Recombinant neurospora crassicola strain for producing astaxanthin as well as construction method and application of recombinant neurospora crassicola strain

Abstract

The invention discloses a recombinant neurospora crassicola strain for producing astaxanthin as well as a construction method and application of the recombinant neurospora crassicola strain. The recombinant neurospora crassa contains key enzymes beta-carotene ketolase and beta-carotene hydroxylase in a heterologous astaxanthin synthetic pathway, and through pathway expression of a targeted subcellular compartment, crosstalk among metabolites is effectively prevented, metabolic flux of a biosynthetic pathway is coordinated, and the activity of the neurospora crassa is improved. The effective contact between the heterologous protein and the substrate in the astaxanthin synthetic route is promoted, the sporulation capability of the heterologous protein is improved, the astaxanthin production and storage capability of the neurospora crassiforme is effectively improved, and the method has relatively high popularization and application values.

Description

Technical Field

[0001] The invention relates to the fields of filamentous fungal biotechnology and synthetic biology, and in particular to a recombinant Neurospora robusta strain for producing astaxanthin, a construction method and an application thereof. Background Art

[0002] Neurospora crassa belongs to the same genus as Neurospora crassa and is usually used as a model strain of eukaryotic microorganisms. It plays an irreplaceable role in basic research fields such as genetics, development and cell biology. In addition, Neurospora crassa has low nutritional requirements, fast mycelial growth, and can produce a large number of conidia during solid-state fermentation. The spores are yellow or light orange and are rich in β-carotene, rhodozyme, neurosporin and other carotenoids (R. Gmoser, J. A. Ferreira, P. R. Lennartsson, M. J. Taherzadeh, Filamentous ascomycetes fungi as a source of natural pigments. Fungal Biology and Biotechnology (2017), 4, 1-4; J. Li, Q. Liu, D. Liu, M. Wu, C. Tian, ​​Advances in metabolic engineering of filamentous fungi. Sheng Wu Gong Cheng Xue Bao (2021), 37, 1637-1658.), compared with the commonly used β-carotene producing strains such as Phycomyces blakesleeanus, Blakesleatrispora and Phaffiarhodozyma (C. Wang et al., Challenges and tackles in metabolic engineering for microbial production of carotenoids. Microb Cell Fact (2019), 18, 1-8.), the carotenoid content of Neurospora robusta in cells is higher, and most of them are β-carotene. Compared with common antioxidants, the carotenoids produced by Neurospora robusta have stronger scavenging ability for three different free radicals, namely DPPH free radical, superoxide free radical and hydroxyl free radical. At the same time, they have stronger free radical scavenging ability than lycopene and β-carotene at the same concentration (R. Gmoser, JA Ferreira, PR Lennartsson, MJ Taherzadeh, Filamentous ascomycetes fungi as a source of natural pigments. Fungal Biology and Biotechnology (2017), 4, 1-4; J. Li, Q. Liu, D. Liu, M. Wu, C.Tian, ​​Advances in metabolic engineering of filamentous fungi. Sheng Wu Gong Cheng Xue Bao (2021), 37, 1637-1658; EBSylvia et al., Metabolic engineering of Neurospora crassa for increasing carotenoids synthesis. African Journal of Biotechnology (2022), 21, 156-166.). As a carotenoid-producing microorganism, Neurospora crassa has been extensively studied at home and abroad. However, there is still a blank in the construction and metabolic modification of Neurospora crassa fermentation to produce astaxanthin, and there is a lot of room for development. .

[0003] At the macroscopic cellular level, the carotenoids in Nervus robustus mainly exist in the form of pigment granules, and are mainly found in the mycelial cell wall and near the spore wall in the spores. The carotenoids contained in the spores (2260μg / g dry spores) are much greater than those in the mycelium (1354μg / g dry mycelium). The carotenoids in the mycelium are synthesized under light induction, while the carotenoids in the spores can be produced when the bacteria grow to a certain stage.

[0004] At the microscopic subcellular organelle level, N. robusta has a rich system of organelles and endomembranes, which divide the cell into numerous compartments (Fungal Genetics Stock Centre). Studies have shown that fat-soluble carotenoids are mostly accumulated in lipid droplets, cell membranes, etc., while the astaxanthin synthesis pathway is a cytoplasmic reaction. It can be seen that the subcellular organelle compartments isolate the enzyme from the substrate, limiting the production of astaxanthin.

