CN108410870B - Kluyveromyces marxianus promoter, secretion signal peptide, preparation and application thereof - Google Patents

Kluyveromyces marxianus promoter, secretion signal peptide, preparation and application thereof Download PDF

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CN108410870B
CN108410870B CN201810153537.XA CN201810153537A CN108410870B CN 108410870 B CN108410870 B CN 108410870B CN 201810153537 A CN201810153537 A CN 201810153537A CN 108410870 B CN108410870 B CN 108410870B
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吕红
余垚
周峻岗
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Abstract

The invention discloses an optimized Kluyveromyces marxianus promoter, which at least comprises the following components: a) a nucleotide sequence shown as SEQ ID No. 1; or b) a nucleotide sequence shown as SEQ ID No. 2; or c) the nucleotide sequence shown as SEQ ID No. 3. Also discloses an optimized Kluyveromyces marxianus secretion signal peptide at least comprising the nucleotide sequence shown as SEQ ID No.4 and an optimized composition at least comprising the nucleotide sequence shown as SEQ ID No. 6. The invention also discloses a recombinant expression vector containing the Kluyveromyces marxianus promoter and/or secretion signal peptide, a genetic engineering bacterium, a preparation method and an application thereof. The technical scheme can obviously improve the capability of Kluyveromyces marxianus for efficiently recombining and expressing the foreign protein, and lays a foundation for promoting the application of a Kluyveromyces marxianus expression system in the field of industrial protein manufacture.

Description

Kluyveromyces marxianus promoter, secretion signal peptide, preparation and application thereof
Technical Field
The invention relates to the technical field of bioengineering, in particular to an optimized Kluyveromyces marxianus promoter, a secretion signal peptide and a composition thereof, a recombinant expression vector containing the Kluyveromyces marxianus promoter and/or the secretion signal peptide, a genetic engineering bacterium, a preparation method and an application thereof.
Background
Kluyveromyces marxianus (hereinafter referred to as KM) is a yeast of the same genus ascomycete, belonging to the genus Kluyveromyces of the family Saccharomycetaceae. KM is commonly found in yogurt, malted milk, cheese, milk, and sugarcane leaves. KM is a commonly recognized food grade yeast. It has passed the GRAS certification of FDA, QPS certification of European Union, and is approved as a new food material by the Ministry of health in China. Because of its high safety, KM has unique advantages in the fields of producing edible, feeding and medicinal proteins. However, the development of KM expression systems is limited due to the lack of efficient expression elements and matched expression vectors in KM.
Disclosure of Invention
In view of the above defects in the prior art, the present invention provides an optimized kluyveromyces marxianus promoter, a secretion signal peptide and a combination thereof, a recombinant expression vector containing the kluyveromyces marxianus promoter and/or the secretion signal peptide, a genetically engineered bacterium, a preparation method and an application thereof. The specific technical scheme is as follows:
the invention provides an optimized Kluyveromyces marxianus promoter in a first aspect, and the nucleotide sequence of the optimized Kluyveromyces marxianus promoter at least comprises:
a) a nucleotide sequence shown as SEQ ID No. 1; or
b) A nucleotide sequence shown as SEQ ID No. 2; or
c) The nucleotide sequence shown as SEQ ID No. 3.
In a second aspect, the invention provides an optimized Kluyveromyces marxianus secretion signal peptide, the nucleotide sequence of which at least comprises the nucleotide sequence shown as SEQ ID No. 4; the amino acid sequence of the polypeptide at least comprises the amino acid sequence shown as SEQ ID No. 5.
In a third aspect, the invention provides an optimized kluyveromyces marxianus promoter and secretion signal peptide assembly, and the nucleotide sequence of the assembly at least comprises the nucleotide sequence shown as SEQ ID No. 6.
In a fourth aspect, the present invention provides a recombinant expression vector comprising the kluyveromyces marxianus promoter provided in the first aspect of the present invention and/or the kluyveromyces marxianus secretion signal peptide provided in the second aspect of the present invention.
Preferably, the recombinant expression vector further comprises an autonomous replication sequence of kluyveromyces marxianus, an inulinase terminator gene sequence of kluyveromyces marxianus and an auxotroph selection marker gene sequence.
More preferably, the auxotrophic marker gene sequence is selected from one or more of URA3 gene, HIS3 gene and ADE2 gene.
Most preferably, the auxotrophic selection marker gene sequence is the URA3 gene.
In a preferred embodiment, the nucleotide sequence of the recombinant expression vector comprises at least:
a) a nucleotide sequence shown as SEQ ID No. 22; or
b) And a nucleotide sequence shown as SEQ ID No. 23.
In a fifth aspect, the present invention provides a genetically engineered bacterium comprising the kluyveromyces marxianus promoter provided in the first aspect and/or the kluyveromyces marxianus secretion signal peptide provided in the second aspect.
The invention also provides a genetically engineered bacterium, which comprises the recombinant expression vector provided by the invention in the fourth aspect.
Preferably, the host bacterium of the genetically engineered bacterium is a yeast.
More preferably, the yeast includes at least Kluyveromyces marxianus, Kluyveromyces lactis, or Saccharomyces cerevisiae.
In a sixth aspect, the present invention provides an application of the kluyveromyces marxianus promoter, which is used for improving the expression of a foreign protein.
Preferably, the foreign protein is feruloyl esterase.
In a seventh aspect, the present invention provides the use of the kluyveromyces marxianus secretion signal peptide for increasing the efficiency of recombinant protein secretion from intracellular secretion to extracellular secretion.
In an eighth aspect, the present invention provides the use of the kluyveromyces marxianus promoter and secretion signal peptide assembly for increasing the expression of foreign proteins and increasing the efficiency of recombinant proteins from intracellular secretion to extracellular secretion.
The present invention provides, in a ninth aspect, a method for producing the above-described kluyveromyces marxianus promoter, wherein the kluyveromyces marxianus promoter is constructed by performing nucleotide mutation or deletion on the basis of a kluyveromyces marxianus inulase promoter, and the method at least comprises:
a) mutating-351 thymine deoxynucleotide in the nucleotide sequence of the Kluyveromyces marxianus inulinase promoter into adenine deoxynucleotide; or
b) Removing 106 nucleotide bases between-8 bp and-115 bp of an inulinase promoter of Kluyveromyces marxianus; or
c) Comprising the combined mutation of step a) and step b).
In a preferred embodiment, the preparation method specifically comprises the following steps:
step 1, taking the genome of Kluyveromyces marxianus FIM-1 as a template, and carrying out PCR amplification on an inulase gene by using a primer INU-F1 with a nucleotide sequence shown as SEQ ID No.7 and a primer INU-R1 with a nucleotide sequence shown as SEQ ID No. 8; adding Taq enzyme into a reaction system, and adding A into a PCR amplification product to obtain a target fragment; recovering a target fragment, and connecting the target fragment with a pMD18-T vector to obtain a plasmid pMD18-T-INU containing a Kluyveromyces marxianus inulase promoter;
step 2, taking the plasmid pMD18-T-INU as a template, removing the sequence of 79-1668bp of the inulase gene by using a primer INU delta-F1 with the nucleotide sequence shown as SEQ ID No.9 and a primer INU delta-R1 with the nucleotide sequence shown as SEQ ID No.10, and simultaneously removing the sequence of 78bp of the inulase gene and a stop codonIntroducing multiple cloning sites to obtain plasmid pMD18-T-PINU-SP-MCS-TINU；
Step 3, using plasmid pMD18-T-PINU-SP-MCS-TINUAs a template, P was amplified using primer INU-F2 whose nucleotide sequence is shown in SEQ ID No.11 and primer INU-R2 whose nucleotide sequence is shown in SEQ ID No.12INU-SP-MCS-TINU fragment;
step 4, taking the pUKD vector as a template, and removing an EcoRI site between pKS and a KD sequence by using a primer pUKD-F1 with a nucleotide sequence shown as SEQ ID No.14 and a primer pUKD-R1 with a nucleotide sequence shown as SEQ ID No.15 to obtain a plasmid pUKD delta E; carrying out enzyme digestion on the plasmid PUKD delta E by using EcoRI, and recovering an enzyme digestion product;
step 5, P in step 3INU-SP-MCS-TINUConnecting the fragment with the enzyme digestion product in the step 4 to obtain a plasmid pUKDN 132;
step 6, using the plasmid pUKDN132 as a template, and mutating thymine deoxynucleotide at 351 bit upstream of the initiation codon of the inulase gene into adenine deoxynucleotide by using a primer T (-351) A-F with a nucleotide sequence shown as SEQ ID No.16 and a primer T (-351) A-R with a nucleotide sequence shown as SEQ ID No.17 to obtain a plasmid pUKDN132-T (-351) A containing the nucleotide sequence shown as SEQ ID No. 1; or
Step 7, taking the plasmid pUKDN132 as a template, and removing a sequence from 8bp upstream to 115bp upstream of an initiation codon of the inulase gene by using a primer UTR delta A-F with a nucleotide sequence shown as SEQ ID No.18 and a primer UTR delta A-R with a nucleotide sequence shown as SEQ ID No.19 to obtain a plasmid pUKDN132-UTR delta A containing the nucleotide sequence shown as SEQ ID No. 2; or
And step 8, taking the plasmid pUKDN132-T (-351) A obtained in the step 6 as a template, and removing a sequence from 8bp upstream to 115bp upstream of the initiation codon of the inulase gene by using a primer UTR delta A-F with a nucleotide sequence shown as SEQ ID No.18 and a primer UTR delta A-R with a nucleotide sequence shown as SEQ ID No.19 to obtain a plasmid pUKDN132-T (-351) A + UTR delta A, wherein the plasmid pUKDN132-T (-351) A contains the nucleotide sequence shown as SEQ ID No. 3.
