CN116004677B - Construction method and application of Trichoderma reesei engineering bacteria for producing itaconic acid - Google Patents
Construction method and application of Trichoderma reesei engineering bacteria for producing itaconic acid Download PDFInfo
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- CN116004677B CN116004677B CN202211494223.9A CN202211494223A CN116004677B CN 116004677 B CN116004677 B CN 116004677B CN 202211494223 A CN202211494223 A CN 202211494223A CN 116004677 B CN116004677 B CN 116004677B
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- 241000499912 Trichoderma reesei Species 0.000 title claims abstract description 75
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- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a construction method and application of Trichoderma reesei engineering bacteria for producing itaconic acid, and belongs to the field of bioengineering. The construction method comprises the following steps: (1) Taking Trichoderma reesei which is a filamentous fungus and does not produce itaconic acid or a derivative bacterium derived from Trichoderma reesei as an initial strain, and introducing exogenous genes for coding cis-aconitic acid decarboxylase and mitochondrial tricarboxylic acid transporter to obtain a strain I; (2) And (3) overexpressing the endogenous membrane transport protein of the strain I to obtain the Trichoderma reesei engineering bacteria producing itaconic acid. The invention obtains the Trichoderma reesei engineering bacteria through genetic modification, and the engineering bacteria can directly ferment and produce a large amount of itaconic acid by taking common substances such as glucose, glycerol, liquefied starch, cellulose hydrolysate and the like as carbon sources, and the maximum yield of shake flask fermentation can reach 60g/L. Therefore, the invention provides a new method for producing itaconic acid from microorganism sources, and can be applied to the industrial production of itaconic acid.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a construction method and application of Trichoderma reesei engineering bacteria for producing itaconic acid.
Background
Itaconic acid, also known as methylsuccinic acid or methylsuccinic acid, is an important unsaturated dicarboxylic acid and is widely used in the production of high-value chemicals such as latex, plastics, resins, etc. Itaconic acid has the characteristics of antibiosis, anti-inflammation, anti-tumor, antivirus and the like, and has good application prospect in the medical science and livestock breeding industry. In addition, itaconic acid is also an intermediate for producing potential biofuel 3-methyltetrahydrofuran, and has great potential for replacing petroleum-based methacrylic acid and acrylic acid.
The industrial production of itaconic acid is realized by microbial fermentation, and the main strain for the industrial production is aspergillus terreus. In a fermentation tank, the yield of itaconic acid produced by aspergillus terreus fermentation reaches 130-160 g/L. The corn black fungus is a basidiomycete and also has the ability to produce itaconic acid.
Advantages of Trichoderma reesei: trichoderma reesei is an important industrial production strain, is GRAS (Generally Regarded as Safe) strain, is very suitable for industrial scale-up production, and has been widely applied to fermentation industries such as food, feed and the like. However, since the Trichoderma reesei strain cannot produce itaconic acid, the invention constructs the Trichoderma reesei engineering strain for fermentation production of itaconic acid through a genetic modification technology.
Disclosure of Invention
The invention aims to provide a construction method and application of Trichoderma reesei engineering bacteria for producing itaconic acid, which are used for solving the problems of the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a construction method of Trichoderma reesei engineering bacteria, which comprises the following steps:
(1) Taking Trichoderma reesei which is a filamentous fungus and does not produce itaconic acid or a derivative bacterium derived from Trichoderma reesei as an initial strain, and introducing exogenous genes for coding cis-aconitic acid decarboxylase and mitochondrial tricarboxylic acid transporter to obtain a strain I;
(2) And (3) overexpressing the endogenous membrane transport protein of the strain I to obtain the Trichoderma reesei engineering bacteria producing itaconic acid.
Preferably, the starting strain comprises Trichoderma reesei QM6a, QM9414, rut-C30, RL-P37, NG14 and PC-3-7.
Preferably, the mitochondrial tricarboxylic acid transporter and the cis-aconitate decarboxylase are derived from a protein-encoding gene annotated by aspergillus terreus having the function of expressing mitochondrial tricarboxylic acid transporter and cis-aconitate decarboxylase.
The amino acid sequence of the cis aconitic acid decarboxylase is shown as SEQ ID NO:1, wherein the gene sequence for encoding the cis-aconitate decarboxylase is shown as SEQ ID NO. 2; the amino acid sequence of the mitochondrial tricarboxylic acid transporter is shown as SEQ ID No.5, and the gene sequence encoding the mitochondrial tricarboxylic acid transporter is shown as SEQ ID No. 6.
