Method for expressing protein through kluyveromyces marxianus bidirectional promoter

Abstract

The invention discloses a method for expressing protein by using a kluyveromyces marxianus bidirectional promoter, which comprises the following steps: using a pHTX bidirectional promoter, specifically constructing a target protein gene and the pHTX bidirectional promoter on a carrier to form a recombinant plasmid, and transferring the recombinant plasmid into a kluyveromyces marxianus strain for target protein expression. The invention provides an efficient protein expression method and provides an effective gene element and an expression system for food-grade microorganism kluyveromyces marxianus by tandem expression of target proteins comprising plant-derived hemoglobin and heme synthesis key enzymes in the same strain.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation, and in particular to a method for expressing proteins using a Kluyveromyces marxianus bidirectional promoter. Background Art

[0002] Kluyveromyces marxianus is a food-grade, non-traditional yeast. It is a strain with great development potential after Saccharomyces cerevisiae[1]. It is widely found in yogurt, fruit, kefir, etc. It is a safe yeast in nature and has been accepted by the U.S. Food and Drug Administration for pharmaceutical and food use[2]. Kluyveromyces marxianus has excellent fermentation performance and can carry out high-density fermentation and utilize various carbon sources. The excellent properties of Kluyveromyces marxianus make it conducive to the application of various biotechnologies[3] and can be used for the production research of compounds such as proteins. Kluyveromyces marxianus has great potential in cell protein production. In solid-state fermentation of microorganisms including Saccharomyces cerevisiae, Kluyveromyces marxianus, and Kefir bacteria, Kluyveromyces marxianus can synthesize high levels of food-grade protein and fat concentrations[4]. In addition, the growth rate of this yeast is very high, reaching 0.86-0.99 / h at 40°C. It is considered to be the eukaryotic organism with the highest growth rate. This characteristic makes it of great significance in biological fermentation, which can improve production efficiency and shorten the fermentation cycle[5]. Therefore, Kluyveromyces marxianus is an excellent chassis microorganism for producing high-value compounds.

[0003] To efficiently regulate Kluyveromyces marxianus, researchers have developed a series of key gene expression elements and constructed several gene expression systems. Bidirectional promoters, such as pHTX, can simultaneously drive the expression of two genes in opposite directions. The advantage of this type of promoter is that it can control the expression of two different genes within a single transcription unit without the need for additional regulatory elements.

[0004] Therefore, those skilled in the art are committed to developing an efficient method for expressing proteins in Kluyveromyces marxianus.

[0005] References:

[0006] [1]GOMBERT AK,MADEIRA JV,CERDáN ME,et al.Kluyveromyces marxianusas a host for

[0007] heterologous protein synthesis[J].Appl Microbiol Biot,2016,100(14):6193-208.

[0008] [2]LEONEL L, ARRUDA P, CHANDEL A, et al. Kluyveromyces marxianus: A potential biocatalyst of

[0009] renewable chemicals and lignocellulosic ethanol production[J]. Critical Reviews in Biotechnology,

[0010] 2021, 41(8): 1131 - 52.

[0011] [3]TITTARELLI F, VARELA J A, GETHINS L, et al. Development and implementation of multilocus

[0012] sequence typing to study the diversity of the yeast in Italian cheeses[J]. Microb Genomics, 2018, 4(2).

[0013] [4]FONSECA G G, HEINZLE E, WITTMANN C, et al. The yeast Kluyveromyces marxianus and its

[0014] biotechnological potential[J]. Appl Microbiol Biot, 2008, 79(3): 339 - 54.

[0015] [5]GROENEVELD P, STOUTHAMER A H, WESTERHOFF H V. Super life–how and why ‘cell

[0016] selection’ leads to the fastest - growing eukaryote[J]. The FEBS journal, 2009, 276(1): 254 - 70. Summary of the Invention

[0017] In view of the above-mentioned defects in the prior art, the technical problem to be solved by the present invention is to provide a method for expressing proteins with high efficiency in Kluyveromyces marxianus.