[0005] Yongshuo Ma (Y. Ma, J. Li, S. Huang, G. Stephanopoulos, Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowia lipolytica. Metab. Eng. (2021), 68, 152-161) et al. effectively reduced the accumulation of metabolic intermediates such as carotene ketones by co-localizing β-carotene ketolase (BTK3) and β-carotene hydroxylase (CrtRb) from Haematococcus pluvialis to the endoplasmic reticulum, lipid droplets and peroxisomes of Yarrowia lipolytica. When these two enzymes were co-targeted to lipid droplets, endoplasmic reticulum and peroxisomes at the same time, the astaxanthin production was the highest, reaching 858 mg / L (Y.Ma, J.Li, S.Huang, G.Stephanopoulos, Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowialipolytica. Metab. Eng. (2021), 68, 152-161). Summary of the invention

[0006] N. robusta is a strain with a strong ability to produce carotenoids, providing sufficient precursor compound β-carotene for astaxanthin synthesis; on this basis, the present invention successfully constructs a recombinant N. robusta strain that produces astaxanthin by introducing heterologous astaxanthin synthesis pathway key enzymes β-carotene ketolase (NcOptBTK3) (SEQ ID NO.5) and β-carotene hydroxylase (NcOptCrtRb) (SEQ ID NO.6), and targets the pathway expression of subcellular compartments to prevent crosstalk between metabolites and coordinate the metabolic flow of the biosynthetic pathway, thereby improving the production and storage capacity of astaxanthin. At the same time, it will also provide a method reference for synthesizing other high value-added compounds using N. robusta.

[0007] The invention provides a method for synthesizing astaxanthin by Neurospora robusta. The method comprises the following steps: overexpressing a beta-carotene ketolase gene and a beta-carotene hydroxylase gene in a starting strain, that is, encoding a NcOptBTK-L1-NcOptCrtRb fusion protein.

[0008] Preferably, the β-carotene ketolase and β-carotene hydroxylase are derived from Haematococcus pluvialis. Preferably, the amino acid sequences encoded by the β-carotene ketolase gene and the β-carotene hydroxylase gene are as shown in SEQ ID NO.1 and SEQ ID NO.3. Specifically, the nucleotide sequences encoding the β-carotene ketolase gene and the β-carotene hydroxylase in Haematococcus pluvialis are as shown in SEQ ID NO.2 and SEQ ID NO.4, respectively.

[0009] Further preferably, the β-carotene ketolase gene and the β-carotene hydroxylase gene are sequence-optimized according to the codon preference of Neurospora robusta. Specifically, their nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0010] In a specific embodiment, the β-carotene ketolase gene and the β-carotene hydroxylase gene are introduced into the starting bacteria by constructing an expression vector and expressed or integrated in the form of a plasmid. Preferably, the β-carotene ketolase gene and the β-carotene hydroxylase gene are connected in series to form a fusion protein gene, more specifically connected by a connecting peptide 1 coding sequence, wherein the connecting peptide 1 is a short peptide L1 (GGGGSGGGGSGGGGS), and the connecting peptide coding sequence is sequence-optimized according to the codon preference of Neurospora robusta. Specifically, its nucleotide sequence is shown in SEQ ID NO.7.

[0011] Particularly preferably, the β-carotene ketolase gene and the β-carotene hydroxylase gene are expressed by constructing expression elements and localizing them to the endoplasmic reticulum, liposomes and / or cell membranes;

[0012] Since astaxanthin and its precursor compound β-carotene are lipophilic molecules, in order to make the fused heterologous protein more fully contact with the substrate, they were targeted to the key organelles in the carotenoid synthesis pathway, the endoplasmic reticulum (KDEL), liposome (Ole) and cell membrane (CLTC) based on the fusion expression of NcOptBTK-L1-NcOptCrtRb. Among them, the endoplasmic reticulum is an important place for lipid synthesis and processing, and the liposome and cell membrane are the storage places of lipid compounds. To this end, the N-terminal localization signal Ole with liposome characterization, the N-terminal signal sequence of the cell membrane protein CLTC and the C-terminal signal sequence KDEL of the endoplasmic reticulum resident protein were connected to the heterologous fusion protein NcOptBTK-L1-NcOptCrtRb through the short peptide L2 (GGGGSGGPGS, SEQ ID NO.14). In order to observe the expression and localization of the heterologous fusion protein, RFP and / or GFP fluorescent protein were added behind the fusion protein NcOptBTK-L1-NcOptCrtRb, and the astaxanthin synthesis pathways targeting the endoplasmic reticulum (NA-ER-GFP), liposomes (NA-LB-GFP) and cell membrane (NA-MM-GFP) were successfully constructed. Figure 2 ).

[0013] Therefore, preferably, the β-carotene ketolase gene and / or β-carotene hydroxylase gene is localized to the endoplasmic reticulum, liposome and / or cell membrane for expression using the coding sequence of the N-terminal localization signal Ole characterized by liposomes, the coding sequence of the N-terminal localization signal CLTC characterized by cell membrane proteins, or the coding sequence of the C-terminal signal fragment KDEL of the endoplasmic reticulum resident protein;

[0014] Specifically, the amino acid sequences of the N-terminal localization signal Ole characterized by liposomes, the N-terminal localization signal CLTC characterized by cell membrane proteins, or the C-terminal signal fragment KDEL of the endoplasmic reticulum resident protein are shown as SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively; their encoding nucleotide sequences are shown as SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13, respectively.

