CN107723253B - Double-plasmid co-transformed exogenous gene high-expression genetic engineering bacterium - Google Patents

Abstract

The invention provides a gene engineering bacterium and a preparation method thereof, wherein a double-auxotroph strain is taken as an expression host, and the expression host is simultaneously transferred into two vectors through one-time electrotransformation, wherein one vector contains an exogenous target gene and a compensating gene for one auxotroph of the expression host, and the other vector contains a UPR key gene HAC1p and a compensating gene for the other auxotroph of the expression host. The engineering bacteria provided by the invention contain functional genes and exogenous target genes with different gene doses, and can obtain a high-yield engineering strain secreting and expressing the exogenous target genes through screening.

Description

Double-plasmid co-transformed exogenous gene high-expression genetic engineering bacterium

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a genetic engineering bacterium and a method for improving high expression of exogenous genes in a yeast system.

Background

In recent years, with intensive studies on the biological properties of Hansenula polymorpha (Hansenula polymorpha), the advantages of Hansenula polymorpha as a foreign target gene expression host have been gradually developed. Hansenula is also a methylotrophic yeast, similar to Pichia. The expression of various key enzymes in the methanol metabolism process, including MOX, DHAS, CAT, etc., is regulated at the transcriptional level, and they are induced by methanol, glycerol and sorbitol, and repressed by glucose and ethanol, but at glucose concentrations below 0.1%, repression is released. Under the condition of complete induction of methanol, peroxisome can account for 80% of the total cell volume, MOX and DHAS can account for 15% of the total cell protein, and the promoters thereof have extremely strong function of promoting the expression of downstream genes and are currently used as common promoters for expressing exogenous target genes in yeast. Inducible promoters are widely applied to the expression of exogenous target genes by virtue of strong starting functions and a strict regulation mechanism, and many exogenous proteins are efficiently expressed in hansenula polymorpha cells under the regulation of the promoters.

In summary of previous studies on high expression levels of proteins in yeast, we have found that there are various disadvantages. Yeast is a low eukaryotic microorganism engineering bacterium with important application value, and is widely applied to the expression of genetic engineering protein drugs, so the research on the process method for improving the protein level of yeast expression exogenous target genes is concerned. The traditional thought of high-efficiency expression mainly aims at improving the copy number of a target gene, adopting a strong promoter element to start the transcription of the target gene, and even adopting methods such as secretory signal peptide optimization screening and the like, researchers have deeply studied the expression improving strategies so far, and a plurality of fruitful research results are obtained. However, many studies have found that the expression level of a part of foreign proteins is not always in a positive correlation with the gene copy number, but rather begins to decrease after the protein expression level reaches a certain degree, which suggests that other factors influence the expression level of the foreign proteins. Later, it was found that when the protein is expressed at a high level, it is easy to cause protein misfolding and accumulation in the endoplasmic reticulum or even precipitation, while the misfolded protein is prohibited from being transported out of the endoplasmic reticulum or transported to the protease body to be eliminated by degradation. Obviously, most studies are limited to the influence level of a certain factor on the protein expression efficiency, but neglect that the protein expression is an ordered whole of coordination and interaction among functional systems with multi-cell localization, and the systems can interact and coordinate with each other in the protein synthesis process to enable the protein polypeptide to be synthesized smoothly and efficiently and further folded into the protein with the correct spatial structure. Moreover, an excessive increase in the transcription level of the target protein gene virtually increases the load of protein transport.

Many inducible or constitutive promoters have been developed and have been used to produce foreign proteins. However, when a gene encoding a foreign protein is expressed at a high level in a cell using a potent promoter, or when a protein that is not easily folded is produced, aggregation of a part of the expression product occurs in the Endoplasmic Reticulum (ER), resulting in a situation where the protein accumulates in the cell. In addition, the secretory protein and the membrane protein are translated into proteins, immediately thereafter, they are moved into the endoplasmic reticulum, specifically modified, and then transferred to the Golgi apparatus. In this case, unfolded proteins may sometimes accumulate in the endoplasmic reticulum for some reason. This is called "endoplasmic reticulum stress (UPR)". Examples of causes of such endoplasmic reticulum stress include interference of modifications occurring in the endoplasmic reticulum, and deterioration of transport from the endoplasmic reticulum. As a gene regulating UPR, Irelp-Haclp is the only gene known as such a gene in the case of yeast, and the Irelp-Haclp gene is related to UPR by a specific mechanism. First, Irelp typically binds to antibody heavy-key binding protein (BiP). However, upon intracellular accumulation of unfolded proteins (UFPs), BiP binds to such UFPs. Irelp dissociated from BiP is activated by autophosphorylation or dimerization, and it exhibits endonuclease activity. Although the HAC1 gene is usually present in an inactivated state, Irelp having endonuclease activity causes splicing of mRNA transcribed from the HAC1 gene and generates active HAClp (Ce11,90: 1031-3139, 1997; the EMBO Journal,18:3119-3132, 1999). Such active HAClp migrates to the nucleus, functions as a transcription factor, and promotes the expression of genes encoding various proteins involved in a series of reactions called UPRs, such as involved sugar bond addition, protein folding, protein degradation, protein sorting, lipid metabolism, etc., thereby relieving "endoplasmic reticulum stress", and increasing the expression amount and activity of the corresponding foreign proteins, particularly secretory expression foreign proteins. The corresponding key gene for UPR, HAC1, and similar mechanisms, are also found in Hansenula.

The obvious advantages of Hansenula over the homologous integration of the Pichia pastoris expression system are: when hansenula polymorpha is used as an expression host of exogenous target genes, the integration of exogenous DNA into its genome can be mainly divided into two recombination mechanisms, random non-homologous integration and homologous integration. Wherein the homologous recombination requires that the introduced DNA must contain a homologous sequence of Hansenula polymorpha. The homologous sequence is completely or partially identical to a DNA sequence in the yeast genome, resulting in the integration of the foreign DNA into the yeast genome at this position by homologous crossover, with a probability of homologous integration of the linearized plasmid of only 1% to 22%. Hansenula polymorpha can integrate multiple copies of exogenous target genes by non-homologous recombination (with a probability of about 50% to 80%), and the maximum copy number can reach over 100. From this, we conclude that Hansenula has significant advantages as an expression host for foreign genes of interest: multiple exogenous target genes can be simultaneously integrated into a yeast genome through one-step transformation and simultaneously or respectively expressed according to a certain dosage relation.

By constructing a co-expression vector, functional genes and exogenous target genes are simultaneously connected to one expression vector, and the purpose of simultaneously expressing the two genes in a transformant can be realized by one-time transformation and screening of the patent; however, the disadvantage of this co-expression method is that the functional gene and the exogenous target gene can only be transferred into the receptor cell with the same gene dosage, and actually the ultimate goal of constructing engineering bacteria is to express the exogenous target gene as high as possible, and the functional gene and the exogenous target gene with the same gene dosage are not good for realizing this goal.

Disclosure of Invention

In view of the above, the present invention provides a new technical route, in which two vectors are simultaneously added to a double auxotrophic host in one electrotransformation, and only transformants in which the two vectors are simultaneously integrated into the host can grow in a selective medium, so as to screen a high-yield engineering strain for secretory expression of an exogenous target gene, which overcomes the endoplasmic reticulum stress, and naturally select the gene dose relationship between a functional gene and the exogenous target gene.

The invention firstly provides a gene engineering bacterium, wherein a double auxotrophic strain is taken as an expression host (or called a receptor strain), two vectors (which can be named as an expression vector and a functional vector respectively) are simultaneously integrated in a transformant of the expression host, wherein one vector (the expression vector) contains an exogenous target gene and a compensating gene of one auxotrophy of the expression host, and the other vector (the functional vector) contains a UPR key gene HAC1p (also called a functional gene herein) and a compensating gene of the other auxotrophy of the expression host.

Wherein, the expression system of the genetic engineering bacteria can select an expression host with a UPR key gene HAC1 and similar mechanisms, preferably a yeast expression system, and more preferably Hansenula polymorpha is adopted as the expression host.

Furthermore, the genetic engineering bacteria take adenine and leucine double auxotroph Hansenula polymorpha (ade-leu-) as an expression host, and two vectors are simultaneously integrated in a transformant of the expression host, wherein one vector (the expression vector) contains an exogenous target gene and a beer yeast leucine gene for compensating the leucine auxotrophy (leu-) of the expression host, and the other vector (a functional vector) contains a UPR key gene HAC1p and a beer yeast adenine gene for compensating the adenine auxotrophy (ade-) of the expression host. The two auxotrophs selected and provided by the invention are easy to mutate, culture, screen and detect in a hansenula polymorpha expression system, have good genetic stability and can be conveniently compensated through a compensation gene.

Wherein the vector (expression vector) containing the exogenous target gene promotes the expression of the exogenous target gene by a strong promoter methanol oxidase promoter (MOX), and the vector (functional vector) containing the UPR key gene HAC1p promotes the expression of the UPR key gene HAC1p by a constitutive promoter glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). The selection of the promoter can ensure that the exogenous target gene and the functional gene can be effectively expressed in a selected expression system.

Wherein, the key gene HAC1p of the UPR is derived from Hansenula polymorpha ATCC34438, and the compensating gene is derived from Saccharomyces cerevisiae S288 c.

Wherein, the genetic engineering bacteria are obtained by screening by using exogenous target gene high expression as a screening index. The two expression vectors are transferred into a receptor cell at different gene doses to obtain a plurality of strains with different expression degrees, and when exogenous target gene high expression is used as a unique screening index, the strain with the highest expression level can be screened.

Wherein, the expression host and the two vectors for constructing the genetic engineering bacteria both contain the same strain autonomous replication sequence fragment, preferably Hansenula polymorpha autonomous replication sequence (HARS) fragment. The existence of the autonomously replicating sequence segments is beneficial to improving the probability of gene integration in an expression system, and particularly improving the variety quantity and copy number of integrated exogenous genes in a Hansenula polymorpha system.

Wherein, the exogenous target gene is derived from the full sequence SEQ ID NO. 18 of Elafin containing MFa leader peptide or the full sequence SEQ ID NO. 19 of Hirudin containing MFa leader peptide. The complete sequences of the two exogenous target genes are subjected to sequence design, can be well integrated into an expression vector and can obtain high-efficiency expression under the action of a promoter.

Wherein the genetic engineering bacteria are Efn17-09 or HRN 9-16. The two obtained strains can continuously and efficiently express two target genes of Elafin and Hirudin by purposefully constructing, transforming and screening an expression vector, the expression amounts are respectively improved by 2 times and 0.5 time, and the method is a great progress in fermentation production.

The invention also provides a vector for constructing the genetic engineering bacteria, which is at least one of the following vectors:

1) expression vector: a compensating gene for an auxotrophy comprising an exogenous gene of interest and an expression host;

2) a functional carrier: contains the UPR key gene HAC1p, and a compensating gene for another auxotrophy of the expression host.

Wherein, the expression vector uses a strong promoter methanol oxidase promoter (MOX) to start the expression of exogenous target genes, and uses a beer yeast leucine gene to compensate and express a host leucine nutritional deficiency (leu-); the functional vector uses a constitutive promoter glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) to start the expression of a UPR key gene HAC1p, and uses a beer yeast adenine gene to compensate and express a host adenine auxotrophy (ade-).