[0018] To achieve the above objectives, the present invention provides a method for expressing proteins using a Kluyveromyces marxianus bidirectional promoter, characterized in that the method uses the pHTX bidirectional promoter, specifically constructing a target protein gene and the pHTX bidirectional promoter on a vector to form a recombinant plasmid, and then transferring the recombinant plasmid into a Kluyveromyces marxianus strain to express the target protein, and the pHTX bidirectional promoter sequence is shown in SEQ ID NO.1.

[0019] In a preferred embodiment of the present invention, the terminators used on both sides of the bidirectional promoter are DAS1 and AOX1 terminators, respectively, wherein the DAS1 terminator sequence is shown as SEQ ID NO.2, and the AOX1 terminator sequence is shown as SEQ ID NO.3. When constructing the recombinant plasmid, the DAS1 and AOX1 terminators are also constructed together on the vector.

[0020] In another preferred embodiment of the present invention, when constructing the vector, the gene fragment of the target protein, the pHTX promoter and the terminator are first amplified separately, and then subjected to agarose gel electrophoresis, purification and recovery. The amplified gene fragment of the target protein, the pHTX promoter, the terminator and the vector are then connected using seamless cloning technology to obtain a recombinant plasmid, and the recombinant plasmid is then transformed into a Kluyveromyces marxianus strain to obtain a recombinant strain.

[0021] In another preferred embodiment of the present invention, when amplifying the pHTX promoter, the genome of Kluyveromyces marxianus is used as a template to amplify the pHTX promoter by PCR, and the primers are:

[0022] Upstream primer SEQ ID NO.6: 5'-TTTTATATTATATTATGTTG-3'

[0023] Downstream primer SEQ ID NO. 7: 5′-TTTTTCTGTATTATTTGGTTA-3′;

[0024] When amplifying the DAS1 and AOX1 terminators, the Pichia Pastoris genome was used as a template to amplify the DAS1 and AOX1 terminators. The primers were:

[0025] AOX1:

[0026] Upstream primer SEQ ID NO. 24: 5'-GGATGTCAGAATGCCATTTG-3'

[0027] Downstream primer SEQ ID NO. 25: 5'-TCTCACTTAATCTTCTGTACT-3'

[0028] DAS1:

[0029] Upstream primer SEQ ID NO. 26: 5'-GACCCTTGTGACTGACACTT-3'

[0030] Downstream primer SEQ ID NO. 27: 5′-ACGGGAAGTCTTTACAGTTT-3′.

[0031] In another preferred embodiment of the present invention, the vector contains the plasmid replication element ARS2 derived from the expression plasmid pMTU-DO-URA-C4 of Kluyveromyces marxianus strain CBS6556, the sequence of which is shown in SEQ ID NO.12, and the vector sequence is shown in SEQ ID NO.23.

[0032] In another preferred embodiment of the present invention, the Kluyveromyces marxianus strain is strains.

[0033] In another preferred embodiment of the present invention, when expressing the target protein in Kluyveromyces marxianus strain, the glucose concentration of the prepared culture medium is 20 g / L.

[0034] In another preferred embodiment of the present invention, the target protein includes hemoglobin, the gene of which is VaHB, and the sequence is shown in SEQ ID NO.13.

[0035] In another preferred embodiment of the present invention, the target protein also includes heme synthesis key enzyme hem1, the gene sequence of which is shown in SEQ ID NO.14, and the recombinant plasmid construction method is specifically as follows:

[0036] First, the gene fragment of hemoglobin VaHB was artificially synthesized and used as a template for PCR amplification to obtain the hemoglobin gene fragment. The genome of Pichia Pastoris was used as a template for PCR amplification to obtain the hem1 gene and amplify the pHTX promoter. The specific primers used for VaHB and hem1 gene amplification are as follows:

[0037] VaHB:

[0038] Upstream primer SEQ ID NO. 15: 5'-TCAATGATGATGATGATGAT-3'

[0039] Downstream primer SEQ ID NO. 16: 5'-ATGGTTGCATTTAGTGATAA-3'

[0040] hem1:

[0041] Upstream primer SEQ ID NO. 17: 5'-ATGGAGTTTGTCGCCCGTCA-3'

[0042] Downstream primer SEQ ID NO. 18: 5′-CTACAATCTGACTCCTGATG-3′;

[0043] The amplified VaHB and hem1 gene fragments, pHTX promoter, DAS1, and AOX1 terminators were then constructed together on a vector and ligated to the vector using seamless cloning technology. The ligation product was transformed into Escherichia coli DH5α competent cells, plated on LB medium solid plates, and cultured in an incubator. Single clones were picked and transferred to LB liquid medium containing ampicillin, cultured on a shaker, and the plasmid was extracted.