[0015] More preferably, the N-terminal localization signal Ole characterized by the liposome, the N-terminal localization signal CLTC characterized by the cell membrane protein or the C-terminal signal fragment KDEL of the endoplasmic reticulum resident protein is connected to the β-carotene ketolase gene and / or the β-carotene hydroxylase gene through the connecting peptide 2 coding sequence, and the specific connecting peptide 2 is a short peptide L2 (GGGGSGGPGS). More preferably, the connecting peptide 2 coding sequence is sequence optimized according to the codon preference of Neurospora robusta, and its nucleotide sequence is shown in SEQ ID NO.14;

[0016] Further preferably, the N-terminal localization signal Ole characterized by the liposome is sequence optimized according to the codon preference of Neurospora robusta. Specifically, its nucleotide sequence is shown in SEQ ID NO.15.

[0017] More preferably, the negative regulatory gene dcc-1 for spore development and carotenoid synthesis is further knocked out or weakened in the starting bacteria;

[0018] Preferably, the method is implemented by gene editing;

[0019] More preferably, the β-carotene ketolase gene and the β-carotene hydroxylase gene are integrated into the site of the negative regulatory gene dcc-1.

[0020] Carotenoids in N. crassa are mainly present in spores. In order to further promote the carotenoid production of N. crassa, we improved the spore production ability of N. crassa. The negative regulatory gene dcc-1 (development and carotenogenesis control, NCU00939) of spore development and carotenoid synthesis was used as the target, and dcc-1 was knocked out using the N. crassa gene editing tool CRISPR (patent: A promoter from N. crassa and its application, ZL 2023 1 1027324.X), and the astaxanthin synthesis pathway expression sequences NcOptBTK-L1-NcOptCrtRb with different targeting signals were introduced into the site respectively.

[0021] The present invention also provides a recombinant Neurospora robusta strain for producing astaxanthin obtained by the construction method.

[0022] The present invention further provides use of the recombinant Neurospora robusta strain producing astaxanthin in producing astaxanthin.

[0023] The present invention also provides a method for producing astaxanthin and / or β-carotene, which comprises the steps of culturing the recombinant Neurospora robusta strain producing astaxanthin and producing astaxanthin and / or β-carotene, and optionally, further comprising the step of collecting the produced astaxanthin and / or β-carotene.

[0024] Specifically, the culture medium used is MM solid culture medium, the culture conditions are dark culture at 28-32° C. for 1-5 days, then culture at room temperature, illuminated by white light to produce spores, and the spores are collected to extract astaxanthin and / or β-carotene.

[0025] Specifically, the collected spores are suspended in water to obtain a suspension, and then centrifuged to discard the supernatant. The spore precipitate is resuspended in water and centrifuged again, washed with water and then centrifuged to obtain wet spores, which are frozen at -80°C for half an hour and then placed in a freeze dryer to freeze-dry spore powder for extracting astaxanthin and / or β-carotene.

[0026] In general, the astaxanthin synthesis pathway of recombinant Neurospora robusta is as follows: on the basis of its endogenous carotenoid synthesis, the source of Haematococcus pluvialis is introduced, and the β-carotene ketolase (NcOptBTK) and β-carotene hydroxylase (NcOptCrtRb) optimized according to the codon preference of Neurospora robusta are constructed to construct a complete astaxanthin synthesis pathway ( Figure 1 ).like Figure 1 As shown, glucose is converted into the precursor isopentenyl pyrophosphate (IPP, C5 module) and its isomer dimethyl propylene pyrophosphate (DMAPP) through glycolysis and mevalonate pathways. Subsequently, IPP and DMAPP are converted into geranylgeranyl pyrophosphate (GGPP, C10 module) by GGPP synthase. GGPP is then converted into β-carotene (C40 module) by dehydrogenase, and finally β-carotene is converted into astaxanthin (ASTA, C40 module) by heterologous enzymes β-carotene ketolase (NcOptBTK) and β-carotene hydroxylase (NcOptCrtRb).

[0027] A robust NcOptBacteria of the present invention contains β-carotene ketolase (NcOptBTK) and β-carotene hydroxylase (NcOptCrtRb) derived from Haematococcus pluvialis and optimized by codon preference of robust NcOptBacteria. In addition, key organelles (endoplasmic reticulum, liposome and cell membrane) in the astaxanthin synthesis pathway are targeted respectively, and the negative regulatory gene ddc-1 of the spore production of recombinant robust NcOptBacteria is knocked out to obtain a recombinant robust NcOptBacteria strain producing astaxanthin. At the same time, a strain transformation method that can effectively improve the astaxanthin synthesis ability of robust NcOptBacteria is provided. It will also provide a method reference for synthesizing other high value-added compounds using robust NcOptBacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is the astaxanthin synthesis pathway of Pseudomonas robusta. Red indicates the astaxanthin biosynthesis pathway of heterologously introduced NcOptBTK and NcOptCrtRb; ASTA, astaxanthin; Acyl-CoA, acetyl-CoA; AA-CoA, acetoacetyl-CoA; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MVA, mevalonate; MVAP, phosphomevalonate; MVAPP, 5-diphosphomevalonate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, trans-Farnesyl pyrophosphate. diphosphate); GGPP, geranylgeranyl diphosphate.

