Construction method of trichoderma reesei engineering bacteria for producing ectoine and application thereof

Abstract

The application discloses a construction method of a trichoderma reesei engineering bacterium for producing ectoine and application thereof, and belongs to the technical field of bioengineering. The application provides a construction method of a trichoderma reesei engineering bacterium for producing ectoine, which comprises the following steps: taking trichoderma reesei as a starting strain, and introducing an ectoine synthesis gene cluster into the trichoderma reesei to construct the trichoderma reesei engineering bacterium; and the nucleotide sequence of the ectoine synthesis gene cluster is shown as SEQ ID NO. 1. The application enables the trichoderma reesei which originally does not have the ectoine synthesis capacity to produce ectoine; and by increasing the number of ectoine gene clusters and optimizing the culture medium, the yield of ectoine is significantly improved, wherein, glucose is used as a carbon source, protein peptone and urea are used as long-acting nitrogen sources, and the addition amount of the nitrogen source is optimized, and the yield of ectoine in flask fermentation can reach 1494.5 mg / L, thereby providing technical support for industrialized production of ectoine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a method for constructing a Trichoderma reesei engineering strain for producing ectoine and application thereof. BACKGROUND

[0002] Ectoine, with the chemical formula of 2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, is a water-soluble zwitterionic amino acid derivative. It was first discovered in 1985 by German scientist Galinski in a halophilic bacterium (Halomonas elongata) in the salt lakes of the Egyptian desert. This substance is a key component for halophilic bacteria to survive in extreme environments such as high salt, high temperature, and strong ultraviolet light. Ectoine can protect the structure and function of microbial cells in extreme environments, maintain the balance of osmotic pressure inside and outside the cell, and prevent the cell from losing water due to high osmotic pressure. It can also stabilize enzyme molecular structure, protect proteins, DNA and cell membranes from damage. Ectoine has strong water molecule complexing ability and can structure the free water in cells, making it an excellent natural moisturizing agent. It can also repair DNA damage caused by ultraviolet light and protect the skin from ultraviolet damage. Ectoine can inhibit the expression of inflammatory factors and block oxidative stress, and has significant anti-inflammatory and antioxidant effects.

[0003] Due to its strong protein protection, cell protection, moisturizing, repair, anti-inflammatory and antioxidant functions, ectoine is widely used in the fields of cosmetics, biological medicine and agriculture. It can be added to various skin care products such as toner, sunscreen lotion and mask liquid. Ectoine can be used to treat atopic dermatitis or allergic skin diseases, and has certain anti-inflammatory and neuroprotective effects. Ectoine can improve the stress resistance of plants in harsh environments such as drought, high temperature and saline-alkali, and promote plant growth.

[0004] In recent years, with the rapid development of synthetic biology, gene engineering bacteria fermentation method is the main method for producing ectoine, which has the advantages of high efficiency, environmental protection and low cost. Due to environmental and cost problems, chemical synthesis method is gradually replaced by biological fermentation method.

[0005] Trichoderma reesei is an important industrial production strain, which meets the GRAS (Generally Regarded as Safe) standard, and its fermentation products have been widely used in food, feed and other industries. There is no report on directly using genetically engineered Trichoderma reesei strain to ferment and produce ectoine. Trichoderma reesei is the most widely used enzyme preparation fermentation strain in industry. Using Trichoderma reesei to ferment and produce ectoine has great significance for enzyme preparation production. SUMMARY

[0006] The application aims to provide a construction method of Trichoderma reesei engineering bacteria for producing ectoine and application thereof, so as to solve the problems in the prior art.

[0007] To achieve the above-mentioned purpose, the application provides the following solutions.

[0008] The application provides a construction method of Trichoderma reesei engineering bacteria for producing ectoine, comprising the following steps: taking Trichoderma reesei as a starting strain, and introducing an ectoine synthesis gene cluster into the Trichoderma reesei to construct the Trichoderma reesei engineering bacteria.

[0009] The nucleotide sequence of the ectoine synthesis gene cluster is shown as SEQ ID NO. 1.

[0010] Optionally, the ectoine synthesis gene cluster is introduced through a recombinant plasmid.

[0011] The recombinant plasmid is constructed by sequentially connecting a pdc promoter, a cbh2 terminator and the ectoine synthesis gene cluster into an LML2.0a skeleton.

[0012] The application also provides a construction method of an optimized Trichoderma reesei strain for producing ectoine, comprising the following steps: taking the Trichoderma reesei engineering bacteria as a starting strain, and introducing the ectoine synthesis gene cluster into the Trichoderma reesei engineering bacteria multiple times to construct the optimized Trichoderma reesei strain.