Wherein, the expression vector and the functional vector both contain the same strain autonomous replication sequence fragment, preferably Hansenula polymorpha autonomous replication sequence (HARS) fragment.

Wherein the expression vector is a vector shown by pMPT-leu2-Elafin or pMPT-leu2-Hirudin, and the functional vector is a vector shown by pGPT-ade2-HAC1 p.

The invention also provides a method for high expression of exogenous target genes, namely screening and culturing expression by adopting the genetic engineering bacteria.

The genetically engineered bacterium can be obtained by a method comprising the following steps: respectively constructing an expression vector and a functional vector, simultaneously transferring the expression vector and the functional vector into double auxotroph recipient bacteria, and screening to obtain the genetic engineering bacteria with high exogenous target gene expression.

Wherein the recipient bacterium is adenine and leucine double auxotroph Hansenula (ade-leu-); the expression vector uses a strong promoter methanol oxidase promoter (MOX) to start the expression of an exogenous target gene, and uses a beer yeast leucine gene to compensate and express a host leucine auxotrophy (leu-); the functional vector uses a constitutive promoter glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) to start the expression of a UPR key gene HAC1p, and uses a beer yeast adenine gene to compensate and express a host adenine auxotrophy (ade-); the expression vector and the functional vector both contain the autonomously replicating sequence segment of the receptor bacterial strain.

In the above method, the construction of the expression vector comprises the following steps:

s1: designing a primer, and amplifying a leucine (LEU2sc) gene by using a beer yeast genome as a template;

s2: cloning a fragment alpha containing a methanol oxidase promoter, a terminator and a hansenula polymorpha autonomous replication sequence (HARS) by taking a plasmid vector pMPT genome as a template;

s3: taking pBluescript SK (+) as a template to clone pBlu as a shuttle vector, and connecting the pBlu with the fragment alpha in the step S2 to obtain a recombinant vector alpha;

s4: respectively inserting the leucine (LEU2sc) gene and a foreign target gene into a recombinant vector alpha to construct an expression vector;

the step S1 is completed before the step S4, that is, the step S1 is not consecutive to the steps S2 and S3.

In the above method, the construction of the functional vector comprises the following steps:

t1: designing a primer, and amplifying an adenine (ADE2sc) gene by using a beer yeast genome as a template;

t2: using Hansenula polymorpha ATCC34438 genome as template to amplify HAC1 gene and mutation HAC1 gene with mutation primer to obtain HAC1p gene with coding activity;

t3: cloning a fragment beta containing a glyceraldehyde-3-phosphate dehydrogenase gene (pGAP) constitutive promoter and a terminator by taking a Hansenula polymorpha ATCC34438 genome as a template;

t4: taking pBluescript SK (+) as a template to clone pBlu as a shuttle vector, and connecting the pBlu with the fragment beta in the step T3 to obtain a recombinant vector beta;

t5: respectively inserting the adenine (ADE2sc) gene and the HAC1p gene into a recombinant vector beta to construct a functional vector;

wherein, the steps T1 and T2 are completed before the step T5, that is, the steps T1 and T2 are not in sequence with the steps T3 and T4.

In order to simplify the operation and improve the efficiency, after obtaining the pre-expression vector/functional vector, the functional vector/expression vector can also be constructed by several steps of replacement method through the expression vector/functional vector.

The construction of the expression vector comprises the following steps:

s1: designing a primer, and amplifying a leucine (LEU2sc) gene by using a beer yeast genome as a template;

s2: cloning a fragment alpha containing a methanol oxidase promoter, a terminator and a hansenula polymorpha autonomous replication sequence (HARS) by taking a plasmid vector pMPT genome as a template;

s3: sequentially carrying out gene replacement on a leucine (LEU2sc) gene, a fragment alpha and an exogenous target gene with an obtained functional vector through three steps to obtain an expression vector;

wherein, the steps S1 and S2 are not in sequence.

The construction of the functional vector comprises the following steps:

t1: designing a primer, and amplifying an adenine (ADE2sc) gene by using a beer yeast genome as a template;

t2: using Hansenula polymorpha ATCC34438 genome as template to amplify HAC1 gene and mutation HAC1 gene with mutation primer to obtain HAC1p gene with coding activity;

t3: cloning a fragment beta containing a glyceraldehyde-3-phosphate dehydrogenase gene (pGAP) constitutive promoter and a terminator by taking a Hansenula polymorpha ATCC34438 genome as a template;

t4: carrying out gene replacement on an adenine (ADE2sc) gene, a fragment beta and a HAC1p gene with the obtained expression vector in sequence by three steps to obtain an expression vector;

wherein, the steps T1, T2 and T3 are not in sequence.

The invention further provides a construction method of adenine and leucine double auxotroph Hansenula polymorpha (ade-leu-) receptor bacteria, which comprises the following steps: the method comprises the steps of designing primers by taking a Hansenula polymorpha genome as a template, respectively amplifying adenine genes and leucine genes, introducing TGGA-inserted four-base mutation products at the starting position of adenine gene structural genes, introducing TGCG-inserted four-base mutation products at the starting position of leucine gene structural genes, and integrating the amplified adenine genes and leucine genes containing the mutation products into the Hansenula polymorpha genome to obtain a target product, namely double-auxotrophic Hansenula polymorpha receptor bacteria.

Wherein, the amplification of the adenine gene adopts a primer ADE-1 and a primer ADE-2; the leucine gene is amplified by adopting a primer LEU-1, a primer LEU-T1, a primer LEU-T2 and a primer LEU-2.

The idea of the invention is that two vectors are transformed into a receptor strain at the same or different gene doses, wherein the functional vector enables the UPR key gene HAC1p to be continuously expressed, and promotes exogenous target genes to overcome endoplasmic reticulum stress to obtain high expression; because different exogenous target genes may need to be matched with UPR key gene HAC1p with different gene doses during high expression, the variable gene doses of the two vectors simultaneously break through the limitation that a co-expression vector (the HAC1p functional gene and the exogenous target gene are simultaneously connected to one expression vector) can only be transferred into a receptor cell with the same gene dose, and a high-yield engineering strain secreting and expressing the exogenous target gene is conveniently obtained by screening.

The invention optimally selects a hansenula polymorpha expression system and auxotrophs thereof to obtain receptor bacteria with stable genetic characters, optimally designs a functional vector capable of enabling a UPR key gene HAC1p to be continuously expressed and an expression vector capable of enabling an exogenous target gene to be continuously and efficiently expressed, successfully obtains and screens high-yield engineering strains secreting and expressing the exogenous target gene, and is an optimized implementation scheme of the concept of the invention. On the basis of the overall concept, different auxotroph expression hosts can be designed according to needs, and different promoters are adopted to start and express a plurality of different exogenous target genes, so that high-yield engineering bacteria capable of overcoming the endoplasmic reticulum stress effect can be obtained through transformation of different gene doses.

"double auxotrophy" as used herein and in the claims is not limited to two auxotrophies in the recipient bacterium, and may be two or more auxotrophies if desired (for example, if it is desired to simultaneously transform a third vector), i.e., "double auxotrophy" means at least two auxotrophies.

Drawings

FIG. 1 is a diagram showing the construction of recombinant vector pUC-HpADE2 delta deficient in Hansenula yeast adenine gene;

FIG. 2 is a schematic diagram showing construction of a recombinant vector pUC-HpADE 2. DELTA. -ARS into which an autonomously replicating sequence of Hansenula polymorpha was further inserted;

FIG. 3 is a schematic diagram of the construction of the recombinant vector KMX-HpADE 2. delta. -ARS with the further insertion of the G418 resistance gene;

FIG. 4 is a diagram showing the construction of a further recombinant vector KMX-HpADE2 delta-HpLEU 2 delta deficient in the leucine gene of Hansenula polymorpha;

FIG. 5 is a schematic structural diagram of the expression vector pMPT-Leu 2-Elafin;

FIG. 6 is a schematic structural diagram of expression vector pMPT-Leu 2-Hirudin;

FIG. 7 is a schematic structural diagram of the functional vector pGPT-ADE2-HAC1 p;

FIG. 8 is a graph comparing fermentation biomass of engineered bacteria expressing Elafin;

FIG. 9 is a graph comparing the fermentation expression level of engineering bacteria expressing Elafin;

FIG. 10 is a comparison graph of transcription levels of HAC 1-related genes of engineered bacteria expressing Elafin;

FIG. 11 is a comparison of fermentation biomass of Hirudin-expressing engineering bacteria;

FIG. 12 is a graph comparing the fermentation expression level of Hirudin-expressing engineering bacteria.

Detailed Description

The present invention will be described in detail below by taking a hansenula polymorpha expression system as an example.

EXAMPLE 1 Dual auxotrophic recipient Strain construction and screening

Firstly, obtaining the Hansenula polymorpha adenine gene (Hp-ADE2 delta) with mutant structural genes and constructing a plasmid vector

Primers were designed based on the reported nucleotide sequence of the adenine (ADE2) gene of Hansenula polymorpha as follows:

primer ADE-1(SEQ ID NO: 1):

(underlined is restriction enzyme cleavage site EcoRI, bold TGGA is ADE2 structural gene insertion mutation base)

Primer ADE-2(SEQ ID NO: 2):

5’-ACGAGCTCCTATTTATTAAGGTATTCTTCAT-3’

(underlined is a restriction enzyme cleavage site SacI)

The mutated Hp-ADE 2. DELTA.was amplified using Hansenula polymorpha (ATCC34438) total DNA as template with primer ADE-1, primer ADE-2, PCR system: premixing 25 mu L of Pfu DNA polymerase, 1 mu L of template, 1 mu L of each primer and complementing 50 mu L of ultrapure water and mixing uniformly; the reaction conditions were set with a PCR machine (PC707-02, Japan): pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃, 10secs, annealing at 60 ℃, 1min, extension at 72 ℃, 2min, and circulation for 30 times; fully extending at 72 ℃ for 10min once; and finishing cooling to 4 ℃ for storage.

The PCR product is precipitated by alcohol, dried and subjected to double digestion with restriction enzymes EcoRI and SacI respectively, a plasmid vector pUC18 is subjected to agarose gel electrophoresis to recover and purify the digestion fragments, the digestion fragments are reacted for 12 to 16 hours at the temperature of 16 ℃ by using T4DNA ligase to be ligated, and the ligation product is chemically transformed into JM109 competent cells. The transformants selected correctly were inoculated into 5mL of LB liquid medium containing ampicillin at a final concentration of 100ug/mL, and cultured at 37 ℃ and 180rpm overnight. Plasmid recovery Kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.2.0) is used for extracting Plasmid to obtain a recombinant vector pUC-HpADE2 delta, and the construction schematic diagram is shown in figure 1. The Hp-ADE2 delta full-length 1720bp (SEQ ID NO:3, the head and tail 6 bases of the sequence are restriction enzyme cutting sites respectively) is introduced into the starting position of a hansenula polymorpha adenine (ADE2) gene structure gene and is inserted with TGGA four base mutation products, the homologous sequence is used for homologous recombination of hansenula polymorpha and is integrated into a yeast genome through homologous exchange, and the knock-out of hansenula polymorpha adenine genes is realized.

