Strain for producing candida antarctica lipase B as well as construction method and application of strain

Abstract

The invention provides a strain for producing candida antarctica lipase B as well as a construction method and application of the strain. The construction method comprises the following steps: S1, constructing a fusion expression cassette containing an MBP gene, a connection sequence and a CALB gene, the connection sequence containing a blood coagulation factor Xa enzyme recognition site; s2, inserting the fusion expression cassette into an expression vector to obtain a recombinant expression vector; and S3, transforming the recombinant expression vector into bacillus subtilis to obtain a recombinant strain. According to the invention, bacillus subtilis is used as a chassis bacterium, so that directional transformation and screening of strains can be rapidly realized, the yield of CALB can be increased, and the cost of industrial production can be reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain of Candida antarctica that produces lipase B, its construction method, and its application. Background Technology

[0002] Candida antarctica lipase B (CALB) is a lipase produced by Candida antarctica and belongs to the hydrolase family. It catalyzes the hydrolysis of esters and fatty acids. CALB exhibits good catalytic efficiency for both water-soluble and water-insoluble substrates, and demonstrates high stability and catalytic activity in non-aqueous solvents. This makes it an indispensable biocatalyst in organic synthesis, with wide applications in industry, including the synthesis of pharmaceutical intermediates, biodiesel, and the catalytic synthesis and degradation of polymers.

[0003] However, a major challenge to the industrial application of CALB is the extremely low enzyme production of wild-type strains, resulting in high costs for natural CALB and making it impossible to meet large-scale demand. To overcome this limitation, existing technologies mainly utilize genetic engineering to construct microbial cell factories to achieve heterologous recombinant expression of CALB. Among these, heterologous recombinant expression is primarily achieved using yeasts (such as Pichia pastoris and Saccharomyces cerevisiae) and filamentous fungi (such as Aspergillus niger and Aspergillus oryzae). By using secretion systems fused with signal peptides or cell surface display systems fused with cell wall proteins, recombinant protease preparations or whole-cell catalysts of CALB can be prepared.

[0004] However, existing recombinant expression systems still have many technical defects and industrialization pain points, which restrict the efficient and low-cost production of CALB. Taking the Pichia pastoris expression system as an example, to achieve efficient secretory expression of CALB, the CALB gene must first be optimized for Pichia pastoris codon preference. Then, the optimized gene is integrated with strong promoters (such as methanol-inducible AOX1 promoter and constitutive pGAP promoter) and signal peptides (such as α-factor) to construct expression vectors such as pPIC9K. After transformation, it is integrated into the Pichia pastoris genome to obtain recombinant strains. Although this system can achieve a high level of CALB expression, its upstream fermentation requires a high-density fermentation process combined with methanol induction. This not only leads to a significant increase in heat production and oxygen consumption during fermentation, but also places stringent requirements on precise process control and production equipment performance. Furthermore, methanol is highly flammable, and its storage, transportation, and production operations all require strict safety protection measures, which significantly increases the complexity and overall cost of production and operation. The expression system of filamentous fungi, represented by Aspergillus niger, has the problem of difficulty in controlling the fermentation process. Its mycelial morphology can easily lead to high viscosity of fermentation broth, which seriously affects the mass transfer and mixing efficiency of the system. As a result, the expression concentration and enzyme activity of CALB are generally lower than those of Pichia pastoris system. At the same time, the genetic manipulation system of filamentous fungi is complex, and the cycle of genetic engineering modification is long and difficult, making it difficult to quickly achieve efficient optimization of strains, which further limits the potential for improving CALB yield and enzyme activity.

[0005] Even if the artificial production of CALB is achieved by relying on the above-mentioned recombinant expression technology, its production cost remains high. For example, CALB produced by Aspergillus oryzae fermentation in the existing technology has a market price of about 16,000 yuan / kg. The high cost directly limits the large-scale application of CALB in various industrial fields.

[0006] In summary, developing a CALB heterologous recombination expression system with high enzyme yield, simple fermentation process, and low production cost, and constructing efficient and stable engineered strains through genetic engineering to achieve low-cost, large-scale production of CALB, is the key to breaking through current technological bottlenecks and promoting the widespread application of CALB in the industrial field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a strain for producing Candida antarcticis lipase B, its construction method, and its applications.