[0013] The nucleotide sequence of the ectoine synthesis gene cluster is shown as SEQ ID NO. 1; the ectoine synthesis gene cluster encodes a pro-ectoine synthesis enzyme, and the amino acid sequence of the pro-ectoine synthesis enzyme is shown as SEQ ID NO. 2.

[0014] The application also provides the Trichoderma reesei engineering bacteria for producing ectoine obtained according to the construction method.

[0015] The application also provides the optimized Trichoderma reesei strain for producing ectoine obtained according to the construction method.

[0016] The application also provides application of the Trichoderma reesei engineering bacteria or the optimized Trichoderma reesei strain in production of ectoine.

[0017] The application also provides a method for producing ectoine, comprising the step of fermenting by using the Trichoderma reesei engineering bacteria or the optimized Trichoderma reesei strain.

[0018] Optionally, the fermentation medium components used in the fermentation include 40 g of carbon source, 3-18 g of peptone, 1-5 g of urea, 4 g of KH2PO4, 3 g of (NH4)2SO4, 0.6 g of MgSO4·7H2O, 0.5 g of CaCl2, 1 mL of Mandels trace element solution, and 1 mL of Tween 80, and water is added to 1 L.

[0019] Optionally, the fermentation medium components used in the fermentation include 40 g of carbon source, 3-18 g of peptone, 1-5 g of urea, 4 g of KH2PO4, 3 g of (NH4)2SO4, 0.6 g of MgSO4·7H2O, 0.5 g of CaCl2, 1 mL of Mandels trace element solution, and 1 mL of Tween 80, and water is added to 1 L.

[0020] Optionally, the carbon source includes glucose or a cellulase inducer.

[0021] When the carbon source is a cellulase inducer, the Trichoderma reesei engineering strain or the Trichoderma reesei optimized strain simultaneously produces ectoine and an enzyme preparation.

[0022] The present application discloses the following technical effects:

[0023] The present application introduces an ectoine gene cluster through genetic engineering modification, so that the Trichoderma reesei originally without ectoine synthesis ability can produce ectoine; and by increasing the number of ectoine gene clusters and optimizing the medium, the yield of ectoine is significantly improved, wherein glucose is used as a carbon source, peptone and urea are used as long-acting nitrogen sources, and the addition amount of the nitrogen source is optimized, and the yield of ectoine in shake flask fermentation can reach 1494.5 mg / L, which provides technical support for the industrial production of ectoine. At the same time, the production of ectoine can not only improve the yield of enzyme preparation, but also significantly improve the heat stability of enzyme preparation, thereby increasing the output value under the same operating conditions and reducing the production cost. The present application has important application value in enzyme preparation production and heat stability protection. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0025] Figure 1 The construction process of the expression plasmid PT1.0, the ectoine synthesis plasmid PT1.0-ectABC, the ectoine engineering strain, and the optimized strain;

[0026] Figure 2The yield of the shake flask fermentation of Trichoderma reesei strains for the production of ectoine;

[0027] Figure 3 The effect of the concentration of proteose peptone (3-18 g / L) and urea (1-5 g / L) on the synthesis of ectoine by Trichoderma reesei;

[0028] Figure 4 The degree of improvement of the activity of the enzyme preparation produced by the four optimized strains compared with the respective starting strains;

[0029] Figure 5 The stability analysis of the human lysozyme produced by the human lysozyme optimized strain and the starting strain at 60°C. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of the application and not as limiting the application. It will be readily apparent to those skilled in the art that varying substitutions and modifications can be made to the application without departing from the scope and spirit of the application.

[0031] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, where a range of values is provided, it is understood that each intervening value, to the extent that it does not adversely affect the end result of the subject matter at hand, between the upper and lower limit of that range is also contended herein. In each instance, any intervening value can be included in the smaller range, if it exists. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also contended herein.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0033] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from consideration of the description and practice of the application disclosed herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0034] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0035] The culture media used in the present application are as follows:

[0036] (1) Luria Bertani (LB) medium formula: yeast powder 5 g, peptone 10 g, sodium chloride 10 g, water to 1 L, natural pH.

[0037] (2) Glucose or xylose PDA medium formula: potato infusion powder 6 g, glucose (or xylose) 20 g, agar 16 g, water to 1 L, natural pH.

[0038] (3) Ectodine fermentation medium formula: carbon source 40 g, peptone 3-18 g, urea 1-5 g, KH2PO4 4 g, (NH4)2SO4 3 g, MgSO4·7H2O 0.6 g, CaCl2 0.5 g, Mandels trace element solution 1 mL, and Tween 80 1 mL, purified water to 1 L.

[0039] (4) Mandels trace element solution (1000x) formula: FeSO4·7H2O 5 g, CoCl·6H2O 2 g, ZnSO4·7H2O 1.4 g, MnSO4·H2O 1.6 g, purified water to 1 L.