Secondly, inserting hansenula polymorpha autonomous replication sequence into the plasmid vector

The plasmid vector pMPT-HPV18L1 (see patent of invention CN201210013236.X, containing Hansenula polymorpha autonomous replication sequence HARS) and the plasmid vector pUC-HpADE 2. delta are subjected to double digestion by restriction enzymes KpnI and XmaI respectively, purified digestion fragments (in this case, 1106bp small fragment and 4407 large fragment respectively) are recovered by agarose gel electrophoresis respectively, and then reacted for 12-16 hours at 16 ℃ by T4DNA ligase to be ligated, and the ligation product is chemically transformed into JM109 competent cells. The transformants selected correctly were inoculated into 5mL of LB liquid medium containing ampicillin at a final concentration of 100ug/mL, and cultured at 37 ℃ and 180rpm overnight. Plasmid recovery Kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.2.0) is used for extracting Plasmid, and the recombinant vector pUC-HpADE2 delta-ARS is obtained. The construction schematic is shown in figure 2.

Thirdly, a G418 resistance gene (KanMX) is further inserted into the plasmid vector

G418 (Geneticin) is an aminoglycoside antibiotic, and is a commonly used resistance selection reagent for stable transfection in molecular genetic tests.

Primers were designed based on the KanMX nucleotide sequence in Cloning vector pMRI-40 as follows:

primer Kan-1(SEQ ID NO: 4):

5’-ATGTCGACGATATCAAGCTTGCCTCGTC-3’

(underlined is a restriction enzyme site SalI)

Primer Kan-2(SEQ ID NO: 5):

5’-ATCCCGGGTCGACACTGGATGGCGGCGTTA-3’

(underlined is restriction enzyme cleavage site XmaI)

The G418 resistance gene (KanMX) was amplified using the plasmid Cloning vector pMRI-40 as a template and the primers Kan-1 and Kan-2, PCR system: premixing 25 mu L of Pfu DNA polymerase, 1 mu L of template, 1 mu L of each primer and complementing 50 mu L of ultrapure water and mixing uniformly; the reaction conditions were set with a PCR machine (PC707-02, Japan): pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃, 10secs, annealing at 60 ℃, 1min, extension at 72 ℃, 2min, and circulation for 30 times; fully extending at 72 ℃ for 10min once; and finishing cooling to 4 ℃ for storage.

The PCR product was alcohol-precipitated, air-dried, and the plasmid vector pUC-HpADE 2. delta. -ARS was double-digested with restriction enzymes SalI and XmaI, respectively, and the purified digested fragments (1453 bp small fragment and 5507bp large fragment in this example) were recovered by agarose gel electrophoresis, respectively, reacted with T4DNA ligase at 16 ℃ for 12-16 hours for ligation, and the ligation product was chemically transformed into JM109 competent cells. The transformants selected correctly were inoculated into 5mL of LB liquid medium containing ampicillin at a final concentration of 100ug/mL, and cultured at 37 ℃ and 180rpm overnight. Plasmid is extracted by a Plasmid recovery Kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.2.0) to obtain the recombinant vector KMX-HpADE2 delta-ARS. The construction schematic is shown in figure 3.

Fourthly, obtaining the Hansenula polymorpha leucine gene (Hp-LEU2 delta) with the structural gene mutation and constructing a plasmid vector

The primers were designed based on the reported nucleotide sequence of the leucine gene (LEU2) of Hansenula polymorpha as follows:

primer LEU-1(SEQ ID NO: 6):

5’-AGGTCGACAGATCTGGGTTTACCACCCGCA-3’

(underlined is a restriction enzyme cleavage site SphI)

Mutation primer LEU-T1(SEQ ID NO: 7):

(bold TGCG is LEU2 structural gene insertion mutation base)

Mutation primer LEU-T2(SEQ ID NO: 8):

(bold CGCA for LEU2 structural gene insertion mutation base)

Primer LEU-2(SEQ ID NO: 9):

5’-ACGCATGCAGATCTGAGTCCCTGAGTACGT-3’

(underlined is a restriction enzyme site SalI)

Using Hansenula polymorpha ATCC34438 total DNA as a template, the mutant LEU2-1 was amplified with primers LEU-1 and LEU-T1, and the mutant LEU2-2 was amplified with mutant primers LEU-T2 and LEU-2, PCR system: mu.L of Pfu DNA polymerase, 1. mu.L of template, 1. mu.L of each primer, and 50. mu.L of ultrapure water were added thereto and mixed. The reaction conditions were set with a PCR machine (PC707-02, Japan): pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃, 10secs, annealing at 60 ℃, 1min, extension at 72 ℃, 2min, and circulation for 30 times; fully extending at 72 ℃ for 10min once; and finishing cooling to 4 ℃ for storage. And (3) carrying out agarose gel electrophoresis on the PCR products LEU2-1 and LEU2-2, recovering and purifying, diluting by 100 times to be used as a template, amplifying the mutant Hp-LEU2 delta, and carrying out PCR system: mu.L of Pfu DNA polymerase was premixed, 1. mu.L of each of the two templates, and 22. mu.L of ultrapure water was added. The reaction conditions were set with a PCR machine (PC707-02, Japan): pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃, 10secs, annealing at 55 ℃, 1min, extension at 72 ℃, 3min, and 5 times of circulation; the temperature is reduced to 4 ℃. Then, 1. mu.L each of the primer LEU-1 and the primer LEU-2 was added to the reaction system. The reaction conditions were set with a PCR machine (PC707-02, Japan): pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃, 10secs, annealing at 60 ℃, 1min, extension at 72 ℃, 2min, and circulation for 30 times; fully extending at 72 ℃ for 10min once; and finishing cooling to 4 ℃ for storage.

The PCR product Hp-LEU2 delta is precipitated by alcohol, dried, and subjected to double digestion with restriction enzymes SphI and SalI respectively in a plasmid vector KMX-HpADE2 delta-ARS, purified digestion fragments are recovered by agarose gel electrophoresis, reacted for 12-16 hours at 16 ℃ by T4DNA ligase and connected, and the connecting product is chemically transformed into JM109 competent cells. The transformants selected correctly were inoculated into 5mL of LB liquid medium containing ampicillin at a final concentration of 100ug/mL, and cultured at 37 ℃ and 180rpm overnight. Plasmid is extracted by a Plasmid recovery Kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.2.0) to obtain a recombinant vector KMX-HpADE2 delta-HpLEU 2 delta. The construction schematic diagram is shown in figure 4, wherein Hp-LEU2 delta (SEQ ID NO:10, the head and tail 6 bases of the sequence are respectively restriction enzyme cutting sites) has the full length of 2481bp, TGCG is inserted into the starting position of the gene structure of Hansenula polymorpha leucine (LEU2) to introduce a mutation product of four bases for homologous recombination of homologous sequences of Hansenula polymorpha, and the homologous recombination is integrated into a yeast genome through homologous exchange to realize the knock-out of Hansenula polymorpha leucine.

Five, two auxotrophic receptor strain preparation and screening

1mL of an overnight culture (OD) of a wild type strain of Hansenula polymorpha (ATCC34438)₆₀₀6.0-10.0) is transferred into 100mL YPD liquid culture medium at 28-30 ℃ and 250-300 r/culture until logarithmic growth phase (OD) of yeast₆₀₀1.0-1.3), taking 1mL of the strain, subpackaging into 1.5mL of EP tubes, centrifuging for 1min at 4 ℃ at 10000g, discarding the supernatant, washing the precipitate with s sterile physiological saline (precooling at 4 ℃), centrifuging under the same condition, and discarding the supernatant. 1mL of a treatment solution [10mM LiAc, 10mM DTTI, 0.6M sorbitol, 10mM TrisHC1(pH7.5) was added]And standing at room temperature for 20 min. Centrifuging, removing supernatant, adding 1mL precooled 1M sorbitol solution, centrifuging at 4 ℃ and 10000g for 1min, removing supernatant, and washing twice. Finally, 80 μ L of 1M sorbitol solution is added and mixed evenly to obtain competent cells, and the competent cells are stored in ice bath or a refrigerator at minus 80 ℃ for later use.

Preparing a recombinant vector KMX-HpADE2 delta-HpLEU 2 delta 10 mu g, transforming the vector into competent cells by electrotransformation (LN-101 type Gene pulse introduction apparatus manufactured by Tianjin Ringji university, setting 1.5kV, 20 mu F, 380. omega. and time constant 4-7 mSec), adding 1M sorbitol after electrotransformation, mixing at 37 ℃ for 45min, diluting 10 times, taking 200. mu.l of YPD plate coated with 0.1mg/mLG418 for 4 days, picking up single colony growing faster, mixing in sterile water, inoculating (1) solid MD (containing 20. mu.g/mL biotin, 1.34% YNB, 2% glucose) culture plate, (2) solid MD culture plate added with 30. mu.g/mL adenine, (3) solid MD culture plate added with 30. mu.g/mL leucine, (4) solid MD culture plate added with 30. mu.g/mL adenine and 30. mu.g/mL leucine and (YPD 5) culture plate, cultured at 37 ℃ for 4 days. Out of 1000 transformants, two strains (1) (2) (3) that did not grow and (4) (5) that grew normally were selected as leucine and adenine double auxotrophic strains and were designated as HTM01 and HTM 02. After 20 times of subculture in complete medium, the results of the above plate tests are consistent, which indicates that HTM01 and HTM02 have no back mutation and are genetically stable.

Example 2 expression vector construction

Firstly, obtaining a MOX promoter terminator and a hansenula polymorpha autonomous replication sequence and constructing a recombinant vector pBlu-MP (18L1) -HARS

The plasmid vector pMPT-HPV18L1 (see patent CN201210013236.X, HPV18L1 gene inserted between MOX promoter and terminator) is digested with restriction enzymes BssH II and KpnI, and the large fragment MP (18L1) + HARS (containing MOX promoter and terminator and Hansenula polymorpha autonomous replication sequence, 4433bp, SEQ ID NO:20) is recovered and purified by agarose gel electrophoresis and used for ligase connection to construct an expression vector.

Using pBluescript SK (+) as a template, designing a primer:

pB-01(SEQ ID NO:11)：

5’-TAGCGCGCTTGGCGTAATCATG-3’

(underlined is restriction enzyme cleavage site BssHI)

pB-02(SEQ ID NO:12)：

5’-GCGGTACCTGACGCGTCACTGGCCGTCGTTTTACAACG-3’

(underlined restriction sites KpnI, MluI)

Using pBluescript SK (+) plasmid to dilute 10000 times as template, using primer pB-01, primer pB-02 to amplify fragment pBlu, PCR system: premixing 25 mu L of Pfu DNA polymerase, 1 mu L of template, 1 mu L of each primer and complementing 50 mu L of ultrapure water and mixing uniformly; the reaction conditions were set with a PCR machine (PC707-02, Japan): pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃, 10secs, annealing at 60 ℃, 1min, extension at 72 ℃, 3min, and circulation for 30 times; fully extending at 72 ℃ for 10min once; and finishing cooling to 4 ℃ for storage.

The PCR product is precipitated by alcohol, dried and cut by restriction enzymes BssHI and KpnI, purified and cut 2800bp pBlu fragment is recovered by agarose gel electrophoresis, cut fragment MP (18L1) + HARS and fragment pBlu are reacted by T4DNA ligase at 16 ℃ for 12-16 hours for ligation, and the ligation product is chemically transformed into JM109 competent cells. The transformants selected correctly were inoculated into 5mL of LB liquid medium containing ampicillin at a final concentration of 100ug/mL, and cultured at 37 ℃ and 180rpm overnight. Plasmid was extracted with a Plasmid recovery Kit (TaKaRa MiniBEST Plasmid Purification Kit Ver.2.0) to obtain recombinant vector pBlu-MP (18L1) -HARS.

