When ScMET15deg, KanMXdeg, patMXdeg and BleMXdeg were used as selection tags, the obtained transformants had a weak fluorescence intensity, and most of them had only one copy (FIG. 3D, FIG. 3F, FIG. 3G and FIG. 3H), and when ScTRR1deg was used as selection tags, most of the transformants had a weak fluorescence intensity (FIG. 3B). While the transformants with high probability of obtaining strong fluorescence signals by using KlURA3deg, KlLEU2deg, SpHIS5deg, natMXdeg and hphMXdeg (FIG. 3C, FIG. 3E and FIG. 3I) have the average fluorescence intensity of 7.9-16.5 times that of the control group, and the strongest one reaches 48.2 times that of the control group. These multicopy integrative plasmids can thus be used to increase the copy number in s.cerevisiae.

Example 3: use of multicopy integrative plasmid kit

The process of synthesis of taxifolin involves the introduction and overexpression of multiple genes and is therefore used to validate the performance of a multicopy integrative plasmid kit. The synthesis route of taxifolin is shown in fig. 4, wherein the module I is glucose to p-coumaric acid, the module II is p-coumaric acid to naringenin, and the module III is naringenin to taxifolin. The multicopy integrative plasmid kit with significantly increased copy number in example 2 was picked for use in the production of taxifolin or related intermediates.

1. Construction of single Ty site integrated recombinant bacterium

Primers Ty4-inte-up/Ty4-inte-down are used for amplifying pcfB4-47LL and pcfB4-P05m4 respectively, PCR products are efficiently transformed by saccharomyces cerevisiae and integrated to Ty4 sites of the saccharomyces cerevisiae strain C800 respectively, and strains C8011 and C805 are obtained respectively. Strain C8011 integrates the genes related to the synthesis of p-coumaric acid in module I, and can synthesize p-coumaric acid from head, and strain C805 integrates the genes related to the synthesis of naringenin to taxifolin in module III, and can synthesize taxifolin from naringenin.

2. Construction of double Ty site integrated recombinant bacteria

And (3) using a primer Ty3-inte-up/Ty3-inte-down amplification plasmid pcfB3-P03, purifying and refining the obtained PCR product, and efficiently converting and integrating the purified PCR product into a Ty3 site of a strain C8011 genome to obtain a strain C824. And (3) using a primer Ty4-inte-up/Ty4-inte-down amplification plasmid pcfB4-P05m4, purifying and refining the obtained PCR product, and efficiently converting and integrating the purified PCR product into a Ty4 site of a genome of a strain C803 through saccharomyces cerevisiae to obtain a strain C857. Strain C824 integrates related genes from glucose to naringenin synthesis in module I and module II, and can synthesize naringenin from head, and strain C857 integrates related genes from p-coumaric acid to taxifolin synthesis in module II and module III, and can synthesize taxifolin from p-coumaric acid.

pT820 was amplified using primers Pf-gal80/Pf-gal80D, and after recovery of PCR products, the PCR products were transformed into strain C803 and strain C857, respectively, by the Saccharomyces cerevisiae high efficiency transformation method, to obtain strain C823 and strain C877, respectively. Strain C823 and strain C877 enhanced the malonyl-CoA pathway on the basis of the starting strains C803 and C857, respectively.

3. Construction of triple Ty site integration recombinant bacteria

Integrating a module-related gene on the basis of the strain C877, and respectively obtaining a strain C900 and a strain C901 according to the expression formula by taking tyrosine or phenylalanine as a precursor: primers Ty2-inte-up/Ty2-inte-down are used for amplifying pcT21-LL and pcT21-LHL, PCR products are recovered and then are transformed to Ty2 sites in a strain C877 through a saccharomyces cerevisiae high-efficiency transformation method, and strains C900 and C901 are obtained respectively. Both the strain C900 and the strain C901 can synthesize taxifolin from the head, wherein the strain C900 takes tyrosine as a precursor and obtains p-coumaric acid through TAL approach to synthesize taxifolin, and the strain C901 takes phenylalanine as a precursor and obtains the p-coumaric acid through PAL/C4H/CPR approach to obtain the taxifolin.

4. RNA expression level of taxifolin synthesis related gene in recombinant strain (detection of integrated copy number of different Ty sites and verification of stability direction)

(1) Copy number detection

Strain culture: single colonies of the strain C900 and the strain C901 which are constructed in the above way are selected and inoculated in a 250mL shake flask containing 5mL YNB medium, and cultured for 24 hours at 30 ℃ and 220rpm, so as to obtain a seed medium. Seed cultures were adjusted to starting OD₆₀₀0.1 to a 250mL shake flask in 50mL YNB liquid medium. Incubated at 30 ℃ and 220rpm for 6-8 hours to early log phase. The fermentation broth was centrifuged at 13500rpm for 3 min. Removing supernatant and collecting yeast cells.