[0040] Some test methods used in the present application are as follows:

[0041] (1) Agrobacterium-mediated transformation of Trichoderma reesei:

[0042] The plasmid is electroporated into Agrobacterium tumefaciens, and then the electroporated Agrobacterium tumefaciens is co-cultured with the Trichoderma reesei host strain on IM plate medium (Covert et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol. Res. 105 (3): 259-264) for Agrobacterium tumefaciens-mediated transformation. After 2 days of co-culture, screening is performed in glucose PDA plates containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL), until mycelium and spores grow, and then PCR verification is performed to prove that the growing cells are correct transformants.

[0043] To make the transformed strain not contain the resistance marker gene, the hygromycin B resistance marker was eliminated using the literature protocol (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6:20761). The principle of resistance marker elimination is as follows: the strain is cultured and subcultured in a xylose PDA plate, and the hygromycin B resistance will be lost, with a loss efficiency close to 70% to 100%; the strain after loss cannot grow in a PDA plate with hygromycin, and this characteristic is used to verify the loss strain.

[0044] (2) Determination method of the content of ectoine:

[0045] a. 1 mL of the fermentation broth after shake flask culture was centrifuged at 14000 x g for 10 min to obtain the supernatant of the fermentation broth, which was filtered with a 0.22 μm filter membrane to obtain the sample to be detected. If the concentration of ectoine in the fermentation broth is higher than 1 g / L, the sample to be detected is prepared by appropriate dilution.

[0046] b. HPLC detection method: HPLC (Agilent 1200 infinity series 1260, Agilent Technologies) was performed using an NH2 chromatographic column (4.6 mm x 150 mm, 5 μm particle size, Japan). Acetonitrile: deionized water (70:30, v / v) was used as the mobile phase, the flow rate was 1.0 mL / min, the injection volume was 5 μL. The produced ectoine was detected at 210 nm, and identified by comparing the retention time with that of the analysis standard (purchased from Sigma). Quantification was performed by dividing the slope of the standard curve by the peak area.

[0047] c. Preparation of ectoine standard curve: 1 g / L ectoine standard solution was prepared as the mother liquor, and serial concentrations of 0.5, 0.25, and 0.1 g / L standard samples (final volume 1 mL) were prepared by dilution; the standard samples were determined by high performance liquid chromatography, and the peak area (y) was linearly regressed against the concentration (x) to obtain the equation y = 15579x, with a correlation coefficient R 2 0.9999, indicating that within the concentration range of 0.1 to 1 g / L, the concentration of ectoine showed a good linear relationship with the chromatographic peak area.

[0048] ​The Trichoderma reesei starting strain used in the present application is Trichoderma reesei QM6a (ATCC 13631), the derivative strain RUT-C30 (ATCC 56765) of QM6a, and the derivative strain of Trichoderma reesei RUT-C30, i.e., the asparaginease engineering strain (disclosed in patent ZL202110667266.1), the lipase engineering strain (disclosed in patent ZL202410105904.4), and the human lysozyme engineering strain (disclosed in patent CN202410045920.9). However, the method of the present application is also applicable to other derivative strains of QM6a (ATCC 13631), such as QM9414 (ATCC 26921), RL-P37 (NRRL 15709), NG14 (ATCC 56767), and PC-3-7 (ATCC 66589).

[0049] Example 1

[0050] 1. Obtaining of ectoine synthesis gene cluster

[0051] The three synthesis enzyme genes (ectABC) in the ectoine synthesis gene cluster derived from H. elongata are codon-optimized based on the codon usage bias of Trichoderma reesei, wherein the ectA, ectB, and ectC genes are connected by a 2A peptide gene. The ectoine synthesis gene cluster sequence is synthesized by Jier company, and the nucleotide sequence is shown in SEQ ID NO. 1.

[0052] SEQ ID NO. 1:

[0053]

[0054] The amino acid sequence encoded by SEQ ID NO. 1 is shown in SEQ ID NO. 2. SEQ ID NO. 2 is an ectoine synthetase precursor encoded by the ectoine gene cluster, in which three enzymes are connected by two 2A peptides, and the underlined part in the sequence is the 2A peptide. The 2A peptide is a short peptide with the characteristic of "self-cleavage". When the 2A peptide connects multiple protein coding sequences, cleavage will occur at the 2A peptide after translation, so that the originally fusion-expressed multiple proteins are separated, and each independently folds and functions.

