CN107058144A - A kind of restructuring yeast strains for producing itaconic acid and its construction method and application - Google Patents

Abstract

The invention discloses a recombinant yeast strain producing itaconic acid, a construction method and application thereof. The recombinant yeast is a recombinant yeast strain obtained by inserting aconitic acid decarboxylase gene into the genome of C. glycerolgenesis by using C. glycerolgenesis as the starting strain. A recombinant plasmid containing the aconitic acid decarboxylase gene expression cassette Pgap-CadA-Tgap-zeocin was constructed by genetic engineering, and inserted into the C. glycerolgenesis genome after linearization to express aconitic acid decarboxylase to catalyze the acquisition of cis-aconitic acid itaconic acid. The recombinant yeast strain provided by the invention can synthesize itaconic acid, which provides a new idea for the production of itaconic acid.

Description

A recombinant yeast strain producing itaconic acid and its construction method and application

technical field

The invention relates to a recombinant yeast strain producing itaconic acid and its construction method and application, belonging to the field of genetic engineering.

Background technique

Itaconic acid is an important chemical product and one of the twelve important high value-added chemicals certified by the US Department of Energy. Itaconic acid is mainly used in the production of resins, plastics, rubber, synthetic fibers, and surfactants. Itaconic acid polymer can also be used to replace the original additive acrylic acid. In addition, studies have found that itaconic acid can act as an antibacterial substance in mammalian immune cells.

At present, the producing strain of itaconic acid is mainly Aspergillus terreus. The metabolic pathway of itaconic acid in Aspergillus terreus is shunted from the TCA cycle, and cis-aconitic acid in the TCA cycle is converted into itaconic acid under the action of cis-aconitic acid decarboxylase (CAD) (Dwiarti ,L.,Yamane,K.,Yamatani,H.,Kahar,P.,Okabe,M.,2002.Purification and characterization of cis-aconic aciddecarboxylase from Aspergillus terreus TN484-M1.J.Biosci.Bioeng.94,29 –33; Kanamasa, S., Dwiarti, L., Okabe, M., Park, E.Y., 2008. Cloning and functional characterization of the cis-aconic acid decarboxylase (CAD) gene from Aspergillus terreus. Appl. Microbiol. Biotechnol. 80, 223-229 ), cis-aconitic acid decarboxylase CAD is a key enzyme in the biosynthetic pathway of itaconic acid. However, during the synthesis of itaconic acid, Aspergillus terreus grows slowly and the fermentation period is long; continuous aeration and high-speed stirring are required to meet the dissolved oxygen demand of the bacteria growth during the fermentation process, but the mechanical pressure generated by high-speed stirring will make the Damage to the mycelium affects the growth of the bacteria. These shortcomings of Aspergillus terreus make it urgent to seek a new host to produce itaconic acid.

Glycerol-producing Candida (C.glycerolgenesis CCTCC AB 204047) is an industrial strain with excellent fermentation performance with independent intellectual property rights in my country. It grows fast and can grow on 55% glucose or 15% Nacl hyperosmotic medium It grows and reproduces normally, has the characteristics of high osmosis resistance and high stress resistance, and has strong tolerance to itaconic acid.

Contents of the invention

The purpose of the present invention is to provide a recombinant yeast strain producing itaconic acid and its construction method and application.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A recombinant yeast strain producing itaconic acid, the strain uses C. glycerolgenesis CCTCC AB204047 as the starting strain, and inserts the aconitic acid decarboxylase expression cassette Pgap-CadA-Tgap-zeocin into the C. glycerolgenesis genome through genetic engineering Obtained recombinant yeast strains.

The cis-aconitic acid decarboxylase expression cassette Pgap-CadA-Tgap-zeocin described in the present invention comprises the DNA that can express the cis-aconitic acid decarboxylase shown in SEQ ID NO.1 in the host cell, starts described cis-aconitic acid decarboxylase C. glycerolgenesis glyceraldehyde-3-phosphate dehydrogenase gene promoter for acid decarboxylase gene transcription, C. glycerolgenesis glyceraldehyde-3-phosphate dehydrogenase gene terminator for cis-aconitic acid decarboxylase gene transcription, also includes A DNA expressing bleomycin, the DNA comprising a promoter and a terminator for initiating and terminating the transcription of the bleomycin gene.

