Recombinant yeast strain for synthesizing astaxanthin as well as construction method and application of recombinant yeast strain

Abstract

The invention belongs to the technical field of gene engineering, and discloses a recombinant yeast strain for synthesizing astaxanthin as well as a construction method and application of the recombinant yeast strain. The recombinant plasmid provided by the invention comprises the following gene segments: a promoter TEF1p, a beta-carotene hydroxylase gene CrtZ, a cis-acting element IGG6, a beta-carotene ketolase gene CrtW and a terminator CYC1t which are spliced in sequence. A specific cis-acting element IGG6, a promoter, a terminator and exogenous CrtZ and CrtW are selected, a gene segment capable of producing astaxanthin is obtained, a recombinant plasmid is constructed from the gene segment, the recombinant plasmid is transformed into a saccharomyces cerevisiae strain, and high transformation of astaxanthin is achieved. Furthermore, the CrtZ and the CrtW are mutated by adopting error-prone PCR (Polymerase Chain Reaction) to obtain forward mutation, so that the conversion efficiency of the astaxanthin is further improved, and a more economic and more efficient way is provided for heterologous production of the astaxanthin by a microbial chassis.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant yeast strain for synthesizing astaxanthin, a construction method thereof, and an application thereof. Background Art

[0002] Currently, astaxanthin-producing strains have been constructed by introducing heterologous genes for the astaxanthin synthase enzymes β-carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) into a bacterial strain that already possesses the foundation for β-carotene synthesis. However, the heterologous enzymes often exhibit insufficient activity in the host, resulting in low catalytic efficiency and incompatibility with the endogenous metabolic network. Error-prone PCR, an in vitro mutagenesis technique, can introduce random mutations during the amplification of specific gene sequences, allowing screening for target mutations and thus optimizing the catalytic performance of key enzymes. This provides a new strategy for efficient astaxanthin synthesis. Combining targeted mutagenesis with high-throughput screening has provided an efficient and cost-effective solution for increasing astaxanthin production in Saccharomyces cerevisiae. By focusing on the directed evolution of key enzymes and integrating systems biology with automated screening platforms, it is hoped that this will overcome existing metabolic bottlenecks and advance the industrialization of microbial astaxanthin synthesis.

[0003] The core of error-prone PCR technology lies in using a low-fidelity DNA polymerase to perform the PCR amplification reaction, while adjusting the ion concentration and changing the dNTP ratio in the reaction system. Optimizing these conditions can promote the random introduction of incorrect bases at a certain frequency during the nucleic acid amplification process, thereby achieving gene mutation. Error-prone PCR has the following advantages:

[0004] (1) Controllability: By adjusting the reaction conditions (such as dNTP concentration, Mg 2∈ / Mn 2+ ratio, number of cycles, etc.), error-prone PCR can achieve flexible regulation of mutation rate.

[0005] (2) High efficiency: Combined with high-throughput screening technology (such as microfluidic droplet sorting or fluorescence-activated cell sorting), mutant enzymes with enhanced activity can be quickly obtained.

[0006] (3) Targetedness: Mutation is performed only on specific genes (such as CrtZ or CrtW), avoiding interference from irrelevant genes and significantly narrowing the screening scope. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a recombinant yeast strain for synthesizing astaxanthin, a construction method and application thereof.

[0008] The first object of the present invention is to provide a recombinant plasmid comprising the following gene fragments: promoter TEF1p, β-carotene hydroxylase gene CrtZ, cis-acting element IGG6, β-carotene ketolase gene CrtW, and terminator CYC1t, which are sequentially spliced ​​together;

[0009] The β-carotene hydroxylase gene crtZ is derived from Agrobacterium aurantiacum;

[0010] The β-carotene ketolase gene crtW is derived from Brevundimonas sp. SD212;

[0011] The nucleic acid sequence of the cis-acting element IGG6 is: CAATCAAAC.

[0012] Preferably, the nucleic acid sequence of the gene fragment is SEQ ID NO.4.

[0013] Preferably, the amino acid sequence of crtZ is SEQ ID NO.6, and the amino acid sequence of crtW is SEQ ID NO.2; or

[0014] The amino acid sequence of crtZ is SEQ ID NO.8, and the amino acid sequence of crtW is SEQ ID NO.10;

[0015] Preferably, the gene sequence of crtZ is SEQ ID NO.5, and the gene sequence of crtW is bases 901-1626 in SEQ ID NO.4;

[0016] Preferably, the gene sequence of crtZ is SEQ ID NO.7, and the gene sequence of crtW is SEQ ID NO.9.

[0017] Preferably, the recombinant plasmid is constructed based on plasmid p416TEF;

[0018] Preferably, the p416TEF plasmid is digested with XbaI restriction nuclease to obtain a linearized vector fragment, which is then ligated to the above gene fragment.

[0019] The second object of the present invention is to provide the use of the above-mentioned recombinant plasmid in preparing an astaxanthin-producing strain.

[0020] The third object of the present invention is to provide a recombinant Saccharomyces cerevisiae strain for synthesizing astaxanthin, which is obtained by transforming the Saccharomyces cerevisiae strain with the above-mentioned recombinant plasmid;

[0021] Preferably, the Saccharomyces cerevisiae is Saccharomyces cerevisiae YZ03; the Saccharomyces cerevisiae YZ03 strain is: CEN.PK113-5D, hoΔ::TADH1-crtYB-PTDH3-PTEF1-crtI-TCYC1;

[0022] HMG1::TADH1-crtE-PTDH3-PTEF1-tHMG1-TCYC1, and the Saccharomyces cerevisiae CEN.PK113-5D strain is: MATaMAL2-8c SUC2 ura3-52.

[0023] The present invention can use any β-carotene-producing Saccharomyces cerevisiae strain, for example, the above-mentioned Saccharomyces cerevisiae YZ03 strain can be selected.

[0024] The fourth object of the present invention is to provide the use of the above-mentioned recombinant Saccharomyces cerevisiae in synthesizing astaxanthin.