Preferably, the endogenous membrane transporter is an mfs superfamily transporter; genes for encoding the endogenous membrane transporters comprise mfs1, mfs2, mfs3, mfs4 and mfs5, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.
The invention also provides trichoderma reesei engineering bacteria which are constructed by the construction method.
The invention also provides a method for producing itaconic acid, which comprises the steps of fermenting by using the Trichoderma reesei engineering bacteria, and collecting fermentation liquor to obtain itaconic acid.
Preferably, the fermentation conditions are: the inoculation amount is 10 8 The fermentation temperature of the spores/50 mL of liquid culture medium is 28-30 ℃ and the rotating speed is 200rpm.
Preferably, the liquid medium comprises the following concentration components: 50-100g/L of carbon source, 1-6g/L of peptone and KH 2 PO 4 0.15g/L，K 2 HPO 4 0.15g/L，CaCl 2 ·2H 2 O 0.10g/L，MgSO 4 ·7H 2 0.10g/L of O, 20-40g/L of calcium carbonate and 0.05g/L of NaCl and 1mL/L of trace element liquid.
Preferably, the carbon source comprises any one of glucose, glycerol, liquefied starch, cellulose hydrolysate;
the microelements comprise the following components in parts by weight: 1.6g MnSO 4 ·4H 2 O，5g FeSO 4 ·7H 2 O，2gCoCl 2 ·6H 2 O，1.4g ZnSO 4 ·7H 2 O, dissolved in water and fixed to a volume of 1L.
The invention also provides a construction method or application of the Trichoderma reesei engineering bacteria in itaconic acid production.
The invention discloses the following technical effects:
the invention takes Trichoderma reesei as an original strain, and introduces exogenous genes through genetic modification, so that the modified strain can express mitochondrial tricarboxylic acid transporter and cis aconitic acid decarboxylase, and the Trichoderma reesei which does not have itaconic acid production capacity originally can obtain itaconic acid synthesis capacity; further, the yield of itaconic acid is improved by over-expressing the endogenous membrane transporter of Trichoderma reesei, and Trichoderma reesei is transformed into an engineering strain capable of efficiently synthesizing and secreting itaconic acid.
Experiments prove that the Trichoderma reesei engineering strain obtained by the invention can directly ferment and produce a large amount of itaconic acid by taking common substances such as glucose, glycerol, liquefied starch, cellulose hydrolysate and the like as carbon sources, and the maximum yield of a shake flask can reach 60g/L. Therefore, the invention provides a new method for producing itaconic acid from microorganism sources, and can be applied to the industrial production of itaconic acid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart showing construction of expression plasmids in the present invention; a: mttA-cad1 double gene expression vector; b: different strains and engineering strains constructed based on genetic modification ferment glucose to produce itaconic acid;
FIG. 2 is a flow chart showing construction of expression plasmids in the present invention; a: mfs expression vector; b: engineering strain TrIA02 and the like, fermenting glucose to produce itaconic acid;
FIG. 3 shows the yield of itaconic acid from the genetically engineered strain TrIA02 when glycerol, liquefied starch, cellulose and cellulose hydrolysate were used as carbon sources in the present invention.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
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 invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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 invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The following examples relate to strains: trichoderma reesei strain QM6a (purchased from American type culture Collection ATCC 13631), QM9414 (purchased from American type culture Collection ATCC 26921), rut-C30 (purchased from American type culture Collection ATCC 56765), RL-P37 (American type agricultural research Collection NRRL 15709), NG14 (purchased from American type culture Collection ATCC 56767), PC-3-7 (purchased from American type culture Collection ATCC 66589).
Example 1 fermentation production of itaconic acid Using genetically engineered bacteria
1. Preparation of ball-milled cellulose suspension
Into a 500 mL-volume triangular flask, 20g of corn stalk powder (capable of passing through a 50 mesh sieve), 200mL of deionized water, 0.5-1cm diameter glass beads (preferably, the bottom is paved), and sterilizing (121 ℃ C., 20 min). The bottle mouth is tied up by using rubber bag to prevent water from volatilizing, then placed in a shaking table, and after shaking for 10-15 days (preferably 12 days) at 200rpm, taken out, and sterilized once again (115 ℃ for 20 min). A 10% ball-milled cellulose suspension (calculated as starting corn stalk solid dry matter dose) was made.