A method for constructing a recombinant yeast strain producing itaconic acid, constructing a recombinant plasmid containing an aconitic acid decarboxylase expression cassette Pgap-CadA-Tgap-zeocin through genetic engineering, and inserting the recombinant plasmid into the C. glycerolgenesis genome after linearization to obtain Recombinant yeast strains.

The application of the recombinant yeast strain producing itaconic acid in the preparation of itaconic acid, the recombinant yeast strain is cultivated in the fermentation medium, and the fermentation medium is: 50g/L glucose, 10g/L yeast powder, 20g /L peptone, zeocin 150μg/mL, the balance is water.

The advantages of the present invention: the method provided by the present invention to make Candida glycerologenus synthesize itaconic acid through genetic engineering transformation has clear purpose and strong operability. The itaconic acid fermentation yield of the recombinant yeast strain provided is generally higher than that of other yeast recombinant strains (Blazeck J, Miller J, Pan A, et al. Metabolic engineering of Saccharomyces cerevisiae for itaconic acid production. [J]. Applied Microbiology and Biotechnology, 2014, 98 (19):8155-64; Blazeck J, Hill A, JamoussiM, et al.Metabolic engineering of Yarrowia lipolytica for itaconic acidproduction.[J].Metabolic Engineering,2015,32:66-73.), and the strain itself has excellent Fermentation performance, and strong tolerance to itaconic acid, has strong application value, and can be used as a potential itaconic acid production strain.

Description of drawings

Fig. 1 is the relevant metabolic pathway of itaconic acid in Aspergillus terreus mentioned in the background art.

Fig. 2 is the plasmid map of the vector pCadA-T provided by the embodiment of the present invention, wherein CadA is aconitic acid decarboxylase gene.

Figure 3 is the plasmid map of the vector pURGAPZ provided by the embodiment of the present invention, Pgap is the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of Candida glycerologenicum, and Tgap is the termination of the gene of C.glycerolgenesis glyceraldehyde-3-phosphate dehydrogenase Son, ZEOCIN is composed of Pzeocin, zeocin and Tzeocin.

Fig. 4 is the plasmid map of the vector pURGAPZC provided by the embodiment of the present invention.

Fig. 5 is the process curve of the fermentation and synthesis of itaconic acid by the recombinant yeast strain.

detailed description

The present invention will be described in further detail below by way of examples.

Embodiment 1 Construction of cis-aconitic acid decarboxylase gene cadA expression cassette

1.1 Cloning of the aconitic acid decarboxylase gene from Aspergillus terreus

The Aspergillus terreus CICC 40205 strain used in the present invention comes from the China Industrial Microorganism Strain Collection Center. Primers cadA-F (5'-AAGGGATCCATGACCAAACAATCTGCGGAC-3') and cadA-R (5'-CGAGGTACCTTATACCAGTGGCGATTTCA-3') were designed according to the information published in the Aspergillus terreus FGSC A1156 genome database, and the cDNA of A. Aconitic acid decarboxylase gene, the length of the target fragment detected by 1.0% agarose gel electrophoresis is about 1.5kb, the target product is recovered and purified by tapping gel and ligated with pMD19-T to obtain the recombinant plasmid pCadA-T containing the cadA gene sequence. The pCadA-T was sequenced, and the sequencing results showed that the cloned DNA fragment was the Aconitate decarboxylase gene of Aspergillus terreus. The sequence homology with the published sequence of Aspergillus terreus FGSC A1156 was 100%.

1.2 Construction of target gene cadA expression vector

Extract the plasmid pCadA-T and the plasmid pURGAPZ (constructed in the laboratory), digest with BamHI and KpnI respectively, and perform rubber tapping recovery and purification, and the digested product of pCadA-T recovers a cadA gene band with a size of about 1.5kb , The target band with a size of about 6.2kb was recovered from the digested product of pURGAPZ. The recovered fragments were ligated to obtain the recombinant plasmid pURGAPZC containing the Pgap-CadA-Tgap-zeocin expression cassette.