[0055] SEQ ID NO. 2:

[0056] MNATTEPFTPSADLAKPSVADAVVGHEASPLFIRKPSPDDGWGIYELVKSCPPLDVNSAYAYLLLATQFRDSCAVATNEEGEIVGFVSGYVKSNAPDTYFLWQVAVGEKARGTGLARRLVEAVMTRPEMAEVHHLETTITPDNQASWGLFRRLADRWQAPLNSREYFSTDQLGGEHDPENLVRIGPFQTDQI APVKQTLNFDLLKLAGDVESNPGP MQTQILERMESDVRTYSRSFPVVFTKARNARLTDEEGREYIDFLAGAGTLNYGHNNPHLKQALLDYIDSDGIVHGLDFWTAAKRDYLETLEEVILKPRGLDYKVHLPGPTGTNAVEAAIRLARVAKGRHNIVSFTNGFHGVTMGALATTGNRKFREATGGVPTQAASFMPFDGYLGSSTDTLDYFEKLLGDKSGGLDVPAAVIVETVQGEGGINVAGLEWLKRLESICRANDILLIIDDIQAGCGRTGKFFSFEHAGITPDIVTNSKSLSGYGLPFAHVLMRPELDKWKPGQYNGTFRGFNLAFATAAAAMRKYWSDDTFERDVQRKARIVEERFGKIAAWLSENGIEASERGRGLMRGIDVGSGDIADKITHQAFENGLIIETSGQDGEVVKCLCPLTIPDEDLVEGLDILETSTKQAFS APVKQTLNFDLLKLAGDVESNPGPMIVRNLEEARQTDRLVTAENGNWDSTRLSLAEDGGNCSFHITRIFEGTETHIHYKHHFEAVYCIEGEGEVETLADGKIWPIKPGDIYILDQHDEHLLRASKTMHLACVFTPGLTGNEVHREDGSYAPADEADDQKPL.

[0057] 2. Construction of blank expression plasmid

[0058] In order to achieve constitutive expression in T. reesei, the present application constructs a blank expression plasmid PT1.0, and the following experiments are carried out:

[0059] (1) Promoter cloning

[0060] The DNA sequence of the pdc promoter (SEQ ID NO. 9) is amplified using primers pdc-F1 and pdc-F2 and the T. reesei genome as a template. The 16 nt long linker required for constructing the expression plasmid is added to the pdc-F1 primer. The sequence identical to the linker sequence exists in the plasmid LML2.0a (published in the literature Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6: 20761), which is used for subsequent seamless ligation by Vazyme One Step Clone Kit. The pdc-F2 primer also adds a translation initiation codon ATG and a restriction site ATTTAAAT, as well as part of the cbh2 terminator sequence as a linker (a total of 19 nt), which is used for subsequent seamless ligation of the cbh2 terminator sequence to construct a blank expression plasmid.

[0061] pdc-F1: 5’- ACTAGTGAGCTCATTT ATGAAAGGAGGGAGCATTCTTCGA-3’ (SEQ ID NO. 3);

[0062] pdc-F2: 5’- CGAAAGCCATTTAAATCAT GATTGTGCTGTAGCTGCGC-3’ (SEQ ID NO. 4);

[0063] The underlined part represents the linker sequence.

[0064] Reaction system: 10x PCR Buffer for KOD-Plus-Neo 5 μL, 2 mM dNTPs 5 μL, 25 mM MgSO4 3 μL, 10 μM primer pdc-F1 / pdc-F2 1.5 μL each, genomic template (~200 ng) 1 μL, KOD-Plus-Neo (1 U / μL) 1 μL.

[0065] Reaction procedure: 94 °C 2 min; 98 °C 10 sec, 68 °C 1 min, run for 30 cycles; 68 °C 5 min.

[0066] (2) Terminator cloning

[0067] Using primers cbh2-F1 and cbh2-F2, the DNA sequence of the cbh2 terminator (SEQ ID NO. 10) was amplified from the Trichoderma reesei genome. A 19 nt length linker was added to the cbh2-F2 primer, which was required for the construction of the expression plasmid. There was an 11 nt sequence in cbh2-F1 that was reverse complementary to the sequence in pdc-F2 (translation start codon ATG and restriction site ATTTAAAT) as a linker for subsequent seamless ligation by Vazyme One Step Clone Kit.

[0068] cbh2-F1: 5’- ATGATTTAAAT GGCTTTCGTGACCGGGCTT-3’ (SEQ ID NO. 5);

[0069] cbh2-F2: 5’- GCCAGTGCCAAGCTTATTT TGGGTATGGTTTCCACGTGCA-3’ (SEQ ID NO. 6);

[0070] The underlined sequence represents the linker sequence.

[0071] Reaction system: 10x PCR Buffer for KOD-Plus-Neo 5 μL, 2 mM dNTPs 5 μL, 25 mM MgSO4 3 μL, 10 μM primer cbh2-F1 / cbh2-F2 1.5 μL each, genomic template (~200 ng) 1 μL, KOD-Plus-Neo (1 U / μL) 1 μL.