[0025] The fifth object of the present invention is to provide a method for constructing the above-mentioned recombinant plasmid, wherein the bases 403-891 in SEQ ID NO.4 are amplified by primers AB7-crtZ-F: GCAA TCTAATCTAAGTTTTatgactaacttcttgatcgttgttgctactg; AB7-crtZ-R: GTTTGATTGttaagttctttcttgag cttcagctctcaaaac to obtain the crtZ-AB7 fragment, and the bases 403-891 in SEQ ID NO.4 are amplified by primers AB7-crtW-F: caagaaagaacttaaCAATCAAACatgtccgctgttactccaatgagtag, AB7-crtW-R: GCCCGGGGGATCCACTAGTTttatgaaataaagaccaccaaggcttccaag The bases 901-1626 in NO.4 were amplified to obtain the crtW-AB7 fragment, and the p416TEF plasmid was digested with XbaI restriction nuclease to obtain a linearized vector fragment, and the above fragments were ligated.

[0026] A sixth object of the present invention is to provide a method for producing astaxanthin, comprising the following steps:

[0027] (1) inoculating the above-mentioned recombinant Saccharomyces cerevisiae strain into a seed culture medium for activation, and then performing expansion culture;

[0028] (2) The expanded cultured strain is inoculated into a fermentation medium for fermentation culture, and after the culture is completed, the bacterial cells are collected for astaxanthin extraction.

[0029] Preferably, in step (1):

[0030] The recombinant Saccharomyces cerevisiae strain according to claim 6 is inoculated into a seed culture medium and cultured at 25-30° C. and 250 rpm for 16-20 hours;

[0031] Preferably, the seed culture medium is an SC nutrient-deficient liquid culture medium containing 10-30 g / L glucose or a YPD liquid culture medium containing 10-30 g / L glucose; and / or

[0032] In step (2): the strain after expansion culture was 600 = 0.2 transferred to fermentation medium, cultured at 25-30°C, 250 rpm for 82-86 hours, and then the bacterial cells were collected to extract astaxanthin;

[0033] Preferably, the seed culture medium is an SC nutrient-deficient liquid culture medium containing 10-30 g / L glucose or a YPD liquid culture medium containing 10-30 g / L glucose.

[0034] The present invention obtains a gene fragment capable of producing astaxanthin by selecting a specific cis-acting element IGG6, a promoter, a terminator, and exogenous CrtZ and CrtW, thereby constructing a recombinant plasmid and transforming it into a cerevisiae strain to achieve a high conversion of astaxanthin. Furthermore, error-prone PCR is used to mutate CrtZ and CrtW in the astaxanthin synthesis pathway, obtaining CrtZ and CrtW with a positive mutant phenotype, thereby further improving the conversion efficiency of astaxanthin and providing a more economical and efficient method for heterologous production of astaxanthin in a microbial chassis. The synthesis pathway of astaxanthin in cerevisiae is shown in Figure 1 .

[0035] Figure 1 This is the synthesis pathway of astaxanthin in Saccharomyces cerevisiae. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0037] Figure 1 This is the synthesis pathway of astaxanthin in Saccharomyces cerevisiae.

[0038] Figure 2 The bands are amplified at different TaqDNA polymerase concentrations.

[0039] Figure 3 For different Mn 2+ The bands were amplified at the concentration.

[0040] Figure 4 This is a map of plasmid p416TEF.

[0041] Figure 5 Maps of plasmids AB7, AB8, AB9, and AC1.

[0042] Figure 6 The effects of different series connection methods on astaxanthin production.

[0043] Figure 7 Schematic diagram of the process of error-prone PCR screening mutants.

[0044] Figure 8 This is the color of the colonies on the plate after transformation of the error-prone PCR library.

[0045] Figure 9 Batch fermentation was performed to initially screen the dominant strains using error-prone PCR.

[0046] Figure 10 These are the fermentation results of the third batch of error-prone PCR with different colored strains.

[0047] Figure 11 Repeat the verification results for rescreening.

[0048] Figure 12 Maps of plasmids huiYKP101, huiYKP102, huiYKP109, and huiYKP1010.

[0049] Figure 13 Verify colony color change for Saccharomyces cerevisiae.

[0050] Figure 14 To verify the stability of the forward mutant strain using reverse mutation. DETAILED DESCRIPTION

[0051] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative tests in the following examples were performed in triplicate, and the results were averaged.

[0052] Example 1

[0053] 1 Experimental Materials and Methods

[0054] 1.1 Strains and plasmids

[0055] The strains and plasmids constructed in the present invention are shown in Table 7, and the primers used in the construction process are listed in Table 8. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0056] The CrtZ gene in the present invention is derived from Agrobacterium aurantiacum, and the CrtW gene is derived from Brevundimonas sp. SD212.

[0057] The plasmid p416TEF in the present invention was purchased from Newpro Biotechnology with the trade name p416TEF vector and the product number V009725.

[0058] 1.2 Main experimental instruments and equipment

[0059] Table 1 Main experimental instruments

[0060] instrument model factory Gel imaging instrument SRB-YS-005 Beijing Oriental Rayleigh Company High-speed refrigerated centrifuge CTH2790R Hitachi Corporation Freeze grinder JXFST-CL-BSC Shanghai Jingxin Co., Ltd. Agarose gel electrophoresis apparatus DYY-6D Beijing Liuyi Biotechnology Co., Ltd. Vortex Oscillator Vortex3000 Haimen Qilin Bell Instrument Manufacturing Co., Ltd. Liquid chromatograph LC-20303D Shimadzu Corporation High-performance liquid chromatography (HPLC) LC-20AT Shimadzu Corporation PCR instrument 621BR36800 Bio-Rad Biomedical Products (Shanghai) Co., Ltd.

[0061] 1.3 Culture medium and astaxanthin extraction method

[0062] The culture media used in the experiments of the present invention include Luria-Bertani (LB) medium, Yeast Extract Peptone Dextrose (YPD) medium, and SC (Synthetic Complete) nutrient-deficient medium, which are mainly used for the cultivation of Escherichia coli and Saccharomyces cerevisiae. Among them, YPD medium and SC nutrient-deficient medium contain 20g / L of glucose. In addition, all solid culture media require the addition of a certain amount of agar powder based on the formula of the liquid culture medium. Generally, 15-20g of agar powder is added to 1L of liquid culture medium. A rotor is added during sterilization to facilitate the subsequent thorough mixing of glucose and culture medium. Finally, high-temperature and high-pressure sterilization is performed at 121°C for 20 minutes.

