An engineered Trichoderma reesei strain secreting and expressing thaumatin and its construction method and application

Abstract

The present invention discloses a Trichoderma reesei engineering bacterium secreting and expressing brazzein, and its construction method and application, which relate to the fields of molecular biology and biotechnology. The construction method includes the step of transforming an expression module into the original strain of Trichoderma reesei to construct the Trichoderma reesei engineering bacterium. The present invention constructs a brazzein fusion gene and an expression module, and transforms the expression module into a variety of Trichoderma reesei strains, and successfully constructs an engineering strain secreting and expressing brazzein. The engineering strain can be used for the production of brazzein. Through experimental verification, the brazzein secreted and expressed by the engineering strain can be used as a sweetener in the production industries such as food and beverage. The present invention provides a new natural sweetener with good safety for industries such as food and beverage, and has important industrial application value.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and biotechnology, and particularly to a Trichoderma reesei engineering bacterium for secreting and expressing brazzein, and a construction method and application thereof. Background Art

[0002] Sweeteners are food additives that impart sweetness to food or feed. According to the source, they can be divided into natural sweeteners and synthetic sweeteners. With the development of organic chemistry, synthetic sweeteners have gradually taken the dominant position. However, due to the suspicion of safety problems of some chemically synthesized sweeteners, many countries in the world have successively prohibited the use of these sweeteners. In view of this, it is of great significance to find sweeteners that are safe, non-toxic, and have pure sweetness.

[0003] In 1968, Kurihara and Beidier isolated a protein from plant fruits that can turn sour taste into sweet taste, and it was later named miraculin. Plant sweet proteins are derived from natural foods and are degraded and metabolized in the stomach and intestines in the same way as conventional proteins, which is completely different from carbohydrate metabolism. They do not cause significant blood sugar changes, nor do they have the possible thrombus risk brought by small molecule sweeteners (such as erythritol in sugar alcohols) entering the blood. Therefore, it has triggered the research and application of natural plant sweet proteins.

[0004] Brazzein is a sweet protein isolated and purified by Ding et al. in 1994 from the fruits of a wild plant in western Africa. Brazzein is a single-chain polypeptide composed of 54 amino acid residues, containing 8 cysteines, which form 4 pairs of intramolecular disulfide bonds. The relative molecular mass of brazzein is 6,500, and its sweetness is 2,000 times that of sucrose of the same mass. Compared with other sweet proteins, brazzein has the smallest molecular weight, the best water solubility, and its aqueous solution still retains sweetness after heat treatment at 80°C for 4 hours, having good thermal stability and pH stability.

[0005] Trichoderma reesei is an important industrial production strain that meets the GRAS (Generally Regarded as Safe) standard. The enzyme preparations produced by its fermentation have been widely used in industries such as food and feed. Although there are successful cases of heterologous expression of enzyme preparations using Trichoderma reesei, it does not mean that thaumatin can also be successfully expressed in Trichoderma reesei. First of all, thaumatin needs to be secreted and expressed, and secretion and expression require a secretion peptide. Commercial strains (such as Escherichia coli and Pichia pastoris) have mature and efficient secretion peptides, while non-commercial strains (such as Trichoderma reesei) require a large amount of experimental and innovative research to discover and prove. Finding the appropriate secretion peptide for thaumatin is the key to success. Secondly, thaumatin needs to be correctly folded in Trichoderma reesei and form a large number (4-8) of disulfide bonds, which also requires a large amount of experimental optimization and innovative work. Therefore, there has not been a report on the fermentation production of thaumatin using Trichoderma reesei strains. Summary of the Invention

[0006] The object of the present invention is to provide an engineered Trichoderma reesei strain for secreting and expressing thaumatin, its construction method and application, so as to solve the problems existing in the above-mentioned prior art. This engineered Trichoderma reesei strain can be used to produce thaumatin, and this thaumatin, as a natural and safe sweetener, can be applied to production industries such as food and beverage.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for constructing an engineered Trichoderma reesei strain for secreting and expressing thaumatin, including the step of transforming an expression module into the original Trichoderma reesei strain to construct the engineered Trichoderma reesei strain;