[0044] The extracted plasmid is then transferred into the Kluyveromyces marxianus strain by electroporation to obtain a recombinant strain.

[0045] In another preferred embodiment of the present invention, the recombinant strain is cultured in a plate culture medium and a liquid culture medium and then transferred to a fermenter for culture. After the fermentation is completed, a portion of the fermentation liquid is centrifuged, crushed and purified to obtain hemoglobin VaHB.

[0046] Technical Effects

[0047] 1. The present invention uses an endogenous bidirectional promoter pHTX to construct a gene expression plasmid, which successfully expresses a fluorescent protein in Kluyveromyces marxianus. By constructing a protein expression plasmid that can work in Kluyveromyces marxianus strains, the effect of protein expression is achieved, enriching the means of achieving exogenous gene expression in Kluyveromyces marxianus.

[0048] 2. This invention compares the intensity of fluorescent protein expression in different Kluyveromyces marxianus strains using the same protein expression plasmid system to identify the strain with the highest protein expression capacity. By verifying the ability of different strains to express fluorescent protein using the same expression system, the strain with the highest protein expression capacity is screened, and Kluyveromyces marxianus chassis cells with strong protein expression capacity are discovered, which is conducive to the construction of an efficient protein production platform.

[0049] 3. By utilizing a bidirectional promoter expression system, plant-derived hemoglobin and hem1, a key enzyme in heme synthesis, were tandemly expressed in the same strain, simultaneously increasing hemoglobin and heme production. By enhancing the synthesis of key heme synthesis enzymes and verifying hemoglobin purification via a histidine tag, heme production was increased, removing restrictions on hemoglobin synthesis. Using a single-plasmid multi-protein co-expression system, hemoglobin and hem1, a key enzyme in heme synthesis, were simultaneously expressed, improving hemoglobin synthesis efficiency. This study provides effective genetic elements and expression systems for the food-grade microorganism Kluyveromyces marxianus, laying a foundation for subsequent applications in synthetic biology and metabolic engineering of Kluyveromyces marxianus.

[0050] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a schematic diagram of the structure of a pHTX bidirectional promoter expressing fluorescent protein system according to a preferred embodiment of the present invention;

[0052] Figure 2 This is a schematic diagram of different fluorescent protein expression levels according to a preferred embodiment of the present invention;

[0053] Figure 3 This is a schematic diagram of the expression level of a fluorescent protein under different glucose concentrations according to a preferred embodiment of the present invention;

[0054] Figure 4 This is a schematic diagram of the expression levels of a fluorescent protein in different strains according to a preferred embodiment of the present invention;

[0055] Figure 5 Schematic diagram of the structure of a hemoglobin expression system according to a preferred embodiment of the present invention;

[0056] Figure 6 This is an electrophoretic diagram of purified hemoglobin according to a preferred embodiment of the present invention;

[0057] Figure 7 The effect of different carbon sources on hemoglobin fermentation titer in a preferred embodiment of the present invention is

[0058] Schematic diagram. DETAILED DESCRIPTION

[0059] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0060] Example 1 Construction and identification of bidirectional promoter pHTX expression system

[0061] To achieve protein expression in Kluyveromyces marxianus using the bidirectional promoter pHTX, plasmid vectors Plasmid 1 and Plasmid 2 were constructed to express the green fluorescent protein gene and the red fluorescent protein gene using pHTX as a bidirectional promoter (sequence shown in SEQ ID NO. 1). Plasmid information is shown in Table 1. The terminators used on both sides of the bidirectional promoter were DAS1 (full name Diacetoxyscirpenol Aldehyde Synthase 1) and AOX1 terminator (full name Aldehyde Oxidase 1), both of which are derived from Pichia Pastoris. The sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0062] Table 1 Plasmid information and genotype

[0063]