Preferably, in the step 1, the kluyveromyces marxianus FIM-1 is preserved in the common microorganism center of the china committee for culture collection and management of microorganisms, with the preservation number of CGMCC No. 10621.
Preferably, in step 2 above, the multiple cloning site includes at least SmaI, SpeI and NotI sites.
In a tenth aspect, the present invention provides a method for producing the kluyveromyces marxianus secretion signal peptide, wherein the method at least comprises mutating cytosine deoxynucleotide at the 29 th position of inulase gene to thymine deoxynucleotide.
In a preferred embodiment, the preparation method specifically comprises the following steps: the plasmid pUKDN132 is used as a template, a primer P10L-F with a nucleotide sequence shown as SEQ ID No.20 and a primer P10L-R with a nucleotide sequence shown as SEQ ID No.21 are used for mutating cytosine deoxynucleotide at the 29 th site of the inulase gene into thymine deoxynucleotide, so that proline at the 10 th site of the inulase signal peptide is changed into leucine.
Preferably, plasmid pUKDN132 is prepared according to steps 1-5 provided in the ninth aspect of the invention.
In an eleventh aspect, the present invention provides a method for preparing the kluyveromyces marxianus promoter/secretion signal peptide assembly, which comprises the following steps: the plasmid pUKDN132-T (-351) A is used as a template, a primer P10L-F with a nucleotide sequence shown as SEQ ID No.20 and a primer P10L-R with a nucleotide sequence shown as SEQ ID No.21 are used for mutating cytosine deoxynucleotide at the 29 th site of the inulase gene into thymine deoxynucleotide, so that proline at the 10 th site of an inulase signal peptide is changed into leucine.
Preferably, plasmid pUKDN132-T (-351) A is prepared according to steps 1-6 of the present invention as provided in the ninth aspect.
In a twelfth aspect, the present invention provides a method for preparing the above recombinant expression vector, comprising the steps of:
step 1, taking the genome of Kluyveromyces marxianus FIM-1 as a template, and carrying out PCR amplification on an inulase gene by using a primer INU-F1 with a nucleotide sequence shown as SEQ ID No.7 and a primer INU-R1 with a nucleotide sequence shown as SEQ ID No. 8; adding Taq enzyme into a reaction system, and adding A into a PCR amplification product to obtain a target fragment; recovering a target fragment, and connecting the target fragment with a pMD18-T vector to obtain a plasmid pMD18-T-INU containing a Kluyveromyces marxianus inulase promoter;
step 2, taking the plasmid pMD18-T-INU as a template, removing the sequence of 79-1668bp of the inulase gene by using a primer INU delta-F1 with the nucleotide sequence shown as SEQ ID No.9 and a primer INU delta-R1 with the nucleotide sequence shown as SEQ ID No.10, and introducing a multiple cloning site between 78bp of the inulase gene and a stop codon to obtain the plasmid pMD18-T-PINU-SP-MCS-TINU；
Step 3, using plasmid pMD18-T-PINU-SP-MCS-TINUAs a template, P was amplified using primer INU-F2 whose nucleotide sequence is shown in SEQ ID No.11 and primer INU-R2 whose nucleotide sequence is shown in SEQ ID No.12INU-SP-MCS-TINU fragment;
step 4, taking the pUKD vector as a template, and removing an EcoRI site between pKS and a KD sequence by using a primer pUKD-F1 with a nucleotide sequence shown as SEQ ID No.14 and a primer pUKD-R1 with a nucleotide sequence shown as SEQ ID No.15 to obtain a plasmid pUKD delta E; carrying out enzyme digestion on the plasmid PUKD delta E by using EcoRI, and recovering an enzyme digestion product;
step 5, P in step 3INU-SP-MCS-TINUConnecting the fragment with the enzyme digestion product in the step 4 to obtain a plasmid pUKDN 132;
step 6, using the plasmid pUKDN132 as a template, and mutating thymine deoxynucleotide at 351 bit upstream of the initiation codon of the inulase gene into adenine deoxynucleotide by using a primer T (-351) A-F with a nucleotide sequence shown as SEQ ID No.16 and a primer T (-351) A-R with a nucleotide sequence shown as SEQ ID No.17 to obtain a plasmid pUKDN132-T (-351) A containing the nucleotide sequence shown as SEQ ID No. 1;
step 7, taking the plasmid pUKDN132-T (-351) A obtained in the step 6 as a template, and removing a sequence from 8bp upstream to 115bp upstream of the initiation codon of the inulase gene by using a primer UTR delta A-F with a nucleotide sequence shown as SEQ ID No.18 and a primer UTR delta A-R with a nucleotide sequence shown as SEQ ID No.19 to obtain a plasmid pUKDN132-T (-351) A + UTR delta A with a nucleotide sequence shown as SEQ ID No. 22; or
And step 8, using the plasmid pUKDN132-T (-351) A obtained in the step 6 as a template, and mutating cytosine deoxynucleotide at the 29 th site of the inulase gene into thymine deoxynucleotide by using a primer P10L-F with a nucleotide sequence shown as SEQ ID No.20 and a primer P10L-R with a nucleotide sequence shown as SEQ ID No.21 to obtain the plasmid pUKDN132-T (-351) A + P10L with a nucleotide sequence shown as SEQ ID No. 23.
The technical scheme provided by the invention can obviously improve the capability of Kluyveromyces marxianus for efficiently recombining and expressing the foreign protein, and lays a foundation for promoting the application of a Kluyveromyces marxianus expression system in the field of industrial protein manufacture.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space. It is therefore contemplated to cover by the present invention, equivalents and modifications that fall within the scope of the invention, and that fall within the scope of the invention.
The present invention will be further described with reference to the accompanying drawings to fully illustrate the objects, technical features and technical effects of the present invention.
Drawings
FIG. 1 shows a schematic representation of a recombinant expression vector pUKDN132 containing the inulase gene promoter and signal peptide in a preferred embodiment of the invention;
FIG. 2 shows the positions of the T (-351) A, UTR Δ A and P10L mutations described in the preferred embodiment of the present invention in the inulase gene promoter and signal peptide;
FIG. 3 shows the effect of the T (-351) A, UTR Δ A and P10L mutations and combinations thereof described in the preferred embodiment of the present invention on the secretory expression of feruloyl esterase: on pUKDN132 carrier, wild type or mutant inulinase promoter and signal peptide drive the expression of feruloyl esterase; the enzyme activity of ferulic acid esterase in the fermentation supernatant of a transformant containing the wild-type vector pUKDN132-EstE was defined as 1, and the bars represent the mean. + -. S.D (n ═ 4);
FIG. 4 shows the effect of the T (-351) A, UTR Δ A mutation described in the preferred embodiment of the invention on mRNA levels: the content of EstE mRNA in pUKDN132-EstE, pUKDN132-T (-351) A-EstE and pUKDN132-UTR delta A-EstE transformants is determined relative to 18s rDNA; the relative amount of EstE mRNA in transformants containing the wild-type plasmid pUKDN132-EstE (WT) was defined as 1 and the bars represent the mean ± S.D (n ═ 4).
FIG. 5 shows the effect of the P10L mutation described in the preferred embodiment of the present invention on the secretory expression of feruloyl esterase; contains pUKDN132-EstE-His6And transformants of pUKDN132 were cultured in YD medium; supernatants and cells were collected at the indicated time points; western detection of EstE-His in supernatants and cell lysates6Histone H3 was used as an internal control.
Detailed Description
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Example 1 construction of recombinant expression vector containing Kluyveromyces marxianus inulase Gene promoter and Signal peptide
The genome of Kluyveromyces marxianus FIM-1 was extracted according to the method of the yeast genome DNA extraction kit (Solarbio, D1900) instructions. The yeast Kluyveromyces marxianus is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.10621 (the preservation unit code is CGMCC, the preservation unit address is China general microbiological culture Collection center of China microbiological culture Collection center No.3, Xilu No.1 Hospital, 3 rd of Beijing, the south Korean district, the preservation date is 2015 year, 3 months and 13 days, and the detection result is survival and classified naming is Kluyveromyces marxianus.
The inulase gene is amplified by using the genome of FIM-1 as a template and primers INU-F1 (the sequence is shown as SEQ ID No. 7) and INU-R1 (the sequence is shown as SEQ ID No. 8), wherein the inulase gene comprises an inulase gene promoter (which starts 1136bp upstream of a start codon), an inulase gene (1671bp) and an inulase gene terminator (842 bp). The PCR procedure was performed according to the product instructions of Phanta Super Fidelity DNA Polymerase (Vazyme, cat. No. P505-d1/d2/d 3). After completion of PCR, 1. mu.L of Taq enzyme (Takara, R001Q) was added to the reaction system, and the reaction was carried out at 72 ℃ for 20min to add A to the PCR product. The PCR amplification product was analyzed by agarose electrophoresis to obtain a target fragment of 3649 bp. The PCR product was recovered according to the protocol of the SanPrep column DNA gel recovery kit (Bio-Rad., Cat. No. B518131-0050). The PCR product was ligated with pMD18-T vector (Takara,6011) by the method described in the product description. The resulting plasmid was designated pMD 18-T-INU.