[0009] The expression module includes a promoter, a fusion gene and a terminator connected in sequence; the fusion gene includes a secretion peptide gene and a thaumatin gene connected in sequence;

[0010] The secretion peptide gene is a gene encoding a secretion peptide, and the amino acid sequence of the secretion peptide is shown as any one of SEQ ID NO.2-4;

[0011] The thaumatin gene is a gene encoding thaumatin, and the amino acid sequence of the thaumatin is shown as SEQ ID NO.1.

[0012] Further, the nucleotide sequence of the secretion peptide gene is shown as any one of SEQ ID NO.14-16; the nucleotide sequence of the thaumatin gene is shown as SEQ ID NO.13.

[0013] Preferably, the nucleotide sequence of the secretion peptide gene is shown as SEQ ID NO.15.

[0014] Furthermore, the original Trichoderma reesei strains include Trichoderma reesei QM6a, QM9414, RUT-C30, RL-P37, NG14 or PC-3-7.

[0015] Preferably, the original Trichoderma reesei strain is PC-3-7.

[0016] The present invention also provides a Trichoderma reesei engineering strain constructed according to the above construction method.

[0017] The present invention also provides the application of the above Trichoderma reesei engineering strain in the preparation of brazzein.

[0018] The present invention also provides a brazzein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0019] The present invention also provides the application of the above brazzein in the preparation of sweeteners, sweet foods or sweet beverages.

[0020] The present invention also provides a sweetener, comprising the above brazzein.

[0021] The present invention discloses the following technical effects:

[0022] The present invention constructs a brazzein fusion gene and an expression module, and transforms the expression module into various Trichoderma reesei strains, successfully constructing an engineering strain that secretes and expresses brazzein. This engineering strain can be used for the production of brazzein. Through experiments, it is verified that the brazzein secreted and expressed by this engineering strain can be used as a sweetener in the production industries such as food and beverage. The present invention provides a new natural sweetener with good safety for industries such as food and beverage, and has important industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 Schematic diagram of the construction process of the brazzein fusion gene (A), eight expression plasmids (B), and the brazzein expression module (C);

[0025] Figure 2Results of analyzing thaumatin produced by different thaumatin fusion genes (A), different expression plasmids (B), and different Trichoderma reesei strains (C) using the sweetness threshold method; among them, "0" represents no sweetness, "1" represents weak sweetness, "2" represents obvious sweetness, and each result is the average value calculated from multiple evaluations.

[0026] Figure 3 Results of analyzing and comparing the sweetness differences between thaumatin solutions at different concentrations and 11.75% (w / v) sucrose solution using the sweetness comparison method; among them, "0" represents less sweet than the sucrose solution, "1" represents similar sweetness to the sucrose solution, "2" represents significantly sweeter than the sucrose solution, and each result is the average value calculated from multiple evaluations.

[0027] Figure 4 Results of sweetness tests on different breads; the smaller the value, the higher the sweetness. Detailed implementation manners

[0028] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0029] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0032] As used herein, terms such as "comprising", "including", "having", "containing", etc. are all open-ended terms, meaning including but not limited to.

[0033] For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989).

[0034] The amino acid sequence of thaumatin is shown as SEQ ID NO.1, and its corresponding nucleotide sequence is shown as SEQ ID NO.13.

[0035] The amino acid sequences of the secretion peptides are shown as SEQ ID NOs. 2 to 4, and their corresponding nucleotide sequences are shown as SEQ ID NOs. 14 to 16.