[0064] First, the gene fragments of green fluorescent protein sfGFP (FPbase ID: B4SOW) and red fluorescent protein mSI3 (FPbase ID: THSBS) were artificially synthesized (the gene sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the amino acid sequences are shown in SEQ ID NO.19 and SEQ ID NO.20, respectively). sfGFP comes from Aequorea victoria, and mSI3 is a synthetic structure with a sequence from FPbase. They were commissioned to Sangon Biotechnology for artificial synthesis. PCR amplification was performed using the two synthetic gene fragments as templates to obtain fluorescent protein gene fragments. Next, the pHTX promoter was amplified using the genome of Kluyveromyces marxianus as a template, and the two terminators DAS1 and AOX1 were amplified using the genome of Pichia Pastoris as a template. The specific primers used for amplification are shown below. Schematic diagram of the Plasmid 1 and Plasmid 2 plasmid expression systems is shown in Figure 2. Figure 1 A, B.

[0065] pHTX:

[0066] Upstream primer SEQ ID NO.6: 5'-TTTTATATTATATTATGTTG-3'

[0067] Downstream primer SEQ ID NO.7: 5'-TTTTTCTGTATTATTTGGTTA-3'

[0068] AOX1:

[0069] Upstream primer SEQ ID NO. 24: 5'-GGATGTCAGAATGCCATTTG-3'

[0070] Downstream primer SEQ ID NO. 25: 5'-TCTCACTTAATCTTCTGTACT-3'

[0071] DAS1:

[0072] Upstream primer SEQ ID NO. 26: 5'-GACCCTTGTGACTGACACTT-3'

[0073] Downstream primer SEQ ID NO. 27: 5'-ACGGGAAGTCTTTACAGTTT-3'

[0074] sfGFP:

[0075] Upstream primer SEQ ID NO.8: 5'-TCACTTGTACAATTCGTCCA-3'

[0076] Downstream primer SEQ ID NO.9: 5'-ATGTCTAAGGGTGAAGAATTGT-3'

[0077] mSI3:

[0078] Upstream primer SEQ ID NO.10: 5'-ATGGATTCCACCGAAGCTGTT-3'

[0079] Downstream primer SEQ ID NO.11: 5'-TCAGGAACCACCGGAACCAC-3'

[0080] The PCR reaction system was as follows: 1.5 μL of each primer, 1 μL of template, 25 μL of KOD Fx enzyme, 22.5 μL of sterile water, and a total system of 50 μL.

[0081] PCR reaction conditions were as follows: 94°C pre-denaturation for 5 min, 95°C denaturation for 20 s, 60°C annealing for 30 s, 72°C extension for 60 s, 30 cycles, and 72°C post-extension for 10 min. The amplified fragments were subjected to agarose gel electrophoresis for purification and recovery.

[0082] The amplified target gene fragment, pHTX promoter, DAS1 and AOX1 terminators, and vector backbone (sequence shown in SEQ ID NO. 23) were connected using seamless cloning technology. The backbone contained the plasmid replication element ARS2 derived from the Kluyveromyces marxianus strain CBS6556 expression plasmid pMTU-DO-URA-C4 (Addgene No.: 160339), the sequence of which is shown in SEQ ID NO. 12 ( Figure 1 A, B, where A: KM expression system; B: KM2 expression system. pHTX: bidirectional promoter; sfGFP: green fluorescent protein gene; mSI3: red fluorescent protein gene; DAS1TT: terminator; AOX1: terminator; ARS2: eukaryotic plasmid replication element; AmpR: ampicillin resistance gene; Ori: plasmid replication origin; G418R: yeast antibiotic selection tag. The ligation product was transformed into Escherichia coli DH5α competent cells, plated onto solid LB medium plates (containing 50 μg / mL ampicillin), and cultured in a 37°C incubator for 16 h. Single colonies were then picked and transferred to LB liquid medium containing 50 μg / mL ampicillin and cultured in a shaking incubator at 220 rpm at 37°C for 12 h. Plasmids were then extracted and stored at -20°C until use. See Table 1 for plasmid information.