According to the method of the QuikChange II Site-Directed Mutagenesis Kit (Agilent,200523), pMD18-T-INU is used as a template, primers INU delta-F1 (shown as SEQ ID No. 9) and INU delta-R1 (shown as SEQ ID No. 10) are used for removing 79-1668bp sequences of the inulase gene, and a multiple cloning Site is introduced between 78bp later and a stop codon (TGA) of the inulase gene. The multiple cloning site includes the sites for SmaI, SpeI and NotI. The obtained plasmid was named pMD18-T-PINU-SP-MCS-TINU。
With pMD18-T-PINU-SP-MCS-TINUAs a template, P was amplified using primers INU-F2 (SEQ ID No. 11) and INU-R2 (SEQ ID No. 12)INU-SP-MCS-TINUAnd (3) fragment. The fragment contains a promoter (from 1136bp upstream of the initiation codon to 1bp upstream of the initiation codon) of the inulase gene (the sequence is shown as SEQ ID No. 13), 1-78bp (containing a sequence for coding a signal peptide) of the inulase gene, a multiple cloning site and an inulase terminator (842 bp). The PCR reaction was performed using Phanta Super Fidelity DNA Polymerase. The PCR amplification product was analyzed by agarose electrophoresis to obtain 2150bp PINU-SP-MCS-TINUAnd (3) fragment. P pair by using SanPrep column type DNA gel recovery kitINU-SP-MCS-TINUThe fragments are recovered.
According to the instructions of the QuikChange II Site-Directed Mutagenesis Kit, the pUKD vector (Appl Microbiol Biotechnol (2003)62: 387-And (3) removing an EcoRI site between a pKS sequence (an escherichia coli replication sequence and an ampicillin resistance gene) and a KD sequence (a yeast autonomous replication sequence) by using primers pUKD-F1 (the sequence is shown as SEQ ID No. 14) and pUKD-R1 (the sequence is shown as SEQ ID No. 15) to obtain a plasmid pUKD delta E. By using EcoRI enzyme-cleaves pUKD delta E, and recovery of enzyme-cleaved products is carried out by utilizing a SanPrep column type DNA gel recovery kit.
Using the Gibson Assembly traceless ligation System (NEB Corp., cat No. E2611S/L), the PINU-SP-MCS-TINU fragment was ligated with pUKD. DELTA.EcoAnd connecting RI enzyme digestion products. The obtained vector is pUKDN132, as shown in FIG. 1.
Example 2 optimization of the promoters and Signal peptides of the Kluyveromyces marxianus inulase Gene
According to the instruction of QuikChange II Site-Directed Mutagenesis Kit, using pUKDN132 as a template, a mutation was introduced into the inulinase promoter using a primer T (-351) A-F (sequence shown in SEQ ID No. 16) and a primer T (-351) A-R (sequence shown in SEQ ID No. 17) to mutate T at position 351 upstream of the initiation codon to A, which is located in the binding Site of a potential transcription factor, and the obtained vector was named pUKDN132-T (-351) A as shown in FIG. 2.
According to the instruction of QuikChange II Site-Directed Mutagenesis Kit, a sequence from 8bp upstream to 115bp upstream of the initiation codon is removed by using a primer UTR delta A-F (the sequence is shown as SEQ ID No. 18) and a primer UTR delta A-R (the sequence is shown as SEQ ID No. 19) by taking pUKDN132 as a template, the sequence is positioned in the 5' UTR of the enzyme gene inulin, and the obtained vector is named as pUKDN132-UTR delta A as shown in FIG. 2.
According to the instruction of QuikChange II Site-Directed Mutagenesis Kit, pUKDN132 was used as a template, and C at position 29 of the inulase gene was changed to T using primer P10L-F (SEQ ID No. 20) and primer P10L-R (SEQ ID No. 21), and the mutation changed proline at position 10 of the inulase signal peptide to leucine. The obtained vector was named pUKDN 132-P10L.
According to the instruction of QuikChange II Site-Directed Mutagenesis Kit, pUKDN132-T (-351) A is used as a template, and a sequence from 8bp upstream to 115bp upstream of the initiation codon is removed by using a primer UTR delta A-F (the sequence is shown in SEQ ID No. 18) and a primer UTR delta A-R (the sequence is shown in SEQ ID No. 19), and the sequence is positioned in the 5' UTR of the inulase gene and is shown in FIG. 2. The obtained vector is named as pUKDN132-T (-351) A + UTR delta A, and the sequence of the vector is shown as SEQ ID No. 22.
According to the instruction of QuikChange II Site-Directed Mutagenesis Kit, pUKDN132-T (-351) A was used as a template, and C at position 29 of the inulase gene was changed to T using primer P10L-F (SEQ ID No. 20) and primer P10L-R (SEQ ID No. 21), and this mutation changed proline at position 10 of the inulase signal peptide to leucine. The obtained vector is named as pUKDN132-T (-351) A + P10L, and the sequence of the vector is shown as SEQ ID No. 23.
Example 3 secretory expression of Feruloyl esterase Using mutated promoter and Signal peptide of Kluyveromyces marxianus inulase Gene
The pUKDN112-EstE (patent application No. 201711086961.9) stored in the laboratory was digested with SpeI and NotI. 754bp enzyme digestion product is recovered by using a SanPrep column type DNA gel recovery kit. This fragment contains the coding sequence of the EstE gene.
The enzyme digestion is respectively carried out on pUKDN132, pUKDN132-T (-351) A, pUKDN132-UTR delta A, pUKDN132-P10L, pUKDN132-T (-351) A + UTR delta A and pUKDN132-T (-351) A + P10L by using SpeI and NotI, and the enzyme digestion product is recovered by using a SanPrep column type DNA glue recovery kit. The cleavage products of pUKDN132, pUKDN132-T (-351) A, pUKDN132-UTR delta A, pUKDN132-P10L, pUKDN132-T (-351) A + UTR delta A and pUKDN132-T (-351) A + P10L were ligated with the cleavage products of EstE, respectively, according to the procedures described in the DNA ligation Kit Ver.2.1(Takara, 6022). The obtained plasmids were named pUKDN132-EstE, pUKDN132-T (-351) A-EstE, pUKDN132-UTR delta A-EstE, pUKDN132-P10L-EstE, pUKDN132-T (-351) A + UTR delta A-EstE and pUKDN132-T (-351) A + P10L-EstE.
The yeast expression host strain adopted by the invention is derived from Kluyveromyces marxianus Fim-1, is preserved in China general microbiological culture Collection center (CGMCC), has the preservation number of CGMCC No.10621 (the preservation unit code is CGMCC, the preservation unit address is China microbiological culture Collection center (China Committee for preservation and management) No.3 of No.1 Homeh No.3 of south China Korean district, Beijing, the preservation date is 2015, 3, 13 days, the detection result is survival, the classification name is Kluyveromyces marxianus, the gene except URA3 is knocked out by a homologous recombination method, and the YPD containing 5-fluoroorotic acid (1.5g/L) is utilized to screen and obtain the uracil-deficient expression host strain which is named as Kluyveromyces marxianus Fim-1URA3 delta. pUKDN132-EstE, pUKDN132-T (-351) A-EstE, pUKDN132-UTR delta A-EstE, pUKDN132-P10L-EstE, pUKDN132-T (-351) A + UTR delta A-EstE and pUKDN132-T (-351) A + P10L-EstE were transferred into Fim-1ura3 delta, respectively, by using a lithium acetate transformation method (World Journal of Microbiology & Biotechnology 16:653-654, 2000). The transformed product was spread on SD plates (0.67% amino acid-free yeast nitrogen source, 2% glucose, 2% agar), and the plates were incubated in a 30 ℃ incubator for 2-4 days until colonies were formed.
4 different clones were selected from each group of transformants, inoculated in 50ml of YD medium (2% yeast extract, 4% glucose), cultured at 30 ℃ and 220rpm for 72 hours, and the supernatant was collected and assayed for the enzyme activity of feruloyl esterase. The assay and enzyme activity definitions are made with reference to the patent (patent application No. 201711086961.9). The enzyme activity of the supernatant of the pUKDN132-T (-351) A-EstE transformant is 2.4 times of that of the wild plasmid pUKDN132-EstE, the enzyme activity of the pUKDN132-UTR delta A-EstE transformant is 2.1 times of that of the wild plasmid, and the enzyme activity of the pUKDN132-P10L-EstE transformant is 5.1 times of that of the wild plasmid. The results show that the mutation of the inulinase promoter and signal peptide of the invention can effectively improve the secretion expression capability driven by the inulinase promoter and signal peptide.
The enzyme activity of pUKDN132-T (-351) A + UTR delta A-EstE combining the T (-351) A mutation and the UTR delta A mutation is 3.5 times of that of a wild plasmid, and is higher than that of the independent T (-351) A and UTR delta A mutants, so that the two mutations have a superimposed effect on the improvement of the enzyme activity. The enzyme activity of pUKDN132-T (-351) A + P10L-EstE combining the T (-351) A and P10L mutations is 5.9 times of that of a wild plasmid, and is higher than that of the T (-351) A and P10L mutants, so that the two mutations have a superimposed effect on the improvement of the enzyme activity.