Secondly, obtaining a saccharomyces cerevisiae leucine (LEU2sc) gene and constructing a recombinant vector pMPT-Leu2-HPV18L1

Primers were designed based on the reported nucleotide sequence of the Saccharomyces cerevisiae leucine (LEU2sc) gene as follows:

primer Le-1(SEQ ID NO: 13):

5’-GCACGCGTGTCGACTACGTCGTTAAG-3’

(underlined is restriction enzyme cleavage site MluI)

Mutant primer Le-T1(SEQ ID NO: 14):

5’-GTCCTAAATGGGGTTCCGGTAGTGTTAG-3’

mutant primer Le-T2(SEQ ID NO: 15):

5’-CTAACACTACCGGAACCCCATTTAGGAC-3’

(underlined is removal of the cleavage site KpnI in LEU2 by base mutation for subsequent cloning operation)

Primer Le-2(SEQ ID NO: 16):

5’-CAGGTACCTCGAGGAGAACTTCTAG-3’

(underlined is the restriction enzyme cleavage site KpnI)

The nested PCR method, using beer yeast (S288c) total DNA as a template: amplifying the mutated Leu-1 by using a primer Le-1 and a mutated primer Le-T1, amplifying the mutated Leu-2 by using a mutated primer Le-T2 and a primer Le-2, wherein the PCR system comprises: mu.L of Pfu DNA polymerase, 1. mu.L of template, 1. mu.L of each primer, and 50. mu.L of ultrapure water were added thereto and mixed. The reaction conditions were set with a PCR machine (PC707-02, Japan): pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃, 10secs, annealing at 60 ℃, 1min, extension at 72 ℃, 2min, and circulation for 30 times; fully extending at 72 ℃ for 10min once; and finishing cooling to 4 ℃ for storage. And (3) carrying out agarose gel electrophoresis on the PCR products Leu-1 and Leu-2, recovering and purifying, diluting by 100 times to be used as a template, amplifying the mutant sc-LEU2, and carrying out PCR system: mu.L of Pfu DNA polymerase was premixed, 1. mu.L of each of the two templates, and 22. mu.L of ultrapure water was added. The reaction conditions were set with a PCR machine (PC707-02, Japan): pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃, 10secs, annealing at 55 ℃, 1min, extension at 72 ℃, 3min, and 5 times of circulation; the temperature is reduced to 4 ℃. Then, 1. mu.L of each of the primer Le-1 and the primer Le-2 was added to the reaction system. The reaction conditions were set with a PCR machine (PC707-02, Japan): pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃, 10secs, annealing at 60 ℃, 1min, extension at 72 ℃, 2min, and circulation for 30 times; fully extending at 72 ℃ for 10min once; and finishing cooling to 4 ℃ for storage. The PCR product is subjected to alcohol precipitation, air-dried, double-enzyme digestion by restriction enzymes MluI and KpnI, and agarose gel electrophoresis is carried out to recover and purify a digestion fragment LEU2sc (SEQ ID NO:17, the head and tail 6 bases of the sequence are respectively restriction enzyme sites) for ligase connection to construct an expression vector.

The recombinant vector pBlu-MP (18L1) -HARS is subjected to double digestion by restriction enzymes MluI and KpnI, the large fragment is recovered and purified by agarose gel electrophoresis, and the large fragment is connected with the prepared fragment LEU2sc to construct the vector pMPT-Leu2-HPV18L1 (9212 bp in the example) by the same transformation and screening method.

Thirdly, foreign target genes are inserted to obtain expression vectors pMPT-Leu2-Elafin and pMPT-Leu2-Hirudin

The invention discloses an Elafin full sequence containing MFa leader peptide (SEQ ID NO:18, wherein, the head and tail of the sequence are respectively 6 bases as restriction enzyme sites, the 7 th to 255 th sites are coding sequences of MFa leader peptide, and the 256 th and 435 th sites are coding sequences of the Elafin), and commissioned to Shanghai Bioengineering limited company to chemically synthesize the full sequence as an exogenous target gene for secretory expression.

The vector pMPT-Leu2-HPV18L1 was double-digested with restriction enzymes EcoRI and BamHI, the fragments were recovered and purified by agarose gel electrophoresis, the above described whole sequence of the Elafin containing MFa leader peptide was double-digested with restriction enzymes EcoRI and BamHI, the fragments were recovered and purified by agarose gel electrophoresis, the two fragments were ligated using the same transformation screening method to construct the expression vector pMPT-Leu2-Elafin (8361 bp in this example), the expression vector structure is shown in FIG. 5.

The expression vector pMPT-Leu2-Hirudin (8385bp) was constructed in the same manner as shown in FIG. 6. Wherein, a Hirudin complete sequence containing MFa leader peptide is artificially designed (SEQ ID NO:19, wherein, the head and tail of the sequence are respectively 6 basic groups which are respectively a restriction enzyme cutting site, the 7 th to 255 th sites are coding sequences of the MFa leader peptide, and the 256 th and 459 th sites are coding sequences of Elafin), and the Hirudin complete sequence is entrusted to the chemical synthesis of Shanghai biological engineering Limited company and is used as an exogenous target gene for secretory expression.

Example 3 functional vector construction

Firstly, obtaining Saccharomyces cerevisiae adenine (ADE2sc) gene and carrying out first-step vector replacement

The primers were designed based on the reported nucleotide sequence of the Saccharomyces cerevisiae adenine (ADE2sc) gene as follows:

primer Ad-01(SEQ ID NO: 20):

5’-CAACGCGTCGCTATCCTCGGTTCTGCATTG-3’

(underlined is a restriction enzyme site MLUI)

Primer Ad-02(SEQ ID NO: 21):

5’-TAGGTACCTAACGCCGTATCGTGATTAACG-3’

(underlined is the restriction enzyme cleavage site KpnI)

The total DNA of beer yeast (S288c) is used as a template, a primer Ad-01 and a primer Ad-02 are used for amplifying the adenine gene ADE2sc of the beer yeast, a PCR product is subjected to alcohol precipitation and then is dried and is subjected to double enzyme digestion by restriction enzymes MluI and KpnI, and agarose gel electrophoresis is used for recovering and purifying an enzyme digestion fragment ADE2sc (SEQ ID NO:22, and the head and tail 6 bases of the sequence are respectively restriction enzyme digestion sites) for replacing the Leu2sc position in an expression vector pMPT-Leu 2-Elafin.

Secondly, obtaining the glyceraldehyde-3-phosphate dehydrogenase gene of the Hansenula polymorpha and carrying out the second step of vector replacement

The primers were designed based on the reported nucleotide sequence of glyceraldehyde-3-phosphate dehydrogenase gene of Hansenula polymorpha as follows:

Gp-01(SEQ ID NO:23)：

5’-GTCCCGGGCCTTTGCTCAATGCCGTTTTGG-3’

(underlined is a restriction enzyme cleavage site Sma I)

Gp-02(SEQ ID NO:24)：

5’-GCGGATCCATCGATGAATTCATTGTTTCTATATTATC-3’

(underlined are restriction enzyme sites BamHI, EcoRI)

Gp-03(SEQ ID NO:25)：

5’-ATGAATTCATCGATGGATCCGCACGGCCTCATCTAC-3’

(underlined restriction sites EcoRI, BamHI)

Gp-04(SEQ ID NO:26)：

5’-TGCGCGCGAGCTCGGACCACAATCCAAATAAAG-3’

(underlined is restriction enzyme cleavage site BssHI)

The same nested PCR method as used for the amplification of the Saccharomyces cerevisiae leucine (LEU2sc) gene in example 2 was used to amplify constitutive promoter and terminator sequences of the glyceraldehyde-3-phosphate dehydrogenase gene (pGAP, SEQ ID NO:27, the first and last 6 bases of the indicated sequence being restriction sites, respectively).

And (3) carrying out alcohol precipitation on the PCR product, airing, carrying out double digestion by using restriction enzymes XmaI and BssHI, and recovering and purifying a digestion fragment GPT by agarose gel electrophoresis for replacing the MOX promoter terminator position in the expression vector pMPT-Leu 2-Elafin. The vector pGPT-ADE2 was constructed by the above two-step replacement.

Thirdly, obtaining hansenula polymorpha HAC1 gene and carrying out third-step vector replacement to obtain a functional vector pGPT-ADE2-HAC1p

A primer is designed according to the reported hansenula polymorpha HAC1 gene nucleotide sequence, the HAC1 gene is amplified by taking a hansenula polymorpha ATCC34438 genome as a template, and the primer sequence is as follows:

HA-01(SEQ ID NO:28)：

(underlined restriction sites EcoRI)

HA-02(SEQ ID NO:29)：

5’-CTGTTAATGGTGCTGCTGCTGGATGATGCAC-3’

HA-03(SEQ ID NO:30)：

5’-GTGCATCATCCAGCAGCAGCACCATTAACAG-3’

HA-04(SEQ ID NO:31)：

5’-CAGGATCCTCAAGACAAATAGTCG-3’

(underlined is a restriction enzyme cleavage site BamHI)

The following HAC1p structural gene (SEQ ID NO:32, positions 3-8 and 987-992 of the indicated sequence are restriction sites, respectively) was amplified by the same nested PCR method as used in example 2 for the Saccharomyces cerevisiae leucine (LEU2sc) gene.

After the PCR product is precipitated by alcohol, the PCR product is dried in the air, the vector pGPT-ADE2 is subjected to double digestion by restriction enzymes EcoRI and BamHI respectively, agarose gel electrophoresis is carried out to recover and purify the digestion fragments, and ligase ligation is carried out to insert HAC1p into the vector pGPT-ADE2 to construct a functional vector pGPT-ADE2-HAC1p (figure 7).

EXAMPLE 4 construction of engineered strains of Hansenula polymorpha secreting and expressing Elfin (Elafin) by double plasmid cotransformation

Preparation of competent cells of the one-leucine-and-adenine-double auxotrophic strain HTM01

1mL of Hansenula polymorpha double auxotrophic strain HTM01 overnight culture (OD)₆₀₀6.0 to 10.0) is rotationally connected to 1Culturing at 28 deg.C and 250-₆₀₀1.0-1.3), taking 1mL of the strain, subpackaging into 1.5mL of EP tubes, centrifuging for 1min at 4 ℃ at 10000g, discarding the supernatant, washing the precipitate with s sterile physiological saline (precooling at 4 ℃), centrifuging under the same condition, and discarding the supernatant. 1mL of a treatment solution [10mM LiAc, 10mM DTTI, 0.6M sorbitol, 10mM TrisHC1(pH7.5) was added]And standing at room temperature for 20 min. Centrifuging, removing supernatant, adding 1mL precooled 1M sorbitol solution, centrifuging at 4 ℃ and 10000g for 1min, removing supernatant, and washing twice. And finally adding 80 mu L of 1M sorbitol solution, mixing uniformly to obtain competent cells HTM01, and storing in ice bath or a refrigerator at-80 ℃ for later use.