[0083] To test whether the constructed plasmid expression system can successfully express fluorescent protein, the strain K. marxianus CICC was first incubated on ice with 1 M sorbitol. Competent cells were prepared using 32423 (purchased from China Industrial Microbiological Culture Collection (CICC)). 500 ng of Plasmid 1 and Plasmid 2 were used with the competent cells for electroporation to obtain strains KM and KM2. The strain information is shown in Table 2.

[0084] Table 2 Strain information

[0085]

[0086]

[0087] The transformed cells were spread onto Yeast Extract Peptone Dextrose Medium (YPD) plates (20 g / L peptone, 10 g / L yeast powder, 20 g / L glucose) containing G418 resistance. After colonies grew on the plates, the strains were picked and inoculated into 10 mL of YPD medium containing 1% (v / v) G418 antibiotics (50 mg / mL) for 96 hours of fermentation. Every 24 hours, 400 μL of bacterial solution was centrifuged in a 1.5 mL centrifuge tube (13000 rpm, 2 min), the supernatant was removed, and the solution was resuspended in ultrapure water. 200 μL of the sample to be tested was added to a 96-well plate and measured using a microplate reader. The measurement wavelength depends on the absorption wavelength of the target protein. (Note: Superfolder GFP, sfGFP, superfolded green fluorescent protein, is derived from the jellyfish Aequorea victoria. It is a very rapidly maturing weak dimer with an excitation wavelength of 488nm and an emission wavelength of 510nm; mScarlet-I3, mSI3, red fluorescent protein is a very rapidly maturing monomer with an excitation wavelength of 568nm and an emission wavelength of 592nm). See the test results. Figure 2 ((A) sfGFP protein expression changes with fermentation time; (B) mSI3 protein expression changes with fermentation time.) The results show that pHTX1 promoter can be successfully expressed in Kluyveromyces marxianus. The strain was used to successfully express green and red fluorescent proteins and detect fluorescence. The fluorescence intensity was not much different after the genes on both sides of the promoter were expressed, indicating that the expression intensity on both sides of the bidirectional promoter was relatively close.

[0088] To identify the effect of carbon source concentration on protein expression, KM and KM2 strains were used as research objects. Fermentation was carried out under glucose concentrations of 20 g / L, 50 g / L and 100 g / L, and samples were taken for detection by enzyme marker at 1, 2, 3, 4, 5, 6 and 7 days during the fermentation process. The fermentation conditions and detection methods were the same as above. The results are shown in Figure 2. Figure 3 The test results showed that between 0 and 7 days of fermentation, the expression of green fluorescent protein sfGFP in KM and KM2 showed a downward trend as time increased ( Figure 3 A, C), while the expression of red fluorescent protein mSI3 showed an upward trend ( Figure 3 B, D), Figure 3(A) KM: sfGFP protein expression changes with fermentation time; (B) KM: mSI3 protein expression changes with fermentation time; (C) KM2: sfGFP protein expression changes with fermentation time; (D) KM2: mSI3 protein expression changes with fermentation time. At the same time point, sfGFP expression decreased with increasing glucose concentration. This may be due to the inhibitory effect of higher glucose concentrations on protein synthesis. Metabolites (such as ethanol) produced by yeast using glucose (a fast-acting carbon source) during mixed carbon source fermentation can inhibit the expression of genes and protein activity related to the metabolism of other non-fast-acting carbon sources, leading to decreased fluorescent protein expression. For the red fluorescent protein mSI3 expressed in KM and KM2, expression levels reached their lowest level after 1 day of fermentation. From 2 to 7 days of fermentation, mSI3 expression levels gradually increased and then stabilized. These results indicate that the differences in expression levels of the two fluorescent proteins with varying glucose concentrations and fermentation time are determined by the characteristics of the two proteins, not by the pHTX promoter. The above results indicate that when using Kluyveromyces marxianus to express foreign proteins, the optimal glucose concentration for preparing the culture medium is 20 g / L. This condition ensures high expression levels of proteins on both sides of the bidirectional promoter while also avoiding waste of excess glucose, thus reducing costs.