Example 4 Effect of T (-351) A and UTR delta A mutations on mRNA levels
4 clones were picked from transformants of pUKDN132-EstE, pUKDN132-T (-351) A-EstE and pUKDN 132-UTR. DELTA.A-EstE, respectively, inoculated into 3mL of SD medium (0.67% without amino acid yeast nitrogen source, 2% glucose) and cultured overnight at 220rpm at 30 ℃. The overnight strain was diluted and inoculated into 50ml of YD medium to obtain the initial OD600Is 0.2. Cells were collected after culturing at 30 ℃ and 220rpm for 9 hours. Total RNA was extracted according to the method described in ZR Fungal/Bacterial RNA MiniPrep (Zymoresearch, R2014). RNA was reverse transcribed into cDNA according to the method described in PrimeScript RT (Takara, RR 037A). Quantitative PCR analysis was performed using the LightCycler 480II Real-Time PCR system (Roche) according to the method described in SYBR Premix Ex TaqII (Takara Inc., RR 820A). The primers for amplifying the EstE gene are EstE-F (the sequence is shown as SEQ ID No. 24) and EstE-R (the sequence is shown as SEQ ID No. 25). The primers for amplifying the internal reference 18S rDNA are 18S-F (shown in SEQ ID No. 26) and 18S-R (shown in SEQ ID No. 27). The relative amount of EstE mRNA was calculated by calculating the ratio of EstE mRNA to 18s rDNA. The relative content of EstE mRNA in the pUKDN132-EstE transformant is defined as 1, the relative content of EstE mRNA in the pUKDN132-T (-351) A transformant is 2.0, and the relative content of EstE mRNA in the pUKDN132-UTR delta A-EstE transformant is 22. This result demonstrates that the T (-351) a and UTR Δ a mutations of the present invention can significantly increase the mRNA level of the driven gene.
Example 5 Effect of the P10L mutation on protein secretion
According to the instruction of QuikChange II Site-Directed Mutagenesis Kit, pUKDN132-EstE and pUKDN132-P10L-EstE are used as templates, and a primer HIS6-F (shown as SEQ ID No. 28) and a primer HIS6-R (shown as SEQ ID No. 29) are used for adding and encoding His at the N end of EstE gene6The sequence of the tag. The obtained vectors were named pUKDN132-EstE-His, respectively6And pUKDN132-P10L-EstE-His6。
pUKDN132-EstE-His was prepared according to the method of example 36And pUKDN132-P10L-EstE-His6Transfer to Fim-1ura3 Δ. pUKDN132-EstE-His6And pUKDN132-P10L-EstE-His6The transformant was inoculated into 3mL of SD medium and cultured overnight at 30 ℃ and 220 rpm. The overnight strain was diluted and inoculated into 50ml of YD medium to obtain the initial OD600Is 0.2. Cells and supernatant were collected after 9h, 24h, 48h and 72h at 30 ℃ and 220 rpm. Western detection of EstE-His in supernatants and cell lysates6The antibody is anti-His6(Abmart corporation, M30111). Meanwhile, histone H3 in cell lysate is detected as a control, and an antibody is anti-H3.1(Abmart corporation, P30266). The Western method was performed according to the instructions of "molecular cloning protocols". As shown in FIG. 5, pUKDN132-P10L-EstE-His6EstE-His in the supernatant of transformants6The content of the protein is obviously more than that of pUKDN132-EstE-His6. In accordance therewith, pUKDN132-P10L-EstE-His6EstE-His in cell lysates of transformants6The content of the extract is obviously less than that of pUKDN132-EstE-His6. This result indicates that the P10L mutation can effectively promote the secretion of the EstE protein.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
<110> university of Compound Dan
Kluyveromyces marxianus promoter and secretion signal peptide as well as preparation and application thereof
<130> 2018
<160> 29
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1136
<212> DNA
<213> Artificial sequence ()
<400> 1
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgtaaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
gttttttatt ttttttttcc attgccatag agaaagaaaa aaaaaaaaaa gagagtttgt 1080
gaagatcttc cattcgaatc ccataagtga cacatttaat tttttttttg ttagat 1136
<210> 2
<211> 1028
<212> DNA
<213> Artificial sequence ()
<400> 2
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgttaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
ggttagat 1028
<210> 3
<211> 1028
<212> DNA
<213> Artificial sequence ()
<400> 3
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgtaaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
ggttagat 1028
<210> 4
<211> 78
<212> DNA
<213> Artificial sequence ()
<400> 4
atgaagttag catactccct cttgcttcta ttggcaggag tcagtgcttc agtgatcaat 60
tacaagagag acggtgac 78
<210> 5
<211> 26
<212> PRT
<213> Artificial sequence ()
<400> 5
Met Lys Leu Ala Tyr Ser Leu Leu Leu Leu Leu Ala Gly Val Ser Ala
1 5 10 15
Ser Val Ile Asn Tyr Lys Arg Asp Gly Asp
20 25
<210> 6
<211> 1214
<212> DNA
<213> Artificial sequence ()
<400> 6
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgtaaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
gttttttatt ttttttttcc attgccatag agaaagaaaa aaaaaaaaaa gagagtttgt 1080
gaagatcttc cattcgaatc ccataagtga cacatttaat tttttttttg ttagatatga 1140
agttagcata ctccctcttg cttctattgg caggagtcag tgcttcagtg atcaattaca 1200
agagagacgg tgac 1214
<210> 7
<211> 28
<212> DNA
<213> Artificial sequence ()
<400> 7
cacaaacaca aacacaaaca caaaaacg 28
<210> 8
<211> 29
<212> DNA
<213> Artificial sequence ()
<400> 8
tagaatgttg gtcagatgtg atgtacacc 29
<210> 9
<211> 107
<212> DNA
<213> Artificial sequence ()
<400> 9
ggagtcagtg cttcagtgat caattacaag agagacggtg accccgggac tagtgcggcc 60
gcttaaggcc gcaagctttg atctgatctg cttactttac taacgac 107
<210> 10
<211> 107
<212> DNA
<213> Artificial sequence ()
<400> 10
gtcgttagta aagtaagcag atcagatcaa agcttgcggc cttaagcggc cgcactagtc 60
ccggggtcac cgtctctctt gtaattgatc actgaagcac tgactcc 107
<210> 11
<211> 55
<212> DNA
<213> Artificial sequence ()
<400> 11
tgccgattcg cacgctgcaa ccgcggcaca aacacaaaca caaacacaaa aacgc 55
<210> 12
<211> 54
<212> DNA
<213> Artificial sequence ()
<400> 12
atggtctttc caatcagaat tcgaccgatc ctagaatgtt ggtcagatgt gatg 54
<210> 13
<211> 1136
<212> DNA
<213> Artificial sequence ()
<400> 13
cacaaacaca aacacaaaca caaaaacgct aaattatgca cacaagggcc ggcggggctg 60
ccggaaaaaa aaagggaaaa atacacagac gagcgcgcac agatggggtt accactgcaa 120
gttacaagtt gcaagttgca cgctggaatc agaattggaa tcagaattgg aattggaatt 180
agaattagaa ttaaacttgg ggtagccacg ggaacgggat aactcaggaa tcgctcgcag 240
gcgtctccgt ctaggcaatc ccaaggtaag cctaggcact cccacagggg aaagaacggt 300
tgaaggcaaa gtagtgctaa caattggtaa cgaatggtaa caagtgtgtc cgtctccacc 360
tgacatttgc tagagctggg gattccacat tcttgtgctc tgaattctca aaccgaaatg 420
gggcgttgtt accccaggta tccggttgta gttggcactg gggatggaaa aaaatgatgt 480
tgatgttgag ttagttgggt tgagtcaatt agtgcgtgaa agtatcacca cttttgtcat 540
ccggcgtttc tgtgcgaatc acacacacac acacagttta ttggagcact tgtttctggc 600
gtattcgtaa ttgttctgcg gtgcggttct gtgtgcattt ttcctggggt gtctgccgca 660
cctactcatc acccacgccg tgggtttgag ccatggcgga ggtacgactg actggctgcc 720
tgcctgcctg actgactgcc tgactgcagg aaaagagggt ttcgaaggaa aaacttttcc 780
tgtgttaatc cggccgtgcg ccgctgctcc aaaatccacc ttcatgagaa ggagtttgaa 840
aaaacaaaaa aattcacata taaaaagcgt atctcgagat ctcaaagtct cccttgaatc 900
gtgtttgcca gttgtaactc atcctttatt cttctattct atctctctct ttccttcccc 960
taatcagcaa ttaaatccgg ggtaaggaag aattactact gtgtgtaacg gttatatttc 1020
gttttttatt ttttttttcc attgccatag agaaagaaaa aaaaaaaaaa gagagtttgt 1080
gaagatcttc cattcgaatc ccataagtga cacatttaat tttttttttg ttagat 1136
<210> 14
<211> 69
<212> DNA
<213> Artificial sequence ()
<400> 14
gtcacgacgt tgtaaaacga cggccagtgc caagcttgca tgcatcacta atgaaaagca 60
tacgacgcc 69
<210> 15
<211> 69
<212> DNA
<213> Artificial sequence ()
<400> 15
ggcgtcgtat gcttttcatt agtgatgcat gcaagcttgg cactggccgt cgttttacaa 60
cgtcgtgac 69
<210> 16
<211> 30
<212> DNA
<213> Artificial sequence ()
<400> 16
aaacttttcc tgtgtaaatc cggccgtgcg 30
<210> 17
<211> 30
<212> DNA
<213> Artificial sequence ()
<400> 17
cgcacggccg gatttacaca ggaaaagttt 30
<210> 18
<211> 63
<212> DNA
<213> Artificial sequence ()
<400> 18