[0063] Astaxanthin is an intracellular metabolite. Therefore, the extraction of astaxanthin from Saccharomyces cerevisiae involves two key steps: first, cell crushing and lysis to release intracellular substances; second, separation and collection of astaxanthin. Astaxanthin extraction:

[0064] (1) Bacterial solution treatment: Place 1 mL of Saccharomyces cerevisiae culture in a 2 mL centrifuge tube and centrifuge at 12,000 rpm for 1 minute. Aspirate the supernatant. Add 1 mL of distilled water, gently pipette to mix, centrifuge at 12,000 rpm for 1 minute, and discard the supernatant.

[0065] (2) Astaxanthin extraction: Add 500 μL of acetone (containing 1% BHT, butylated hydroxytoluene, and antioxidant) and approximately 400 μL of glass beads (0.4 mm-0.6 mm) to a centrifuge tube and grind thoroughly using a cryo-grinder. Place the centrifuge tube in a 30°C incubator for 10-15 minutes (astaxanthin has the highest solubility in acetone at 30°C).

[0066] (3) Extraction treatment: Centrifuge at 12,000 rpm for 5 minutes to precipitate cell debris. Aspirate the supernatant with a 1 mL sterile syringe and filter through a 0.22 μm organic filter to remove impurities. The entire process must be performed in the dark to prevent astaxanthin degradation.

[0067] 1.4 Fermentation method

[0068] The shake flask fermentation culture is adopted. First, a single colony is picked and activated on a plate. The brewer's yeast is inoculated on a SC nutrient-deficient solid medium containing 20g / L glucose or a YPD solid medium containing 20g / L glucose. It is cultured in a 30°C constant temperature incubator for 2-3 days until obvious single colonies appear on the plate. Use a sterile inoculation loop to pick a single colony and transfer it to a shake tube containing the corresponding liquid culture medium (such as SC nutrient-deficient medium containing 20g / L glucose or YPD medium containing 20g / L glucose). Culture overnight in a shaker at 30°C and 250rpm as a seed solution. According to OD 600 The 0.2% agar-agar was transferred to 10 mL of SC nutrient-deficient medium containing 20 g / L glucose or a 100 mL shake flask containing 20 g / L glucose of YPD medium. The fermentation process was carried out at a constant temperature of 30°C, the dissolved oxygen level was maintained by an oscillation frequency of 250 rpm, the culture cycle was set to 84 hours, and the bacterial cells were collected to extract astaxanthin.

[0069] 1.5 Error-prone PCR reaction procedures

[0070] The specific reaction system and experimental procedure settings are shown in Tables 2 and 3.

[0071] Table 2 System components of error-prone PCR

[0072] Components dose 10×PCRBuffer 5μL <![CDATA[25mMMgCl2]]> 10 μL Topprimer (10 μM) 2μL Bottom primer (10 μM) 2μL dNTPMixture 4 μL <![CDATA[MnCl2]]> Adjust according to concentration TaKaRaTaq 1 μL plasmids 1 μL <![CDATA[Nuclease-freeH2O]]> Make up to 50 μL

[0073] Table 3 Reaction procedures for error-prone PCR

[0074]

[0075]

[0076] 2 Results and Discussion

[0077] 2.1 Determination of error-prone PCR conditions

[0078] We first systematically evaluated the dosage of Taq DNA polymerase. By setting up a gradient of different concentrations (1, 2, 3, and 4 μL), plasmid AB7 was constructed according to the method in step 2.2. The Aa_CrtZ gene from Paracoccus aquaticus and the BDC263_CrtW gene from Brevundimonas were amplified in their entirety using the primers AB7-PCR-F and AB7-PCR-R listed in Table 8. The results showed that, as shown in Table 8, Figure 2 As shown in the figure, all gradient settings were able to successfully amplify the target band. Therefore, 1 μL of Taq DNA polymerase was selected as the standard for subsequent experiments.

[0079] Figure 2 The bands are amplified at different Taq DNA polymerase concentrations.

[0080] In addition, we chose to keep Mg 2+ The concentration is stable to ensure the overall stability of the PCR reaction and by adjusting the Mn 2+ The frequency of base mutations can be precisely controlled by adjusting the concentration. 2+ ions as a kind of 2+ Similar metal ions have been shown to promote the mismatch effect of Taq DNA polymerase, thereby increasing the generation of mismatch mutations. 2+ Concentration adjustment can effectively control the mutation rate to meet the needs of the experiment. Through error-prone PCR technology, the base mismatch rate of CrtZ and CrtW genes can be effectively increased to achieve random mutation. 2+ ions and set a concentration gradient of 0.01, 0.02, 0.03 and 0.04 mM. PCR amplification was performed according to the system in Table 2. The primers used were AB7-PCR-F and AB7-PCR-R in Table 8. DNA agarose gel electrophoresis results ( Figure 3 ) shows that in Mn 2+ The target band was successfully amplified in the concentration range of 0.01 to 0.04 mM. 2+ With the increase of concentration, the brightness of the amplified band gradually weakened. 2+ To investigate the effect of CrtZ and CrtW gene mutations, we sequenced the amplified PCR products. The sequencing results showed that 0.01 and 0.02 mM Mn 2+ No gene mutation was observed at 0.03 mM Mn 2+ Can cause 1-3 base mutations in CrtZ and CrtW, 0.04mM Mn 2+ Based on this result, 0.03mM Mn 2+The mutation rate standard of 3-5 base mutations in a 1 kb fragment amplified by error-prone PCR was met, and this concentration was selected as the reaction condition for subsequent experiments.

[0081] Figure 3 For different Mn 2+ The bands were amplified at the concentration.