[0072] Reaction procedure: 94 °C 2 min; 98 °C 10 sec, 68 °C 1 min, run for 30 cycles; 68 °C 5 min.

[0073] (3) Construction of blank expression plasmid

[0074] The expression vector was constructed with LML2.0a as a backbone. LML2.0a was subjected to SmiI single enzyme digestion, and the amplified pdc promoter and amplified cbh2 terminator were connected by using Vazyme One Step Clone Kit for seamless connection to construct a blank expression plasmid PT1.0 Figure 1 )。

[0075] 3. Construction of ectoine synthesis plasmid

[0076] (1) Cloning of ectABC gene cluster

[0077] The DNA sequence of SEQ ID NO. 1 was amplified using primers ectABC-F1 and ectABC-F2 with synthesized DNA as a template. The 15nt length of linker sequence was added to the primers ectABC-F1 and ectABC-F2 for the construction of the expression plasmid. The blank expression plasmid PT1.0 contains the same sequence as the linker sequence, which is used for subsequent seamless connection by Vazyme One Step Clone Kit.

[0078] ectABC-F1: 5’- TACAGCACAATCATG AACGCGACGACGGAGCC-3’ (SEQ ID NO. 7);

[0079] ectABC-F2: 5’- TCACGAAAGCCATTT TCACAGCGGCTTTTGGTCATCG-3’ (SEQ ID NO. 8);

[0080] The underlined part represents the linker sequence.

[0081] Amplification reaction system: 10×PCR Buffer for KOD-Plus-Neo 5μL, 2mM dNTPs 5μL, 25mM MgSO4 3μL, 10μM primers ectABC-F1 / ectABC-F2 1.5μL each, genomic template (~200ng) 1μL, KOD-Plus-Neo (1U / μL) 1μL.

[0082] Reaction procedure: 94℃ 2min; 98℃ 10sec, 68℃ 1.5min, 30 cycles; 68℃ 5min.

[0083] (2) Construction of ectoine synthesis plasmid

[0084] Ectoine synthesis plasmid was constructed by using PT1.0 as a backbone. PT1.0 was subjected to SmiI single enzyme digestion, and the amplified ectABC gene cluster was connected by using Vazyme One Step Clone Kit for seamless connection to construct ectoine synthesis plasmid PT1.0-ectABC. Figure 1

[0085] SEQ ID NO. 9 (pdc promoter sequence):

[0086] ​

[0087] SEQ ID NO. 10 (cbh2 terminator sequence):

[0088] GGCTTTCGTGACCGGGCTTCAAACAATGATGTGCGATGGTGTGGTTCCCGGTTGGCGGAGTCTTTGTCTACTTTGGTTGTCTGTCGCAGGTCGGTAGACCGCAAATGAGCAACTGATGGATTGTTGCCAGCGATACTATAATTCACATGGATGGTCTTTGTCGATCAGTAGCTAGTGAGAGAGAGAGAACATCTATCCACAATGTCGAGTGTCTATTAGACATACTCCGAGAATAAAGTCAACTGTGTCTGTGATCTAAAGATCGATTCGGCAGTCGAGTAGCGTATAACAACTCCGAGTACCAGCAAAAGCACGTCGTGACAGGAGCAGGGCTTTGCCAACTGCGCAACCTTGCTTGAATGAGGATACACGGGGTGCAACATGGCTGTACTGATCCATCGCAACCAAAATTTCTGTTTATAGATCAAGCTGGTAGATTCCAATTACTCCACCTCTTGCGCTTCTCCATGACATGTAAGTGCACGTGGAAACCATACCCA.

[0089] 4. Construction of Ectoine engineering strain

[0090] The ectoine synthesis plasmid PT1.0-ectABC was electroporated into Agrobacterium, and the transformed Agrobacterium was co-cultured with the Trichoderma reesei starting strain QM6a on an IM plate to perform rhizobium-mediated conjugation transfer, and the PT1.0-ectABC was randomly inserted into the genome of the Trichoderma reesei QM6a strain. After co-culturing for two days, the transformant was transferred to a PDA plate containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) for screening, until the plate grew mycelium and spores, and then the successful transformation was verified. In order to make the transformed strain not contain the resistance marker gene, the hygromycin B resistance marker was eliminated using the literature scheme (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6: 20761), that is, the ectoine engineering strain.

[0091] 5. Construction of an ectoine optimization strain

[0092] The ectoine engineering strain obtained in the above 4, construction of an ectoine engineering strain, has lost the hygromycin B resistance marker, and is consistent with QM6a in the resistance phenotype. Therefore, the obtained ectoine engineering strain can be used as a starting strain for transformation again.