[0029] Figure 2 Schematic diagram of the construction of the organelle-targeting plasmid for astaxanthin synthesis in Neurospora robusta. ER, endoplasmic reticulum; LB, lipid body; MM, membrane.

[0030] Figure 3 Molecular identification of astaxanthin-producing strains of Neurospora robusta (A) and organelle-targeted fluorescence localization observation (B). A, molecular identification results of astaxanthin-producing strains of Neurospora robusta. Lanes 1-8 are the identification results of NA-RFP (1,2), NA-ER-GFP (3,4), NA-LB-GFP (5,6) and NA-MM-GFP (7,8), respectively; B, organelle-targeted fluorescence localization observation. ER, endoplasmic reticulum; LB, lipid droplet; MM, membrane.

[0031] Figure 4 The standard curves of astaxanthin (A) and β-carotene (B) are shown.

[0032] Figure 5 The production of carotenoids (A), β-carotene (B) and astaxanthin (C) by the astaxanthin-producing strain of Neurospora robusta.

[0033] Figure 6 The HPLC chromatograms of astaxanthin production by strains NA-RFP, NA-ER-GFP, NA-LB-GFP and NA-MM-GFP show that the present invention successfully constructs astaxanthin production strains of Neurospora robusta. DETAILED DESCRIPTION

[0034] In order to further illustrate the technical means and effects adopted by the present invention, the technical scheme of the present invention is further described below in combination with the preferred embodiments of the present invention. It should be pointed out that the specific implementation methods described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0035] The strains and culture conditions used in the examples are as follows:

[0036] The strain W3 of Neurospora robusta (CGMCC No. 40045) was used as the material. MM solid medium (20 g / L glucose, 1× Vogel's salt, Fungal Genetics Stock Centre) was used for routine culture at 30°C.

[0037] Example 1. Sequence acquisition of β-carotene ketolase (NcOptBTK3) and β-carotene hydroxylase (NcOptCrtRb) in the astaxanthin synthesis pathway

[0038] from Figure 1 From the perspective of biosynthesis, astaxanthin is the terminal product of the carotenoid synthesis metabolic pathway. β-carotene needs to be catalyzed by β-carotene ketolase (BTK) and β-carotene hydroxylase (CrtRb) to be finally converted into astaxanthin. However, according to the order of ketolysis and hydroxylation reactions in the biosynthesis of β-carotene to astaxanthin, several intermediates such as echinenone, canthaxanthin, 3-hydroxyechinenone and adonirubin can be synthesized. There are multiple pathways from β-carotene to the final product astaxanthin ( Figure 1). Studies have shown that β-carotene ketolase BTK from Haematococcus pluvialis has better catalytic activity for β-carotene and echinenone, but poor catalytic activity for hydroxylated β-carotene (T. Lotan, J. Hirschberg, Cloning and expression in Escherichia coli of the gene encoding beta-C-4-oxygenase, that converts beta-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis. FEBS Lett. (1995), 364, 125-128.), so the synthetic pathway of converting β-carotene to canthaxanthin via β-carotene ketolase (BTK) and then converting it to astaxanthin via β-carotene hydroxylase (CrtRb) was selected. In natural evolution, the differences in protein nucleic acid sequences between different species are large, but there is a certain degree of conservation in protein amino acid sequences. Therefore, the amino acid sequences of β-carotene ketolase (BTK3) (SEQ ID NO.1) and β-carotene hydroxylase (CrtRb) (SEQ ID NO.3) from Haematococcus pluvialis were used as references, and sequence optimization was performed according to the codon preference of Neurospora robusta to obtain the optimized sequences β-carotene ketolase (NcOptBTK3) (SEQ ID NO.5) and β-carotene hydroxylase (NcOptCrtRb) (SEQ ID NO.6).

[0039] Example 2: Design and construction of expression vector for astaxanthin production strain

[0040] Based on the endogenous carotenoid synthesis of Neurospora robusta, a complete astaxanthin synthesis pathway was constructed by introducing β-carotene ketolase (NcOptBTK) and β-carotene hydroxylase (NcOptCrtRb) from Haematococcus pluvialis and optimizing them according to the codon preference of Neurospora robusta. Figure 1 ).like Figure 1 As shown, glucose is converted into the precursor isopentenyl pyrophosphate (IPP, C5 module) and its isomer dimethyl propylene pyrophosphate (DMAPP) through glycolysis and mevalonate pathways. Subsequently, IPP and DMAPP are converted into geranylgeranyl pyrophosphate (GGPP, C10 module) by GGPP synthase, and then GGPP is converted into β-carotene (C40 module) by dehydrogenase, and finally β-carotene is converted into astaxanthin (C40 module) by heterologous enzymes β-carotene ketolase (NcOptBTK) and β-carotene hydroxylase (NcOptCrtRb).