2. Preparation of saccharified starch solution
0.5g of calcium chloride was dissolved in 1kg of hot water at 60℃and then 1kg of corn starch was added, followed by 0.5g of high Wen-amylase (commercial enzyme, ningxia Seisakusho Co., ltd.) to prepare a batter, and the pH of the batter was adjusted to 6.0. Then maintained at 97-98deg.C for 1.5h, then cooled to 60-62deg.C (preferably 60deg.C), the pH is adjusted to 4.0, 0.8mL of glucoamylase (commercial enzyme, ningxia Co., ltd.) is added, and maintained at 60-62deg.C for 27-28h ((preferably 60deg.C), h). Distilled water is added to a volume of 2.5L, and then the mixture is subjected to wet heat sterilization at 115 ℃ for 30min, so that a saccharified starch solution with a sugar content of about 400g/L is obtained.
3. Preparation of cellulose hydrolysate
The dry corn stalks subjected to alkali pretreatment contain 62.6% of cellulose, 21.4% of hemicellulose and 8.2% of lignin. Cellulose hydrolysis experiments can be performed in shake flasks with pretreated corn stover as substrate with a substrate loading of 15% (150 g straw dry matter/total reaction volume of 1L). Two enzymes were added to hydrolyze cellulose: cellulases and beta-glucosidases. Cellulase (commercial enzyme, ningxia Hospital, inc.) at a loading of 20FPU/g dry biomass; the beta-glucosidase (commercial enzyme, ningxia Hospital, inc.) loading was 40CBU/g dry biomass. The reaction flask was placed at 50 ℃ (100 rpm) and reacted at pH 5.0 for 96h. After the reaction is finished, insoluble substances are removed by centrifugation, the volume of the supernatant is fixed to 1.5L, and then the supernatant is subjected to wet heat sterilization at 115 ℃ for 30min. 10% cellulose hydrolysate (calculated as dry corn stalk solid matter dosed starting alkaline pretreatment) was prepared.
4. Genetically engineered bacteria fermentation production of itaconic acid
Respectively inoculating genetically engineered bacteria into 50mL culture medium (formula: carbon source 50-100g/L, peptone 6g/L, KH) with glucose, glycerol, liquefied starch, cellulose, and cellulose hydrolysate as carbon source in 250mL triangular flask 2 PO 4 0.15g/L，K 2 HPO 4 0.15g/L，CaCl 2 ·2H 2 O 0.10g/L，MgSO 4 ·7H 2 0.10g/L of O, 20-40g/L of calcium carbonate, 0.05g/L of NaCl and 1mL/L of trace element liquid. Microelement liquid formula (1000 mL:1.6g MnSO) 4 ·4H 2 O，5g FeSO 4 ·7H 2 O，2g CoCl 2 ·6H 2 O，1.4g ZnSO 4 ·7H 2 O, dissolved in water, fixed volume to 1L) with an inoculum size of 10 8 The spores were cultured in 50mL of medium at 28℃and 220rpm, and samples were taken on the eighth day to determine the itaconic acid content. When glucose and liquefied starch are used as carbon sources, the final carbon source concentration is 100g/L (calculated by the glucose molecular content), and the calcium carbonate concentration is 40g/L; when glycerol is used as a carbon source, the final carbon source concentration is 80g/L, and the calcium carbonate concentration is 40g/L; cellulose and cellulose hydrolysate are used as carbon sources, the final carbon source concentration is 50g/L (calculated by the amount of solid dry matter of the starting corn straw), and the calcium carbonate concentration is 20g/L.
Example 2 determination of itaconic acid content
Taking a fermentation according to example 1Adding 1 time volume of 2mol/L H into a centrifuge tube 2 SO 4 Placing into a water bath shaker at 80deg.C and 100rpm for shaking for 30min, mixing fermentation broth with water droplets on the tube wall after calcium carbonate in the fermentation broth is completely dissolved, taking 1mL of liquid, centrifuging in a 1.5mL centrifuge tube at 14000 Xg for 30min, sucking the supernatant, and measuring itaconic acid content by High Performance Liquid Chromatography (HPLC).
High Performance Liquid Chromatography (HPLC) to determine itaconic acid content: mobile phase: 5mM H 2 SO 4 The method comprises the steps of carrying out a first treatment on the surface of the Flow rate: 0.5mL/min; column temperature: 30 ℃; a detector: an ultraviolet detector; wavelength: 210nm; column: amineX HPX-87X, (300 mm. Times.7.8 mm).