[0093] Agrobacterium, electroporated with the ectoin synthetic plasmid PT1.0-ectABC, was co-cultured with the ectoin engineered strain on IM plates for Agrobacterium-mediated conjugation transfer. PT1.0-ectABC randomly inserted into the genome of the ectoin engineered strain. After two days of co-culture, transformants were transferred to PDA plates containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) for selection until hyphae and spores grew on the plates, and then the transformation was verified as successful. To ensure that the transformed strain did not contain the resistance marker gene, the hygromycin B resistance marker was removed using the protocol described in the literature (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6: 20761), resulting in the ectoin optimized strain (first generation). At this point, the ectoine-optimized strain (generation 1) has lost the hygromycin B resistance marker and is phenotypically identical to QM6a in terms of resistance. Therefore, it can be used as the starting strain for subsequent transformations to obtain: ectoine-optimized strain (generation 2), ectoine-optimized strain (generation 3), and so on, until the ectoine-optimized strain (generation N).

[0094] Example 2: Ectocin yield detection of engineered and optimized ectocin strains

[0095] The engineered and optimized ectoine strains were inoculated into 50 mL of ectoine fermentation medium (carbon source: glucose 40 g / L, peptone 3 g / L, urea 1 g / L, KH2PO4 4 g / L, (NH4)2SO4 3 g / L, MgSO4·7H2O 0.6 g / L, CaCl2 0.5 g / L, Mandels trace element solution 1 mL / L, and Tween 80 1 mL / L), with an inoculation size of 10. 7 One spore per 50 mL culture medium, cultured at 28℃ and 200 rpm. On day 6, 1 mL of fermentation broth was collected and centrifuged at 14000×g for 10 min to obtain the supernatant. The ectoine content was then determined to verify whether increasing the number of ectoine gene clusters could improve ectoine yield. The results are as follows: Figure 2 As shown.

[0096] Depend on Figure 2As can be seen, the starting strain QM6a cannot synthesize ectoine; the ectoine engineering strain transformed with the ectoine synthesis plasmid PT1.0-ectABC and the optimized strain can synthesize ectoine; the optimized strain with the increased ectoine gene cluster can significantly improve the yield of ectoine, and with the increase of generations, the yield of ectoine will also increase. The yield of ectoine in the ectoine engineering strain can reach 435.03 mg / L; the yield of ectoine in the ectoine optimized strain (first generation) can reach 625.2 mg / L, the yield of ectoine in the ectoine optimized strain (second generation) can reach 765.2 mg / L, and the yield of ectoine in the ectoine optimized strain (third generation) can reach 845.6 mg / L.

[0097] Example 3 Optimization of fermentation medium

[0098] Ectoine is an amino acid, and the synthesis of amino acids by microorganisms has specific requirements for the composition of nitrogen sources in the culture medium. Ammonium sulfate as an inorganic readily available nitrogen source does not need to be optimized. Therefore, the long-term nitrogen source formula (peptone 3-18 g / L, urea 1-5 g / L) of the culture medium is further optimized to study the effect of nitrogen source on the synthesis of ectoine by Trichoderma reesei.

[0099] The ectoine optimized strain (third generation) was inoculated into 50 mL ectoine fermentation medium (carbon source: 40 g / L glucose, peptone 3-18 g / L, urea 1-5 g / L, KH2PO4 4 g / L, (NH4)2SO4 3 g / L, MgSO4·7H2O 0.6 g / L, CaCl2 0.5 g / L, Mandels trace element solution 1 mL / L, and Tween 80 1 mL / L). The inoculation amount was 10 7 spores / 50 mL medium, and after 5 days of culture at 28°C and 200 rpm, 1 mL of fermentation broth was collected, and the fermentation broth supernatant was obtained by centrifugation at 14,000 x g for 10 min.

[0100] Firstly, the concentration of long-term nitrogen source peptone in the fermentation medium formula was selected as 3, 6, 9, 12, and 18 g / L, respectively, and other medium formula conditions were unchanged. Secondly, the concentration of long-term nitrogen source urea in the fermentation medium formula was selected as 1, 3, and 5 g / L, and other medium formula conditions were unchanged. Finally, the concentration of long-term nitrogen source peptone in the fermentation medium formula was set to 12 g / L, the concentration of urea was set to 3 g / L, and other medium formula conditions were unchanged. The results are shown in Figure 3 .