[0041] The intermediate canthaxanthin of this pathway accumulated more, while the accumulation of intermediate compounds was significantly reduced when BTK-CrtRb was fused and expressed, indicating that the conversion of canthaxanthin to astaxanthin is a bottleneck in the synthesis pathway due to the substrate limitation around β-carotene hydroxylase (CrtRb). Therefore, we connected NcOptBTK and NcOptCrtRb optimized according to the codon preference of Neurospora robusta (NcOptBTK-L1-NcOptCrtRb) through a short peptide L1 (GGGGSGGGGSGGGGS, SEQ ID NO.7) to shorten the distance between β-carotene hydroxylase and its substrate ( Figure 2 ), increasing the conversion rate of canthaxanthin to astaxanthin.

[0042] Since β-carotene is a lipophilic molecule, in order to make the fused heterologous protein contact the substrate more fully, we then targeted the key organelles in the carotenoid synthesis pathway, the endoplasmic reticulum (KDEL), liposomes (NcOptOle, SEQ ID NO.15) and cell membranes (CLTC, SEQID NO.12), based on the fusion expression of NcOptBTK-L1-NcOptCrtRb. Among them, the endoplasmic reticulum is an important place for lipid synthesis and processing, and liposomes and cell membranes are storage places for lipid compounds. To this end, we used the N-terminal localization signal Ole characterized by liposomes, the N-terminal signal sequence of the CLTC protein characterized by the cell membrane, and the C-terminal signal sequence KDEL of the endoplasmic reticulum resident protein, and connected them to the heterologous fusion protein NcOptBTK-L1-NcOptCrtRb through the short peptide L2 (GGGGSGGPGS, SEQ ID NO.14). In order to observe the expression and localization of heterologous fusion proteins, RFP and / or GFP fluorescent proteins were added to the rear of the fusion protein NcOptBTK-L1-NcOptCrtRb. Figure 2 ).

[0043] Carotenoids in N. crassa are mainly present in spores. In order to further promote the carotenoid production of N. crassa, we improved the spore production ability of N. crassa. The negative regulatory gene dcc-1 (development and carotenogenesis control, NCU00939) of spore development and carotenoid synthesis was used as the target, and the N. crassa gene editing tool CRISPR was used to knock it out (patent: A promoter derived from N. crassa and its application, ZL 2023 1 1027324.X), and the heterologous protein expression sequence NcOptBTK-L1-NcOptCrtRb was introduced into this site.

[0044] The above sequence was amplified by PCR and then connected to the linearized pAN52-1N expression vector using the Gibson Assembly technology system to construct the astaxanthin expression cassette plasmid pAN52-Ncef1-NcOptBTK-L1-NcOptCrtRb-GFP-L2-KDEL / Ole / CL TC-neo ( Figure 2 ). The PCR primer sequences required for constructing the expression vector are shown in Table 1.

[0045] The PCR reaction system was: 25 μL of 2×Vazyme buffer, 1 μL of 10 mM dNTPs, 2 μL of upstream / downstream primers (10 mM), 1 μL of template DNA, 1 μL of Vazyme DNA polymerase, and 19 μL of ddH2O.

[0046] The PCR reaction conditions were as follows: first 95°C for 3 min; then 95°C for 15 s, 57°C for 15 s, and 72°C for 1 min, for 34 cycles; and finally 72°C for 5 min and 4°C for 10 min.

[0047] The Gibson Assembly reaction system is: 5 μL of 2×Gibson Mix solution and 5 μL of PCR fragment mixed solution.

[0048] The Gibson Assembly reaction conditions are: 50°C for 30-60 min.

[0049] Example 3: Fluorescence localization observation of astaxanthin synthesis strain of Neurospora robusta

[0050] All strains were verified by PCR and fluorescence expression ( Figure 3 ). Using the extracted genomic DNA as a template, the transformants were verified by PCR using primers seq-dcc1up-5F and neo-R1 ( Figure 3 The sizes of the PCR bands of the successfully constructed NA-RFP, NA-ER-GFP, NA-LB-GFP and NA-MM-GFP strains were ∼9.8 kb ( Figure 3 Lane A, 1, 2), ~9.8kb ( Figure 3 Lane A, 3, 4), ~10.4 kb ( Figure 3 Lane A, 5, 6) and ~13 kb ( Figure 3 Lanes 7 and 8 in A), and no bands in the wild-type strain ( Figure 3 A). Fluorescence localization observation was performed on the spores of the strains verified by PCR, and it was found that all of them expressed fluorescence, among which the fluorescence of the endoplasmic reticulum-targeted strain NA-ER-GFP was the strongest ( Figure 3 (B).

[0051] The PCR amplification system, amplification conditions and assembly conditions were consistent with those described in Example 2.