Example 3 simultaneous expression of A.terreus-derived mitochondrial tricarboxylic acid transporter coding gene mttA and cis-aconitate decarboxylase coding gene cad1 in Trichoderma reesei
1. construction of cad1 Single Gene expression vector (pOEcad 1)
1) The Ppdc sequence was amplified using the primers Ppdc-F and Ppdc-R and Trichoderma reesei genome as a template.
Ppdc-F：5’-ACTAGTGAGCTCATTTATGAAAGGAGGGAGCATTCTTCGA-3’；
Ppdc-R：5’-CATGATTGTGCTGTAGCTGCGC-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.M each); genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
2) The primers cad1-1 and cad1-2 were used to amplify the cad1 sequence using the codon-optimized cad1 plasmid as template.
cad1-1：5’-AGCTACAGCACAATCATGACGAAGCAGAGCGCCG-3’；
cad1-2：5’-CCGGTCACGAAAGCCTCAGACGAGGGGGCTCTTGACG-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.M each); 1. Mu.L of synthetic plasmid template (10 ng); KOD-Plus-Neo(1U/μL)1μL。
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
3) Amplifying a Tcbh2 sequence by using the primers Tcbh2-1 and Tcbh2-2 and using the Trichoderma reesei genome as a template;
Tcbh2-1：5’-GGCTTTCGTGACCGGGCTT-3’；
Tcbh2-2：5’-AGTGCCAAGCTTATTTTGGGTATGGTTTCCACGTGCA-3’。
amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.M each); genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 15 sec; and at 68℃for 5min.
4) An expression vector was constructed by using LML2.0a (Zhang et al light-inducible genetic engineering and control of non-homologo end-joining in industrial eukaryotic microorganisms: LML 3.0and OFN 1.0.Scientific Reports.2016,6:20761), single enzyme digestion was performed on restriction enzyme SwaI on the existing plasmid LML2.0a, and homologous recombination was performed by Vazyme One Step Clone Kit to construct a Ppdc-cad1-Tcbh2 expression cassette, thereby obtaining a cad1 single gene expression vector pOEcad1 (FIG. 1A).
Wherein the amino acid sequence of cad1 is shown as SEQ ID NO.1, the nucleotide sequence of cad1 is shown as SEQ ID NO.2, the nucleotide sequence of Ppdc is shown as SEQ ID NO.3, and the nucleotide sequence of Tcbh2 is shown as SEQ ID NO. 4.
2. Construction of mttA-cad1 double gene expression vector (pOEmtA-cad 1).
1) And (3) amplifying the Peno sequence by using primers Peno-F and Peno-R and using the Trichoderma reesei genome as a template.
Peno-F：5’-GATTACGAATTCTTAATTAATGCCAACTCCTTGACGCCAA-3’；
Peno-R：5’-CATTTTGAAGCTATTTCAGGT-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; primer (10. Mu.M each) 15. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
2) The mttA sequence was amplified using the primers mttA-1 and mttA-2, with the codon-optimized mttA plasmid as template. mttA-1:5'-TGAAATAGCTTCAAAATGTCTAAGAAGCACATTGTTATCATTG-3';
mttA-2：5’-TTTCGCCACGGAGCTTCAGATGACGTTGGAGTTGTGG-3’。
amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.M each); 1. Mu.L of synthetic plasmid template (10 ng); KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
3) The primer Tcbh1-1 and Tcbh1-2 are used to amplify Tcbh1 sequence with Trichoderma reesei genome as template.
Tcbh1-1：5’-AGCTCCGTGGCGAAAGCC-3’；
Tcbh1-2：5’-CATTATACGAAGTTATTCTAGAATTTCCACTGTTGCTATTATGCTGT-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.M each); genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 15 sec; and at 68℃for 5min.
4) The cad1 single gene expression vector pOEcad1 (figure 1A) is used as a framework to construct an expression vector. Double digestion is carried out on the restriction enzyme PacI/XbaI on the existing plasmid pOEcad1, homologous recombination is carried out by utilizing Vazyme One Step Clone Kit, and a Peno-mttA-Tcbh1 expression frame is constructed, so that an mttA-cad1 double-gene expression vector pOEmttA-cad1 is obtained (FIG. 1A).
Wherein the amino acid sequence of mttA is shown as SEQ ID NO.5, the nucleotide sequence of mttA is shown as SEQ ID NO.6, the nucleotide sequence of Peno is shown as SEQ ID NO.7, and the nucleotide sequence of Tcbh1 is shown as SEQ ID NO. 8.