gaattactac tgtgtgtaac ggttatattt cggttagata tgaagttagc atactccctc 60
ttg 63
<210> 19
<211> 63
<212> DNA
<213> Artificial sequence ()
<400> 19
caagagggag tatgctaact tcatatctaa ccgaaatata accgttacac acagtagtaa 60
ttc 63
<210> 20
<211> 40
<212> DNA
<213> Artificial sequence ()
<400> 20
agcatactcc ctcttgcttc tattggcagg agtcagtgct 40
<210> 21
<211> 40
<212> DNA
<213> Artificial sequence ()
<400> 21
agcactgact cctgccaata gaagcaagag ggagtatgct 40
<210> 22
<211> 11280
<212> DNA
<213> Artificial sequence ()
<400> 22
cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 60
catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 120
attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 180
ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 240
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 300
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 360
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 420
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 480
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 540
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 600
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 660
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 720
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 780
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 840
ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 900
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 960
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 1020
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 1080
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 1140
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 1200
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 1260
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 1320
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 1380
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 1440
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 1500
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 1560
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 1620
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 1680
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 1740
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 1800
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 1860
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 1920
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 1980
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 2040
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 2100
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 2160
tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 2220
gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 2280
ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 2340
gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 2400
actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt 2460
ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc 2520
aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt 2580
aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact 2640
acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg 2700
gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag 2760
aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac 2820
gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg 2880
ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 2940
aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 3000
tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 3060
ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct tgcatgcatc 3120
actaatgaaa agcatacgac gcctgcgtct gacatgcact cattctgaag aagattctgg 3180
gcgcgtttcg ttctcgtttt cctctgtata ttgtactctg gtggacaatt tgaacataac 3240
gtctttcacc tcgccattct caataatggg ttccaattct atccaggtag cggttaattg 3300
acggtgctta agccgtatgc tcactctaac gctaccgttg tccaaacaac ggaccccttt 3360
gtgacgggtg taagacccat catgaagtaa aacatctcta acggtatgga aaagagtggt 3420
acggtcaagt ttcctggcac gagtcaattt tccctcttcg tgtagatcag aggctatata 3480
catgccgagg tattcgatca ctctacgatg acggtctgtt agctcaacaa cttcttctaa 3540
atgctccata accgtaacgt aagaagcata actgtcaata ctgaagtcat cccagtttat 3600
tggtgctcct gttgaacagt catccactat atgttcgaat agcccaggat cacgaggagg 3660
tcctacaaac ggatacggta cagtcttctt tttatagtct gcaaattcta gaatagcatt 3720
ttttatccaa tagtgtcgaa tcgtcctggc cgttctaccg ataaaggatc caatgtgatt 3780
attagctcca ctacacgata tgttaagttt gatcgatgtc ttgttaacaa acgctaaact 3840
caagttcggc atttccaaca gcgagaagaa atcatcaatt ccatcggcta tctcttgata 3900
agtcattaga tcatatacct tctcgggatg tcgttgagtt actttatgac tagaaatctt 3960
caggttatca tcaacgtaat tgttctccaa tagctctgga gagggacata acaatacttt 4020
gattttttcc atggcctgga cttgtttccg taggaaatac ttgttctttt gtagacgttc 4080
catgatgagt ttgtatacct ctgctggaga tatccattct agatctttga tataagtttg 4140
gtatggtaaa gagttgattt tgtaggacac gtaaatctgc gctagataag tacattgtgc 4200
aaatgcctct ggtacttcgt aagacccatg ctgcgtaatt atagtattat tgagtggatc 4260
ataagcgttg tactcgtttt tgaatttaaa actgtctaat aaggccctgt aaatctctct 4320
gacttgttgt acacctttct gctcttcggg actgagatcg gatagcaatg gagcagcagt 4380
ttctgagctt tctgatgggg ctgacatggc agatgcctat tcaatgctgc cttttgtttg 4440
ggaggttatg aaatgcatct gtttacattg tatgtaatac ccttactagg caatgttata 4500
agcaaaaatc ctttgatcac atggaatatc actttatacg tgttgaaata tgcaaaaaaa 4560
cagtccccct gagctcaggg ggtggtttac gcttttgagg ctcagcagcg cgaattctct 4620
cttggggctg aagtgaaatt taaaaaagtc gcttgaggct cagccggaat tataaaacat 4680
cacctgagtc ttgagagcgc tttcactcac ctgaggctca gctgaaattt caaaaagtca 4740
cttgagccca gaaggagtgt ttcaccccct gaggctataa cgttcgttat tttaatacct 4800
aaataaacaa aaatatatgg tacaggaacg cgaggcaacg cgccgataca gggtcaatgg 4860
gtacacgaga gggtgacact aggcgtagaa agtcattagt ataaaataca gtggtatata 4920
gtagatattt agtttgtttt ccttttcttt ttctccaaaa cgatatcaga catttgtctg 4980
ataatgaagc attatcagac aaatgtctga tatcgttttt caataataat atacatcatc 5040
acaaaacaaa caaacatagc atcgcaagcc ccatcatgcc accaccgtcc gctgtgatcg 5100
caactcatgt ttccggcggt attctgcaat gaattggaga acctcgtctg agataattcc 5160
atgccattgt tcgaacaact ggaggctagg atgagctgag aaggattgag cgaccaagcg 5220
cggacttgac ggtgggctga gtggtgggct accagggctg ttaccctcct cttcaagtag 5280
ctcctcgcga gataaaggtt tattagaagg atccttcaaa acatatattt cactgcccaa 5340
tggggcttcc ttgtaaaaac ctgatataaa ggcaaataca cggtcatcta cagtcacact 5400
accatgactg tagtgtgatc tagccactgc actttgaatt tcacggtccc cagcccaatt 5460
gcccagtgag gagacatatt ttcccttctc agatctcgat aaaaaggtcg ccattaaatg 5520
tcttccaaaa tgagacttcg ggccgttcca gatcttgaag actggttcat cgacatgctg 5580
ggtaagaaac ctagagaacg ttctggccaa tgactctggt aaaaactgat gagtttgatt 5640
agttggtcta ttactggata ctgttttttc aataggcgag caaacacgca aatagtcgta 5700
taatgatatc agaagatcgc aatcaccatt cacaggatag aagttaacgt accgttcagt 5760
tctgcttttc gtttctgtca cagtagcacg cacaattggg cccagaaatg aattgttgta 5820
gatctcaaaa gtccttggat ctagattctt cagatcgctg tatctgcagc aatttccaac 5880
agctcccaga agtagcaatc ggtattccgc tcgttttgta gtggttacgc aggactgatc 5940
gaagaagcag gcaatcctgg agacaatctt ccaaatatct ttttcttttg acagaatatt 6000
agtgaattgt aatccaacca tagaagcatc gtatttatgt gtttcctcgt agcgatcaaa 6060
caaggaaact tcttgatttt taaatgggct aacaacaacc ttgtaagaag gcaatgctga 6120
ttcgatatcc ttttgcagag actctgtctt tcttagtcta acagtgaatt tgataatttt 6180
gtcatcctta tcaaaagaca gagatttgcc aattgcgctc ttgtaagagc ggtaggtatt 6240
gattttcatc tcgcgtcgga tagatagcga ctgcattgtc aagatagaga atagggacgc 6300
cagcttattt ctaatttctt tcgcatttat attaaaggtg tcagattcca gaatttcatt 6360
aatttcatta gcacactgat gaggtgtgag gtgagcagcc tccgcaaagg tagacatagg 6420
ggcattggtt ggaggccttt gaggtaccac tagagtgctg caaacatagc accgttcgag 6480
actttaaaat cttcagtttt aaaattatga aaaaaaacat cgtcctgagt tgaaacggtc 6540
gtttcaacct ccgtgtacag aaagatacat agcatatggc aagctgcacg cagcgtaaac 6600
atgccggaca actgtcattt cgtcagatca gttgatctac tctctgtgat actgcttcgt 6660
ttgtccacgg aggtcggact aactctcacc acgcttccac ggcattcgaa agaactaata 6720