[0052] Table 1 PCR amplification primers used in the present invention

[0053]

[0054]

[0055] Example 4: Transformation of Neurospora robusta

[0056] Collect conidia of the recipient bacteria for about 10 days and make a spore suspension with 50 mL of pre-cooled sterile 1M sorbitol (filter sterilized). Filter the spore suspension with sterilized lens paper, centrifuge at 2500 rpm for 5 minutes at 4°C to remove the supernatant. Resuspend the conidia with 20 mL of pre-cooled 1M sorbitol, centrifuge at 2500 rpm for 5 minutes at 4°C to remove the supernatant. Repeat this step once. Adjust the conidia concentration to 2.5×10 9 / mL. Take 40μL of spore suspension to a pre-cooled 1.5mL centrifuge tube, add 2-5μL of linearized plasmid (1-2μg), mix gently, and let stand on ice for 5min. Transfer the spore mixture after the transformation fragments are mixed in equal proportions to a 0.2cm electric shock cup (Bio-Rad), wipe the cup wall dry, and shock it instantly at 1.5kV. Immediately add 1mL of pre-cooled 1M sorbitol, blow and mix with a gun tip, and then transfer to a centrifuge tube. In a 15mL centrifuge tube, mix 1mL of spore suspension with 10mL of solid transformation medium (containing 90mg / L G418) that is not solidified at about 50℃, spread it on the transformation plate (containing 90mg / L G418), and culture it in the dark at 28℃ for 4-7 days to see the transformants. Transformants were picked onto MM slants containing 90 mg / L G418, cultured in the dark at 28°C for 2-3 days, and then cultured at room temperature under light to promote sporulation. Genomes can be extracted from spores / hyphae for PCR molecular verification.

[0057] Example 5: Extraction of the genome of Psoralea robusta and verification of transformants

[0058] The genomic DNA was extracted from the transformants selected in the above transformation process using the phenol-chloroform method, which specifically included the following operations:

[0059] The mycelium of the robust nerve spores growing on the plate was picked up and placed in a 2.0 mL sterile DNA extraction tube, zirconium beads and 1 mL of lysis solution (formula: 0.2 M Tris·HCl (pH 7.5), 0.5 M NaCl, 10 mM EDTA, 1% SDS (w / v)), all DNA extraction tubes were placed on a grinder, oscillated for 30 seconds, and repeated twice; 75°C water bath for 20 minutes, vortexed every few minutes during the water bath; after the water bath, the tube was taken out and 80 μL Tris·HCl (1 M, pH 7.5) was added to each tube for neutralization; 400 μL of phenol: chloroform (1:1) was added, vortexed to mix thoroughly, and centrifuged at 13000 rpm for 5 minutes after standing for 5 minutes; 300 μL of supernatant was taken into a new 1.5 mL EP tube, and 600 μL 95% ethanol (DNA grade), precipitate at -20℃ for one hour, then centrifuge at 4℃, 13000rpm, and white DNA precipitate can be seen at the bottom of the EP tube; then use 75% alcohol (DNA grade) 400μL to wash the precipitate, centrifuge at 4℃, 13000rpm, and gently aspirate the supernatant; place the EP tube in a vacuum concentrator to remove residual alcohol; add 50μL ddH2O to dissolve the DNA, measure the DNA concentration with NanoDrop, and store in a -20℃ refrigerator for later use.

[0060] The genomic DNA extracted above was used as a template, and the transformants were verified by gene PCR using primers seq-dcc1up-5F and neo-R1. The PCR amplification products were subjected to 1% agarose gel electrophoresis (130V voltage, 30min). Obvious gene amplification bands were observed under the gel imaging system. The sizes of the PCR bands of the successfully constructed NA-RFP, NA-ER-GFP, NA-LB-GFP and NA-MM-GFP strains were ~9.8kb ( Figure 3 Lane A, 1, 2), ~9.8kb ( Figure 3 Lane A, 3, 4), ~10.4 kb ( Figure 3 Lane A, 5, 6) and ~13 kb ( Figure 3 Lanes 7 and 8 in A, the wild-type strain has no bands, and the expression results of fluorescent proteins traced by RFP / eGFP are shown in Figure 3 As shown in B, this indicates that the recombinant Neurospora robusta astaxanthin-producing strain was successfully constructed.

[0061] Example 6. Phenotypic analysis of transformants and determination of biomass

[0062] The above-verified transformants were inoculated into conventional MM solid medium, cultured in the dark at 30°C for 2-3 days, then cultured at room temperature and illuminated with white light to produce spores. After 7-10 days, spores were collected to extract astaxanthin, β-carotene and other carotenoids.

[0063] The collected spore suspension was centrifuged at 4000 rpm for 5 min. After discarding the supernatant, the spore precipitate was resuspended in water and centrifuged again. It was washed with water twice and the wet spores were obtained after centrifugation. They were frozen at -80°C for half an hour and then placed in a freeze dryer for about 16 hours. The cell dry weight was measured and the results were calculated as spore dry weight.