[0036] The nucleotide sequence of the CBHI promoter-terminator is shown as SEQ ID NO.5; the nucleotide sequence of the CBHII terminator-terminator is shown as SEQ ID NO.6; the nucleotide sequence of the EGLI promoter-terminator is shown as SEQ ID NO.7; the nucleotide sequence of the EGLII promoter-terminator is shown as SEQ ID NO.8; the nucleotide sequence of the EGLIII promoter-terminator is shown as SEQ ID NO.9; the nucleotide sequence of the XYNI promoter-terminator is shown as SEQ ID NO.10; the nucleotide sequence of the XYNII promoter-terminator is shown as SEQ ID NO.11; the nucleotide sequence of the XYNIII promoter-terminator is shown as SEQ ID NO.12.

[0037] Example 1 Construction of the Thaumatin Fusion Gene

[0038] In order to achieve the efficient secretion and expression of thaumatin in Trichoderma reesei, it is necessary to fuse an endogenous fungal secretion peptide at the N-terminus of thaumatin to direct the secretion and expression of the heterologous thaumatin. In addition to secretion, rapid cleavage and separation between the secretion peptide and thaumatin need to be achieved, so a lysine-arginine tandem structure needs to be inserted between the two to achieve cleavage. Therefore, the amino acid sequence structure encoded by the fusion gene is: secretion peptide - thaumatin, as shown in Figure 1 A in the figure. The present invention provides 3 kinds of secretion peptides (SEQ ID NOs. 2 to 4) and 1 kind of thaumatin (SEQ ID NO.1), and 3 different amino acid sequence-encoded thaumatin fusion genes can be arranged and combined. For example, the fusion gene g3.1 is the gene sequence encoding the amino acid sequence SEQ ID NO.3 (secretion peptide) + SEQ ID NO.1 (thaumatin).

[0039] The method for obtaining the fusion gene is as follows: providing an amino acid sequence and entrusting a conventional gene company to synthesize the corresponding nucleotide coding sequence. The synthesis rule of the present invention is based on the codon preference of Trichoderma reesei host. Due to the existence of codon degeneracy (codon degeneracy refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid), degenerate sequences with as low as about 92% homology to the nucleotide sequences in SEQ ID NOs. 13-16 can also encode the corresponding amino acid sequences. Therefore, the term "fusion gene sequence" also includes nucleotide sequences with at least 92% homology to the nucleotide sequences in SEQ ID NOs. 13-16.

[0040] Example 2 Construction of the brazzein expression module

[0041] To transcribe and express the fusion gene in Example 1 in Trichoderma reesei, a promoter and a terminator are required. The present invention provides eight endogenous promoters with the highest expression levels in Trichoderma reesei and the sequences of their terminators SEQ ID NOs. 5-12. The construction scheme is as follows:

[0042] 1) Using the overlap extension PCR scheme, connect the promoter and the terminator to synthesize the sequences of SEQ ID NOs. 5-12, and construct eight DNA structures, as Figure 1 shown in B. Restriction enzyme sites are added at both ends of the sequence. Three restriction enzyme sites - PacI (TTAATTAA), EcoRI (GAATTC), XbaI (TCTAGA) - are added upstream of the promoter; a restriction enzyme site - PmlI (CACGTG) - is inserted at the junction of the promoter and the terminator to facilitate the subsequent insertion of the fusion gene; three restriction enzyme sites - SpeI (ACTAGT), SacI (GAGCTC), SwaI (ATTTAAAT) - are added downstream of the terminator, as Figure 1 shown in B. When synthesizing DNA, 5 sequence-length protection bases (AAACC, GGTTT) are added to the 5' ends upstream of the promoter and downstream of the terminator respectively to facilitate subsequent DNA digestion.