[0008] This invention provides a method for constructing a strain that produces Candida antarcticis lipase B, comprising the following steps: S1: Construct a fusion expression cassette containing the MBP gene, a linker sequence, and the CALB gene. The linker sequence includes a coagulation factor Xa enzyme recognition site; S2: Insert the fusion expression cassette into the expression vector to obtain the recombinant expression vector; S3: Transform the recombinant expression vector into Bacillus subtilis to obtain a recombinant strain.

[0009] In some embodiments, the amino acid sequence encoded by the MBP gene is shown in SEQ ID NO.1; The amino acid sequence encoded by the CALB gene is shown in SEQ ID NO.2; The amino acid sequence of the linker sequence is shown in SEQ ID NO.3.

[0010] In some embodiments, the nucleotide sequence of the fusion expression cassette described in S1 is shown in SEQ ID NO.4.

[0011] In some embodiments, the expression vector described in S2 is a pMK4 plasmid vector.

[0012] In some implementations, the construction of the expression vector in S2 specifically involves: Using pMK4 plasmid as a template, PCR was performed with primers shown in SEQ ID NO.5~6 to obtain the linear vector fragment; Using the MBP-CALB fusion protein gene as a template, PCR amplification was performed using the primers shown in SEQ ID NO.7~8 to obtain the linear target fragment; The linear target fragment and the linear vector fragment are homologously recombinated to obtain the recombinant expression vector.

[0013] The present invention also provides strains obtained by the construction method described above.

[0014] The present invention also provides a method for producing Candida antarcticis lipase B, wherein the strain is fermented and cultured, an inducer is added to induce fermentation, and Candida antarcticis lipase B is obtained from the fermentation supernatant.

[0015] In some embodiments, the inducer is xylose, and the final concentration of xylose in the fermentation system is 0.5% to 2%.

[0016] In some implementations, the fermentation culture temperature is 30~37℃ and the time is 18~30h.

[0017] The present invention also provides the application of the strain in the production of Candida antarcticis lipase B.

[0018] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) This invention uses Bacillus subtilis as the substrate bacteria, which can quickly achieve targeted modification and screening of strains, which is beneficial to increase CALB yield and reduce the cost of industrial production. Bacillus subtilis is an industrial-grade microorganism. Its culture has simple requirements for culture medium components and a fast growth and reproduction rate, which greatly reduces the basic culture cost compared with expression hosts such as yeast. At the same time, the industrial fermentation process of this strain has been developed and can be directly adapted to the existing industrial fermentation system without the need for complex process modification, which effectively reduces the overall operating cost and technical investment of upstream and downstream fermentation processes.

[0019] (2) Bacillus subtilis is a food-grade prokaryotic microorganism. In this invention, this strain can achieve soluble secretory expression of CALB fusion protein, directly obtain the target protein with biological activity, avoid the technical difficulties and cost losses of inclusion body formation and subsequent protein refolding commonly encountered in Escherichia coli expression systems; and its expressed protein product does not contain endotoxin, which greatly simplifies the downstream purification process, shortens the purification cycle, and further saves the time and economic cost of protein separation and purification. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a map of the pMK4-MBP-CALB recombinant plasmid of the present invention.

[0022] Figure 2 This is a diagram of the MBP-CALB gene amplification of the present invention, where M is the DNA molecular weight standard (Trans5K); 1, 2, and 3 are the PCR amplification products of the MBP-CALB gene.

[0023] Figure 3 This is an SDS-PAGE image of the supernatant of the MBP-CALB gene induced expression of the present invention; M is the protein molecular weight standard, wt is the induced expression product of Bacillus subtilis, CALB+ is the induced expression product of the engineered strain, and CALB- is the non-induced expression product of the engineered strain.

[0024] Figure 4 This is a standard curve showing the p-NP concentration versus absorbance at 405 nm, as presented in this invention.

[0025] Figure 5wt represents the concentration of p-NP produced by hydrolyzing p-NPP of CALB fusion protein in the fermentation supernatant of this invention; wt represents the induced expression supernatant of Bacillus subtilis; CALB- represents the non-induced expression supernatant of the engineered strain; and CALB+ represents the induced expression supernatant of the engineered strain.