Two, co-transformation and screening of high expression strain

The DNA concentration was measured, and 10. mu.g of the expression vector pMPT-Leu2-Elafin prepared in example 2 and 10. mu.g of the expression vector functional vector pGPT-ADE2-HAC1p prepared in example 3 (the sum of the volumes of the two plasmid vectors is less than 6. mu.L) were added to 80. mu.L of the competent cell HTM01 prepared above and mixed well. The gene was transformed into the competent cells simultaneously by using LN-101 type gene pulse introduction instrument manufactured by Tianjin Risk university with setting of 1.5kV, 20. mu.F, 380. omega. and time constant of 4-7 mSec. After the electric transformation, 700. mu.L of 1M sorbitol was added, mixed well, left to stand at 37 ℃ for 45min, diluted 3 times, and 200. mu.L of a spread solid MD (containing 20. mu.g/ml biotin, 1.34% YNB, 2% glucose) was applied to a culture plate and cultured at 37 ℃ for 4 days.

300 single colonies of the transformants are picked and subcultured for 10 times by using a solid MD culture medium, and then are subcultured by transferring into a liquid MD culture medium for about 200 generations. During the passage, the yeast genome was extracted by a glass bead preparation method (A. Adams et al, guide to Yeast genetics methods, science publishers, 2000) using PCR primers: an upstream primer: 5'-TCAAAAGCGGTATGTCCTTCCACGT-3' and the downstream primer: 5'-GCACGGTGGTGACATCAATCTAAAG-3' PCR screening was performed to obtain 26 high copy strains. The high copy strain is placed in 10mL of glycerol induction medium, and 200uL of inducer (25% glycerol as inducer) is added every 12h for induction expression for 72 h. The activity measurement result shows that the 17# strain has the highest expression activity, and the induction screening is repeated three times. The strain # 17 was inoculated into YPD medium (liquid medium containing 2% glucose, 2% soybean peptone and 1% yeast extract powder) for subculture for 3 days to remove free plasmid. The strain # 17 after subculture was diluted and plated on MD solid medium (1.34% YNB, 2% glucose, 2% agar powder) for subcloning. 30 subclones were picked and induced to express in 10mL glycerol medium, with the highest expressing strain being Elafin 17-09.

The activity measuring method comprises the following steps: the activity of Porcine Pancreatic Elastase (PPE) can be calculated by measuring the absorbance value at 410nm of a fluorescent substance produced by its hydrolysis substrate Suc-Ala-Ala-Pro-Abu-PNA (Electron Abscissubstrate IV, S4). Elafin is a PPE specific inhibitor that is able to react with PPE in a 1: 1, the higher the content of rhElafin in the fermentation supernatant, the stronger the inhibitory activity on PPE. This experiment is described in reference to the reference document [ Eur J Biochem,2004,271(12):2370-8] which comprises incubating a fermentation broth supernatant (containing rhElafin) and PPE in an incubator at 37 ℃ for 20min, adding S4 to the reaction mixture, incubating in the incubator at 37 ℃ for 30min, and measuring the absorbance of the reaction product of PPE and S4 remaining after the action of rhElafin at 410nm to determine the amount of rhElafin in the supernatant, using HEPES having a pH of 8.0 as a buffer. The activity of the Elafin is detected by measuring the A410 value of the recombinant Elafin in the drug shake flask induction or the supernatant of the fermentation liquor after the recombinant Elafin acts on the porcine pancreatic elastase, and the Hansenula polymorpha engineering bacteria with high expression of the rhElafin can be screened out or the fermentation expression level can be detected by the method.

Engineering strain fermentation process

A high-expression strain screened by transforming HU11 (uracil single-nutrition deficiency receptor strain) with a pMPT-URA3-Elafin expression vector is used as a reference strain Efn2-13 (the construction embodiment of the engineering strain is the same as that of the invention patent CN201210013236.X, and a functional gene HAC1p is not transferred), and the high-expression strain is compared with the high-expression strain Efn17-09 (a functional gene HAC1p is transferred) screened. The biomass and product activity were compared using the same fermentation process conditions as follows.

1) Preparation of a culture medium:

seed culture medium: 2g/100ml glucose, 1.34g/100ml YNB; 200mL of primary seed culture medium and 1600mL of secondary seed culture medium, and autoclaving.

A supplemented medium: dissolving NH4H2PO 487 g in 540mL of water, and autoclaving; another 260g of glycerol was autoclaved and combined on a clean bench after sterilization.

Derepression medium: 3L, NH in total₄H₂PO₄ 27g、MgSO₄·7H₂Dissolving O6 g, KCl 6.8g and NaCl 0.7g in 660mL of water, and autoclaving; another 1.8L of glycerol was autoclaved. After sterilization, the components were combined in a clean bench.

Fermentation medium: NH (NH)₄H₂PO₄ 175g、MgSO₄·7H₂40g of O, 44g of KCl, 4.4g of NaCl, 520g of glycerol and 4mL of antifoaming agent are added, the initial volume of fermentation is 12L, and the autoclave is sterilized for 30 minutes at the temperature of 110 ℃ and 115 ℃.

2) Fermentation process

Taking a frozen Hansenula polymorpha recombinant strain 14-13(1mL, OD) from a freezer at-70 DEG C₆₀₀About 100) is inoculated into 200mL of primary seed culture medium, the mixture is cultured at 31 ℃ for 22 hours and then transferred into 1600mL of secondary seed culture medium for amplification culture, and the OD600 of the cells is over 10 after the mixture is cultured at 31 ℃ for 22 hours and then the cells are used as fermentation seeds.

The secondary seeds were inoculated into a 30L fermentor (Japan pellet physicochemical apparatus research institute, model: MSJ-J2) containing 12L of sterilized fermentation medium to start fermentation.

The fermentation process mainly comprises two phases, namely a growth phase and a derepression phase.

Growth phase: controlling the pH value of the fermentation tank to be 5.4-5.5; controlling the fermentation temperature to be 30 ℃; when the dissolved oxygen in the fermentation tank (monitored by an FC-280 type dissolved oxygen monitor of the biological engineering college of university of eastern science and technology) slowly drops to about 60 percent (about 16 hours of fermentation) under the pressure of 0.5Kg/cm, a material supplementing pipeline is connected, and about 400mL of material supplementing solution is pumped into the fermentation tank at a constant speed by a peristaltic pump. When the dissolved oxygen level in the fermentation tank is reduced to below 40%, the air flow is adjusted to 15L/min, and the stirring speed is adjusted to 600 rpm. In the later growth phase (about 22 hours of fermentation), if the cell growth state is good, the dissolved oxygen level in the fermentation tank can be reduced to below 20 percent; cell OD₆₀₀Up to more than 80.

Derepression phase: before the dissolved oxygen rises back, connecting a power plug of the peristaltic pump with a signal output power supply of a dissolved oxygen monitoring instrument; the dissolved oxygen of the monitor is controlled by 40 percent, the dead zone is 2 percent, and the high end is opened; the peristaltic pump power switch is turned off. When the dissolved oxygen of the fermentation tank is observed to rise back to more than 60% from the lowest point, entering a derepression time phase; at this point, the peristaltic pump power switch is turned on, the pump speed is set at 22rpm, and the flow of the de-repressed solution is initiated. In the post derepression stage, OD if cell growth is good₆₀₀Can reach more than 350, and the fermentation time is about 96 hours. After the fermentation was completed, centrifugation was performed using 500mL centrifuge cups, Sigma 6K-15 centrifuge 7000rpm, and the supernatant was collected for subsequent purification treatment.

Sampling every 24h in the fermentation process, and measuring OD (origin-destination) of fermentation liquor at different time points₆₀₀Comparison of biomass, the results are shown in FIG. 8; the supernatant was diluted 10 times, 60. mu.l each was used to determine the activity of Elafin, which was converted to mg/L expression amount to observe the relative relationship between the expression product Elafin and the induction time, and the results are shown in FIG. 9.

Fourth, comparison of functional Gene HAC1p related Gene expression

Control strain Efn2-13 (without transferred functional gene HAC1p) and strain Efn17-09 (with transferred functional gene HAC1p) were transferred into MD (containing 20. mu.g/ml biotin, 1.34% YNB, 2% glucose) and cultured at 31 ℃ for 1 day, replaced with MM (containing 20. mu.g/ml biotin, 1.34% YNB) culture medium, 200uL inducer (25% glycerol) was added for induction expression, 200uL inducer was added every 12 hours, samples were taken after 1 day of culture at 31 ℃, total RNA was extracted from the cells, cDNA was obtained by reverse transcription of the obtained RNA, and relative quantitative detection was performed on functional gene HAC1p and secretory key gene by RT-PCR.

RT-PCR mainly selects protein folding related genes KAR2, ERO1 and HAC1 p; protein degradation related genes PRB1 and BUL 1; several genes of protein transport related gene SEC31 were examined as representatives. As a result, as shown in FIG. 10, it was found that the transcription level of the functional gene HAC1p was significantly increased, and the downstream gene was significantly regulated in a direction favorable for secretory expression. This provides a theoretical basis for the above increase of cell growth rate and the obvious increase of fermentation secretion expression. The expression quantity of exogenous target gene Elafin is improved by 2 times and the fermentation performance of engineering strains is also improved by transferring functional genes into continuous expression or even over-expression.

EXAMPLE 5 construction of engineered Strain of Hansenula polymorpha secreting and expressing Hirudin (Hirudin) by two plasmid Co-transformation

Preparation of competent cells of the one-leucine-and-adenine-double auxotrophic strain HTM01

Competent cells HTM01 were prepared as in example 4 above and stored in ice or in a freezer at-80 ℃ until use.

Two, co-transformation and screening of high expression strain

The DNA concentration was measured, and 10. mu.g of the expression vector pMPT-Leu2-Hirudin prepared in example 2 and 10. mu.g of the expression vector functional vector pGPT-ADE2-HAC1p prepared in example 3 (the sum of the volumes of the two plasmid vectors is less than 6. mu.L) were added to 80. mu.L of the competent cell HTM01 prepared above and mixed well. The gene was transformed into the competent cells simultaneously by using LN-101 type gene pulse introduction instrument manufactured by Tianjin Risk university with setting of 1.5kV, 20. mu.F, 380. omega. and time constant of 4-7 mSec. After the electric transformation, 700. mu.L of 1M sorbitol was added, mixed well, left to stand at 37 ℃ for 45min, diluted 3 times, and 200. mu.L of a spread solid MD (containing 20. mu.g/ml biotin, 1.34% YNB, 2% glucose) was applied to a culture plate and cultured at 37 ℃ for 4 days.

200 single colonies of the transformants are picked and subcultured for 10 times by a solid MD culture medium, and then are subcultured by a liquid MD culture medium for about 200 generations. During the passage, the yeast genome was extracted by a glass bead preparation method (A. Adams et al, guide to Yeast genetics methods, science publishers, 2000) using PCR primers: an upstream primer: 5'-TCAAAAGCGGTATGTCCTTCCACGT-3' and the downstream primer: 5'-GCACGGTGGTGACATCAATCTAAAG-3' PCR screening was performed to obtain 18 high copy strains. The high copy strain is placed in 10mL of glycerol induction medium, and 200uL of inducer (25% glycerol as inducer) is added every 12h for induction expression for 72 h. The chromogenic substrate method activity measurement result shows that the 9# strain has the highest expression activity, and the induction screening is repeated three times. The 9# strain was inoculated into YPD medium (liquid medium containing 2% glucose, 2% soybean peptone and 1% yeast extract powder) for subculture for 3 days to remove free plasmid. The subcultured strain # 9 was diluted and plated on MD solid medium (1.34% YNB, 2% glucose, 2% agar powder) for subcloning. 30 subclones were picked and induced to express in 10mL glycerol medium, with the highest expressing strain being HRN 9-16.