[0043] 2) The above eight DNA structures are ligated into the backbone plasmid pPK1s by DNA ligation to construct eight expression plasmids, namely CBHI expression plasmid, CBHII expression plasmid, EGLI expression plasmid, EGLII expression plasmid, EGLIII expression plasmid, XYNI expression plasmid, XYNII expression plasmid, and XYNIII expression plasmid. The purpose of ligating into the plasmid is, firstly, to better preserve and replicate the above eight DNA structures for subsequent large-scale acquisition of this structure; secondly, because there are resistance screening markers such as hygromycin on the plasmid, which is convenient for subsequent transformation of Trichoderma reesei. The backbone plasmid pPK1s, ligation method, and experimental principle are based on the inventor's previous research papers ("Zhang et al. Enhanced production of heterologous proteins by the filamentous fungus Trichoderma reesei via disruption of the alkaline serine protease SPW combined with a pH control strategy. Plasmid 71(2014)16 - 22.", "Wang et al. Construction of a promoter collection for genes co-expression in filamentous fungus Trichoderma reesei. J Ind Microbiol Biotechnol(2014)41:1709 - 1718."). Double digestion is performed at the restriction enzyme SpeI / SwaI sites on the existing backbone plasmid pPK1s, and the eight DNA structures are double digested with XbaI / SwaI sites, and then ligated using T4 DNA ligase. Utilizing the property of SpeI and XbaI being isocaudomers, the above eight DNA structures can be ligated into pPK1s respectively to construct the corresponding eight expression plasmids( Figure 1 in B).

[0044] 3) The fusion gene is ligated into the expression plasmid to construct an expression module for the fusion gene. Double digestion and linearization are performed at the restriction enzyme PmlI / MauBI sites of the eight expression plasmids; a pair of primers is designed according to the requirements of the Vazyme One Step Clone Kit, and the synthesized fusion gene is subjected to PCR cloning; then seamless ligation is carried out using the Vazyme One Step Clone Kit - ligating the linearized expression plasmid and the PCR fragment together, that is, ligating the fusion gene into the expression plasmid to construct expression modules for various fusion genes( Figure 1In C). For example, the CBHI-g3.1 expression module is as follows: using the CBHI expression plasmid to express the g3.1 fusion gene. Design primers for cloning and amplifying the fusion gene according to the specific sequence of the commissioned synthetic fusion gene. When performing seamless cloning, the PCR fragment does not require enzymatic digestion, so the primers do not require protection bases.

[0045] (a) Using primers CBHI-g2.1-F and CBHI-g2.1-R, amplify g2.1 respectively with the synthesized DNA of the g2.1 fusion gene as the template.

[0046] CBHI-g2.1-F: 5’-GGACTGCGCATCATGTACCGCAAGCTGGCCGTT-3’ (SEQ ID NO.17);

[0047] CBHI-g2.1-R: 5’-CACGGAGCTCTATTAGTACTCGCAGTAGTCGCAGAT-3’ (SEQ ID NO.18).

[0048] The amplified fragment is seamlessly ligated into the PmlI site of the CBHI expression plasmid, which is the CBHI-g2.1 expression module.

[0049] Using primers CBHI-g3.1-F and CBHI-g2.1-R, amplify g3.1 and g4.1 respectively with the synthesized DNA of the g3.1 and g4.1 fusion genes as the template.

[0050] CBHI-g3.1-F: 5’-GGACTGCGCATCATGCAGCTTGCGCAGCTGCTC-3’ (SEQ ID NO.19).

[0051] The amplified fragment is seamlessly ligated into the PmlI site of the CBHI expression plasmid, which is the CBHI-g3.1 and CBHI-g4.1 expression modules.

[0052] (b) Using primers CBHII-g2.1-F and CBHII-g2.1-R, amplify g2.1 respectively with the synthesized DNA of the g2.1 fusion gene as the template.

[0053] CBHII-g2.1-F: 5’-GTGTATTGCACCATGTACCGCAAGCTGGCCGTT-3’ (SEQ ID NO.20);

[0054] CBHII-g2.1-R: 5’-ACGAAAGCCCTATTAGTACTCGCAGTAGTCGCAGAT-3’ (SEQ ID NO.21).

[0055] The amplified fragment was seamlessly ligated into the PmlI site of the CBHII expression plasmid, which is the CBHII-g2.1 expression module.