[0026] Figure 6 This is a standard curve showing the concentration of the commercial CALB enzyme and the absorbance at 405 nm, as presented in this invention. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0029] 1. Design of target protein expression sequences and gene synthesis The amino acid sequence of MBP was obtained from the snapgene website as SEQ ID NO.1 (Plasmids MBP), and the amino acid sequence of CALB was obtained from the NCBI website as SEQ ID NO.2 (GeneBank Z30645.1). A linker sequence SEQ ID NO.3 was added between the two proteins, containing the coagulation factor Xa restriction site (marked in red). The designed complete amino acid sequence was given to the gene synthesis company (Genewiz) for codon optimization based on the codon preference of Bacillus subtilis to generate the nucleotide sequence. The optimized gene sequence is SEQ ID NO.4, which was synthesized by the company and constructed into a vector plasmid carrying the T7 promoter.

[0030] 2. Construct the protein expression plasmid pMK4-MBP-CA and transform it into Bacillus subtilis. PCR was performed using primers shown in SEQ ID NO. 5-6 with the vector plasmid pMK4 (ATCC 37315) as a template (see primer list). After the reaction, the linear vector fragment was recovered. Primers shown in SEQ ID NO. 7-8 were designed to amplify the target gene, and repeat sequences approximately 15 bp from both ends of the linear vector were added to the ends of the primers as homologous arms. After the reaction, the linear target fragment was recovered. The recovered target fragment and the linear vector were subjected to homologous recombination to obtain the pMK4-MBP-CALB recombinant plasmid, as shown below. Figure 1 As shown. The reaction product was transformed into an *E. coli* cloning host, and single clones carrying the vector plasmid were obtained after antibiotic selection. PCR identification results showed that the expression vector carrying the target gene was successfully constructed. The expression vector was extracted from the cloning host and sequenced by Genewiz, and the gene sequence was found to be consistent with the designed plasmid sequence. The expression plasmid pMK4-MBP-CALB was transformed into *Bacillus subtilis* (…). Bacillus subtilis ATCC 6051a, disclosed in Engineering Bacillus subtilis (ATCC 6051a for the production of recombinant catalases), was used to select single clones grown in resistant plates for PCR identification. The PCR product bands were consistent with expectations, and the results are as follows. Figure 2 As shown, this indicates that the expression plasmid was successfully transformed into Bacillus subtilis, resulting in an engineered strain expressing the CALB fusion protein.

[0031] 3. CALB fusion protein induced expression Single clones of Bacillus subtilis carrying the CALB fusion protein expression plasmid were selected from resistance plates and inoculated into LB medium, and cultured overnight in a shaker at 37°C. Then, 1 mL of the overnight culture was inoculated into 100 mL of fresh LB medium, and cultured in a shaker at 37°C for 3 hours. Xylose was added to a final concentration of 1% as an inducer, and fermentation continued for 24 hours. The CALB fusion protein was induced and secreted into the fermentation supernatant. The supernatant was collected by centrifugation and filtered using a 0.22 μm filter. SDS-PAGE protein electrophoresis was performed on the fermentation supernatant. Unconverted Bacillus subtilis (wt) and engineered bacteria without inducer (CALB-) served as negative controls.

[0032] The results are as follows Figure 3 As shown, a specific band was observed near the 70 kDa protein molecular weight band in the engineered bacteria (CALB+) induced expression group, indicating that the CALB fusion protein was successfully expressed in soluble form and secreted into the fermentation supernatant.

[0033] 4. CALB fusion protein esterase activity assay Lipase activity in fermentation supernatant was determined using a 96-well plate high-throughput p-nitrophenol method. This method uses p-p-NPP as the reaction substrate. The ester bonds in the substrate are hydrolyzed by lipase, releasing free p-nitrophenol (p-NP) and the corresponding carboxylic acid. p-NP is a yellow, soluble product with a maximum absorption peak at 405 nm. Therefore, lipase activity can be determined by measuring the absorbance at 405 nm after a certain reaction time, and by using a standard curve of different absorbance values ​​at 405 nm for different concentrations of p-NP. The results are shown below. Figure 4 As shown in the figure. The concentration of p-NP generated by the hydrolysis reaction was determined by a standard curve, and the concentration of p-NPP consumed was calculated. The reaction system consisted of 75 μL of Tris-HCl buffer (50 mmol / L, pH=8.5), 5 μL of p-NPP dissolved in acetonitrile (10 mmol / L), and 25 μL of fermentation supernatant. After shaking and mixing, the reaction system was placed in a 45°C water bath for 15 minutes. After the reaction was completed, 80 μL of the reaction system was transferred to a 96-well plate for absorbance measurement, and the results are shown in the figure. Figure 5 As shown, the CALB fusion protein in the supernatant successfully hydrolyzed p-NPP to generate p-NP, demonstrating esterase activity.