[0082] 2.2 Verification of the Serial Connection of CrtZ and CrtW

[0083] We first evaluated the effects of CrtZ and CrtW expression patterns on astaxanthin production. By adjusting the order of the CrtZ and CrtW genes and employing different cis-acting elements, four different expression plasmids were constructed: AB7 (pTEF-CrtZ-IGG6-CrtW), AB8 (pTEF-CrtW-IGG6-CrtZ), AB9 (pTEF-CrtZ-P2A-CrtW), and AC1 (pTEF-CrtW-P2A-CrtZ). These plasmids were subsequently transformed into Escherichia coli for amplification and then expressed in the β-carotene-producing Saccharomyces cerevisiae strain YZ03 (genotype description shown in Table 4). The resulting engineered Saccharomyces cerevisiae strains, YKAB7, YKAB8, YKAB9, and YKAC1, were then analyzed by HPLC to assess the effects of different expression patterns on astaxanthin production.

[0084] Plasmid AB7, namely pTEF1-CrtZ-IGG6-CrtW, is a promoter of TEF1p that expresses two genes, CrtZ and CrtW, in tandem through the IGG6 cis-acting element, with the terminator being CYC1t.

[0085] Plasmid AB8, i.e. pTEF-CrtW-IGG6-CrtZ, is a promoter of TEF1p that expresses two genes, CrtW and CrtZ, in tandem through the IGG6 cis-acting element, with the terminator being CYC1t.

[0086] Plasmid AB9, namely pTEF-CrtZ-P2A-CrtW, is a promoter of TEF1p that expresses two genes, CrtZ and CrtW, in tandem through the P2A cis-acting element, with the terminator being CYC1t.

[0087] Plasmid AC1, i.e. pTEF-CrtW-P2A-CrtZ, is a promoter of TEF1p that expresses two genes, CrtW and CrtZ, in tandem through the P2A cis-acting element, with the terminator being CYC1t.

[0088] in,

[0089] The wild-type CrtZ amino acid sequence is as follows (SEQ ID NO. 1):

[0090] MTNFLIVVATVLVMELTAYSVHRWIMHGPLGWGWHKSHHEEHDHALEKNDLYGL VFAVIATVLFTVGWIWAPVLWWIALGMTVYGLIYFVLHDGLVHQRWPFRYIPRKGYARR LYQAHRLHHAVEGRDHCVSFGFIYAPPVDKLKQDLKMSGVLRAEAQERT.

[0091] The wild-type CrtW amino acid sequence is as follows (SEQ ID NO. 2):

[0092] MSAVTPMSRVVPNQALIGLTLAGLIAAAWLTLHIYGVYFHRWTIWSVLTVPLIVAGQTWLSVGLFIVAHDAMHGSLAPARPRLNTAIGSLALALYAGFRFTPLKTAHHAHHAAPGTADDPD FHADAPRAFLPWFYGFFRTYFGWRELAVLTVLVAVAVLILGARMPNLLVFWAAPALLSALQLFTFGTWLPHRHTDDAFPDNHNARTSPFGPVLSLLTCFHFGRHHEHHLTPWKPWWSLFS.

[0093] The nucleic acid sequence of the IGG6 cis-acting element is as follows: CAATCAAAC.

[0094] Reference for the IgG6 cis-acting element sequence: Yue Q, Meng J, Qiu Y, Yin M, Zhang L, Zhou W, et al. A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi. Nat Commun. 2023;14:4267.

[0095] The nucleic acid sequence of the P2A cis-acting element is as follows (SEQ ID NO. 3): GGTTCTGGTGGTGCTACTA ATTTTTCTTTGTTGAAATTGGCTGGTGATGTTGAATTGAATCCAGGTCCA.

[0096] 2.2.1 Construction of AB7 plasmid

[0097] The wild-type crtZ gene (Genebank: P54973.1) was codon-optimized for Saccharomyces cerevisiae and synthesized by the company. The synthesized crtZ gene was cloned using primers AB7-crtZ-F: GCAATCTAATCTAAGTTTTatgactaacttcttgatcgttgttgctactg in Table 8; AB7-crtZ-R: GTTTGATTGttaagttctttcttgagcttcagctctcaaaac in Table 8 to obtain the crtZ-AB7 fragment. The wild-type crtW gene (Genebank: ABC50116.1) was codon-optimized for Saccharomyces cerevisiae and synthesized by the company. The crtW-AB7 fragment was amplified using primers AB7-crtW-F and AB7-crtW-R in Table 8 to obtain the crtW-AB7 fragment. The p416TEF plasmid was digested with XbaI restriction nuclease to obtain a linearized vector fragment, which was then cloned using Norvegian ClonExpress Ultra One Step Cloning. Kit (Cat. No. C115-01) was used to ligate plasmid AB7. The promoter TEF1p, β-carotene hydroxylase gene CrtZ, cis-acting element IGG6, β-carotene ketolase gene CrtW, and terminator CYC1t were sequentially spliced. The following is the nucleotide sequence of the sequentially spliced ​​TEF1p-CrtZ-IGG6-CrtW-CYC1t (SEQ ID NO. 4):

[0098] aatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttccc

[0099] ctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggc

[0100] aataaaaatttttatcacgtttctttttcttgaaaatttttttttttgattttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaat

[0101] ttctcaagtttcagtttcatttttcttgttctattacaacttttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagttttaattacaaa

[0102] atgactaacttcttgatcgttgttgctactgttttggttatggaattgactgcttactctgttcacagatggatcatgcacggtccattgggttggg

[0103] gttggcacaagtctcaccacgaagaacacgaccacgctttggaaaagaacgacttgtacggtttggttttcgctgttatcgctactgttttgttc

[0104] actgttggttggatctgggctccagttttgtggtggatcgctttgggtatgactgtttacggtttgatctacttcgttttgcacgacggtttggttca

[0105] ccaaagatggccattcagatacatcccaagaaagggttacgctagaagattgtaccaagctcacagattgcaccacgctgttgaaggtaga

[0106] gaccactgtgtttctttcggtttcatctacgctccaccagttgacaagttgaagcaagacttgaagatgtctggtgttttgagagctgaagctca

[0107] agaaagaacttaaCAATCAAACatgtccgctgttactccaatgagtagagttgtccctaatcaagcattgattggtttgactttagcag