[0056] Using primers CBHII-g3.1-F and CBHII-g2.1-R, g3.1 and g4.1 were amplified separately with the DNA of the synthesized g3.1 and g4.1 fusion genes as templates.

[0057] CBHII-g3.1-F: 5’-GTGTATTGCACCATGCAGCTTGCGCAGCTGCTC-3’ (SEQ ID NO.22). The amplified fragment was seamlessly ligated into the PmlI site of the CBHII expression plasmid, which are the CBHII-g3.1 and CBHII-g4.1 expression modules.

[0058] (c) Using primers EGLI-g2.1-F and EGLI-g2.1-R, g2.1 was amplified separately with the DNA of the synthesized g2.1 fusion gene as a template.

[0059] EGLI-g2.1-F: 5’-TGTTGTCCCAAAATGTACCGCAAGCTGGCCGTT-3’ (SEQ ID NO.23);

[0060] EGLI-g2.1-R: 5’-TCACTCCCGCTATTAGTACTCGCAGTAGTCGCAGAT-3’ (SEQ ID NO.24).

[0061] The amplified fragment was seamlessly ligated into the PmlI site of the EGLI expression plasmid, which is the EGLI-g2.1 expression module.

[0062] Using primers EGLI-g3.1-F and EGLI-g2.1-R, g3.1 and g4.1 were amplified separately with the DNA of the synthesized g3.1 and g4.1 fusion genes as templates.

[0063] EGLI-g3.1-F: 5’-TGTTGTCCCAAAATGCAGCTTGCGCAGCTGCTC-3’ (SEQ ID NO.25).

[0064] The amplified fragment was seamlessly ligated into the PmlI site of the EGLI expression plasmid, which are the EGLI-g3.1 and EGLI-g4.1 expression modules.

[0065] (d) Using primers EGLII-g2.1-F and EGLII-g2.1-R, g2.1 was amplified separately with the DNA of the synthesized g2.1 fusion gene as a template.

[0066] EGLII-g2.1-F: 5'-CCGTCATCGACAATGTACCGCAAGCTGGCCGTT-3' (SEQ ID NO.26);

[0067] EGLII-g2.1-R: 5'-CTCAGAGTGCTATTAGTACTCGCAGTAGTCGCAGAT-3' (SEQ ID NO.27).

[0068] The amplified fragment was seamlessly ligated into the PmlI site of the EGLII expression plasmid, which is the EGLII-g2.1 expression module.

[0069] Using the primers EGLII-g3.1-F and EGLII-g2.1-R, g3.1 and g4.1 were amplified separately with the DNA of the synthesized g3.1 and g4.1 fusion genes as templates.

[0070] EGLII-g3.1-F: 5'-CCGTCATCGACAATGCAGCTTGCGCAGCTGCTC-3' (SEQ ID NO.28).

[0071] The amplified fragment was seamlessly ligated into the PmlI site of the EGLII expression plasmid, which are the EGLII-g3.1 and EGLII-g4.1 expression modules.

[0072] (e) Using the primers EGLIII-g2.1-F and EGLIII-g2.1-R, g2.1 was amplified separately with the DNA of the synthesized g2.1 fusion gene as a template.

[0073] EGLIII-g2.1-F: 5'-CATAGCGTCGCAATGTACCGCAAGCTGGCCGTT-3' (SEQ ID NO.29);

[0074] EGLIII-g2.1-R: 5'-TTCCAGGTTCTATTAGTACTCGCAGTAGTCGCAGAT-3' (SEQ ID NO.30).

[0075] The amplified fragment was seamlessly ligated into the PmlI site of the EGLIII expression plasmid, which is the EGLIII-g2.1 expression module.

[0076] Using the primers EGLIII-g3.1-F and EGLIII-g2.1-R, g3.1 and g4.1 were amplified separately with the DNA of the synthesized g3.1 and g4.1 fusion genes as templates.