RT-PCR procedure: yeast total RNA was extracted using the RNAprep pure Plant Kit (TIANGEN, Beijing, China)). Using the total RNA as a template, the DNA was removed using a PrimeScript RT kit, and then reverse transcription was performed to obtain cDNA. RT-PCR was performed using SYBR Premix Ex Taq (Tli RNAseH Plus) kit and RT-PCR was performed using cDNA as a template. The internal reference gene ACT1 for homogenization was subjected to RT-PCR using the primer pair ACT1-F/ACT 1-R. RT-PCR was performed on a LightCycler 480II instrument. All results were normalized according to the reference gene ACT 1. The expression intensity of the gene was determined according to 2^-ΔΔCtMethod (Livak K J, Schmitgen T D. analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]Methods 2001,25(4), 402-8). The integrated copy number of the genes of the modules I, II and III is determined by using primers qEGFP-F/R, qCHS-F/R and qF3H-F/R respectivelyAmount of the compound (A).

According to RT-PCR verification, the module one, the module two and the module three genes in the strain C900 are respectively integrated at the Ty2 site, the Ty3 site and the Ty4 site, and the copy numbers are 4, 5 and 7 in sequence, while the copy numbers of the strain C901 at the Ty2 site, the Ty3 site and the Ty4 site are 8, 5 and 7 in sequence (FIG. 7).

(2) Stability verification

Inoculating strains C900 and C901 into 250mL shake flasks containing 20mL of liquid YPD medium, culturing at 30 ℃ and 220rpm, inoculating 1% (mL/100mL) of the strains into 250mL shake flasks containing fresh 20mL of liquid YPD medium every 12h of culture, carrying out passage for 60 passages, respectively inoculating the two strains obtained after passage and the corresponding starting strains into 250mL shake flasks containing 20mL of liquid YPD medium after 30 days of total passage, respectively, culturing at 30 ℃ and 220rpm for 14-16h, inoculating the strains into 250mL shake flasks containing 20mL of YPD medium according to an inoculation ratio of 1mL/100mL, culturing at 30 ℃ and 220rpm, fermenting for 72h, and respectively adding 0.5% (0.5mL/100mL) of 95% ethanol at 12h, 24h, 36h and 48h, and measuring the yield of the taxifolin after the completion. The results showed that the yields of taxifolin of the starting strains C900 and C901 were 50.93mg/L and 33.83mg/L, respectively, and the yields of taxifolin of the strains after passage of strains C900 and C901 were 52.34mg/L and 33.48mg/L, respectively, and it was apparent that the ability of the strains after passage to produce taxifolin did not significantly change, thereby demonstrating that the stability of the three-site integration strain based on the Ty transposon is good.

5. Recombinant bacterium fermentation production of taxifolin

(1) The strain C8011, the strain C803, the strain C805, the strain C824, the strain C857 and the strain C877 which are obtained by construction are utilized: respectively selecting single colony, inoculating into 250mL shake flask of 20mL YPD medium, controlling shaking table at 30 deg.C and 220rpm, culturing for 14-16h to strain OD₆₀₀Seed solutions were obtained around 25, when the strain was in mid-log phase. The mixture was inoculated into a 250mL shake flask containing 20mL YPD medium at an inoculation ratio of 1mL/100mL, cultured at 30 ℃ and 220rpm, and 0.5% (0.5mL/100mL) of 500g/L glucose solution or 0.5% (0.5mL/100mL) of 95% ethanol was added at 12h, 24h, 36h and 48h, respectively, as required. Sampling after fermentation, detecting the end product andcontent of intermediate product.

Strain C8011 has integrated the genes involved in module one, and it was possible to synthesize 544.01mg/L of p-coumaric acid starting from glucose by fermentation in a 250mL shake flask for 72h (FIG. 5A). Strain C803 integrated the gene related to module two, and fermented in a 250mL shake flask for 72h could synthesize 623.60mg/L naringenin from 1000mg/L p-coumaric acid (FIG. 5B). Strain C805, which incorporates the genes related to Module three, was fermented in 250mL for 72h to synthesize 603.90mg/L of taxifolin from 1000mg/L of naringenin, while 392.30mg/L of eriodictyol was accumulated, and the remainder was 83.80mg/L of naringenin (FIG. 5A). Constitutive strains with stable gene phenotypes can be obtained based on single-site integration of the Ty transposon.

The strain C824 is a strain C8011 integrated with the related gene of module II, and can synthesize naringenin from glucose as a substrate, and synthesize 264.05mg/L naringenin by 72h fermentation in a 250mL shake flask (FIG. 5A). Strain C857 is a strain C803 integrated with related genes of module two, can synthesize taxifolin from p-coumaric acid as a substrate, can synthesize 346.43mg/L taxifolin from 1000mg/L p-coumaric acid after 72h of fermentation in a 250mL shake flask, meanwhile, the accumulation amount of intermediate products of naringenin and eriodictyol is 66.32mg/L and 142.66mg/L, and the rest of substrate p-coumaric acid is 76.52mg/L (FIG. 5C). The double-site integration based on the Ty transposon can still obtain a constitutive strain with good stability.