[0064] Example 7: Extraction and determination of astaxanthin from Neurospora robusta

[0065] 2 mL of acetone and an appropriate amount of zircon beads (0.5 mm in diameter) were added to the spore powder obtained after freeze-drying, and ground with a high-speed tissue grinder (Servicebio) for 2 min, allowed to stand at room temperature for 5 to 10 min, and centrifuged at 4000 rpm for 5 min. The supernatant of carotenoids extracted with acetone was transferred to a new centrifuge tube, and the remaining residue was further extracted with 2 mL of acetone, allowed to stand at room temperature for 5 to 10 min, and centrifuged at 4000 rpm for 5 min to collect the supernatant until the color of the supernatant was colorless, and the collected supernatants were combined to obtain a carotenoid extract. The carotenoid sample extracted with acetone was filtered through a 0.22 μm organic phase filter and then subjected to HPLC detection.

[0066] HPLC detection conditions: DAD detector, Symmetry C18 chromatographic column (250mm×4.6mm, 5μm), column oven controlled at 30°C, mixed gradient flow (mobile phase A and mobile phase B), flow rate of 0.8mL / min. Mobile phase A is a mixed organic solvent (a liquid mixed with acetonitrile, methanol and dichloromethane in a volume ratio of 21:21:8), and mobile phase B is a 10% methanol aqueous solution by volume. The liquid obtained by mixing mobile phase A and mobile phase B is used as the mobile phase for gradient elution, and the conditions are:

[0067] Mobile phase A: 80% to 100% by volume (0-18min), 100% by volume (18-40min), 100% to 80% by volume (40-45min), 80% by volume (45-50min); Mobile phase B: 20% to 0% by volume (0-18min), 0% by volume (18-40min), 0% to 20% by volume (40-45min), 20% by volume (45-50min).

[0068] The injection volume of each sample was 20 μL, the detection time was 50 min, the detection wavelength was 476 nm, the peak time of astaxanthin was 11.8 min, and the peak time of β-carotene was 39.8 min ( Figure 6 ).

[0069] Astaxanthin standard and β-carotene standard were purchased from Aladdin. A certain amount of astaxanthin and β-carotene standard were accurately weighed, dissolved in mobile phase A, and diluted at different times. The peak areas of the standards with different concentrations were determined by HPLC using the above method. The standard curves of astaxanthin and β-carotene were drawn according to the concentration and peak area of ​​the standards. Figure 4 shown.

[0070] Astaxanthin and β-carotene content determination: The peak area of ​​the acetone extract sample of the test bacteria determined by HPLC was substituted into the standard curve to obtain the astaxanthin and β-carotene content of the test bacteria (mg / L, specifically the β-carotene content in the extracted sample).

[0071] Astaxanthin yield (mg / g) = astaxanthin content (mg / L) / cell dry weight (g / L)

[0072] β-carotene yield (mg / g) = β-carotene content (mg / L) / cell dry weight (g / L)

[0073] The absorbance was measured at 461 nm using a UV-visible spectrophotometer, and the carotenoid content was calculated according to the following formula:

[0074] CY=A×D×V1(0.16M)

[0075] CY, carotenoid production (μg / g DCW);

[0076] A, absorbance of the diluted acetone extract at 461 nm;

[0077] D, dilution multiple; V, volume of acetone used for extraction;

[0078] 0.16, carotenoid molar extinction coefficient; M, cell dry weight (g).

[0079] Neurospora robusta has a strong ability to produce carotenoids. On this basis, a complete astaxanthin synthesis pathway was constructed by introducing heterologous β-carotene ketolase (NcOptBTK3) and β-carotene hydroxylase (NcOptCrtRb). After clarifying the expression of these two enzymes, the two enzymes were further regulated by targeting the subcellular compartments of the endoplasmic reticulum (ER), liposomes (LB) and cell membranes (MM). At the same time, the spore production capacity of Neurospora robusta was improved by knocking out the carotenoid negative regulatory gene dcc-1. Compared with the astaxanthin synthesis cytoplasmic pathway (NA-RFP, 6.96μg / gDCW), the astaxanthin production of strains NA-ER-GFP, NA-LB-GFP and NA-MM-GFP targeting subcellular compartments such as the endoplasmic reticulum, liposomes and cell membranes increased, reaching 10.24, 8.19, and 7.22μg / gDCW, respectively. Figure 5 ), the conversion rates of astaxanthin synthesis strains NA-RFP, NA-ER-GFP, NA-LB-GFP and NA-MM-GFP from β-carotene to astaxanthin were 4.66%, 5.56%, 4.89% and 4.59%, respectively ( Figure 5 ). The results show that by targeting the expression of pathways in subcellular compartments, crosstalk between metabolites can be effectively prevented, metabolic flows in the biosynthetic pathway can be coordinated, effective contact between heterologous proteins and substrates in the astaxanthin synthesis pathway can be promoted, and astaxanthin production and storage capacity can be improved. Among them, the endoplasmic reticulum-targeted strain NA-ER-GFP has the highest astaxanthin production. This indicates that the present invention successfully constructs an astaxanthin-producing recombinant Neurospora robusta strain, and organelle targeting during the synthesis process effectively increases astaxanthin production.