Chromogenic substrate method activity assay: the antithrombin activity of the hirudin can be indirectly detected by measuring the TH amount which is not neutralized by the hirudin in a reaction system by taking a chromogenic reagent Chromozym TH as a substrate and hydrolyzing the chromogenic reagent by thrombin to release nitrobenzene which has specific absorption at 405 nm. The method comprises the following specific steps: 50uL of the diluted standard solution and the sample are respectively taken by a 1.5mL centrifuge tube; adding 50uL of thrombin solution, and keeping the temperature at 37 ℃ for 5 minutes; 50uL of chromogen reagent Chromozym TH is added into each tube, and the reaction is carried out for 2 minutes at 37 ℃; 50uL of acetic acid stop solution is added into each tube; immediately after 600uL of distilled water was added to each tube, the OD was measured at 405 nm. Sample activity (ATU/mL) was calculated from the standards.

By measuring the activity of recombinant hirudin in drug shake flask induction or fermentation liquor supernatant, the method can screen out the engineering bacteria of the hansenula polymorpha with high hirudin expression or detect the fermentation expression level.

Engineering strain fermentation process

A high-expression strain screened by transforming HU11 (uracil single nutrition deficiency receptor strain) with a pMPT-URA3-Hirudin expression vector is used as a reference strain 205-17 (functional gene HAC1p is not transferred, see patent CN200810103154, and the strain preservation number is CGMCC No.2424), and is compared with the high-expression strain HRN9-16 (functional gene HAC1p is transferred). 30L scale fermentations were carried out using the same fermentation process conditions as in example 4.

Sampling every 24h in the fermentation process, and measuring OD (origin-destination) of fermentation liquor at different time points₆₀₀Comparison of biomass, the results are shown in FIG. 11; the supernatant was diluted 10-fold, and the activity of Hirudin was determined, and the expression level was compared as shown in FIG. 12. As can be seen by comparison, the functional gene HAC1p is transferred into the engineering strain for continuous expression and even over-expression, the fermentation performance of the engineering strain is unchanged, and the expression level of the exogenous target gene Hirudin is improved by 0.5 times.

Example 6 real-time quantitative PCR detection of copy number of functional gene and exogenous target gene of engineering strain

Hansenula polymorpha engineered strains Efn17-09 and HRN9-16 overnight cultures (OD)₆₀₀6.0-10.0)), taking about 1mL of 10OD yeast to a 1.5mL centrifuge tube, centrifuging at 6000rpm for 5min, removing supernatant, adding acid-washed glass beads with the same volume as the precipitated bacteria and 100 mu L of cell disruption solution, and placing on an oscillator for oscillation for 5 min-10 min.

After the cell disruption was completed, 100. mu.L of sterile water was added to the centrifuge tube, 100. mu.L of phenol/chloroform extract was added thereto, the mixture was left for ten minutes at 4 ℃ after being mixed, and centrifuged for 5 minutes at 10000rpm in a desk centrifuge, and the supernatant was taken for dilution as a template for amplification reaction. The prepared DNA is not suitable for long-term storage in a refrigerator, and generally does not exceed 24 hours.

The MOX gene is known to be a single copy gene of Hansenula polymorpha, the detection method takes the MOX gene as an internal standard, designs primers for simultaneously amplifying the MOX gene, the HAC1p gene and an exogenous target gene of the same sample, detects the genes by using a fluorescent quantitative PCR instrument, determines the copy numbers of the HAC1p gene and the exogenous target gene contained in a single cell through relative comparison, and compares the doses of the two genes.

The MOX gene primer sequences were designed as follows:

MXcr-1(SEQ ID NO:33)：5’-AACCTCACAGTTGCCCTGA-3’

MXcr-2(SEQ ID NO:34)：5’-CGATGGTCTGGACGAGTAGAA-3’

the HAC1p primer was designed as follows:

HAcr-1(SEQ ID NO:35)：5’-GAAATCGCCCAAATCGTTATC-3’

HAcr-2(SEQ ID NO:36)：5’-TCGGTTGCTTCTTGTCCTG-3’

the Elafin target gene primers were designed as follows:

Elcr-1(SEQ ID NO:37)：5’-TCTTGCCCAATCATCCTGA-3’

Elcr-2(SEQ ID NO:38)：5’-AACGAAGCACGCCATACC-3’

the primers of the Hirudin target gene are designed as follows:

HRNcr-1(SEQ ID NO:39)：5’-AGGACGAGACCGCTCAGAT-3’

HRNcr-2(SEQ ID NO:40)：5’-GAGGCAATAGTGGTGTTGATGA-3’

a fluorescence quantitative PCR instrument of Rotor-Gene 3000 type was used. Real-time quantitative PCR reaction thermal cycle parameters: pre-denaturation at 94 ℃ for 5 min; 15secs denaturation at 94 ℃, 50secs annealing and extension at 60 ℃ and 35 cycles; changes in fluorescence intensity were detected by the Rotor-Gene detection system at the end of each cycle extension phase and the data were analyzed by Rotor-Gene 5.0.28(Corbett Research) software after the amplification reaction was completed.

The detection results of the copy numbers of functional genes and exogenous target genes of the hansenula polymorpha engineering strains Efn17-09 and HRN9-16 are shown in Table 1:

TABLE 1

Strain number	Ct (functional gene)	Ct(MOX)	ΔCt	N
					Efn17-09	16.07±0.09	19.26±0.06	3.19±0.11	9.1
HRN9-16	15.76±0.11	19.81±0.08	4.05±0.14	16.6
					Strain number	Ct (target gene)	Ct(MOX)	ΔCt
Efn17-09	14.06±0.10	19.26±0.06	5.20±0.12	36.7
					HRN9-16	13.76±0.15	19.81±0.08	6.05±0.17	66.3

Through relative quantification, the copy number of the functional gene HAC1p of the Hansenula polymorpha engineering strain Efn17-09 is about 9, and the copy number of the target gene Elafin is about 37; the copy number of functional gene HAC1p of Hansenula polymorpha engineering strain HRN9-16 is about 17, and the copy number of target gene Hirudin is about 66.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Xindada

<120> double plasmid cotransformation exogenous gene high expression gene engineering bacterium

<130> XYD1701

<160> 40

<170> SIPOSequenceListing 1.0

<210> 1

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cagaattcat ggatggactc gaaggtcgtt ggaat 35

<210> 2

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

acgagctcct atttattaag gtattcttca t 31

<210> 3

<211> 1720

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaattcatgg atggactcga aggtcgttgg aattttgggc ggcggccagc tcggccgcat 60

gatggtcgag gcagctagcc gactgaacat caaaacagtg attcttgaga acggcgcaga 120

ttcgccggcc aagcagatca attccagtgc agaacacatc gacggctcct tcaacgatga 180

ggcggccatc cgcaagctcg ccgagaaatg cgacgtgctg accgttgaga ttgagcacgt 240

tgatgttgag gccttgaaga aggtgcagga gcaaacttcc gtcaagatct atccatctcc 300

tgagaccatt gctcttatca aggacaaata cttgcagaaa gagcatttga tcaggaacca 360

gatcgccgtt gctgagtcca ctgctgttga aagcactgct caagctttgc aatctgtggg 420

acagaagtat ggatatccgt acatgctcaa gtccagaacg atggcttatg atggtagggg 480

taactttgtt gttgaggatg tttccaagat cccagaggct ttggaagctt tgaaggacag 540

accgctctat gctgaaaaat gggctccctt taccaaggag ctagcagtga tggtggtgcg 600

gggtcttggc ggagacgtcc atgcctaccc aacagtagag actatccaca aaaacaatat 660

ctgccacacg gtgtttgcgc ctgcgcgtgt caatgacacc atacagaagc gcgcgcaact 720

tttggcggag aaggctgtgt ctgcattccc gggagcaggc atctttggtg tcgaaatgtt 780

cctgcttcca aatgacgagc tgttgatcaa cgagattgct cctagaccgc acaactctgg 840

acattacaca atcgatgcgt gtgtgacgag ccagtttgag gcacacatcc gtgccgtttg 900

cagtctgccg ctgccaaaga actttacttc tctatccaca ccatctaccc atgctatcat 960

gttaaacgtg ttgggcagcc ctgatccaga agagtggttg caaaagtgca agagagcgct 1020

tgaaactccg cacgcgtcgg tttatctgta cggaaaatcc aacagaccgg gccggaaact 1080

gggtcatatc aacattgtct cccagtcgat ggacgactgc atccgtcgtc tagagtacat 1140

agatggcaaa tctggcactc tgaaagagcc agaaaacaac acaggtgttg caggaaccag 1200

cagcaaacct ctagtcggcg tgataatggg ctcagactcg gatctgcctg tgatgtccct 1260

tggttgcaat attctgaaat cttttggcgt tcctttcgag gttaccattg tgtccgccca 1320

cagaacgcct cagagaatgg tcaagtacgc tgccgaagcc ccagagaggg gaatacggtg 1380

catcatcgct ggcgctggtg gagctgccca tctaccagga atggttgctg ccatgactcc 1440

attgccggtc attggtgttc ccgtcaaggg atcgactctc gacggagtcg actcgctgta 1500

ttcgatagtt cagatgccta gaggagttcc tgtggccact gttgccatca acaatgccac 1560

caacgctgcg cttctggccg tgcgtattct tggctcgttc gacccagtgt atttcagcaa 1620

aatggcgaaa tacatgagcg agatggaaaa tgaggttctt gaaaaggctg aacggttggg 1680

ctctgttggc tatgaagaat accttaataa ataggagctc 1720

<210> 4

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgtcgacga tatcaagctt gcctcgtc 28

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atcccgggtc gacactggat ggcggcgtta 30

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aggtcgacag atctgggttt accacccgca 30

<210> 7

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gccccgaggt tgttgcgtgc ggaggccgtc aaggttct 38

<210> 8

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agaaccttga cggcctccgc acgcaacaac ctcggggc 38

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acgcatgcag atctgagtcc ctgagtacgt 30