[0108] gtttgattgctgctgcttggttgacattgcatatatacggtgtctacttccacagatggacaatctggtctgttttgaccgtcccattaattgtagct

[0109] ggtcaaacttggttgtccgtaggtttattcatagttgctcatgatgcaatgcacggtagtttggcaccagccagacctagattaaacacagcaa

[0110] tcggttctttggctttagcattgtatgccggttttagattcaccccattgaaaactgcccatcacgctcatcacgctgcaccaggtacagcagat

[0111] gaccctgattttcatgccgacgctccaagagcatttttgccttggttctatggtttctttagaacctacttcggttggagagaattagcagttttga

[0112] ctgtattagttgcagtcgccgtattaatattgggtgctagaatgccaaatttgttagtcttttgggccgctcctgcattgttgtctgccttacaattg

[0113] ttcactttcggtacatggttgccacatagacacaccgatgacgcctttcctgataatcataacgctagaacatctccattcggtcctgttttatca

[0114] ttgttgacctgtttccatttcggtagacatcacgaacatcacttgacaccttggaagccttggtggtctttattttcataaACTAGTGGAT

[0115] CCCCCGGGCTGCAGGAATTCGATATCAAGCTTacaggccccttttcctttgtcgatatcatgtaattagttatgtca

[0116] cgcttacattcacgccctcctcccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttattttttttaata

[0117] gttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgc.

[0118] The 1st-402nd base sequence is the promoter TEF1p sequence, the 403th-891th base sequence is the crtZ sequence, the 892th-900th base sequence is the IGG6 sequence, the 901st-1626th base sequence is the crtW sequence, and the 1627th-1943rd base sequence is the terminator CYC1t sequence.

[0119] 2.2.2 Construction of AB8 plasmid

[0120] The wild-type crtZ gene (Genebank: P54973.1) was codon-optimized for Saccharomyces cerevisiae and synthesized by the company (the optimized sequence was identical to the corresponding sequence in step 2.2.1). The synthesized crtZ gene was cloned using primers AB8-crtZ-F and AB8-crtZ-R in Table 8 to obtain the crtZ-AB8 fragment. The wild-type crtW gene (Genebank: ABC50116.1) was codon-optimized for Saccharomyces cerevisiae and synthesized by the company (the optimized sequence was identical to the corresponding sequence in step 2.2.1). The crtW-AB8 fragment was amplified using primers AB8-crtW-F and AB8-crtW-R in Table 8 to obtain the crtW-AB8 fragment. The p416TEF plasmid was digested with XbaI restriction nuclease to obtain a linearized vector fragment, which was then ligated using the Norvegian ClonExpress Ultra One Step Cloning Kit (Cat. No. C115-01) to obtain plasmid AB8.

[0121] 2.2.3 Construction of AB9 plasmid

[0122] The wild-type crtZ gene (Genebank: P54973.1) was codon-optimized for Saccharomyces cerevisiae and synthesized by the company (the optimized sequence was identical to the corresponding sequence in step 2.2.1). The synthesized crtZ gene was cloned using primers AB9-crtZ-F and AB9-crtZ-R in Table 8 to obtain the crtZ-AB9 fragment. The wild-type crtW gene (Genebank: ABC50116.1) was codon-optimized for Saccharomyces cerevisiae and synthesized by the company (the optimized sequence was identical to the corresponding sequence in step 2.2.1). The crtW-AB9 fragment was amplified using primers AB9-crtW-F and AB9-crtW- in Table 8 to obtain the crtW-AB9 fragment. The p416TEF plasmid was digested with XbaI restriction nuclease to obtain a linearized vector fragment, which was then ligated using the ClonExpress Ultra One Step Cloning Kit (Cat. No. C115-01) to obtain plasmid AB9.

[0123] 2.2.4 Construction of AC1 plasmid

[0124] The wild-type crtZ gene (Genebank: P54973.1) was codon-optimized for Saccharomyces cerevisiae and synthesized by the company (the optimized sequence was identical to the corresponding sequence in step 2.2.1). The synthesized crtZ gene was cloned using primers AC1-crtZ-F and AC1-crtZ-R in Table 8 to obtain the crtZ-AC1 fragment. The wild-type crtW gene (Genebank: ABC50116.1) was codon-optimized for Saccharomyces cerevisiae and synthesized by the company (the optimized sequence was identical to the corresponding sequence in step 2.2.1). The crtW-AC1 fragment was amplified using primers AC1-crtW-F and AC1-crtW-R in Table 8 to obtain the crtW-AC1 fragment. The p416TEF plasmid was digested with XbaI restriction nuclease to obtain a linearized vector fragment, which was then ligated using the Norvegian ClonExpress Ultra One Step Cloning Kit (Cat. No. C115-01) to obtain plasmid AC1.

[0125] Table 4 YZ03 construction description

[0126]

[0127] Figure 4 This is a map of plasmid p416TEF.

[0128] Figure 5 Maps of plasmids AB7, AB8, AB9, and AC1.

[0129] Figure 6The effects of different series connection methods on astaxanthin production.

[0130] From the HPLC test results ( Figure 6 ), it can be seen that changing the order of the CrtZ and CrtW genes affects gene expression efficiency. This suggests that changes in gene order may regulate metabolic flux by altering protein folding or interaction patterns, thereby affecting the final metabolite. In addition, the use of different cis-acting elements in series also had a significant impact on astaxanthin production. When the IGG6 cis-acting element was used in series, changes in the order of the CrtZ and CrtW genes led to significant differences in astaxanthin production, with a maximum difference of 2.5 times; while when the P2A cis-acting element was used in series, changes in gene order resulted in a difference of approximately 1.9 times in astaxanthin production. This shows that the choice of cis-acting elements has a significant impact on the efficiency of gene expression and the accumulation of metabolites.

[0131] Based on the above results, we found that among all the expression methods evaluated, the tandem IGG6 cis-acting element method with CrtZ in front and CrtW in the back (i.e., AB7 plasmid expression method) significantly improved astaxanthin production and achieved the highest expression level. Therefore, the pTEF-CrtZ-IGG6-CrtW expression method is the optimal combination, indicating that the gene order and the choice of cis-acting elements play a crucial role in optimizing astaxanthin synthesis.