Claims

1. A method for constructing a recombinant Neurospora robusta strain for producing astaxanthin, characterized in that: The method comprises the following steps: overexpressing a beta-carotene ketolase gene and a beta-carotene hydroxylase gene in a starting strain.

2. The construction method according to claim 1, characterized in that: The β-carotene ketolase and β-carotene hydroxylase are derived from Haematococcus pluvialis. Preferably, the amino acid sequences encoded by the β-carotene ketolase gene and the β-carotene hydroxylase gene are as shown in SEQ ID NO.1 and SEQ ID NO.

3. Specifically, the nucleotide sequences encoding the β-carotene ketolase gene and the β-carotene hydroxylase in Haematococcus pluvialis are as shown in SEQ ID NO.2 and SEQ ID NO.4, respectively. Further preferably, the β-carotene ketolase gene and the β-carotene hydroxylase gene are sequence-optimized according to the codon preference of Neurospora robusta. Specifically, their nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

3. The construction method according to claim 1 or 2, characterized in that: The β-carotene ketolase gene and the β-carotene hydroxylase gene are introduced into the starting bacteria by constructing an expression vector to be expressed or integrated in the form of a plasmid. Preferably, the β-carotene ketolase gene and the β-carotene hydroxylase gene are connected in series to form a fusion protein gene, more specifically connected by a connecting peptide 1 coding sequence, wherein the connecting peptide 1 is a short peptide L1 (GGGGSGGGGSGGGGS), and the connecting peptide coding sequence is sequence-optimized according to the codon preference of Neurospora robusta. Specifically, its nucleotide sequence is shown in SEQ ID NO.

7.

4. The construction method according to any one of claims 1 to 3, characterized in that: The β-carotene ketolase gene and the β-carotene hydroxylase gene are expressed by localizing to the endoplasmic reticulum, liposome and / or cell membrane through constructing expression elements; Preferably, the β-carotene ketolase gene and / or β-carotene hydroxylase gene is localized to the endoplasmic reticulum, liposome and / or cell membrane for expression using the coding sequence of the N-terminal localization signal Ole characterized by liposomes, the coding sequence of the N-terminal localization signal CLTC characterized by cell membrane proteins, or the coding sequence of the C-terminal signal fragment KDEL of the endoplasmic reticulum resident protein; Specifically, the amino acid sequences of the N-terminal localization signal Ole characterized by liposomes, the N-terminal localization signal CLTC characterized by cell membrane proteins, or the C-terminal signal fragment KDEL of the endoplasmic reticulum resident protein are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively; their encoding nucleotide sequences are shown in SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13, respectively; More preferably, the N-terminal localization signal Ole characterized by the liposome, the N-terminal localization signal CLTC characterized by the cell membrane protein or the C-terminal signal fragment KDEL of the endoplasmic reticulum resident protein is connected to the β-carotene ketolase gene and / or the β-carotene hydroxylase gene through the connecting peptide 2 coding sequence, and the specific connecting peptide 2 is a short peptide L2 (GGGGSGGPGS). More preferably, the connecting peptide 2 coding sequence is sequence optimized according to the codon preference of Neurospora robusta, and its nucleotide sequence is shown in SEQ ID NO.14; Further preferably, the N-terminal localization signal Ole characterized by the liposome is sequence optimized according to the codon preference of Neurospora robusta. Specifically, its nucleotide sequence is shown in SEQ ID NO.

15.

5. The construction method according to any one of claims 1 to 3, characterized in that: Further knock out or weaken the negative regulatory genes of spore development and carotenoid synthesis in the starting bacteria dcc-1 ; Preferably, the method is implemented by gene editing; More preferably, the β-carotene ketolase gene and the β-carotene hydroxylase gene are integrated into the negative regulatory gene dcc-1 The location.

6. A recombinant Neurospora robusta strain producing astaxanthin obtained by the construction method according to any one of claims 1 to 5.

7. Use of the recombinant Neurospora robusta strain producing astaxanthin as claimed in claim 6 in producing astaxanthin.

8. A method for producing astaxanthin and / or β-carotene, characterized in that: The method comprises the steps of culturing the recombinant Neurospora robusta strain for producing astaxanthin as claimed in claim 6 and producing astaxanthin and / or β-carotene, and optionally, also comprises the step of collecting the produced astaxanthin and / or β-carotene.

9. The method according to claim 8, characterized in that The culture medium used is MM solid culture medium, the culture conditions are dark culture at 28-32°C for 1-5 days, then culture at room temperature, illuminated with white light to produce spores, and collect spores to extract astaxanthin and / or β-carotene.

10. The method according to claim 9, characterized in that The collected spores are suspended in water to obtain a suspension, and then centrifuged to discard the supernatant. The spore precipitate is resuspended in water and centrifuged again, washed with water and then centrifuged to obtain wet spores. After freezing at -80°C for half an hour, the spore powder is placed in a freeze dryer and freeze-dried for extracting astaxanthin and / or β-carotene.

Images