<210> 10

<211> 2481

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gtcgacagat ctgggtttac cacccgcaaa cctcctgtcg ggcactattc ggcggcaccg 60

cggctccagc ggcagcgtcc cggcaacgaa ccgtcgctga accgtctcga cgtgcccggc 120

atgtccagtc tgtcgtcctc ggacatgaat ccgaatgcgg ttggctccga ttcctgctgc 180

cgcacctcgc gagccagctg gaggctgacg ggaagacgac gccaaaatgc tggatgcgcg 240

cttttcgcta aggttgtctc tgtcgtcgcg ctggcgtctg cgccaaaaca ggaacaggag 300

caggagtccc aagagcagca aagctccgag gatcgacccg acagcgattc cggccttcgc 360

gcccttgctg agccccgtgg acctgttgtt ggcgctcgag gcacttgagg acgacgcggc 420

agccgacgcc gaggtggctg tagcgacgga ctgcgtgatg tagatgatcg aggtctgcat 480

gtttccaggt gaaacggacg tagttatccg cactgattcg gatgagggtg tggacgaaga 540

ggtggttgaa gacgacgacg agctctggga gaaagaaaag gacgaaatcg agcttgttgt 600

agtcgagcta gcggacagac ttgaggacgt tggagacgag tctgtggaag acatggtgga 660

gctctgtggg gaggaacttt gcttggagga agacgatgtt gaggagctgg acagactgga 720

actggaggag gaggtggtgg aggtggagga ggtggaagtg gcatcgtctg tggacgagtg 780

cgacgactgc gccgacgaac tgtcgtcgtc gttttcctca gactcgtcaa catagacgga 840

ataagagtcg ctaccaccac acttctccat gggataacca gtgcaggacg ttgtgcacga 900

gccgtcgggg ttgtccgaag gtacgtcgtc tccgcaataa cagtcctggc cgttgatcaa 960

tgcagcaact gcgtgtcccg aacactgctc cgtgcaatgg ctggagctct gccatgtgta 1020

cgagtccttc ttggtcaagc cggaaatggc atccgagctg tagcaaccta aatacgtaaa 1080

gtctgctagg acttgggtga tcacgaatac gacaccgaat ctcatgttga catggaaaag 1140

ctaaaaaata aacaatagag cgtcgcgtcg aaaaaaataa aatctgtgag gcgtgcgtgt 1200

ttaggatttt tgggattatt taggaattat tcgagatcct actttttttt ctcccttatt 1260

tagttcttga gtagcttggc cacctcctgc gcaacagcat caccaacttc ctgtgttgag 1320

gagcttccac ccaaatcggc agtcatgata cctgcatcca ggacgttctt gacggcctgc 1380

tcgatcgcac ggccggcatc caccaagtcc agcgacagct tcagcatcat ggcggcagac 1440

aaaattgtgg ccaatggatt gaccttgccc gggcccaaat ctggcgccga gccgtggcag 1500

ggctcgtaaa gaccaaacgc cttgtttgtg tctggcagag acgccaacga ggcagaaggc 1560

agcaggccca gagacccagg aatcacactg gcctcgtcac tgatgatgtc gccaaacatg 1620

ttgttggtga caatgacacc gttgagtttc gttggcgact tgaccaaaat catggctgcc 1680

gagtcgatca gctggtgctg caccgtcagc tgcgggaact cgttcttgat ggtctcctca 1740

acagtcttcc gccacaaacg cgaggatgca agcacgttgg ctttgtccag cgaccacagt 1800

gggagcggtg ggtcgctctg cagcgccaaa aaggccgcca ttctcgtgat tctctgcacc 1860

tctggaacag aatagctctc agtgtcgctg gcaactccgt cgccggcatc ctccttgcgg 1920

tcaccaaagt agattccacc aaccaactca cgcacaacaa caaagtcagt gcccttgacg 1980

atttctgatt tcagtggaga tagcttcaga agagcgtcgg aagcaaaact gcatggacgc 2040

aggttcgcgt acaagttgag ctcttttctg atcttcaaca gaccctgctc aggacgcacg 2100

gagccggttc cccacttagg tcctccgacg gctccaagca aaacggcgtc agccttcttg 2160

gcggcttcga gggcctcgtc ggacaatggc accccataag catcgatcga ggcaccgccg 2220

atcaggtgct tggaaaagtt gaacttaacg ccgattgccg acgagacagc ctcgagaacc 2280

ttgacggcct ccgcacgcaa caacctcggg gcccacgtga tcaccaggga gaagcacaat 2340

gttcttactc atgattgcaa aatgatgcaa ctattttgcg ccggtaccgg aaaaattgaa 2400

aaaccatcca cttactcatt cctgtctttt tatttcgtat taccaaaccg cttacgtact 2460

cagggactca gatctgcatg c 2481

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tagcgcgctt ggcgtaatca tg 22

<210> 12

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gcggtacctg acgcgtcact ggccgtcgtt ttacaacg 38

<210> 13

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gcacgcgtgt cgactacgtc gttaag 26

<210> 14

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gtcctaaatg gggttccggt agtgttag 28

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ctaacactac cggaacccca tttaggac 28

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

caggtacctc gaggagaact tctag 25

<210> 17

<211> 2232

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

acgcgtgtcg actacgtcgt taaggccgtt tctgacagag taaaattctt gagggaactt 60

tcaccattat gggaaatggt tcaagaaggt attgacttaa actccatcaa atggtcaggt 120

cattgagtgt tttttatttg ttgtattttt ttttttttag agaaaatcct ccaatatcaa 180

attaggaatc gtagtttcat gattttctgt tacacctaac tttttgtgtg gtgccctcct 240

ccttgtcaat attaatgtta aagtgcaatt ctttttcctt atcacgttga gccattagta 300

tcaatttgct tacctgtatt cctttacatc ctcctttttc tccttcttga taaatgtatg 360

tagattgcgt atatagtttc gtctacccta tgaacatatt ccattttgta atttcgtgtc 420

gtttctatta tgaatttcat ttataaagtt tatgtacaaa tatcataaaa aaagagaatc 480

tttttaagca aggattttct taacttcttc ggcgacagca tcaccgactt ccgtggtact 540

gttggaacca cctaaatcac cagttctgat acctgcatcc aaaacctttt taactgcatc 600

ttcaatggcc ttaccttctt caggcaagtt caatgacaat ttcaacatca ttgcagcaga 660

caagatagtg gcgatagggt tgaccttatt ctttggcaaa tctggagcag aaccgtggca 720

tggttcgtac aaaccaaatg cggtgttctt gtctggcaaa gaggccaagg acgcagatgg 780

caacaaaccc aaggaacctg ggataacgga ggcttcatcg gagatgatat caccaaacat 840

gttgctggtg attataatac catttaggtg ggttgggttc ttaactagga tcatggcggc 900

agaatcaatc aattgatgtt gaaccttcaa tgtagggaat tcgttcttga tggtttcctc 960

cacagttttt ctccataatc ttgaagaggc caaaacatta gctttatcca aggaccaaat 1020

aggcaatggt ggctcatgtt gtagggccat gaaagcggcc attcttgtga ttctttgcac 1080

ttctggaacg gtgtattgtt cactatccca agcgacacca tcaccatcgt cttcctttct 1140

cttaccaaag taaatacctc ccactaattc tctgacaaca acgaagtcag tacctttagc 1200

aaattgtggc ttgattggag ataagtctaa aagagagtcg gatgcaaagt tacatggtct 1260

taagttggcg tacaattgaa gttctttacg gatttttagt aaaccttgtt caggtctaac 1320

actaccggaa ccccatttag gaccacccac agcacctaac aaaacggcat caaccttctt 1380

ggaggcttcc agcgcctcat ctggaagtgg gacacctgta gcatcgatag cagcaccacc 1440

aattaaatga ttttcgaaat cgaacttgac attggaacga acatcagaaa tagctttaag 1500

aaccttaatg gcttcggctg tgatttcttg accaacgtgg tcacctggca aaacgacgat 1560

cttcttaggg gcagacatta gaatggtata tccttgaaat atatatatat atattgctga 1620

aatgtaaaag gtaagaaaag ttagaaagta agacgattgc taaccaccta ttggaaaaaa 1680

caataggtcc ttaaataata ttgtcaactt caagtattgt gatgcaagca tttagtcatg 1740

aacgcttctc tattctatat gaaaagccgg ttccgcggct ctcacctttc ctttttctcc 1800

caatttttca gttgaaaaag gtatatgcgt caggcgacct ctgaaattaa caaaaaattt 1860

ccagtcatcg aatttgattc tgtgcgatag cgcccctgtg tgttctcgtt atgttgagga 1920

aaaaaataat ggttgctaag agattcgaac tcttgcatct tacgatacct gagtattccc 1980

acagttaact gcggtcaaga tatttcttga atcaggcgcc ttagaccgct cggccaaaca 2040

accaattact tgttgagaaa tagagtataa ttatcctata aatataacgt ttttgaacac 2100

acatgaacaa ggaagtacag gacaattgat tttgaagaga atgtggattt tgatgtaatt 2160

gttgggattc catttttaat aaggcaataa tattaggtat gtagatatac tagaagttct 2220

cctcgaggta cc 2232

<210> 18

<211> 450

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gaattcatga gattcccttc gatctttacc gccgtgctgt tcgcagcatc ttccgccctg 60

gccgctccag tcaataccac gacagaggac gagaccgctc agatccccgc tgaggccgtc 120

atcggttact ctgatcttga gggagacttc gacgtggctg ttttgccatt ttccaactcg 180

actaataacg gacttctgtt catcaacacc actattgcct cgattgccgc gaaagaagag 240

ggcgtcagcc tcgagaagag agcgcaggaa cctgttaaag gtccggtttc taccaaaccg 300

ggttcttgcc caatcatcct gatccgttgc gcgctgctga acccgccgaa ccgttgcctg 360

aaagacaccg actgcccggg tatcaaaaag tgctgcgaag gctcttgcgg tatggcgtgc 420

ttcgttccgc agtaatgaag atctggatcc 450

<210> 19

<211> 474

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gaattcatga gattcccttc gatctttacc gccgtgctgt tcgcagcatc ttccgccctg 60