[0132] 2.3 Screening of CrtZ and CrtW forward mutant strains

[0133] First, we digested the vector plasmid p416TEF with XbaI to obtain the corresponding vector fragment. In order to remove the influence of the template plasmid, we then used DpnI enzyme to digest it to ensure that the template plasmid was completely removed. Subsequently, we also digested the fragment containing the target genes CrtZ and CrtW obtained by error-prone PCR (amplified using primers AB7-yicuo-F / R with plasmid AB7 as a template) with DpnI to remove the residual template plasmid. Next, using the homologous recombination mechanism of Saccharomyces cerevisiae, we connected the target gene fragment to the vector plasmid (upstream homologous arm: Ctaatctaagttttaattacaaa; downstream homologous arm: ACTAGTGGATCCCCCGGGC) and used the yeast transformation method. This method constructs plasmids through homologous recombination in yeast cells.

[0134] The detailed steps and experimental procedures of the entire screening process are shown in Figure 7 , ensuring the efficiency and accuracy of screening. In order to screen the strains with positive mutations, we screened by the color difference of the colonies on the plate (such as Figure 8In this way, yeast strains that have been successfully transformed with the mutant gene can be effectively identified.

[0135] Figure 7 Schematic diagram of the process of error-prone PCR screening mutants.

[0136] Figure 8 This is the color of the colonies on the plate after transformation of the error-prone PCR library.

[0137] We performed 15 rounds of error-prone PCR to ensure a sufficiently large mutant library. Approximately 200 plates were plated in each round to ensure a sufficient number of colonies. All darker clones were streaked and subsequently analyzed by HPLC. Due to the large sample size, the initial screening process was divided into four rounds to ensure comprehensive coverage. Single colonies that exhibited color changes were initially identified and then transferred to fresh SC-URA plates for rescreening. Given the inhibitory effect of astaxanthin on cell growth, which could potentially lead to strain instability, re-streaking effectively eliminated strains with unstable astaxanthin production. Strains with stable color changes were inoculated into SC-URA liquid medium for fermentation, and strains with improved production were retested. After confirming that their production was improved compared to the control strain YAB7 (obtained by transforming the AB7 plasmid into yeast), we selected the forward mutant strains to extract yeast plasmids for further sequencing analysis to identify mutations in the CrtZ and CrtW genes.

[0138] like Figure 9 As shown, the initial screening results showed that we obtained a strain with a higher yield than the control strain YAB7, and further rescreened it.

[0139] Figure 9 Batch fermentation was performed to initially screen the dominant strains using error-prone PCR.

[0140] By analyzing the liquid phase results of the initial screening strains, we found that some dark-colored monoclones did not produce astaxanthin. During the fermentation process, in order to verify whether there is a clear correlation between color and astaxanthin production, we recorded the color of each seed liquid before the third round of initial screening fermentation, such as Figure 10 As shown, and corresponding to its HPLC analysis results ( Figure 9 AB7 plasmid expression-error-prone PCR batch 3).

[0141] Figure 10 These are the fermentation results of the third batch of error-prone PCR with different colored strains.

[0142] The results showed that strains 4, 5, 6, 12, 16, and 17 with yellowish colors had extremely low astaxanthin production, while strains 1, 3, 7, 9, 10, 13, and 14 with reddish colors had higher astaxanthin production, or did not produce astaxanthin at all. Therefore, we can use color changes as the basis for preliminary screening, among which strains with reddish colors are more likely to screen out high-yield strains with positive mutations. Although these strains have darker colors, no astaxanthin was found in the HPLC test. After in-depth analysis, the possible reason is that the process of CrtZ and CrtW catalyzing the production of astaxanthin from β-carotene is more complicated, involving multiple reaction steps, and is accompanied by the production of a large number of intermediate by-products, such as zeaxanthin, canthaxanthin, lycopene, etc. The dark color phenomenon may not be due to the accumulation of astaxanthin, but the color difference change caused by the accumulation of these by-products in the reaction. We further re-screened the strains whose astaxanthin production was higher than that of the control strain YAB7 obtained by the initial screening fermentation. The HPLC results are as follows Figure 11 shown.

[0143] Figure 11 Repeat the verification results for rescreening.

[0144] Due to the experimental duration, we split the error-prone PCR screening of positive lines for CrtZ and CrtW into two separate rescreening steps. Combining the HPLC results from these two rescreenings, we ultimately identified six strains with improved yields: YKP1001, YKP102, YKP103, YKP104, YKP109, and YKP1010. We then streaked these six strains, isolated single colonies, and cultured them overnight. Yeast plasmids from these six strains were then extracted and sequenced to verify mutations in the CrtZ and CrtW genes and to identify the mutation sites. Detailed sequencing results and statistical data are shown in Table 5.

[0145] Table 5 CrtZ and CrtW mutations

[0146]

[0147] According to the sequencing results, we found that only 4 strains had missense mutations. According to the sequencing results, we found that only 4 strains had missense mutations, namely YKP101, YKP02, YKP09, and YKP1010.

[0148] In order to verify the stability of these missense mutation sites, we conducted a reverse mutation experiment and successfully constructed four corresponding plasmids, corresponding to huiYKP101, huiYKP102, huiYKP109 and huiYKP1010. By designing primers (only for missense mutation sites), mutations were introduced at specific sites, and then the mutant fragments and vector fragments were assembled by Gibson in Escherichia coli. The homologous recombination reaction was carried out using the homologous recombination enzyme ClonExpress MultiS One Step Cloning Kit (from Novozymes Biotech Co., Ltd.) and the reaction was carried out according to the instructions. The plasmid map is shown in the figure below. Figure 12 As shown in Table 6, the primers used are shown in Table 6, and the detailed process is as follows:

[0149] (1) Using primers YKP101-F / back mutation-R and plasmid AB7 as template, PCR amplification was performed to obtain the fragment huiYKP101-1, with a size of 2953 bp. Using primers back mutation-F / YKP101-R and plasmid AB7 as template, PCR amplification was performed to obtain the fragment huiYKP101-2, with a size of 3745 bp. The gene fragments of huiYKP101-1 and huiYKP101-2 will be used to construct the huiYKP101 plasmid.