ttgtatcatt gtacatatga ggaacacgca gttgaactga gcaaaccagg actcaggaaa 6780
gcaggaggta agtgctcgct tttcgtggat ccagaggaac gtgaaaattc gccttctcct 6840
cctataccgc cgtatcagat atcagagatg ccccttcatg aacttctcga gtcaggcaat 6900
gctaaattgg ttccaaatcc cgagtttgat ctaactgatc cagacgactt tcataagtgt 6960
ttctcggtca cctattcagc attatcttta atggtaccat atctgcccag agctgctcta 7020
aaggctgctc gagtgttttg taaagatcat tcaatattaa caacggatat gcttgatttg 7080
aattatcttg aagagctaat tgagttctca aaggaaactg tgaacaaaat cccagctaga 7140
atccctatag aggacatgct tctcgagcgg ggatatgtgc taccatgggt tcatggtggt 7200
acagtgaagg gaggaaagct actgaccccc aacgattgat tctttaccga atcattgcat 7260
aattcattgc ataattcatt gcagaatacc gccggaaaca tgagttgcga tcacagcgga 7320
cggtggtggc atgatggggc ttgcgatgct atgtttgttt gttttgtgat gatgtatatt 7380
attattgaaa aacgatatca gacatttgtc tgataatgct tcattatcag acaaatgtct 7440
gatatcgttt tggagaaaaa gaaaaggaaa acaaactaaa tatctactat ataccactgt 7500
attttatact aatgactttc tacgcctagt gtcaccctct cgtgtaccca ttgaccctgt 7560
atcggcgcgt tgcctcgcgt tcctgtacca tatatttttg tttatttagg tattaaaatt 7620
tactttcctc atacaaatat taaattcacc aaacttctca aaaactaatt attcgtagtt 7680
acaaactcta ttttacaatc acgtttattc aaccattcta catccaataa ccaaaatgcc 7740
catgtacctc tcagcgaagt ccaacggtac tgtccaatat tctcattaaa tagtctttca 7800
tctatatatc agaaggtaat tataattaga gatttcgaat cattaccgtg ccgattcgca 7860
cgctgcaacc gcggcacaaa cacaaacaca aacacaaaaa cgctaaatta tgcacacaag 7920
ggccggcggg gctgccggaa aaaaaaaggg aaaaatacac agacgagcgc gcacagatgg 7980
ggttaccact gcaagttaca agttgcaagt tgcacgctgg aatcagaatt ggaatcagaa 8040
ttggaattgg aattagaatt agaattaaac ttggggtagc cacgggaacg ggataactca 8100
ggaatcgctc gcaggcgtct ccgtctaggc aatcccaagg taagcctagg cactcccaca 8160
ggggaaagaa cggttgaagg caaagtagtg ctaacaattg gtaacgaatg gtaacaagtg 8220
tgtccgtctc cacctgacat ttgctagagc tggggattcc acattcttgt gctctgaatt 8280
ctcaaaccga aatggggcgt tgttacccca ggtatccggt tgtagttggc actggggatg 8340
gaaaaaaatg atgttgatgt tgagttagtt gggttgagtc aattagtgcg tgaaagtatc 8400
accacttttg tcatccggcg tttctgtgcg aatcacacac acacacacag tttattggag 8460
cacttgtttc tggcgtattc gtaattgttc tgcggtgcgg ttctgtgtgc atttttcctg 8520
gggtgtctgc cgcacctact catcacccac gccgtgggtt tgagccatgg cggaggtacg 8580
actgactggc tgcctgcctg cctgactgac tgcctgactg caggaaaaga gggtttcgaa 8640
ggaaaaactt ttcctgtgta aatccggccg tgcgccgctg ctccaaaatc caccttcatg 8700
agaaggagtt tgaaaaaaca aaaaaattca catataaaaa gcgtatctcg agatctcaaa 8760
gtctcccttg aatcgtgttt gccagttgta actcatcctt tattcttcta ttctatctct 8820
ctctttcctt cccctaatca gcaattaaat ccggggtaag gaagaattac tactgtgtgt 8880
aacggttata tttcggttag atatgaagtt agcatactcc ctcttgcttc cattggcagg 8940
agtcagtgct tcagtgatca attacaagag agacggtgac cccgggacta gtgcggccgc 9000
ttaaggccgc aagctttgat ctgatctgct tactttacta acgacaaaaa aaaatcaaaa 9060
aaaaaaaaac aatcagtcct tctcttctta cgatatgata tgattaaatg atgctatgaa 9120
atcatcttct tcttaacttt cttaaatctt acgcgtcact tactctatat acccgtttag 9180
ctttgcctgg tcacagcgac attttatata agtgtacgta ttttcttttt ttttttaaaa 9240
atttctattc taaccttaga aaagtgccct ttaaaccagc tgtcctggca ctatatcttt 9300
atcatgtgcc ggtcgctttc cctttccgtt tcccttttcc tttcaattgg tggcctggaa 9360
ttccgaactc attttcgcat ctgaaactaa ttctcgaaac ctttaacatc aaacaattga 9420
aaagatcatc atcaccagaa ataagaaaaa gatcaacaca acagctaata acagtacgaa 9480
agaaagatcg ctcgagtgaa aaggcagcca agaaaggtca ttcgatttgg gtctagactg 9540
attatagaca taccaattgc actcagtaag aaaatgagtt tcaaatttga cgatgacggt 9600
gtggtaaaag aatttcacgg caacaccatc atatgccata ttcctcaaca aaccgaattc 9660
ttcaacaaat tgttggactt ctaccgtttt gcgaaacgac tttccttcta cgacaagatc 9720
accctacttc ctccttcaag ctaccacgtt acgatcatga attgctgcca cgaacacgat 9780
cgttctgagg gccactggcc caaaggaatc gatccggaca caagcatgct gcggtgtaca 9840
tcacatctga ccaacattct aggatcggtc gaattctgat tggaaagacc attctgcttt 9900
acttttagag catcttggtc ttctgagctc attatacctc aatcaaaact gaaattaggt 9960
gcctgtcacg gctctttttt tactgtacct ttgacttcct ttcttatttc caaggatgct 10020
catcacaata cgcttctaga tctattatgc attataatta atagttgtag ctacaaaagg 10080
taaaagaaag tccggggcag gcaacaatag aaatcggcaa aaaaaactac agaaatacta 10140
agagcttctt ccccattcag tcatcgcatt tcgaaacaag aggggaatgg ctctggctag 10200
ggaactaacc accatcgact gactctatgc actaaccacg tgactacata tatgtgatcg 10260
tttttaacat ttttcaaagg ctgtgtgtct ggctgtttcc attaattttc actgattaag 10320
cagtcatatt gaatctgagc tcatcaccaa caagaaattc taccgtaaaa gtgtaaaagt 10380
tcgtttaaat catttgtaaa ctggaacagc aagaggaagt atcatcagct agccccataa 10440
actaatcaaa ggaggatgtc gactaagagt tactcggaaa gagcagctgc tcatagaagt 10500
ccagttgctg ccaagctttt aaacttgatg gaagagaaga agtcaaactt atgtgcttct 10560
cttgatgttc gtaaaacagc agagttgtta agattagttg aggttttggg tccatatatc 10620
tgtctattga agacacatgt agatatcttg gaggatttca gctttgagaa taccattgtg 10680
ccgttgaagc aattagcaga gaaacacaag tttttgatat ttgaagacag gaagtttgcc 10740
gacattggga acactgttaa attacaatac acgtctggtg tataccgtat cgccgaatgg 10800
tctgatatca ccaatgcaca cggtgtgact ggtgcgggca ttgttgctgg tttgaagcaa 10860
ggtgccgagg aagttacaaa agaacctaga gggttgttaa tgcttgccga gttatcgtcc 10920
aaggggtctc tagcgcacgg tgaatacact cgtgggaccg tggaaattgc caagagtgat 10980
aaggactttg ttattggatt tattgctcaa aacgatatgg gtggaagaga agagggctac 11040
gattggttga tcatgacgcc aggtgttggt cttgatgaca aaggtgatgc tttgggacaa 11100
caatacagaa ctgtggatga agttgttgcc ggtggatcag acatcattat tgttggtaga 11160
ggtcttttcg caaagggaag agatcctgta gtggaaggtg agagatacag aaaggcggga 11220
tgggacgctt acttgaagag agtaggcaga tccgcttaag aggggtaccg agctcgaatt 11280
<210> 23
<211> 11388
<212> DNA
<213> Artificial sequence ()
<400> 23
cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 60
catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 120
attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 180
ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 240
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 300
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 360
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 420
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 480
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 540
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 600
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 660
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 720
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 780
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 840
ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 900
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 960
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 1020
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 1080
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 1140
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 1200
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 1260
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 1320
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 1380
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 1440
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 1500
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 1560
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 1620
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 1680
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 1740
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 1800
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 1860
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 1920
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 1980
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 2040
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 2100
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 2160
tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 2220
gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 2280
ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 2340
gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 2400
actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt 2460
ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc 2520
aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt 2580
aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact 2640
acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg 2700
gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag 2760
aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac 2820
gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg 2880
ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 2940
aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 3000
tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 3060
ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct tgcatgcatc 3120
actaatgaaa agcatacgac gcctgcgtct gacatgcact cattctgaag aagattctgg 3180
gcgcgtttcg ttctcgtttt cctctgtata ttgtactctg gtggacaatt tgaacataac 3240
gtctttcacc tcgccattct caataatggg ttccaattct atccaggtag cggttaattg 3300
acggtgctta agccgtatgc tcactctaac gctaccgttg tccaaacaac ggaccccttt 3360
gtgacgggtg taagacccat catgaagtaa aacatctcta acggtatgga aaagagtggt 3420
acggtcaagt ttcctggcac gagtcaattt tccctcttcg tgtagatcag aggctatata 3480
catgccgagg tattcgatca ctctacgatg acggtctgtt agctcaacaa cttcttctaa 3540
atgctccata accgtaacgt aagaagcata actgtcaata ctgaagtcat cccagtttat 3600
tggtgctcct gttgaacagt catccactat atgttcgaat agcccaggat cacgaggagg 3660
tcctacaaac ggatacggta cagtcttctt tttatagtct gcaaattcta gaatagcatt 3720
ttttatccaa tagtgtcgaa tcgtcctggc cgttctaccg ataaaggatc caatgtgatt 3780
attagctcca ctacacgata tgttaagttt gatcgatgtc ttgttaacaa acgctaaact 3840
caagttcggc atttccaaca gcgagaagaa atcatcaatt ccatcggcta tctcttgata 3900
agtcattaga tcatatacct tctcgggatg tcgttgagtt actttatgac tagaaatctt 3960
caggttatca tcaacgtaat tgttctccaa tagctctgga gagggacata acaatacttt 4020
gattttttcc atggcctgga cttgtttccg taggaaatac ttgttctttt gtagacgttc 4080
catgatgagt ttgtatacct ctgctggaga tatccattct agatctttga tataagtttg 4140
gtatggtaaa gagttgattt tgtaggacac gtaaatctgc gctagataag tacattgtgc 4200
aaatgcctct ggtacttcgt aagacccatg ctgcgtaatt atagtattat tgagtggatc 4260
ataagcgttg tactcgtttt tgaatttaaa actgtctaat aaggccctgt aaatctctct 4320
gacttgttgt acacctttct gctcttcggg actgagatcg gatagcaatg gagcagcagt 4380
ttctgagctt tctgatgggg ctgacatggc agatgcctat tcaatgctgc cttttgtttg 4440
ggaggttatg aaatgcatct gtttacattg tatgtaatac ccttactagg caatgttata 4500
agcaaaaatc ctttgatcac atggaatatc actttatacg tgttgaaata tgcaaaaaaa 4560
cagtccccct gagctcaggg ggtggtttac gcttttgagg ctcagcagcg cgaattctct 4620
cttggggctg aagtgaaatt taaaaaagtc gcttgaggct cagccggaat tataaaacat 4680
cacctgagtc ttgagagcgc tttcactcac ctgaggctca gctgaaattt caaaaagtca 4740
cttgagccca gaaggagtgt ttcaccccct gaggctataa cgttcgttat tttaatacct 4800
aaataaacaa aaatatatgg tacaggaacg cgaggcaacg cgccgataca gggtcaatgg 4860
gtacacgaga gggtgacact aggcgtagaa agtcattagt ataaaataca gtggtatata 4920
gtagatattt agtttgtttt ccttttcttt ttctccaaaa cgatatcaga catttgtctg 4980
ataatgaagc attatcagac aaatgtctga tatcgttttt caataataat atacatcatc 5040
acaaaacaaa caaacatagc atcgcaagcc ccatcatgcc accaccgtcc gctgtgatcg 5100
caactcatgt ttccggcggt attctgcaat gaattggaga acctcgtctg agataattcc 5160
atgccattgt tcgaacaact ggaggctagg atgagctgag aaggattgag cgaccaagcg 5220
cggacttgac ggtgggctga gtggtgggct accagggctg ttaccctcct cttcaagtag 5280
ctcctcgcga gataaaggtt tattagaagg atccttcaaa acatatattt cactgcccaa 5340
tggggcttcc ttgtaaaaac ctgatataaa ggcaaataca cggtcatcta cagtcacact 5400
accatgactg tagtgtgatc tagccactgc actttgaatt tcacggtccc cagcccaatt 5460
gcccagtgag gagacatatt ttcccttctc agatctcgat aaaaaggtcg ccattaaatg 5520
tcttccaaaa tgagacttcg ggccgttcca gatcttgaag actggttcat cgacatgctg 5580
ggtaagaaac ctagagaacg ttctggccaa tgactctggt aaaaactgat gagtttgatt 5640
agttggtcta ttactggata ctgttttttc aataggcgag caaacacgca aatagtcgta 5700
taatgatatc agaagatcgc aatcaccatt cacaggatag aagttaacgt accgttcagt 5760
tctgcttttc gtttctgtca cagtagcacg cacaattggg cccagaaatg aattgttgta 5820
gatctcaaaa gtccttggat ctagattctt cagatcgctg tatctgcagc aatttccaac 5880
agctcccaga agtagcaatc ggtattccgc tcgttttgta gtggttacgc aggactgatc 5940
gaagaagcag gcaatcctgg agacaatctt ccaaatatct ttttcttttg acagaatatt 6000
agtgaattgt aatccaacca tagaagcatc gtatttatgt gtttcctcgt agcgatcaaa 6060
caaggaaact tcttgatttt taaatgggct aacaacaacc ttgtaagaag gcaatgctga 6120
ttcgatatcc ttttgcagag actctgtctt tcttagtcta acagtgaatt tgataatttt 6180
gtcatcctta tcaaaagaca gagatttgcc aattgcgctc ttgtaagagc ggtaggtatt 6240
gattttcatc tcgcgtcgga tagatagcga ctgcattgtc aagatagaga atagggacgc 6300
cagcttattt ctaatttctt tcgcatttat attaaaggtg tcagattcca gaatttcatt 6360
aatttcatta gcacactgat gaggtgtgag gtgagcagcc tccgcaaagg tagacatagg 6420
ggcattggtt ggaggccttt gaggtaccac tagagtgctg caaacatagc accgttcgag 6480
actttaaaat cttcagtttt aaaattatga aaaaaaacat cgtcctgagt tgaaacggtc 6540
gtttcaacct ccgtgtacag aaagatacat agcatatggc aagctgcacg cagcgtaaac 6600
atgccggaca actgtcattt cgtcagatca gttgatctac tctctgtgat actgcttcgt 6660
ttgtccacgg aggtcggact aactctcacc acgcttccac ggcattcgaa agaactaata 6720
ttgtatcatt gtacatatga ggaacacgca gttgaactga gcaaaccagg actcaggaaa 6780
gcaggaggta agtgctcgct tttcgtggat ccagaggaac gtgaaaattc gccttctcct 6840
cctataccgc cgtatcagat atcagagatg ccccttcatg aacttctcga gtcaggcaat 6900
gctaaattgg ttccaaatcc cgagtttgat ctaactgatc cagacgactt tcataagtgt 6960
ttctcggtca cctattcagc attatcttta atggtaccat atctgcccag agctgctcta 7020
aaggctgctc gagtgttttg taaagatcat tcaatattaa caacggatat gcttgatttg 7080
aattatcttg aagagctaat tgagttctca aaggaaactg tgaacaaaat cccagctaga 7140
atccctatag aggacatgct tctcgagcgg ggatatgtgc taccatgggt tcatggtggt 7200
acagtgaagg gaggaaagct actgaccccc aacgattgat tctttaccga atcattgcat 7260
aattcattgc ataattcatt gcagaatacc gccggaaaca tgagttgcga tcacagcgga 7320
cggtggtggc atgatggggc ttgcgatgct atgtttgttt gttttgtgat gatgtatatt 7380
attattgaaa aacgatatca gacatttgtc tgataatgct tcattatcag acaaatgtct 7440
gatatcgttt tggagaaaaa gaaaaggaaa acaaactaaa tatctactat ataccactgt 7500
attttatact aatgactttc tacgcctagt gtcaccctct cgtgtaccca ttgaccctgt 7560
atcggcgcgt tgcctcgcgt tcctgtacca tatatttttg tttatttagg tattaaaatt 7620
tactttcctc atacaaatat taaattcacc aaacttctca aaaactaatt attcgtagtt 7680
acaaactcta ttttacaatc acgtttattc aaccattcta catccaataa ccaaaatgcc 7740
catgtacctc tcagcgaagt ccaacggtac tgtccaatat tctcattaaa tagtctttca 7800
tctatatatc agaaggtaat tataattaga gatttcgaat cattaccgtg ccgattcgca 7860
cgctgcaacc gcggcacaaa cacaaacaca aacacaaaaa cgctaaatta tgcacacaag 7920
ggccggcggg gctgccggaa aaaaaaaggg aaaaatacac agacgagcgc gcacagatgg 7980
ggttaccact gcaagttaca agttgcaagt tgcacgctgg aatcagaatt ggaatcagaa 8040
ttggaattgg aattagaatt agaattaaac ttggggtagc cacgggaacg ggataactca 8100
ggaatcgctc gcaggcgtct ccgtctaggc aatcccaagg taagcctagg cactcccaca 8160
ggggaaagaa cggttgaagg caaagtagtg ctaacaattg gtaacgaatg gtaacaagtg 8220
tgtccgtctc cacctgacat ttgctagagc tggggattcc acattcttgt gctctgaatt 8280
ctcaaaccga aatggggcgt tgttacccca ggtatccggt tgtagttggc actggggatg 8340
gaaaaaaatg atgttgatgt tgagttagtt gggttgagtc aattagtgcg tgaaagtatc 8400
accacttttg tcatccggcg tttctgtgcg aatcacacac acacacacag tttattggag 8460
cacttgtttc tggcgtattc gtaattgttc tgcggtgcgg ttctgtgtgc atttttcctg 8520
gggtgtctgc cgcacctact catcacccac gccgtgggtt tgagccatgg cggaggtacg 8580
actgactggc tgcctgcctg cctgactgac tgcctgactg caggaaaaga gggtttcgaa 8640
ggaaaaactt ttcctgtgta aatccggccg tgcgccgctg ctccaaaatc caccttcatg 8700
agaaggagtt tgaaaaaaca aaaaaattca catataaaaa gcgtatctcg agatctcaaa 8760
gtctcccttg aatcgtgttt gccagttgta actcatcctt tattcttcta ttctatctct 8820
ctctttcctt cccctaatca gcaattaaat ccggggtaag gaagaattac tactgtgtgt 8880
aacggttata tttcgttttt tatttttttt ttccattgcc atagagaaag aaaaaaaaaa 8940
aaaagagagt ttgtgaagat cttccattcg aatcccataa gtgacacatt taattttttt 9000
tttgttagat atgaagttag catactccct cttgcttcta ttggcaggag tcagtgcttc 9060
agtgatcaat tacaagagag acggtgaccc cgggactagt gcggccgctt aaggccgcaa 9120
gctttgatct gatctgctta ctttactaac gacaaaaaaa aatcaaaaaa aaaaaaacaa 9180
tcagtccttc tcttcttacg atatgatatg attaaatgat gctatgaaat catcttcttc 9240