[0089] Example 2 Co-expression of fluorescent proteins in different Kluyveromyces marxianus strains using the pHTX bidirectional promoter expression system

[0090] To verify the expression strength of the pHTX bidirectional promoter expression system in different Kluyveromyces marxianus strains, Kluyveromyces marxianus strains purchased from China Industrial Microbiological Culture Collection (CICC) were used to express K. marxianus. 1276, K. marxianus CICC 1953, K. marxianus CICC 31992, and K. marxianus CICC 33373 was used as the host, and Plasmid 1 was transformed into the above strains to obtain strains KM1276, KM1953, KM31992, and KM33373. The transformation method was the same as above, and the strain information is shown in Table 2. Fermentation was carried out under a glucose concentration of 20 g / L (30°C). Samples were taken at 24, 48, 72, and 96 hours of the fermentation process for fluorescence intensity detection using the same method as above. The results of the microplate reader test are shown in Figure 2. Figure 4As shown, (A) sfGFP protein expression changes with fermentation time; (B) mSI3 protein expression changes with fermentation time. The test results show that the gene expression system designed in the experiment effectively played a role in the five strains of Kluyveromyces marxianus. Although the protein expression capacity varies, the expression intensity of the same fluorescent protein in different strains has basically the same trend over time; the sfGFP gene was effectively expressed in all five yeasts ( Figure 4 A), while the mSI3 gene in strain K. marxianus CICC 33373 is not fully expressed ( Figure 4 B); By comparing the expression intensity of fluorescent proteins in different strains, it can be preliminarily concluded that Kluyveromyces marxianus

[0091] The protein expression ability of K.marxianus CICC is stronger. 33373 has poor ability to express foreign proteins.

[0092] Example 3 Application of pHTX bidirectional promoter expression system in expressing hemoglobin

[0093] VaHB is a hemoglobin expression gene from Vigna angularis that can express hemoglobin (NCBI sequence number: XP_017409386.1). It has been successfully expressed in Pichia pastoris (Chinese invention patent: CN119060170A). The hemoglobin is composed of a polypeptide chain, heme and iron ions, with a molecular weight of about 15kDa. In order to realize the expression of the hemoglobin gene VaHB from Vigna angularis in Kluyveromyces marxianus To express hemoglobin and the key enzyme hem1 in heme synthesis in strains, an expression plasmid Plasmid 3 (NCBI sequence number: NC_012964.1) under the control of the pHTX bidirectional promoter was constructed. First, the gene fragment of hemoglobin VaHB was artificially synthesized (its gene sequence is shown in SEQ ID NO.13, and its amino acid sequence is shown in SEQ ID NO.21), and PCR amplification was performed using it as a template to obtain the hemoglobin gene fragment. Secondly, the genome of Pichia Pastoris was used as a template, and PCR amplification was performed to obtain the hem1 gene (its gene sequence is shown in SEQ ID NO.14, and its amino acid sequence is shown in SEQ ID NO.22). The specific primers used for amplification are shown below.

[0094] VaHB:

[0095] Upstream primer SEQ ID NO. 15: 5'-TCAATGATGATGATGATGAT-3'

[0096] Downstream primer SEQ ID NO. 16: 5'-ATGGTTGCATTTAGTGATAA-3'

[0097] hem1:

[0098] Upstream primer SEQ ID NO. 17: 5'-ATGGAGTTTGTCGCCCGTCA-3'

[0099] Downstream primer SEQ ID NO. 18: 5'-CTACAATCTGACTCCTGATG-3'

[0100] The PCR reaction system and conditions were the same as above, and the gene fragment ligation and plasmid transformation methods were the same as above. Figure 5 Schematic diagram of the structure of the hemoglobin plasmid expression system.