[0077] EGLIII-g3.1-F: 5’-CATAGCGTCGCAATGCAGCTTGCGCAGCTGCTC-3’ (SEQ ID NO.31).

[0078] The amplified fragment was seamlessly ligated into the PmlI site of the EGLIII expression plasmid, which is the EGLIII-g3.1 and EGLIII-g4.1 expression modules.

[0079] (f) Using primers XYN I-g2.1-F and XYN I-g2.1-R, g2.1 was amplified separately using the DNA of the synthesized g2.1 fusion gene as a template.

[0080] XYN I-g2.1-F: 5’-CAAATAATCATCATGTACCGCAAGCTGGCCGTT-3’ (SEQ ID NO.32);

[0081] XYN I-g2.1-R: 5’-TGAACGATCCTATTAGTACTCGCAGTAGTCGCAGAT-3’ (SEQ ID NO.33).

[0082] The amplified fragment was seamlessly ligated into the PmlI site of the XYN I expression plasmid, which is the XYN I-g2.1 expression module.

[0083] Using primers XYN I-g3.1-F and XYN I-g2.1-R, g3.1 and g4.1 were amplified separately using the DNA of the synthesized g3.1 and g4.1 fusion genes as templates.

[0084] XYN I-g3.1-F: 5’-CAAATAATCATCATGCAGCTTGCGCAGCTGCTC-3’ (SEQ ID NO.34).

[0085] The amplified fragment was seamlessly ligated into the PmlI site of the XYN I expression plasmid, which is the XYN I-g3.1 and XYN I-g4.1 expression modules.

[0086] (g) Using primers XYN II-g2.1-F and XYN II-g2.1-R, g2.1 was amplified separately using the DNA of the synthesized g2.1 fusion gene as a template.

[0087] XYN II-g2.1-F: 5’-GAAGACATCAACATGTACCGCAAGCTGGCCGTT-3’ (SEQ ID NO.35;

[0088] XYNII-g2.1-R: 5'-GAGCCCCCTCTATTAGTACTCGCAGTAGTCGCAGAT-3' (SEQ ID NO.36).

[0089] The amplified fragment was seamlessly ligated into the PmlI site of the XYNII expression plasmid, which is the XYNII-g2.1 expression module.

[0090] Using primers XYNII-g3.1-F and XYNII-g2.1-R, g3.1 and g4.1 were amplified separately with the DNA of the synthesized g3.1 and g4.1 fusion genes as templates.

[0091] XYNII-g3.1-F: 5'-GAAGACATCAACATGCAGCTTGCGCAGCTGCTC-3' (SEQ ID NO.37).

[0092] The amplified fragment was seamlessly ligated into the PmlI site of the XYNII expression plasmid, which are the XYNII-g3.1 and XYNII-g4.1 expression modules.

[0093] (h) Using primers XYNIII-g2.1-F and XYNIII-g2.1-R, g2.1 was amplified separately with the DNA of the synthesized g2.1 fusion gene as a template.

[0094] XYNIII-g2.1-F: 5'-GAGGCGGACAATATGTACCGCAAGCTGGCCGTT-3' (SEQ ID NO.38);

[0095] XYNIII-g2.1-R: 5'-GCAGAGCAGCTATTAGTACTCGCAGTAGTCGCAGAT-3' (SEQ ID NO.39).

[0096] The amplified fragment was seamlessly ligated into the PmlI site of the XYNIII expression plasmid, which is the XYNIII-g2.1 expression module.

[0097] Using primers XYNIII-g3.1-F and XYNIII-g2.1-R, g3.1 and g4.1 were amplified separately with the DNA of the synthesized g3.1 and g4.1 fusion genes as templates.

[0098] XYNIII-g3.1-F: 5'-GAGGCGGACAATATGCAGCTTGCGCAGCTGCTC-3' (SEQ ID NO.40).