gccgctccag tcaataccac gacagaggac gagaccgctc agatccccgc tgaggccgtc 120

atcggttact ctgatcttga gggagacttc gacgtggctg ttttgccatt ttccaactcg 180

actaataacg gacttctgtt catcaacacc actattgcct cgattgccgc gaaagaagag 240

ggcgtcagcc tcgagaagag aatcacctac accgactgca ccgagtccgg acagaacctg 300

tgcctgtgcg agggctccaa cgtctgcggc cagggcaaca agtgcatcct gggcagagac 360

ggcgagaaga accagtgcgt caccggcgag ggaaccccaa agccacagtc ccacaacgac 420

ggcgacttcg aggagatccc agaggagtac ctgcagtaat gaagatctgg atcc 474

<210> 20

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

caacgcgtcg ctatcctcgg ttctgcattg 30

<210> 21

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

taggtaccta acgccgtatc gtgattaacg 30

<210> 22

<211> 2695

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

acgcgtcgct atcctcggtt ctgcattgag ccgccttata tgaactgtat cgaaacgtta 60

tttttttaat cgcagactta agcaggtaat tattccttgc ttcttgttac tggatatgta 120

tgtatgtata ataagtgatc ttatgtatga aattcttaaa aaaggacacc tgtaagcgtt 180

gatttctatg tatgaagtcc acatttgatg taatcataac aaagcctaaa aaataggtat 240

atcattttat aattatttgc tgtacaagta tatcaataaa cttatatatt acttgttttc 300

tagataagct tcgtaaccga cagtttctaa cttttgtgct ttgacaagaa cttcttcttc 360

ttgctttaat aaaaactgtt ccattttcgt tgtataactt gaatcataag cgccaagcag 420

tctgacagcc aacagcgcag cgttcgtact attattaata gcgacggtag ctactggaac 480

acctctaggc atttgcacaa ttgaatgtaa agaatctact ccatctagac aagaaccttt 540

tacgggcaca ccgatgacag gaagtggtgt cattgcagcc accatacctg gcaagtgagc 600

agccccacca gctccagcga taattgtttt aattccacgc ttgcttgcgg aaatagcata 660

tgctgacatc ctatgtggag ttctatgagc agagactatt gtcacttcaa atggaacgcc 720

aaaatctttt aaaaccgcac atgcggcaga cattaccggc aagtcagagt ctgatcccat 780

gatgattcca accaatggtt tgaccattgc ttccaagtcc aacttttgag cgacagagat 840

tttgattgga atatcagttc tacctgtaat gtagttcagc ctttgttcac attccgccat 900

actggaggca ataatattta tgtgacctac ttttctgtta ggtctagact cttttccata 960

taagtacact gaggaacctg gagtcgccaa tgctctttcg caagtttcta gctctttatc 1020

ttttgtatgt ttgtctccaa gaacatttag cataatggcg ttcgttgtaa tggtggagaa 1080

agatgtgaaa ttctttggca ttggcaaatc caatattgat ctcaaatgag cttcaaattg 1140

agaagtgacg caagcatcaa tggtataatg tccagagttg tgaggccttg gggcaatttc 1200

gttaataagc aattcccctg tttctaaata gaacatttcc acaccaaata taccacaacc 1260

gggaaaagat ttgattgcat tttctgccaa caacttcgcc ttaagttgaa cggagtccgg 1320

aactctagca ggcgcataac ataagtcaca aatattgtcc ttgtggatag tctctacaat 1380

tgggtaagaa aacactaaac cgttaacaga tctcacaatc atgactgcta attctttagt 1440

aaatggtgcc catttttcgg cgtacaaagg acgatccttc agtacttcca aagcttccgg 1500

aatcatttcc ttattcttta caacgaagtt acctcttcca tcgtatgcca aagtcctcga 1560

cttcaagacg aatggaaaac ccaaatctct tccaacattc aatagggacg tctcactggc 1620

ttgttccaca ggaacacttt gggtaactgc tataccattt ttgattaaat gctctttttg 1680

aatatatttg tcttgtatca atctgattgt ttctggagaa gggtaaattt ttaatttggg 1740

atgttttact tgaagattct ttagtgtagg aacatcaaca tgctcaatct caatcgttag 1800

cacatcacat ttttcagcta gtttttcgat atcaagagga ttggaaaagg agccattaac 1860

gtggtcattg gagttgctta tttgtttggc aggagaattt tcagcatcta gtattaccgt 1920

cttaatgttg agcctgtttg ctgcctcaac aatcatacgt cccaattgtc cccctcctaa 1980

tataccaact gttctagaat ccatacttga ttgttttgtc cgattttctt gtttttcttg 2040

attgttatag taggatgtac ttagaagaga gatccaacga ttttacgcac caatttatac 2100

atgaaatgct ccataatatt gtccatttag ttcttaataa aaggtcagca agagtcaatc 2160

acttagtatt acccggttcg tagccatgca acaagagtca tttgtcagca tagctgtaat 2220

aatcaatcat gacgtaagaa atgtatcata attaaaagtt gttaaagatg tcagtgttat 2280

gttggtgtta caaaattctc ggcttctcac taatatttaa tatctcttaa attttatctg 2340

tctttgattc ttttaagaaa agttatgtat tattcaagaa aaagtcaatt ccgcatcaaa 2400

aggtaaaatt tatataaact gctttaaaat ttcatgaaac taggcaactt ttcgaaatga 2460

tctttttcga gcatgaagtt tcttttataa taacctggtc aaaagctttc aatatataat 2520

acatttggta tttacggaaa tgagatgata tactggtagt gcgctagtca agttctaatt 2580

aaattaaaat gcaaaatagt aatgtcagat ggacacattg ggcgaaatag ctaacccata 2640

agcatgaatc ctgtaactta tgtaatacgt taatcacgat acggcgttag gtacc 2695

<210> 23

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gtcccgggcc tttgctcaat gccgttttgg 30

<210> 24

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gcggatccat cgatgaattc attgtttcta tattatc 37

<210> 25

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

atgaattcat cgatggatcc gcacggcctc atctac 36

<210> 26

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tgcgcgcgag ctcggaccac aatccaaata aag 33

<210> 27

<211> 1025

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

cccgggcctt tgctcaatgc cgttttggtg atatcgacca attgctggtt aacggtgtcg 60

ttgccgtact tgtaataata agtaccgcag ccaaaggcta atcttggaat gcccgaggag 120

gtggttggaa tgctcatttc gaatacagtc aacaatcaat tgagtttgta tttatagcca 180

attggtcatt aataatcagg cttcctgcat ggtttagagg ttggtctaga ctacatccgt 240

gcaccagaaa agaggcgggc cacggaggaa aaggtgacaa ctcgcaaagt tgcaacaact 300

gctatggctc cagcacggtg cgtggggtaa agacaatctc cgggaaccgg tcccgaaacc 360

gagaaagagg gttttaagcc tgtgtcctct gcggaggtgg tgtagcactt cttattgtcc 420

tttgggccgc tccggcggta gagcttccat ggaacaacct tgcacggaca ggcaagtccc 480

cgagacgcct tgttgggtga tgtccacttc tggctataca gagctttata tcaccttact 540

gaacgctaga gtagacccaa ttcccggctc acaccaccct tacatgcaga gctaaccaat 600

aaggtaatta attaacacta tatagctcgt ggtgaacact ggcccggagt agtcatacgt 660

gtaggttttt ggcgtgatga aaatcaggtg gcgcacgact tttcgtaaag ttcgggaggg 720

agtgctgcaa acggcatata aggaccagtt tttctcgcac attatcaatt gctctttagt 780

acaaagataa tatagaaaca atgaattcat cgatggatcc gcacggcctc atctacatat 840

ttacggctta actgattttt atagttaagg agaaaaaaag ttcaacatac gtcattatta 900

ttgtacgcgc tttcgtgttt caaacttggc tgccatgata aataaatcta ttgttgcttg 960

ctatgtaaaa attatttgat tacttcttcc atgcactttc tttatttgga ttgtggtccg 1020

cgcgc 1025

<210> 28

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cagaattcat gactgctctc aacag 25

<210> 29

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ctgttaatgg tgctgctgct ggatgatgca c 31

<210> 30

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gtgcatcatc cagcagcagc accattaaca g 31

<210> 31

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

caggatcctc aagacaaata gtcg 24

<210> 32

<211> 994

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

cagaattcat gactgctctc aacagctctg tccagcacca agaagtctct tcggacttgc 60

cttttggaac tctacctcca agaaagcgtg ccaagaccga agaggagaaa gagcaaagaa 120

gagttgaaag aatcttgaga aacagaaggg cagctcacgc gtctagagaa aagaagagaa 180

gacatgttga gtacctggaa aactatgtga ctgatctgga gtctgcgctg gcgacacatg 240

agggcaatta tcggaagatg gccaaaattc aatcgagcct gatatctctg ttgtctgaac 300

atggaatcga ttactcgtct gtggatttag ctgttgaacc atgtcctaaa gttgaaagac 360

cggaaggttt ggagttgact ggttcaattc cagtgaaaaa acagaaaatc gcctcggcga 420

aatcgcccaa atcgttatcg agaaaatcga agtcggaaat cccatcacca agttttgatg 480

agaatatttt ttctgaggag gaaaacgaac atgacgatgg tattgaggaa tacgggaaag 540

caggacaaga agcaaccgag gctccatcct tgtctcacaa ccgcaaaaga aaggcgcaag 600

atgcttatat ctcgcctccg ggctccacct ccccatccaa gttgaaactt gaagaagacg 660

aaaggatctc caaacatgaa tacagtaact tgtttgatga caccgatgac attttcccgt 720

cggagaagtc atcaagtctc gagctgtata aacaggatga tctgaccatg gcatcatttg 780

tgaaacaaga agaggaagaa atggtgccat ttgtgaaaca ggaagacgag ttcaagtttc 840

ctgattcggg tttcaacgct gacgattgtc atcttatcca agtggaagac ctctgctctt 900

ttaatagcgt gcatcatcca gcagcagcac cattaacagc agagagtatc gacaaccact 960

ttgaatttga cgactatttg tcttgaggat cctg 994

<210> 33

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

aacctcacag ttgccctga 19

<210> 34

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

cgatggtctg gacgagtaga a 21

<210> 35

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gaaatcgccc aaatcgttat c 21

<210> 36

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

tcggttgctt cttgtcctg 19

<210> 37

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

tcttgcccaa tcatcctga 19

<210> 38

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

aacgaagcac gccatacc 18

<210> 39

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

aggacgagac cgctcagat 19

<210> 40

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

gaggcaatag tggtgttgat ga 22

Claims (6)

1. A method for obtaining a genetic engineering bacterium with high expression exogenous genes comprises the following steps: respectively constructing an expression vector and a functional vector, simultaneously transferring the expression vector and the functional vector into double auxotroph receptor bacteria, and screening to obtain exogenous target gene high-expression genetic engineering bacteria;

the recipient bacterium is Hansenula polymorpha with adenine and leucine double auxotrophy; the expression vector uses a strong promoter methanol oxidase promoter to start the expression of an exogenous target gene, and uses a beer yeast leucine gene to compensate and express the leucine auxotrophy of a host; the functional vector uses a constitutive promoter glyceraldehyde-3-phosphate dehydrogenase promoter to start the expression of a UPR key gene HAC1p, and uses a beer yeast adenine gene to compensate and express the adenine auxotrophy of a host; the expression vector and the functional vector both contain the autonomously replicating sequence segment of the receptor strain; the expression vector and the functional vector are simultaneously transferred into recipient bacteria through one-time electrotransformation, and the compensation of adenine and leucine double nutrition deficiency and the expression of functional genes and target genes are simultaneously realized;

the exogenous target gene is derived from an Elafin full sequence SEQ ID NO. 18 containing MFa leader peptide or a Hirudin full sequence SEQ ID NO. 19 containing MFa leader peptide.

2. The genetically engineered bacterium obtained by the method of claim 1, wherein a double auxotrophic strain is used as an expression host, and two vectors are simultaneously integrated into a transformant of the expression host, wherein one vector contains an exogenous target gene and a compensating gene for one auxotrophy of the expression host, and the other vector contains a UPR key gene HAC1p and a compensating gene for the other auxotrophy of the expression host.

3. The genetically engineered bacterium of claim 2, wherein the UPR key gene HAC1p is derived from Hansenula polymorpha ATCC34438, and the complementing gene is derived from Saccharomyces cerevisiae S288 c.

4. The genetically engineered bacterium of claim 2, wherein the autonomously replicating sequence fragment is derived from Hansenula polymorpha and is a Hansenula polymorpha autonomously replicating sequence fragment.

5. The vector for constructing the genetically engineered bacterium according to any one of claims 2 to 4, comprising the following vectors:

1) expression vector: a compensating gene for an auxotrophy comprising an exogenous gene of interest and an expression host; the exogenous target gene is derived from an Elafin full sequence SEQ ID NO. 18 containing MFa leader peptide or a Hirudin full sequence SEQ ID NO. 19 containing MFa leader peptide;

2) a functional carrier: contains the UPR key gene HAC1p, and a compensating gene for another auxotrophy of the expression host.

6. The carrier of the genetic engineering bacteria as claimed in claim 5, wherein the expression carrier uses strong promoter methanol oxidase promoter to start the expression of exogenous target gene, uses beer yeast leucine gene to compensate and express host leucine nutrition deficiency; the functional vector uses a constitutive promoter glyceraldehyde-3-phosphate dehydrogenase promoter to start the expression of a UPR key gene HAC1p, and uses a beer yeast adenine gene to compensate and express a host adenine auxotrophy.

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