[0101] From the above experiments, it can be seen that the yield of ectoine in the ectoine engineering strain can reach 435.03 mg / L; the yield of ectoine in the ectoine optimized strain (first generation) can reach 625.2 mg / L, the yield of ectoine in the ectoine optimized strain (second generation) can reach 765.2 mg / L, and the yield of ectoine in the ectoine optimized strain (third generation) can reach 845.6 mg / L. Figure 3As can be seen, in terms of proteose peptone, the production of ectoine can be effectively promoted when the concentration of proteose peptone is increased from 3 g / L to 12 g / L, and the yield is as high as 1145.6 mg / L; in terms of urea, the production of ectoine can be effectively promoted when the concentration of urea is increased from 1 g / L to 3 g / L, and the yield is as high as 1065.6 mg / L. Considering comprehensively, 12 g / L proteose peptone and 3 g / L urea are selected as the long-acting nitrogen source, and the yield of ectoine is as high as 1494.5 mg / L.

[0102] Example 4

[0103] Studies have shown that the addition of ectoine can improve the stress resistance of cells and protect the metabolic activity of cells; the addition of ectoine can protect the tool enzymes of molecular operation and increase the heat resistance and stability of enzymes. In order to verify whether the ectoine produced by Trichoderma reesei can affect the production of enzyme preparations by enzyme preparation engineering strains, the following tests were carried out in this embodiment.

[0104] 1. Transforming ectoine synthesis plasmid PT1.0-ectABC into enzyme preparation engineering strains

[0105] The agrobacterium electroporated with the ectoine synthesis plasmid PT1.0-ectABC was co-cultured with four enzyme preparation engineering strains, i.e., Trichoderma reesei RUT-C30 (ATCC 56765) capable of producing cellulase and xylanase, asparaginase engineering strain (patent ZL202110667266.1) capable of producing asparaginase, lipase engineering strain (patent ZL202410105904.4) capable of producing lipase, and human lysozyme engineering strain (patent CN202410045920.9) capable of producing human lysozyme, as the starting strains on IM plates, and the agrobacterium-mediated conjugation was carried out. PT1.0-ectABC will be randomly inserted into the genome of the enzyme preparation engineering strains. After co-culturing for two days, the transformants were transferred to PDA plates containing cefotaxime (300 μg / mL) and hygromycin B (75 μg / mL) for screening, until mycelium and spores grew on the plates, and then the successful transformation was verified. In order to make the transformed strains not contain the resistance marker gene, the hygromycin B resistance marker was eliminated according to the literature scheme (Zhang et al. Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Scientific Reports. 2016, 6: 20761), i.e., the enzyme preparation optimization strain.

[0106] Finally, four enzyme preparation optimization strains were obtained: RUTC30-ectABC, asparaginase-ectABC, lipase-ectABC, and human lysozyme-ectABC. The respective starting strains were: RUTC30, asparaginase engineering strain, lipase engineering strain, and human lysozyme engineering strain.

[0107] 2. Detection of enzyme preparation yield of the four enzyme preparation optimization strains

[0108] The above four optimization strains were inoculated into 50 mL of Ektosin fermentation medium (carbon source: cellulase inducer - transformed sophorose: 40 g / L transformed sophorose, peptone 12 g / L, urea 3 g / L, KH2PO4 4 g / L, (NH4)2SO4 3 g / L, MgSO4·7H2O 0.6 g / L, CaCl2 0.5 g / L, Mandels trace element solution 1 mL / L, and Tween 80 1 mL / L) at an inoculation amount of 10 7 spores / 50 mL medium, and cultured at 28°C, 200 rpm. Among them, transformed sophorose is catalyzed by glucosidase to glucose, and partially transformed into sophorose, and finally a mixture of sophorose-containing sugars, the preparation method is described in the reference: Li et al. Overproduction of cellulase by Trichoderma reesei RUT C30 through batch-feeding of synthesized low-cost sugar mixture. Bioresource Technology 216 (2016) 503-510.

[0109] The enzyme preparation starting strains and optimization strains were fermented for 6 days, and the Ektosin yield (Ektosin yield was 300-600 mg / L) and the activity of the five enzyme preparations (cellulase, xylanase, asparaginase, lipase, and human lysozyme) were determined. The enzyme preparation activity of the four enzyme preparation starting strains and the four enzyme preparation optimization strains was compared, and the improvement degree (%) of the enzyme preparation activity of the optimization strains was calculated to investigate whether the simultaneous production of Ektosin and enzyme preparation by introducing an Ektosin gene cluster would in turn affect the yield of enzyme preparation? The results are shown in Figure 4 Figure 4 It can be seen that by introducing an Ektosin gene cluster, the simultaneous production of Ektosin and enzyme preparation can indeed increase the yield of enzyme preparation. The yield of enzyme preparation in the optimization strain transformed by PT1.0-ectABC increased by 10%-35% compared with the starting strain.