[0099] The amplified fragments were seamlessly ligated into the PmlI site of the XYNIII expression plasmid, namely the XYNIII-g3.1 and XYNIII-g4.1 expression modules.

[0100] Example 3: The expression module was introduced into Trichoderma reesei to obtain an engineered strain expressing brazzein, and brazzein was produced.

[0101] In the present invention, through conventional experimental operations such as electrotransformation, protoplast transformation or Agrobacterium tumefaciens-mediated conjugation transfer, the expression module constructed in Example 2 was introduced into the Trichoderma reesei strain and integrated into the Trichoderma reesei genome. Through the selection marker on the expression module, the transformants were screened. The present invention takes the CBHI-g3.1 expression module and the Agrobacterium tumefaciens-mediated conjugation transfer scheme as an example for illustration.

[0102] 1) The CBHI-g3.1 expression module was electrotransformed into Agrobacterium tumefaciens, and then the successfully electrotransformed Agrobacterium tumefaciens was co-cultured with the Trichoderma reesei host strains QM6a (ATCC 13631), QM9414 (ATCC 26921), RUT-C30 (ATCC 56765), RL-P37 (NRRL 15709), NG14 (ATCC 56767) and PC-3-7 (ATCC 66589) on an IM plate (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105(3):259-264) for Agrobacterium tumefaciens-mediated conjugation transfer. After two days of co-culture, it was screened on a PDA plate containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) until hyphae and spores grew, and then PCR verification was carried out to prove that the grown bacteria were correct transformants.

[0103] 2) Taking the transformation of the Trichoderma reesei RUT-C30 strain with the CBHI-g3.1 expression module as an example for illustration: The transformant CBHI-g3.1 (RUT-C30) was inoculated into 50 mL in a 250 mL Erlenmeyer flask for verification culture using microcrystalline cellulose as the carbon source. The formula of the verification medium was: microcrystalline cellulose 10 g / L, Tryptone 1 g / L, yeast extract 0.5 g / L, (NH 4 ) 2 SO 4 1.4 g / L, Urea 0.3 g / L, KH 2 PO 4 2 g / L, CaCl 2 ·2H 2 O 0.3 g / L, MgSO4 ·7H 2 O 0.3 g / L, FeSO 4 ·7H 2 O 5 mg / L, MnSO 4 ·H 2 O 1.6 mg / L, ZnSO 4 ·7H 2 O 1.4 mg / L and CoCl 2 ·6H 2 O 2 mg / L. The inoculum size is 10 8 spores / 50 mL of medium, cultured at 28 °C and 200 rpm. Take the supernatant of the fermentation broth on the 4th day. Since the sweetening mechanism of brazzein is different from that of sugars, it cannot be measured by an electronic tongue. Therefore, according to the conventional gustatory sensory threshold method analysis, 10 volunteers were selected to evaluate the sweet taste perception of the fermentation broth diluted 1 - 1000 times, and the results are shown in Figure 2 .

[0104] 3) Inoculate the CBHI - g3.1 (PC - 3 - 7) engineering strain into the fermentation medium (microcrystalline cellulose 40 g / L, corn steep liquor 15 g / L, Tryptone 2 g / L, (NH 4 ) 2 SO 4 2.8 g / L, CaCl 2 ·2H 2 O 0.5 g / L, MgSO 4 ·7H 2 O 1 g / L, FeSO 4 ·7H 2 O 5 mg / L, MnSO 4 ·H 2 O 1.6 mg / L, ZnSO 4 ·7H 2 O 1.4 mg / L and CoCl 2 ·6H 2 O 2 mg / L, and culture at 28 °C for 10 days. After the fermentation is completed, the fermentation broth is roughly filtered using a glass column filled with cotton packing, and then secondary filtered with a 0.45 - micron filter membrane to finally obtain a sterile filtrate, which is liquid brazzein. The liquid brazzein is freeze - dried to make powdered brazzein.