[0150] (2) Using primers YKP102-F / back mutation-R and plasmid AB7 as template, PCR amplification yielded the huiYKP102-1 fragment, which was 2741 bp in size. Using primers back mutation-F / YKP102-R and plasmid AB7 as template, PCR amplification yielded the huiYKP102-2 fragment, which was 3970 bp in size. The gene fragments of huiYKP102-1 and huiYKP102-2 were used to construct the huiYKP102 plasmid.

[0151] (3) Using primers YKP109-F / back mutation-R and plasmid AB7 as template, PCR amplification yielded the huiYKP109-1 fragment, which was 2851 bp in size. Using primers back mutation-F / YKP109-R and plasmid AB7 as template, PCR amplification yielded the huiYKP109-2 fragment, which was 3843 bp in size. The gene fragments of huiYKP109-1 and huiYKP109-2 were used to construct the huiYKP109 plasmid.

[0152] (4) Using YKP1010-1-F / YKP1010-2-R as primers and plasmid AB7 as template, PCR amplification was performed to obtain the fragment huiYKP1010-1, with a fragment size of 500 bp. Using YKP106-2-F / back mutation-R as primers and plasmid AB7 as template, PCR amplification was performed to obtain the fragment huiYKP1010-2, with a fragment size of 2238 bp. Using back mutation-F / YKP106-1-R as primers and plasmid AB7 as template, PCR amplification was performed to obtain the fragment huiYKP1010-3, with a fragment size of 3987 bp. The gene fragments of huiYKP1010-1, huiYKP1010-2, and huiYKP1010-3 will be used to construct the huiYKP1010 plasmid.

[0153] Of course, the CrtZ and CrtW gene sequences can also be directly synthesized according to the base sequences at positions 901-1626 in SEQ ID NO. 5 and SEQ ID NO. 4 in the sequence listing, and then the above-mentioned plasmid huiYKP101 can be directly constructed according to the construction method of the AB7 plasmid in step 2.2.1.

[0154] Alternatively, the CrtZ and CrtW gene sequences can be directly synthesized according to SEQ ID NO. 7 and SEQ ID NO. 9 in the sequence listing, and then the above-mentioned plasmid huiYKP1010 can be directly constructed according to the construction method of the AB7 plasmid in step 2.2.1.

[0155] Figure 12 Maps of plasmids huiYKP101, huiYKP102, huiYKP109, and huiYKP1010.

[0156] Table 6 Primers required for reverse mutation plasmid construction

[0157]

[0158] Subsequently, the obtained plasmid was transformed into the Saccharomyces cerevisiae strain YZ03 using the lithium acetate (LiAc) transformation method, and fermentation was carried out according to the method in step 1.4, and astaxanthin was extracted according to the method in step 1.3. We observed the color change of the strain (such as Figure 13 ) Combined with HPLC detection, the performance, effect and stability of the reverse mutation in yeast were further evaluated. Figure 14As shown in the figure, YKP101 and YKP1010 showed stable characteristics after reverse mutation, with astaxanthin production being effective and continuously expressed respectively; while strains YKP102 and YKP109 failed to maintain the stability of the forward mutation after reverse mutation, failing to maintain the expected mutation effect.

[0159] Figure 13 Verify colony color change for Saccharomyces cerevisiae.

[0160] Figure 14 To verify the stability of the forward mutant strain using reverse mutation.

[0161] YKP101 and YKP1010 still showed stable characteristics after reverse mutation, with astaxanthin production of 0.23 mg / L / OD 600 , 0.22mg / L / OD 600 , the astaxanthin production increased by 15% and 10% respectively compared to the control strain YAB7. The corresponding sequences of CrtZ and CrtW are as follows (the bold and underlined parts are the mutation sites):

[0162] The nucleic acid sequence of the mutated CrtZ in strain YKP101 is (SEQ ID NO.5 in the sequence listing):

[0163]

[0164] The amino acid sequence of the mutated CrtZ in strain YKP101 is (SEQ ID NO.6 in the sequence listing):

[0165]

[0166] The underlined part is the mutation site.

[0167] The nucleic acid sequence of CrtW in strain YKP101 is bases 901 to 1626 in SEQ ID NO. 4 of the sequence listing.

[0168] The amino acid sequence of CrtW in strain YKP101 is identical to that of wild-type CrtW, and is the sequence shown in SEQ ID NO.2.

[0169] The nucleic acid sequence of the mutated CrtZ in strain YKP1010 is (SEQ ID NO.7 in the sequence listing):

[0170]

[0171] The amino acid sequence of the mutated CrtZ in strain YKP1010 is (SEQ ID NO. 8 in the sequence listing):

[0172] MTNFLIVVATVLVMELTAYSVHRWIMHGPLGWGWHKSHHEEHDHALEKNDLYGL VFAVIATVLFTVGWIWAPVLWWIALGMTVYGLIYFVLHDGLVHQRWPFRYIPRKGYARR LYQAHRLHHAVEGRDHCVSFGFFIYAPPVDKLKQDLKMSGVLRAEAQ G RT.

[0173] The nucleic acid sequence of the mutated CrtW in strain YKP1010 is (SEQ ID NO.9 in the sequence listing):

[0174]

[0175] The amino acid sequence of the mutated CrtW in strain YKP1010 is (SEQ ID NO.10 in the sequence listing):

[0176] 3 Conclusion

[0177] The present invention uses error-prone PCR technology to mutate the CrtZ and CrtW genes, and finally obtains forward mutants of CrtZ and CrtW. The specific operation is as follows:

[0178] (1) First, we need to determine the appropriate amount of enzyme in the reaction. We set up different concentration gradients of rTaq enzyme for the reaction. Based on the experimental results, we chose 1uL as the amount of rTaq enzyme in error-prone PCR. Next, we adjusted Mn 2+ The concentration of Mn was used to evaluate its effect on the mutation rate of CrtZ and CrtW genes. 2+ as conditions for CrtZ and CrtW mutations.

