A mutant of lycopene cyclase and its application

Abstract

This invention belongs to the field of enzyme mutant technology and discloses a mutant of lycopene cyclase and its application. The mutant is obtained by mutating the glutamic acid at position 321 to lysine, the phenylalanine at position 319 to leucine, and the cysteine ​​at position 323 to alanine from the wild-type lycopene cyclase with the amino acid sequence shown in SEQ ID NO.1, which is derived from Arabidopsis thaliana. The result is a lycopene cyclase mutant with the amino acid sequence shown in SEQ ID NO.2. BTS1, CrtB, and CrtI were integrated into *Saccharomyces cerevisiae* strain BY4741 to construct a chassis strain ZA1 that stably produces lycopene, the precursor of α-carotene. A mutant expression plasmid was constructed, and positive clones were screened. The mutant enzyme showed significantly stronger ε-cyclization ability than β-cyclization ability. Using lycopene as a substrate, the expression vector PET10 was constructed. Genes related to TAG synthesis, including PAH1, DGA1, and Cat2, along with PET10, were constructed into the expression vector and transformed into ZA1. This improved the efficiency of catalytic production of α-carotene when lycopene was used as a substrate.

Description

Technical Field

[0001] This invention belongs to the field of enzyme mutant technology, and relates to a mutant of lycopene cyclase and its application, particularly to a mutant of lycopene cyclase, its encoding gene, amino acid sequence and its application in the production of α-carotene. Background Technology

[0002] Alpha-carotene, an oxygen-free carotenoid, is widely found in nature and possesses physiological activities such as antioxidation, prevention of cardiovascular disease and Alzheimer's disease, immune enhancement, and anti-tumor activity. It has broad application prospects in the food, animal feed, health products, and cosmetics industries. Alpha-carotene is a natural pigment that is orange or red in color, with strong coloring ability and is safe and non-toxic. It can be used to color foods such as candies, ice cream, cream, and seasonings, providing excellent coloring effects and preservation. Adding alpha-carotene to animal feed can not only improve the growth rate of animals and enhance the reproductive capacity of livestock such as cattle, horses, and pigs, but also enhance the skin color of seafood such as salmon and shrimp. Alpha-carotene has strong antioxidant capacity and can effectively scavenge free radicals; therefore, adding alpha-carotene to functional nutritional products can help slow down aging. However, due to the difficulty of synthesis, the current production of alpha-carotene is low and expensive. Therefore, facing the increasing market demand for alpha-carotene, finding an economically feasible method for its production will expand its overall application in food ingredients.

[0003] Microbial fermentation for α-carotene synthesis offers advantages such as high conversion efficiency, low cost, stable production, and environmental friendliness, providing an attractive and sustainable alternative for α-carotene production. The synthesis of α-carotene in *Saccharomyces cerevisiae* requires the participation of lycopene ε- and β-cyclases. These two cyclases catalyze the ε- and β-cyclization of the substrate lycopene, simultaneously producing the byproduct β-carotene. To increase α-carotene yield and reduce byproduct formation, it is necessary to enhance the catalytic activity of lycopene ε-cyclase and weaken the catalytic activity of lycopene β-cyclase. Therefore, the cyclases are designed to balance their activities, promoting the synthesis of α-carotene from lycopene. Arabidopsis thaliana lycopene cyclase (AtLCYE) is a bifunctional cyclase and a key enzyme in the α-carotene production pathway. This enzyme selectively catalyzes the conversion of lycopene to α-carotene. Therefore, how to improve the substrate selectivity of lycopene cyclase to increase the efficiency of α-carotene production and provide an effective way to synthesize α-carotene by microbial fermentation is an urgent technical problem to be solved.