3. Introducing mttA-cad1 double-gene expression vector pOEmttA-cad1 into Trichoderma reesei to obtain genetically engineered strain TrIA01
The invention relates to expression or heterologous expression, which is agrobacterium-mediated trichoderma reesei transformation and clone screening, and integrates related genes into a trichoderma reesei genome for expression. The transformation method related by the invention is agrobacterium tumefaciens mediated combination transfer.
1) Plasmid pOEmtA-cad 1 was electrotransferred to Agrobacterium, then Agrobacterium containing plasmid pOEmtA-cad 1 was co-cultured with Trichoderma reesei host strain QM6a (ATCC 13631), QM9414 (ATCC 26921), rut-C30 (ATCC 56765), RL-P37 (NRRL 15709), NG14 (ATCC 56767), PC-3-7 (ATCC 66589) in IM plates (cover et al. Agrobacterium tumefaciens-mediated transformation of fusarium. Res.105 (3): 259-264), agrobacterium tumefaciens mediated binding transfer was performed, transformants were transferred after two days of co-culture to PDA plates containing cefotaxime (300. Mu.g/mL) and hygromycin B (75. Mu.g/mL) for selection and spore development, and then selection and validation were performed.
2) The transformant verified above was inoculated into 50mL of a medium containing glucose as a carbon source (see example 1) in a 250mL flask in an amount of 10 8 The spores were incubated at 28℃and 220rpm per 50mL of medium, and samples were taken on day 8 to determine the itaconic acid content.
3) When mttA-cad1 is co-expressed in Trichoderma reesei strains, itaconic acid can be obviously produced by fermentation. Among them, the strain with the highest yield was the transformant of QM6a, named as TrIA01, and the yield of itaconic acid was 42.5g/L when glucose was the carbon source (FIG. 1B).
Example 4 overexpression of the self Membrane Transporter encoding Gene in Trichoderma reesei
The amino acid sequence (SEQ ID NO. 9) of main accelerator superfamily protein (major facilitator superfamily protein) mfsA from aspergillus terreus is taken as a template, and the coding genes of 5 endogenous membrane transporters of trichoderma reesei are identified through homologous comparison, and are named mfs1, mfs2, mfs3, mfs4 and mfs5, and the gene sequences are respectively shown as SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14.
1. Construction of endogenous Membrane Transporter encoding Gene overexpression vector pOEmfs1
1) Using primers mfs1-F1 and mfs1-F2, the upstream sequence mae1-F of mfs1 was amplified using Trichoderma reesei genome as a template (the nucleotide sequence is shown as SEQ ID NO. 15).
mfs1-F1：5’-ATTACGAATTCTTAATTAACTGTTGGCAATGGCTGGATGA-3’；
mfs1-F2：5’-CATTATACGAAGTTATTCTAGACGGCAGCACAGGACGATGAA-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 10. Mu.M primer mfs1-F1/mfs1-F2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 25 sec; and at 68℃for 5min.
2) The promoter Ppdc sequence (nucleotide sequence shown in SEQ ID NO. 3) was amplified using Trichoderma reesei genome as a template using the primers pdc-F and pdc-R and amplification conditions in example 3.
3) Using primers mfs1-1 and rmfs1-2, a partial mfs1 gene sequence mfs1-O (the nucleotide sequence is shown as SEQ ID NO. 16) was amplified using Trichoderma reesei genome as a template.
mfs1-1：5’-AGCTACAGCACAATCATGAAGCAAGATGAAGCGATACAGC-3’；
mfs1-2：5’-AGTGCCAAGCTTATTTGCGACGAAGATGGCACAGAAC-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL;10 mu M primer mfs1-1/mfs1-2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
4) Constructing an expression vector by taking lcNG (Wu et al efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei.J.Gen.appl.Microbiol.,2019, 65:301-307.) as a framework, performing double digestion by using restriction enzymes PacI/XbaI on the existing plasmid lcNG, performing seamless connection by using Vazyme One Step Clone Kit, and connecting to an upstream sequence mfs1-F; single enzyme digestion is carried out by SwaI, seamless connection is carried out by Vazyme One Step Clone Kit, and the promoter Ppdc and partial gene sequence mfs1-O are connected; an overexpression plasmid pOEmfs1 of mfs1 was constructed (FIG. 2A).