[0034] 5. Determination of CALB fusion protein concentration in fermentation supernatant A commercially available recombinant CALB enzyme (62288, Sigma-Aldrich) derived from *Aspergillus oryzae* was purchased, and a series of CALB protein solutions of different concentrations were prepared. The yield of CALB fusion protein in the fermentation supernatant was determined using a relative quantification method. 25 μL of the protein solution was reacted with the substrate p-NPP, with the reaction system and time identical to those in step 4. After reacting different CALB protein concentrations with the p-NPP substrate, the absorbance was measured at 405 nm, and calibration curves were plotted accordingly. The results are shown below. Figure 6 As shown in the figure. Then, the relative content of CALB fusion protein in the fermentation supernatant was calculated using the calibration curve, and it was found that the protein yield in the fermentation supernatant was approximately 4 g / L.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0036] sequence list SEQ ID NO.1 MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT SEQ ID NO.2 MKLLSLTGVAGVLATCVAATPLVKRLPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPTTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSALRSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTPHHHHHH SEQ ID NO.3 NSSSNNNNNNNNNNLGIEGRGSACEL SEQ ID NO.4 SEQ ID NO.5 tgagaattcctgctaacaaa SEQ ID NO.6 tttcgcgggatcgagatc SEQ ID NO.7 ctcgatcccgcgaaattaatacgactcactatagg SEQ ID NO.8 tagcaggaattctcattaatgatggtggtgatgat

Claims

1. A method for constructing a strain that produces Candida antarcticis lipase B, characterized in that, Includes the following steps: S1: Construct a fusion expression cassette containing the MBP gene, a linker sequence, and the CALB gene. The linker sequence includes a coagulation factor Xa enzyme recognition site; S2: Insert the fusion expression cassette into the expression vector to obtain the recombinant expression vector; S3: Transform the recombinant expression vector into Bacillus subtilis to obtain a recombinant strain.

2. The construction method according to claim 1, characterized in that, The amino acid sequence encoded by the MBP gene is shown in SEQ ID NO.1; The amino acid sequence encoded by the CALB gene is shown in SEQ ID NO.2; The amino acid sequence of the linker sequence is shown in SEQ ID NO.

3.

3. The construction method according to claim 1, characterized in that, The nucleotide sequence of the fusion expression cassette described in S1 is shown in SEQ ID NO.

4.

4. The construction method according to claim 1, characterized in that, The expression vector described in S2 is the pMK4 plasmid vector.

5. The construction method according to claim 4, characterized in that, The construction of the expression vector in S2 is as follows: Using pMK4 plasmid as a template, PCR was performed with primers shown in SEQ ID NO.5~6 to obtain the linear vector fragment; Using the MBP-CALB fusion protein gene as a template, PCR amplification was performed using the primers shown in SEQ ID NO.7~8 to obtain the linear target fragment; The linear target fragment and the linear vector fragment are homologously recombined to obtain the recombinant expression vector.

6. The strain obtained by the construction method according to any one of claims 1 to 5.

7. A method for producing Candida antarcticis lipase B, characterized in that, The strain described in claim 6 was fermented and cultured, and an inducer was added to induce fermentation. Antarctic Candida lipase B was obtained from the fermentation supernatant.

8. The method according to claim 7, characterized in that, The inducer is xylose, and the final concentration of xylose in the fermentation system is 0.5%~2%.

9. The method according to claim 7, characterized in that, The fermentation culture temperature is 30~37℃, and the time is 18~30h.

10. The use of the strain according to claim 6 in the production of Candida antarcticis lipase B.

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