[0179] (2) In order to screen suitable CrtZ and CrtW mutants, we evaluated four different tandem methods. The results showed that different cis-acting elements and changes in gene order had a significant effect on astaxanthin production. Under the conditions of using IGG6 cis-acting elements in tandem, changes in the gene order of CrtZ and CrtW led to significant differences in astaxanthin production, with a maximum difference of 2.5 times. Under the conditions of using P2A cis-acting elements in tandem, changes in gene order resulted in a difference of about 1.9 times in astaxanthin production. Based on the HPLC results, we finally chose the IGG6 cis-acting element tandem method with CrtZ in front and CrtW in the back (i.e., AB7 plasmid expression method) as the strategy for screening CrtZ and CrtW mutants.

[0180] (3) Finally, based on the overall experimental cycle, we performed HPLC testing on the dark-colored clones obtained by error-prone PCR, and divided the screening process into four rounds of primary screening and two rounds of secondary screening according to the experimental progress. Ultimately, we obtained four positive mutants and constructed Saccharomyces cerevisiae strains YKP101, YKP102, YKP109, and YKP1010. Further verification by reverse mutation confirmed that the mutation sites corresponding to YKP101 and YKP1010 were indeed positive mutation points, and these mutations could effectively increase astaxanthin production.

[0181] Table 7 Plasmids used in Escherichia coli constructed by the present invention

[0182]

[0183]

[0184] Table 8 Primers used in the present invention

[0185]

[0186] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A recombinant plasmid, characterized in that: It includes the following gene fragments: promoter TEF1p, β-carotene hydroxylase gene CrtZ, cis-acting element IGG6, β-carotene ketolase gene CrtW, and terminator CYC1t are spliced ​​in sequence; The β-carotene hydroxylase gene crtZ is derived from Agrobacterium aurantiacum; The β-carotene ketolase gene crtW is derived from Brevundimonas sp. SD212; The nucleic acid sequence of the cis-acting element IGG6 is: CAATCAAAC.

2. The recombinant plasmid according to claim 1, characterized in that The nucleic acid sequence of the gene fragment is SEQ ID NO.

4.

3. The recombinant plasmid according to claim 1, characterized in that The amino acid sequence of crtZ is SEQ ID NO.6, and the amino acid sequence of crtW is SEQ ID NO.2; or The amino acid sequence of crtZ is SEQ ID NO.8, and the amino acid sequence of crtW is SEQ ID NO.10; Preferably, the gene sequence of crtZ is SEQ ID NO.5, and the gene sequence of crtW is bases 901-1626 in SEQ ID NO.4; Preferably, the gene sequence of crtZ is SEQ ID NO.7, and the gene sequence of crtW is SEQ ID NO.

9.

4. The recombinant plasmid according to any one of claims 1 to 3, characterized in that The recombinant plasmid is constructed based on plasmid p416TEF; Preferably, the p416TEF plasmid is digested with XbaI restriction nuclease to obtain a linearized vector fragment, which is then ligated to the gene fragment according to claim 1 or 2.

5. Use of the recombinant plasmid according to any one of claims 1 to 4 in preparing an astaxanthin-producing strain.

6. A recombinant Saccharomyces cerevisiae strain for synthesizing astaxanthin, characterized in that: Obtained by transforming a Saccharomyces cerevisiae strain with the recombinant plasmid according to any one of claims 1 to 4; Preferably, the cerevisiae yeast is cerevisiae yeast YZ03; the cerevisiae yeast YZ03 strain is: CEN.PK113-5D, hoΔ::TADH1-crtYB-PTDH3-PTEF1-crtI-TCYC1; HMG1::TADH1-crtE-PTDH3-PTEF1-tHMG1-TCYC1, and the Saccharomyces cerevisiae CEN.PK113-5D strain is: MATaMAL2-8c SUC2 ura3-52.

7. Use of the recombinant Saccharomyces cerevisiae according to claim 6 in synthesizing astaxanthin.

8. The method for constructing the recombinant plasmid according to any one of claims 1 to 4, characterized in that: The 403-891 bases in SEQ ID NO. 4 were amplified using primers AB7-crt ZF: GCAATCTAATCTAAGTTTTatgactaacttcttgatcgttgttgctactg; AB7-crtZ-R: GTTTGATT Gttaagttctttcttgagcttcagctctcaaaac to obtain the crtZ-AB7 fragment. The 901-1626 bases in SEQ ID NO. 4 were amplified using primers AB7-crtW-F: caagaaagaacttaaCAATCAAACatgtccgctgttactccaatgagtag; AB7-crtW-R: GCCCGGGGGATCCACTAGTTttatgaaaataaagaccaccaaggcttccaag to obtain the crtW-AB7 fragment. The p416TEF plasmid was digested with XbaI restriction nuclease to obtain a linearized vector fragment, and the above fragments were ligated.

9. A method for producing astaxanthin, characterized in that: The following steps are involved: (1) inoculating the recombinant Saccharomyces cerevisiae strain according to claim 6 into a seed culture medium for activation, and then performing expansion culture; (2) The expanded cultured strain is inoculated into a fermentation medium for fermentation culture, and after the culture is completed, the bacterial cells are collected for astaxanthin extraction.

10. A method for producing astaxanthin according to claim 9, characterized in that, In step (1): The recombinant Saccharomyces cerevisiae strain according to claim 6 is inoculated into a seed culture medium and cultured at 25-30° C. and 250 rpm for 16-20 hours; Preferably, the seed culture medium is an SC nutrient-deficient liquid culture medium containing 10-30 g / L glucose or a YPD liquid culture medium containing 10-30 g / L glucose; and / or In step (2): the strain after expansion culture was 600 = 0.2 transferred to fermentation medium, cultured at 25-30°C, 250 rpm for 82-86 hours, and then the bacterial cells were collected to extract astaxanthin; Preferably, the seed culture medium is an SC nutrient-deficient liquid culture medium containing 10-30 g / L glucose or a YPD liquid culture medium containing 10-30 g / L glucose.

Images