Example 2 prepares the recombinant yeast strain containing Pgap-CadA-Tgap-zeocin expression cassette

2.1 Obtaining the recombinant yeast strain containing Pgap-CadA-Tgap-zeocin expression cassette

1) Extract the recombinant plasmid pURGAPZC, and linearize it with HindIII restriction enzyme.

2) Pick one ring of glycerologenic Candida strain and insert it into the seed medium (yeast powder 10g/L, peptone 20g/L, glucose 20g/L, zeocin 150μg/mL, the balance is water), at 30°C, Under the condition of 200r/min, shake culture for 18h to obtain liquid seeds. Insert the obtained liquid seeds into the seed medium at an inoculum amount of 1% (v/v), the liquid filling volume is 10mL/100mL, the fermentation temperature is controlled to be 30°C, and the rotation speed is 200r/min, and cultured until the _OD600 is 1 . Take 1 mL of bacteria, collect the cells by centrifugation, discard the supernatant, resuspend the cells in 1 mL of sterile water, and wash twice by centrifugation; add "transformation buffer" in the following order: 240 μL PEG, 36 μL 1.0mol/L LiAc, 50 μL linear Add water to make up to 360 μL; after mixing well, bathe in water at 42°C for 1 h; collect the cells by centrifugation, resuspend in 1 mL liquid YEPD, and culture with shaking at 200 r/min at 30°C for 2 h; collect cells by centrifugation, wash with 1M sorbitol 2 times; 100 μL of cell suspension was spread on zeocin screening plate, and cultured at 30°C for 2-3 days to obtain transformants.

2.2 Genotype verification of recombinant yeast strains containing Pgap-CadA-Tgap-zeocin expression cassette

Transformants were picked from the screening plate, streaked and purified on the YEPD plate, and cultured at 30°C for 18 hours. Pick a single colony and inoculate it in the seed medium, culture it with shaking at 30°C and 200r/min for 18 hours, collect the bacteria to extract the genome, and use GAP-F(5'-ATAGGGCCCCACCACAGCAGCACCAAC-3') and ura5-R(5 '-CCGCGGATCTCGAGTGAACACCATTGTACCAATG-3') was used as a primer to amplify the Pgap-CadA-Tgap-zeocin expression cassette in recombinant yeast by PCR. The results of 0.8% agarose gel electrophoresis showed that the target fragment of about 4.2 kb could be detected in the selected transformants, and the genomic DNA template of the starting strain was used as a negative control, and no DNA fragment was amplified. Therefore, it can be proved that the Pgap-CadA-Tgap-zeocin expression cassette was successfully integrated in the transformant, that is, the desired recombinant yeast strain.

Example 3 Recombinant Yeast Strain Production of Itaconic Acid

1) Preparation of seed medium: glucose 20g/L, yeast powder 10g/L, peptone 5g/L, zeocin 150μg/mL, the balance is water; itaconic acid fermentation medium: glucose 50g/L, yeast powder 10g/L , peptone 20g/L, zeocin 150μg/mL, and the balance is water.

2) Inoculate the recombinant yeast strain obtained above into the seed culture medium, and vibrate at 30° C. and 200 r/min for 18 hours to obtain liquid seeds. The obtained liquid seeds were inserted into the fermentation medium at an inoculum size of 4% (v/v), filled with 30mL/250mL, the controlled fermentation temperature was 30°C, the rotating speed was 200r/min, the time was 48h, and the fermentation ended.

The content of itaconic acid in the fermentation broth was detected by high performance liquid chromatography (HPLC). Instrument: Agilent high performance liquid chromatography (with UV-visible detector, differential detector and workstation); chromatographic column: Bio-RAD Aminex HPX-87Hcolumn 300mm×7.8mm; mobile phase: 5mmol/L sulfuric acid; flow rate: 0.6mL/ min, column temperature: 60°C, UV detector (210nm), injection 10μL.