2. Construction of endogenous Membrane Transporter encoding Gene overexpression vector pOEmfs2
1) Amplifying an upstream sequence mfs2-F of mfs2 by using primers mfs2-F1 and mfs2-F2 and using a Trichoderma reesei genome as a template (a nucleotide sequence is shown as SEQ ID NO. 17);
mfs2-F1：5’-ATTACGAATTCTTAATTAACTGTTGGCAATGGCTGGATGA-3’；
mfs2-F2：5’-CATTATACGAAGTTATTCTAGACGGCAGCACAGGACGATGAA-3’。
amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 10. Mu.M primer mfs2-F1/mfs2-F2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 25 sec; and at 68℃for 5min.
2) Amplifying a promoter Ppdc sequence (the nucleotide sequence is shown as SEQ ID NO. 3) by using the primers pdc-F and pdc-R in the example 3 and amplification conditions and using the Trichoderma reesei genome as a template;
3) Using primers mfs2-1 and mfs2-2, the partial mfs2 gene sequence mfs2-O (the nucleotide sequence is shown as SEQ ID NO. 18) was amplified using Trichoderma reesei genome as a template.
mfs2-1：5’-AGCTACAGCACAATCATGAAGCAAGATGAAGCGATACAGC-3’；
mfs2-2：5’-AGTGCCAAGCTTATTTGCGACGAAGATGGCACAGAAC-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL;10 mu.M primer mfs2-1/mfs2-2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
4) Constructing an expression vector by taking lcNG (Wu et al efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei.J.Gen.appl.Microbiol.,2019, 65:301-307.) as a framework, performing double digestion by using restriction enzymes PacI/XbaI on the existing plasmid lcNG, performing seamless connection by using Vazyme One Step Clone Kit, and connecting to an upstream sequence mfs2-F; single enzyme digestion is carried out by SwaI, seamless connection is carried out by Vazyme One Step Clone Kit, and the promoter Ppdc and partial gene sequence mfs2-O are connected; an overexpression plasmid pOEmfs2 of mfs2 was constructed (FIG. 2A).
3. Construction of endogenous Membrane Transporter encoding Gene overexpression vector pOEmfs3
1) The primers mfs3-F1 and mfs3-F2 are used, and the Trichoderma reesei genome is used as a template to amplify the mfs3-F (the nucleotide sequence is shown as SEQ ID NO: 19).
mfs3-F1：5’-ATTACGAATTCTTAATTAAGAAGCGTCTCCAGGACATTCC-3’；
mfs3-F2：5’-CATTATACGAAGTTATTCTAGAGGGCGAATCTGAAAGCGGATG-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 10. Mu.M primer mfs3-F1/mfs3-F2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 25 sec; and at 68℃for 5min.
2) The promoter Ppdc sequence (nucleotide sequence shown in SEQ ID NO. 3) was amplified using Trichoderma reesei genome as a template using the primers pdc-F and pdc-R and amplification conditions in example 3.
3) Using primers mfs3-1 and mfs3-2, the genome of Trichoderma reesei was used as a template to amplify mfs3-O, part of the gene sequence mfs3 (the nucleotide sequence is shown as SEQ ID NO. 20).
mfs3-1：5’-AGCTACAGCACAATCATGTCATCAAAATTTGACAATGAACAAGA-3’；
mfs3-2：5’-AGTGCCAAGCTTATTTCGGAACGGACCAAGCACATC-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL;10 mu M primer mfs3-1/mfs3-2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
5) Constructing an expression vector by taking lcNG (Wu et al efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei.J.Gen.appl.Microbiol.,2019, 65:301-307.) as a framework, performing double digestion by using restriction enzymes PacI/XbaI on the existing plasmid lcNG, performing seamless connection by using Vazyme One Step Clone Kit, and connecting to an upstream sequence mfs3-F; single enzyme digestion is carried out by SwaI, seamless connection is carried out by Vazyme One Step Clone Kit, and the promoter Ppdc and partial gene sequence mfs3-O are connected; an overexpression plasmid pOEmfs3 of mfs3 was constructed (FIG. 2A).
4. Construction of endogenous Membrane Transporter encoding Gene overexpression vector pOEmfs4
1) Using primers mfs4-F1 and mfs4-F2, the upstream sequence mfs4-F of mfs4 was amplified using Trichoderma reesei genome as template (the nucleotide sequence is shown as SEQ ID NO. 21).
mfs4-F1：5’-ATTACGAATTCTTAATTAAGCTGTGGAGTTGCCGATAAGG-3’；
mfs4-F2：5’-CATTATACGAAGTTATTCTAGACAGACGACGGCGGTATCAT-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 10. Mu.M primer mfs4-F1/mfs4-F2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 25 sec; and at 68℃for 5min.