[0101] Plasmid 3 was transformed into Kluyveromyces marxianus by the same electroporation method as above. The transformed cells were plated onto YPD plates containing G418 resistance. After colonies grew on the plates, the strains were picked and inoculated into 10 mL of YPD medium containing 1% (v / v) G418 antibiotic (50 mg / mL) for fermentation, and fermented for 24 hours under a glucose concentration of 20 g / L. After fermentation, the absorbance of the bacterial solution at 600 nm was first measured. The bacterial solution was then centrifuged at 8000 g for 10 minutes, the bacteria were collected, resuspended in 50 mM phosphate buffered saline (PBS) with a pH of 8.0, and then ultrasonically disrupted for 25 minutes (400 W, 10 seconds on, 10 seconds off) in an ice-water bath. The supernatant was then centrifuged again at 8000 g for 15 minutes, and the crude protein solution was collected. Based on the His tag fusion protein, the crude protein solution was purified by affinity chromatography using a Ni-NTA column. A low concentration of 10mM imidazole solution (10mM imidazole, 500mM NaCl, 50mM Tris-HCl) was used to balance the column. Then, 20mM imidazole solution (20mM imidazole, 500mM NaCl, 50mM Tris-HCl) was used to elute weakly bound impurities. The target protein was eluted with a 200mM imidazole solution (200mM imidazole, 500mM NaCl, 50mM Tris-HCl). The buffer elution component of this part was collected to obtain a purified hemoglobin solution. The protein purity and molecular weight were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE). The results are as follows: Figure 6 As shown, after electrophoresis, a lighter target band was observed in the fermentation sample, with a size of approximately 15 kDa, consistent with the theoretical value of hemoglobin VaHB, indicating that trace amounts of hemoglobin were produced. Protein color development was performed using Brandford reagent, and a protein concentration standard curve was drawn using bovine serum albumin. 100 μL of Brandford reagent was added to 10 μL of sample, and the absorbance at 595 nm was measured to determine the protein concentration. The results showed that the hemoglobin production was 0.05 g / L.

[0102] In addition, the effect of carbon source type on hemoglobin production was investigated. Fermentation was carried out for 24 h using 20 g / L glucose, lactose, galactose, sucrose, and glycerol as carbon sources. After fermentation, cells were broken and protein was purified (specific operations were the same as above), and the hemoglobin concentration was measured. The results showed that the hemoglobin production was the highest when 20 g / L glucose was used as the carbon source, and OD600 indicated that the strain grew best at this time. When 20 g / L glycerol was used as the carbon source, the hemoglobin production was the lowest, and OD600 indicated that the strain grew poorly at this time. Figure 7 shown.

[0103] Example 4 Expression of hemoglobin VaHB in a 1L fermenter

[0104] By optimizing the carbon source concentration and type, the hemoglobin yield was successfully increased to 0.05 g / L, and its yield was further optimized using a fermentor. After KM3 was activated on a YPD (G418 resistance) plate, a single colony was picked and inoculated into 1 mL of YPD liquid medium (G418 resistance) to prepare a primary seed. The colony was cultured overnight at 30°C and 250 rpm for 24 hours until the OD reached between 1 and 2. The colony was then transferred to a secondary seed bottle containing YPD liquid medium (G418 resistance) at a 1% (v / v) inoculation rate. The colony was cultured at 30°C and 250 rpm for 12 hours. When the OD was between 1 and 2, the colony was inoculated into a 1 L fermentor at a 5-10% (v / v) inoculation rate. The culture medium consisted of YG medium (20 g / L yeast extract, 20 g / L peptone, 40.0 g / L glucose, and 2 mL of trace element PTM1 / L) at 30°C. The fermentor was aerated at 1.6 L / min, with pH automatically controlled at 5.5 using aqueous ammonia. Dissolved oxygen was regulated in series with the fermentor's stirrer (200-800 rpm). When the glucose in the minimal salt medium was depleted, 1% (w / v) glucose was added. Supplementation was stopped when the bacterial OD600 reached 80, and the fermentation was allowed to proceed for 96-120 hours. After fermentation, a portion of the fermentation broth was centrifuged (10,000 rpm for 5 minutes), disrupted, and purified as described above. The hemoglobin VaHB titer was determined to be 0.3 g / L.

[0105] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for expressing a protein using a Kluyveromyces marxianus bidirectional promoter, characterized in that: The method uses the pHTX bidirectional promoter. Specifically, the target protein gene and the pHTX bidirectional promoter are constructed on a vector to form a recombinant plasmid, which is then transferred into the Kluyveromyces marxianus strain for target protein expression. The pHTX bidirectional promoter sequence is shown in SEQ ID NO.

1.

2. The method according to claim 1, wherein The terminators used on both sides of the bidirectional promoter are DAS1 and AOX1 terminators, respectively. The DAS1 terminator sequence is shown in SEQ ID NO.2, and the AOX1 terminator sequence is shown in SEQ ID NO.