(2) In order to verify the relationship between the synthesis capacity of the taxifolin and the copy number of a module gene, 10 strains with different fluorescence intensities are selected from a C900 and C901 transformant library, and fermentation verification is carried out by using a 48-depth orifice plate, and the result proves that the higher the fluorescence intensity is, the higher the accumulation amount of the taxifolin and other flavone related compounds is (FIG. 6A, the number in the horizontal coordinate bracket is the fluorescence intensity). Wherein the strain C901 using phenylalanine as a precursor obtains the highest accumulated amount of taxifolin of 20.66mg/L, and the accumulated amounts of coumaric acid, naringenin and eriodictyol are respectively 56.67, 31.24 and 19.01 mg/L. The highest accumulation of taxifolin obtained by the strain C900 taking tyrosine as a precursor is 19.77mg/L, and the accumulation of p-coumaric acid, naringenin and eriodictyol are 31.42, 23.67 and 15.56mg/L respectively.

Selecting single colonies of C900 and C901, inoculating into 250mL shake flask of 20mL YPD medium, controlling shaking table at 30 deg.C and 220rpm, culturing for 14-16h to strain OD₆₀₀Seed solutions were obtained around 25, when the strain was in mid-log phase. The seed solution was inoculated into a 250mL shake flask containing 20mL YPD medium at an inoculation ratio of 1mL/100mL, cultured at 30 ℃ and 220rpm, fermented for 72 hours, and 0.5% (0.5mL/100mL) of 95% ethanol was added at 12h, 24h, 36h, and 48h, respectively. The transformants with the highest accumulation of taxifolin obtained were named C900 and C901. The optimal fermentation carbon source was then checked at the shake flask level by rescreening and adding 500g/L glucose solution at 0.5% (0.5mL/100mL) or 95% ethanol at 0.5% (0.5mL/100mL) at 12h, 24h, 36h and 48h, respectively. At 72h, the amounts of p-coumaric acid, naringenin, eriodictyol and taxifolin accumulated by fermenting strain C900 with glucose/ethanol as carbon source were 11.88/8.78, 13.35/10.85, 22.72/13.40 and 50.93/33.83mg/L, respectively; the amounts of p-coumaric acid, naringenin, eriodictyol and taxifolin accumulated by fermentation of strain C901 using glucose/ethanol as a carbon source were 15.97/13.27, 24.40/20.72, 45.43/24.56 and 70.54/49.47mg/L, respectively (FIG. 6B).

(3) Strain C901 was fermented in a 5L fermentor: the seed medium was transferred at 1% to a 5L fermenter containing 2.5L YPD medium as above. The temperature was controlled at 30. + -. 0.1 ℃, the speed 600rpm, and the aeration rate 3 vvn. Starting a feed pump when the glucose concentration is reduced to 0g/L, setting the speed of feeding the culture medium to be 5.0mL/L, controlling the pH to be 5.5 +/-0.1 at the same time, and measuring the concentration of various substances in the fermentation liquid every 12h during the fermentation process till the end of 72h of fermentation. As a result, as shown in FIG. 8, 135.83mg/L of taxifolin could be synthesized de novo within 72 hours while p-coumaric acid, naringenin and eriodictyol were accumulated in amounts of 40.37, 10.32 and 41.10 mg/L.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> a set of plasmid kits for saccharomyces cerevisiae multicopy integration

<130> BAA210539A

<160> 57

<170> PatentIn version 3.3

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tacagggcgt atatatacat aacattttaa gataagcaag tgaatgttga ttcccgtttc 180

ttagtcaaca cttctttcta ttttacccgg tcgttaccct attaaaaaaa caacttacaa 240

tcattgttcg ccccttccat acttactgcc actcgcaaaa gggcccaacc agggcaatta 300

cgtatcaaaa aatcatgaca ggctgggtaa taaatattcg tgaagaaaga agaaattaaa 360

aaaagaaacg aagaagcaaa aaaaagaaaa gactccgttt aatcactttc aaccgcggtt 420

tatccggccc cacccatgca taaccctaaa ttattagatc acttagcacg tgaaaaagaa 480

acgtttttaa tgtttttttt ttttttttct ttttcttttt ttgcgttggt gaaaattttt 540

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aaaccttttg catcaaaatt ttgtagaata tctctttttc ttacgctctc tttctttcct 660

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gttcccaaca ggaagaggac aatagacccc gtgatagcta ggaagataag ttcactagtc 180

aaagcctaca agaacagttc aggaacaccc aagtacctag taggagtttt taggaaccag 240

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gtatttccca aggattgtaa tatcctcctt tccgccgcaa gtcaaaagcc ccactcattc 420

atacccctct tcgacagtga ggccggagga acaggagagc ttctagactg gaacagtata 480

tcggattggg taggacgaca agagagtccc gagagtcttc atttcatgct agctggagga 540

ctaacacccg agaacgtcgg agatgcccta cgactaaatg gagtaatagg agtagacgtt 600

agtggagggg tagagacaaa tggagtgaag gacagtaata aaatagctaa tttcgtaaaa 660

aatgcaaaga aggcttgtaa aaattggttc tcttctttgt ctcatttcgt tattcatttg 720

taa 723

<210> 11

<211> 723

<212> DNA

<213> Artificial sequence

<400> 11