[0105] Example 4 Application of Brazzein in Foods such as Beverages and Bread

[0106] 1) Beverage application experiment. 0.01 - 1 g of the powdery brazzein prepared in Example 3 was dissolved in 100 mL of ultrapure water to prepare a test solution. Positive control: 11.75 g of sucrose was dissolved in 100 mL of ultrapure water to prepare an 11.75% (w / v) sucrose aqueous solution. According to the conventional gustatory sensory comparison method, 10 volunteers were selected to compare the sweetness levels of the test solution and the control solution. The results are shown in Figure 3 . It can be seen from Figure 3 that the powdery brazzein provided by the present invention has an obvious sweetness, and is significantly higher than the sweetness of sucrose under the same mass, and can replace sucrose as a sweetener in beverages.

[0107] 2) Baking application experiment. The flour variety selected was Ningxia Saibei Snow high-gluten flour, and the bread was made by the experimental method of single fermentation.

[0108] 1) The ingredients were: 400 g of flour, 5.5 g of yeast, 5 g of glucose, 1 g of the powdery brazzein prepared in Example 3 (the control was 60 g of sucrose), 10 g of salt, 32 g of butter, and 228 g of water. The ingredients were mixed to make a dough, fermented at 38 °C and 80% humidity for 30 min, and then baked in an oven at 180 °C for 20 min. According to the conventional gustatory sensory comparison method, 10 volunteers were selected to compare the sweetness levels of the bread. The results are shown in Figure 4 . It can be seen from Figure 4 that the powdery brazzein provided by the present invention can replace sucrose to produce sweetness in baked foods; the remaining sweetness of 1 g of the powdery brazzein after high-temperature baking still exceeds that of 60 g of sucrose.

[0109] In summary, the present invention discloses a method and application for secreting and producing brazzein by Trichoderma reesei. The sweetness of the brazzein produced by Trichoderma reesei disclosed by the present invention is significantly higher than that of sucrose of the same mass.

[0110] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing an engineered bacterium of Trichoderma reesei that secretes and expresses brasiliensis, characterized in that: The method comprises the steps of transforming the expression module into the original strain of Trichoderma reesei to construct the engineered strain of Trichoderma reesei; The expression module includes a promoter, a fusion gene and a terminator connected in sequence; the fusion gene includes a secretory peptide gene and a brasilien gene connected in sequence; The secretory peptide gene is a gene encoding a secretory peptide, and the amino acid sequence of the secretory peptide is shown in any one of SEQ ID NOs. 2 to 4; The brazilian thauma protein gene is a gene encoding brazilian thauma protein, and the amino acid sequence of the brazilian thauma protein is shown as SEQ ID NO.

1.

2. The construction method according to claim 1, characterized in that: The nucleotide sequence of the secretory peptide gene is shown in any one of SEQ ID NOs. 14 to 16; the nucleotide sequence of the brasiliensis gene is shown in SEQ ID NO.

13.

3. The construction method according to claim 2, characterized in that: The nucleotide sequence of the secretory peptide gene is shown as SEQ ID NO.

15.

4. The construction method according to claim 1, characterized in that: The original strain of Trichoderma reesei includes Trichoderma reesei QM6a, QM9414, RUT-C30, RL-P37, NG14 or PC-3-7.

5. The construction method according to claim 4, characterized in that: The original strain of Trichoderma reesei is PC-3-7.

6. An engineered strain of Trichoderma reesei constructed according to the construction method according to any one of claims 1 to 5.

7. Use of the engineered bacteria of Trichoderma reesei as claimed in claim 6 in the preparation of brasiliensis.

8. A Brazilian sweet protein, characterized in that The amino acid sequence of the brazilian tamarind protein is shown in SEQ ID NO.

1.

9. Use of the brasiliensis protein as claimed in claim 8 in the preparation of sweeteners, sweet foods or sweet beverages.

10. A sweetener, characterized in that The method comprises the Brazilian sweet protein as described in claim 8.

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