ttaactttct taaatcttac gcgtcactta ctctatatac ccgtttagct ttgcctggtc 9300
acagcgacat tttatataag tgtacgtatt ttcttttttt ttttaaaaat ttctattcta 9360
accttagaaa agtgcccttt aaaccagctg tcctggcact atatctttat catgtgccgg 9420
tcgctttccc tttccgtttc ccttttcctt tcaattggtg gcctggaatt ccgaactcat 9480
tttcgcatct gaaactaatt ctcgaaacct ttaacatcaa acaattgaaa agatcatcat 9540
caccagaaat aagaaaaaga tcaacacaac agctaataac agtacgaaag aaagatcgct 9600
cgagtgaaaa ggcagccaag aaaggtcatt cgatttgggt ctagactgat tatagacata 9660
ccaattgcac tcagtaagaa aatgagtttc aaatttgacg atgacggtgt ggtaaaagaa 9720
tttcacggca acaccatcat atgccatatt cctcaacaaa ccgaattctt caacaaattg 9780
ttggacttct accgttttgc gaaacgactt tccttctacg acaagatcac cctacttcct 9840
ccttcaagct accacgttac gatcatgaat tgctgccacg aacacgatcg ttctgagggc 9900
cactggccca aaggaatcga tccggacaca agcatgctgc ggtgtacatc acatctgacc 9960
aacattctag gatcggtcga attctgattg gaaagaccat tctgctttac ttttagagca 10020
tcttggtctt ctgagctcat tatacctcaa tcaaaactga aattaggtgc ctgtcacggc 10080
tcttttttta ctgtaccttt gacttccttt cttatttcca aggatgctca tcacaatacg 10140
cttctagatc tattatgcat tataattaat agttgtagct acaaaaggta aaagaaagtc 10200
cggggcaggc aacaatagaa atcggcaaaa aaaactacag aaatactaag agcttcttcc 10260
ccattcagtc atcgcatttc gaaacaagag gggaatggct ctggctaggg aactaaccac 10320
catcgactga ctctatgcac taaccacgtg actacatata tgtgatcgtt tttaacattt 10380
ttcaaaggct gtgtgtctgg ctgtttccat taattttcac tgattaagca gtcatattga 10440
atctgagctc atcaccaaca agaaattcta ccgtaaaagt gtaaaagttc gtttaaatca 10500
tttgtaaact ggaacagcaa gaggaagtat catcagctag ccccataaac taatcaaagg 10560
aggatgtcga ctaagagtta ctcggaaaga gcagctgctc atagaagtcc agttgctgcc 10620
aagcttttaa acttgatgga agagaagaag tcaaacttat gtgcttctct tgatgttcgt 10680
aaaacagcag agttgttaag attagttgag gttttgggtc catatatctg tctattgaag 10740
acacatgtag atatcttgga ggatttcagc tttgagaata ccattgtgcc gttgaagcaa 10800
ttagcagaga aacacaagtt tttgatattt gaagacagga agtttgccga cattgggaac 10860
actgttaaat tacaatacac gtctggtgta taccgtatcg ccgaatggtc tgatatcacc 10920
aatgcacacg gtgtgactgg tgcgggcatt gttgctggtt tgaagcaagg tgccgaggaa 10980
gttacaaaag aacctagagg gttgttaatg cttgccgagt tatcgtccaa ggggtctcta 11040
gcgcacggtg aatacactcg tgggaccgtg gaaattgcca agagtgataa ggactttgtt 11100
attggattta ttgctcaaaa cgatatgggt ggaagagaag agggctacga ttggttgatc 11160
atgacgccag gtgttggtct tgatgacaaa ggtgatgctt tgggacaaca atacagaact 11220
gtggatgaag ttgttgccgg tggatcagac atcattattg ttggtagagg tcttttcgca 11280
aagggaagag atcctgtagt ggaaggtgag agatacagaa aggcgggatg ggacgcttac 11340
ttgaagagag taggcagatc cgcttaagag gggtaccgag ctcgaatt 11388
<210> 24
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 24
agagcgacgg caagtttgag ga 22
<210> 25
<211> 24
<212> DNA
<213> Artificial sequence ()
<400> 25
cagcaggtga tagtggaatg agtg 24
<210> 26
<211> 24
<212> DNA
<213> Artificial sequence ()
<400> 26
tagttggtgg agtgatttgt ctgc 24
<210> 27
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 27
ctcgctggct ccgtcagtgt ag 22
<210> 28
<211> 62
<212> DNA
<213> Artificial sequence ()
<400> 28
atgcttgagc agattgctaa acaccatcat caccaccatt aagcggccgc ttaaggccgc 60
aa 62
<210> 29
<211> 62
<212> DNA
<213> Artificial sequence ()
<400> 29
ttgcggcctt aagcggccgc ttaatggtgg tgatgatggt gtttagcaat ctgctcaagc 60
at 62

Claims (7)

1. A recombinant expression vector, which is characterized in that the recombinant expression vector comprises a Kluyveromyces marxianus promoter and a Kluyveromyces marxianus secretion signal peptide; the recombinant expression vector also comprises an autonomous replication sequence of Kluyveromyces marxianus, an inulinase terminator gene sequence of Kluyveromyces marxianus and a nutritional defect screening marker gene sequence; the nucleotide sequence of the recombinant expression vector at least comprises:
a) a nucleotide sequence shown as SEQ ID No. 22; or
b) And a nucleotide sequence shown as SEQ ID No. 23.
2. The recombinant expression vector of claim 1, further comprising a gene of interest operably linked to the kluyveromyces marxianus promoter.
3. The recombinant expression vector according to claim 2, wherein the gene of interest is a feruloyl esterase gene.
4. A genetically engineered bacterium comprising the recombinant expression vector according to any one of claims 1 to 3.
5. The genetically engineered bacterium of claim 4, wherein the host bacterium of the genetically engineered bacterium is a yeast.
6. The genetically engineered bacterium of claim 5, wherein the yeast is at least one of Kluyveromyces marxianus, Kluyveromyces lactis, and Saccharomyces cerevisiae.
7. The method for producing a recombinant expression vector according to claim 1, comprising the steps of:
step 1, taking the genome of Kluyveromyces marxianus FIM-1 as a template, and carrying out PCR amplification on an inulase gene by using a primer INU-F1 with a nucleotide sequence shown as SEQ ID No.7 and a primer INU-R1 with a nucleotide sequence shown as SEQ ID No. 8; adding Taq enzyme into a reaction system, and adding A into a PCR amplification product to obtain a target fragment; recovering the target fragment, and connecting the target fragment with a pMD18-T vector to obtain a plasmid pMD18-T-INU containing a Kluyveromyces marxianus inulinase promoter; the Kluyveromyces marxianus FIM-1 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 10621;
step 2, taking the plasmid pMD18-T-INU as a template, removing the sequence of the inulase gene 79-1668bp by using a primer INU delta-F1 with the nucleotide sequence shown as SEQ ID No.9 and a primer INU delta-R1 with the nucleotide sequence shown as SEQ ID No.10, and introducing a polyclonal site between the 78bp later of the inulase gene and a stop codon to obtain the plasmid pMD18-T-PINU-SP-MCS-TINU；
Step 3, preparing the plasmid pMD18-T-PINU-SP-MCS-TINUAs a template, P was amplified using primer INU-F2 whose nucleotide sequence is shown in SEQ ID No.11 and primer INU-R2 whose nucleotide sequence is shown in SEQ ID No.12INU-SP-MCS-TINUA fragment;
step 4, taking the pUKD vector as a template, and removing an EcoRI site between pKS and a KD sequence by using a primer pUKD-F1 with a nucleotide sequence shown as SEQ ID No.14 and a primer pUKD-R1 with a nucleotide sequence shown as SEQ ID No.15 to obtain a plasmid pUKD delta E; carrying out enzyme digestion on the plasmid PUKD delta E by using EcoRI, and recovering an enzyme digestion product;
step 5, the P in the step 3 is addedINU-SP-MCS-TINUConnecting the fragment with the enzyme digestion product in the step 4 to obtain a plasmid pUKDN 132;
step 6, using the plasmid pUKDN132 as a template, and mutating thymine deoxynucleotide at 351 bit upstream of the initiation codon of the inulase gene into adenine deoxynucleotide by using a primer T (-351) A-F with a nucleotide sequence shown as SEQ ID No.16 and a primer T (-351) A-R with a nucleotide sequence shown as SEQ ID No.17 to obtain a plasmid pUKDN132-T (-351) A which contains the nucleotide sequence shown as SEQ ID No. 1;
step 7, taking the plasmid pUKDN132-T (-351) A obtained in the step 6 as a template, and removing a sequence from 8bp upstream to 115bp upstream of the initiation codon of the inulase gene by using a primer UTR delta A-F with a nucleotide sequence shown as SEQ ID No.18 and a primer UTR delta A-R with a nucleotide sequence shown as SEQ ID No.19 to obtain a plasmid pUKDN132-T (-351) A + UTR delta A with a nucleotide sequence shown as SEQ ID No. 22; or
And step 8, using the plasmid pUKDN132-T (-351) A obtained in the step 6 as a template, and mutating cytosine deoxynucleotide at the 29 th site of the inulase gene into thymine deoxynucleotide by using a primer P10L-F with a nucleotide sequence shown as SEQ ID No.20 and a primer P10L-R with a nucleotide sequence shown as SEQ ID No.21 to obtain the plasmid pUKDN132-T (-351) A + P10L with a nucleotide sequence shown as SEQ ID No. 23.
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