2) The primers pdc-F and pdc-R and amplification conditions in example 3 were used to amplify a promoter Ppdc sequence using Trichoderma reesei genome as a template (nucleotide sequence shown in SEQ ID NO: 3).
3) Using primers mfs4-1 and mfs4-2, the partial mfs4 gene sequence mfs4-O (the nucleotide sequence is shown as SEQ ID NO. 22) was amplified using Trichoderma reesei genome as a template.
mfs4-1：5’-AGCTACAGCACAATCATGGGCGCCTCAGAAGACAC-3’；
mfs4-2：5’-AGTGCCAAGCTTATTTGCATCTGCCATCCTGACCTGTA-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL;10 mu.M primer mfs4-1/mfs4-2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
4) Constructing an expression vector by taking lcNG (Wu et al efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei.J.Gen.appl.Microbiol.,2019, 65:301-307.) as a framework, performing double digestion by using restriction enzymes PacI/XbaI on the existing plasmid lcNG, performing seamless connection by using Vazyme One Step Clone Kit, and connecting to an upstream sequence mfs4-F; single enzyme digestion is carried out by SwaI, seamless connection is carried out by Vazyme One Step Clone Kit, and the promoter Ppdc and partial gene sequence mfs4-O are connected; an overexpression plasmid pOEmfs4 of mfs4 was constructed (FIG. 2A).
5. Construction of endogenous Membrane Transporter encoding Gene overexpression vector pOEmfs5
1) Using primers mfs5-F1 and mfs5-F2, the upstream sequence mfs5-F of mfs5 was amplified using Trichoderma reesei genome as template (the nucleotide sequence is shown as SEQ ID NO. 23).
mfs5-F1：5’-ATTACGAATTCTTAATTAACCTCCGAGTTGGCTATCACTGA-3’；
mfs5-F2：5’-CATTATACGAAGTTATTCTAGAGGGTCCTGGATGTATGCGATGA-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 10. Mu.M primer mfs5-F1/mfs5-F2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 25 sec; and at 68℃for 5min.
2) The promoter Ppdc sequence (nucleotide sequence shown in SEQ ID NO. 3) was amplified using Trichoderma reesei genome as a template using the primers pdc-F and pdc-R and amplification conditions in example 3.
3) Using primers mfs5-1 and mfs5-2, the partial mfs5 gene sequence mfs5-O (the nucleotide sequence is shown as SEQ ID NO. 24) was amplified using Trichoderma reesei genome as a template.
mfs5-1：5’-AGCTACAGCACAATCATGTCGTCAACACCCGACCC-3’；
mfs5-2：5’-AGTGCCAAGCTTATTTGAGGATGCGGAAGACGATGA-3’。
Amplification reaction system: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL;10 mu M primer mfs5-1/mfs5-2 each 1.5. Mu.L; genome template (200 ng) 1 μl; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
4) Constructing an expression vector by taking lcNG (Wu et al efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei.J.Gen.appl.Microbiol.,2019, 65:301-307.) as a framework, performing double digestion by using restriction enzymes PacI/XbaI on the existing plasmid lcNG, performing seamless connection by using Vazyme One Step Clone Kit, and connecting to an upstream sequence mfs5-F; single enzyme digestion is carried out by SwaI, seamless connection is carried out by Vazyme One Step Clone Kit, and the promoter Ppdc and partial gene sequence mfs5-O are connected; an overexpression plasmid pOEmfs5 of mfs1 was constructed (FIG. 2A).
Example 5
The expression vectors pOEmfs1 to pOEmfs5 constructed in example 4 were introduced into Trichoderma reesei engineering strain TrIA01, respectively, to obtain engineering strain TrIA02 with improved itaconic acid yield.
The expression of the invention is agrobacterium-mediated trichoderma reesei transformation and clone screening, and trichoderma reesei endogenous genes are integrated into trichoderma reesei genome for expression. The transformation method of the invention is Agrobacterium tumefaciens mediated binding transfer.
1) The expression plasmids pOEmfs1 to pOEmfs5 were electrotransferred to agrobacteria, respectively, and the agrobacteria containing the expression plasmids were co-cultured with Trichoderma reesei host strain TrIA01, respectively (culture condition reference: the transformant was screened after culturing for two days in PDA plates containing cefotaxime (300. Mu.g/mL) and G418 (400. Mu.g/mL) until the transformants developed hyphae and spores, and then verified.