3. When constructing the recombinant plasmid, the DAS1 and AOX1 terminators are also constructed together on the vector.

3. The method according to claim 2, wherein When constructing the vector, the gene fragment of the target protein, the pHTX promoter and the terminator are first amplified separately, and then subjected to agarose gel electrophoresis, purification and recovery. The amplified gene fragment of the target protein, the pHTX promoter, the terminator and the vector are then connected using seamless cloning technology to obtain a recombinant plasmid. The recombinant plasmid is then transformed into a Kluyveromyces marxianus strain to obtain a recombinant strain.

4. The method according to claim 3, wherein When amplifying the pHTX promoter, the genome of Kluyveromyces marxianus was used as a template to amplify the pHTX promoter by PCR. The primers were: Upstream primer SEQ ID NO.6: 5'-TTTTATATTATATTATGTTG-3' Downstream primer SEQ ID NO. 7: 5′-TTTTTCTGTATTATTTGGTTA-3′; When amplifying the DAS1 and AOX1 terminators, the Pichia Pastoris genome was used as a template to amplify the DAS1 and AOX1 terminators. The primers were: AOX1: Upstream primer SEQ ID NO. 24: 5'-GGATGTCAGAATGCCATTTG-3' Downstream primer SEQ ID NO. 25: 5'-TCTCACTTAATCTTCTGTACT-3' DAS1: Upstream primer SEQ ID NO. 26: 5'-GACCCTTGTGACTGACACTT-3' Downstream primer SEQ ID NO. 27: 5′-ACGGGAAGTCTTTACAGTTT-3′.

5. The method according to claim 1, wherein The vector contains the plasmid replication element ARS2 derived from the expression plasmid pMTU-DO-URA-C4 of Kluyveromyces marxianus strain CBS6556, the sequence of which is shown in SEQ ID NO.12, and the vector sequence is shown in SEQ ID NO.

23.

6. The method according to claim 1, wherein The Kluyveromyces marxianus strain is 32423 strains.

7. The method according to claim 1, wherein When expressing the target protein in Kluyveromyces marxianus, the glucose concentration of the prepared culture medium is 20 g / L.

8. The method according to claim 4, wherein The target protein includes hemoglobin, the gene of which is VaHB, and the sequence is shown in SEQ ID NO.

13.

9. The method according to claim 8, wherein The target protein also includes heme synthesis key enzyme hem1, the gene sequence of which is shown in SEQ ID NO.

14. The recombinant plasmid construction method is specifically as follows: First, the gene fragment of hemoglobin VaHB was artificially synthesized and used as a template for PCR amplification to obtain the hemoglobin gene fragment. The genome of Pichia Pastoris was used as a template for PCR amplification to obtain the hem1 gene and amplify the pHTX promoter. The specific primers used for VaHB and hem1 gene amplification are as follows: VaHB: Upstream primer SEQ ID NO. 15: 5'-TCAATGATGATGATGATGAT-3' Downstream primer SEQ ID NO. 16: 5'-ATGGTTGCATTTAGTGATAA-3' hem1: Upstream primer SEQ ID NO. 17: 5'-ATGGAGTTTGTCGCCCGTCA-3' Downstream primer SEQ ID NO. 18: 5′-CTACAATCTGACTCCTGATG-3′; The amplified VaHB and hem1 gene fragments, pHTX promoter, DAS1, and AOX1 terminators were then constructed together on a vector and ligated to the vector using seamless cloning technology. The ligation product was transformed into Escherichia coli DH5α competent cells, plated on LB medium solid plates, and cultured in an incubator. Single clones were picked and transferred to LB liquid medium containing ampicillin, cultured on a shaker, and the plasmid was extracted. The extracted plasmid is then transferred into the Kluyveromyces marxianus strain by electroporation to obtain a recombinant strain.

10. The method according to claim 9, wherein The recombinant strain is cultured in a plate culture medium and a liquid culture medium, and then transferred to a fermenter for culture. After the fermentation is completed, a portion of the fermentation liquid is centrifuged, crushed and purified to obtain hemoglobin VaHB.

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