The fermented liquid obtained after the fermentation was finished was tested for itaconic acid content, and analyzed by HPLC, the concentration of itaconic acid in the fermented liquid was about 200 mg/L.

The above content is only a basic description of the concept of the present invention, and any equivalent transformation made according to the technical solution of the present invention shall fall within the protection scope of the present invention.

SEQ ID NO.1

MTKQSADSNAKSGVTSEICHWASNLATDDIPSDVLERAKYLILDGIACAWVGARVPWSEKYVQATMSFEPPGACRVIGYGQKLGPVAAAMTNSAFIQATELDDYHSEAPLHSASIVLPAVFAASEVLAEQGKTISGIDVILAAIVGFESGPRIGKAIYGSDLLNNGWHCGAVYGAPAGALATGKLLGLTPDSMEDALGIACTQACGLMSAQYGGMVKRVQHGFAARNGLLGGLLAHGGYEAMKGVLERSYGGFLKMFTKGNGREPPYKEEEVVAGLGSFWHTFTIRIKLYACCGLVHGPVEAIENLQGRYPELLNRANLSNIRHVHVQLSTASNSHCGWIPEERPISSIAGQMSVAYILAVQLVDQQCLLSQFSEFDDNLERPEVWDLARKVTSSQSEEFDQDGNCLSAGRVRIEFNDGSSITESVEKPLGVKEPMPNERILHKYRTLAGSVTDESRVKEIEDLVLGLDRLTDISPLLELLNCPVKSPLV

Claims (8)

1. A recombinant yeast strain producing itaconic acid, characterized in that: the recombinant yeast uses C.glycerolgenesisCCTCC AB 204047 as the starting strain, and inserts the cis-aconitic acid decarboxylase gene in the C.glycerolgenesis genome through genetic engineering A recombinant yeast strain obtained by expressing the cassette Pgap-CadA-Tgap-zeocin. 2. according to the described recombinant yeast strain of right 1, it is characterized in that described aconitic acid decarboxylase expression box Pgap-CadA-Tgap-zeocin is by Aspergillus terreus aconitic acid decarboxylase gene, C.glycerolgenesis glyceraldehyde- 3-phosphate dehydrogenase gene promoter and terminator, bleomycin zeocin gene, promoter and terminator composition. 3. The recombinant yeast strain according to rights 1 to 2, characterized in that: said Aspergillus terreus cis-aconitic acid decarboxylase gene encodes a protein: a. A protein consisting of the amino acid sequence shown in SEQ ID NO.1. 4. The recombinant yeast strain according to rights 1 to 2, characterized in that: the C.glycerolgenesis glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator are respectively connected to the gene encoding cis-aconitic acid decarboxylase The two ends of the nucleotide sequence respectively initiate and terminate the transcription of the aconitic acid decarboxylase gene. 5. according to the described recombinant yeast strain of right 1 to 2, it is characterized in that: described bleomycin zeocin is used as the resistance selection marker in the expression cassette, and the promotor and terminator of this gene are respectively connected at the two ends of the gene sequence. to initiate and terminate gene transcription, respectively. 6. according to the construction method of the recombinant yeast strain that produces itaconic acid described in claim 1 to 5, it is characterized in that: construct the recombinant plasmid containing cis-aconitic acid decarboxylase expression cassette by genetic engineering transformation, after recombinant plasmid linearization It is inserted into the genome of C. glycerolgenesis to obtain a recombinant yeast strain containing the expression cassette of aconitic acid decarboxylase. 7. The application of the recombinant yeast strain according to claims 1 to 6 in the preparation of itaconic acid, characterized in that: the recombinant yeast is cultivated in a fermentation medium; and the medium is collected to separate itaconic acid . 8. The application of the recombinant yeast strain according to claim 7 in the preparation of itaconic acid, characterized in that: the fermentation medium is: 50g/L glucose, 10g/L yeast powder, 20g/L peptone, zeocin 150 μg /mL, the balance is water.