2) The transformant was inoculated into 50mL of a fermentation medium (see example 1) containing glucose as a carbon source in a 250mL flask in an amount of 10 8 The spores were cultured in a 50mL culture medium at 28-30deg.C (preferably 30deg.C) at 200rpm, and the itaconic acid content was measured by sampling on day 8 to verify the biological function.
3) The membrane transport protein may transport itaconic acid out of the cell. Thus, after the membrane transport protein is expressed in large amounts in Trichoderma reesei engineering strain TrIA01, the strain can significantly accumulate more itaconic acid (except mfs 4) in the fermentation broth. Among them, the transformant with the highest yield was the transformant of mfs1 gene, named as TrIA02, and the yield of itaconic acid was 60g/L when glucose was the carbon source (FIG. 2B).
4) The engineering strain TrIA02 can directly ferment and produce a large amount of itaconic acid by using common carbon sources such as glycerol, liquefied starch, cellulose hydrolysate and the like (figure 3). Experiments show that the trichoderma reesei can be subjected to itaconic acid fermentation by various carbon sources after genetic modification.
From the results of the above examples, it is understood that the present invention successfully ferments to produce itaconic acid after genetic modification of Trichoderma reesei. The research result of the invention shows that although the original Trichoderma reesei strain can not produce itaconic acid for the first time, common carbon sources such as glucose, glycerol, liquefied starch, cellulose hydrolysate and the like can be used as substrates after genetic engineering modification to ferment and produce itaconic acid. And it was confirmed by experiments that: the potential of the Trichoderma reesei engineering strain for producing the itaconic acid by fermentation in a shake flask provides an excellent strain for the industrial large-scale fermentation production of the itaconic acid.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (7)
1. The construction method of the Trichoderma reesei engineering bacteria is characterized by comprising the following steps of:
(1) Taking Trichoderma reesei which is a filamentous fungus and does not produce itaconic acid or a derivative bacterium derived from Trichoderma reesei as an initial strain, and introducing exogenous genes for coding cis-aconitic acid decarboxylase and mitochondrial tricarboxylic acid transporter to obtain a strain I;
(2) Overexpressing the endogenous membrane transport protein of the strain I to obtain Trichoderma reesei engineering bacteria producing itaconic acid;
the starting strain is trichoderma reesei QM6a;
the amino acid sequence of the cis-aconitate decarboxylase is shown as SEQ ID NO.1, and the nucleotide sequence of the gene for encoding the cis-aconitate decarboxylase is shown as SEQ ID NO. 2; the amino acid sequence of the mitochondrial tricarboxylic acid transport protein is shown as SEQ ID NO.5, and the nucleotide sequence of the gene for encoding the mitochondrial tricarboxylic acid transport protein is shown as SEQ ID NO. 6;
the endogenous membrane transporter is an mfs superfamily transporter; the gene for encoding the endogenous membrane transport protein is mfs1, mfs2, mfs3 or mfs5, and the nucleotide sequences of the gene are respectively shown as SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO. 14.
2. The Trichoderma reesei engineering bacterium is characterized by being constructed by the construction method of claim 1.
3. A method for producing itaconic acid, characterized by fermenting with the trichoderma reesei engineering bacterium according to claim 2, and collecting the fermentation liquid to obtain itaconic acid.
4. As claimed in claim 3The method is characterized in that the fermentation conditions are as follows: the inoculation amount is 10 8 The fermentation temperature of the individual spores/50 mL liquid medium is 28-30 ℃ and the rotating speed is 200rpm.
5. The method of claim 4, wherein the liquid medium comprises the following concentration components: 50-100 parts of carbon source g/L, 1-6 parts of peptone g/L and KH 2 PO 4 0.15 g/L，K 2 HPO 4 0.15 g/L，CaCl 2 ·2H 2 O 0.10 g/L，MgSO 4 ·7H 2 O0.10 g/L, calcium carbonate 20-40g/L, naCl 0.05g/L and 1mL/L microelement liquid.
6. The method of claim 5, wherein the carbon source comprises any one of glucose, glycerol, cellulose, and cellulose hydrolysate;
the microelement liquid comprises the following components in parts by weight: 1.6g MnSO 4 ·4H 2 O，5 g FeSO 4 ·7H 2 O，2 gCoCl 2 ·6H 2 O and 1.4 g ZnSO 4 ·7H 2 O, dissolved in water and fixed to volume 1L.
7. The construction method according to claim 1 or the application of the Trichoderma reesei engineering bacteria according to claim 2 in the production of itaconic acid.
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