[0110] ​The enzyme preparation starting strain and the optimized strain were fermented for 6 days, the fermentation supernatant was taken and placed at 60℃ for 8 hours, and the activity of five enzyme preparations (cellulase, xylanase, asparaginase, lipase and human lysozyme) was determined at different time points to investigate whether the simultaneous production of ectoine and enzyme preparation by introducing ectoine gene cluster can affect the heat resistance and stability of enzyme preparation. The results show that the simultaneous production of ectoine and enzyme preparation by introducing an ectoine gene cluster can indeed increase the heat resistance and stability of enzyme preparation (Table 1). Among them, the improvement of the heat resistance of human lysozyme is the most significant: the fermentation supernatant of the human lysozyme engineering strain without introducing the ectoine gene cluster is placed at 60℃ for 8 hours, and almost completely loses the lysozyme activity; and the supernatant of the human lysozyme optimized strain with introducing the ectoine gene cluster is placed at 60℃ for 8 hours, and the activity is still preserved more than 50% (Table 1). Figure 5

[0111] Table 1

[0112]

[0113] According to the above results, it can be known that by genetic engineering modification, the present application introduces the ectoine gene cluster to enable the Trichoderma reesei originally without ectoine synthesis capacity to produce ectoine; and by increasing the number of ectoine gene clusters and optimizing the culture medium, the yield of ectoine is significantly improved, wherein, using glucose as carbon source, protein peptone and urea as long-acting nitrogen source, and optimizing the addition amount of nitrogen source, the ectoine can reach 1494.5mg / L in shake flask fermentation, which provides technical support for the industrial production of ectoine. At the same time, the production of ectoine can not only improve the yield of enzyme preparation, but also significantly improve the heat resistance and stability of enzyme preparation, increase the output value under the same operating conditions, and reduce the production cost. The present application has important application value in enzyme preparation production and heat stability protection.

[0114] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application defined by the claims.​

Claims

1. A method for constructing an engineered Trichoderma reesei strain for production of ectoine, characterized in that, The step of introducing the ectoine synthesis gene cluster into the Trichoderma reesei strain to construct the Trichoderma reesei engineering strain; The nucleotide sequence of the ectoine synthesis gene cluster is shown as SEQ ID NO. 1; the ectoine synthesis gene cluster encodes a pro-ectoine synthetase, and the amino acid sequence of the pro-ectoine synthetase is shown as SEQ ID NO. 2; The Trichoderma reesei engineering strain produces ectoine and enzyme preparation simultaneously; the ectoine improves the yield and heat-resistant stability of the enzyme preparation.

2. The construction method of claim 1, wherein, The ectoine synthesis gene cluster is introduced by a recombinant plasmid; The recombinant plasmid is constructed by sequentially connecting a pdc promoter, a cbh2 terminator and the ectoine synthesis gene cluster into a LML2.0a backbone.

3. A method of construction of an optimized strain of Trichoderma reesei for production of ectoine, characterized by, The step of introducing the ectoine synthesis gene cluster into the Trichoderma reesei engineering strain multiple times to construct the Trichoderma reesei optimized strain; The nucleotide sequence of the ectoine synthesis gene cluster is shown as SEQ ID NO.

1.

4. The Trichoderma reesei engineering strain producing ectoine obtained by the construction method of claim 1 or 2.

5. The Trichoderma reesei optimized strain producing ectoine obtained by the construction method of claim 3.

6. The application of the Trichoderma reesei engineering strain of claim 4 or the Trichoderma reesei optimized strain of claim 5 in producing ectoine.

7. A method of producing an ectoine, characterized in that, The step of fermenting by using the Trichoderma reesei engineering strain of claim 4 or the Trichoderma reesei optimized strain of claim 5; When the fermentation medium used in the fermentation uses a cellulase inducer as a carbon source, the Trichoderma reesei engineering strain or the Trichoderma reesei optimized strain produces ectoine and enzyme preparation simultaneously.

8. The method of claim 7, wherein, The fermentation medium used in the fermentation includes 40 g of a carbon source, 3-18 g of peptone, 1-5 g of urea, 4 g of KH2PO4, 3 g of (NH4)2SO4, 0.6 g of MgSO4·7H2O, 0.5 g of CaCl2, 1 mL of Mandels trace element solution and 1 mL of Tween 80, and water is added to 1 L.

9. The method of claim 8, wherein, The fermentation medium used in the fermentation includes 40 g of a carbon source, 12 g of peptone, 3 g of urea, 4 g of KH2PO4, 3 g of (NH4)2SO4, 0.6 g of MgSO4·7H2O, 0.5 g of CaCl2, 1 mL of Mandels trace element solution and 1 mL of Tween 80, and water is added to 1 L.

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