[0004] In addition, lipid droplets (LDs), also known as liposomes, are organelles that store lipids and are widely found in plants, insects, bacteria, and yeast. As dynamic organelles, lipid droplets have their own life cycle, first forming in the endoplasmic reticulum and then maturing in the cytoplasm. The stored lipids can be used as energy and carbon sources through fatty acid oxidation. Lipid droplets are mainly composed of triglycerides (TAGs) and cholesterol esters (SEs), with TAGs being the main component. Overexpression of genes related to TAG synthesis can expand the storage space of lipid droplets, increasing the intracellular accumulation of lipophilic compounds. Acetyl-CoA is the initiating molecule for TAG synthesis and a core metabolite in fatty acid biosynthesis. ACC1 encodes acetyl-CoA carboxylase to form malonyl-CoA, the first key step in the TAG synthesis pathway; PAH1 encodes phosphatase catalyzing the synthesis of diglycerides (DAG) from phosphatidylic acid; and DGA1 encodes acyl-CoA diacylglycerol acyltransferase 1 catalyzing the synthesis of TAG from DAG. Furthermore, carnitine acetyltransferase Cat2 can transfer acetyl-CoA produced in mitochondria to the cytoplasm, participating in TAG synthesis. Currently, fat-soluble carotenoids such as α-carotene, β-carotene, and lycopene are believed to be stored in cell membranes or lipid droplets, but their distribution ratio is still unclear. Bu et al. isolated lipid droplets and cell membrane components in β-carotene-producing Saccharomyces cerevisiae and quantitatively determined β-carotene separately, finding that the β-carotene content in lipid droplets was 85.5%, while that in the cell membrane was only 9.4%, indicating that carotenoids are mainly stored in lipid droplets. Therefore, regulating the composition and size of lipid droplets can effectively improve the storage and production efficiency of carotenoids.

[0005] The limitations of existing LCYE gene technology make it difficult to solve the following problems: Non-specific cyclization by enzymes: Lycopene is a straight-chain molecule with two identical ends. Theoretically, lycopene cyclases could catalyze cyclization at both ends. However, the problem is that the catalysis of the two ends by the cyclase is neither concerted nor directional. It may catalyze the cyclization of one end to form δ-carotene; the other end may also cyclize, but possibly to form a β-ring or an ε-ring. Therefore, when a single lycopene cyclase acts on a lycopene substrate, it usually produces a mixture of intermediates such as β-carotene, α-carotene, uncyclized lycopene, and unilaterally cyclized δ-carotene. Furthermore, enzymes that specifically and efficiently catalyze the formation of the ε-ring are rarer in nature and are generally less efficient than β-cyclization, making it difficult to precisely control the reaction toward α-carotene formation. Summary of the Invention

[0006] To address the aforementioned issues, this invention systematically solves the core problem of the non-specific cyclization of natural enzymes, which prevents the selective synthesis of α-carotene. By developing an LCYE mutant, this invention enhances the enzyme's ε-cyclization ability, enabling efficient and specific accumulation of α-carotene. Furthermore, by co-expressing lipid droplet-related genes, it provides a key tool for the biosynthesis of α-carotene.

[0007] The technical solution of the present invention is as follows: A mutant of lycopene cyclase was obtained by mutating glutamic acid at position 321 to lysine, phenylalanine at position 319 to leucine, and cysteine ​​at position 323 to alanine from wild-type lycopene cyclase with the amino acid sequence shown in SEQ ID NO.1, thus obtaining a lycopene cyclase mutant with the amino acid sequence shown in SEQ ID NO.2.

[0008] A method for constructing a mutant of lycopene cyclase includes the following steps: (1) Construction of lycopene chassis strain ZA1 BTS1, CrtB and CrtI were integrated into the Saccharomyces cerevisiae BY4741 strain to construct a chassis strain ZA1 that can stably produce lycopene, the precursor of α-carotene. (2) Construct mutant expression plasmids; (3) Construct the expression vector surrounding lipid droplet protein PET10; (4) The genes PAH1, DGA1 and Cat2 related to TAG synthesis were constructed into the expression vector, transformed into ZA1, positive clone strains were screened, and lycopene in the fermentation product was detected.

[0009] Furthermore, in step (1), BTS1, CrtB and CrtI represent three gene fragments: PPGK1-BTS1-TPGK1, PTDH3-CrtB-TPRM9, and PTEF1-CrtI-TCPS1-Right-ty4. Before constructing the three gene fragments, the Left-ty4-URA3 gene fragment was constructed first.

[0010] Furthermore, in step (2), the mutant expression plasmids are pYES2-Kan-AtLCYE-E321K, pYES2-Kan-AtLCYE-F319L, or pYES2-Kan-AtLCYE-C321A.

[0011] Furthermore, the method for constructing the expression vector perilipid protein PET10 in step (3) is as follows: using the genomic DNA of Saccharomyces cerevisiae BY4741 as a template, the PET10 gene is cloned using primer PET10-BamH IF / R, pYES2-Kan is digested with BamH I, and the PET10 gene is ligated into the pYES2-Kan vector to obtain the recombinant plasmid pYES2-Kan-PET10.

[0012] Furthermore, in step (4), the TAG synthesis-related genes PAH1, DGA1, and Cat2 were constructed into a recombinant plasmid: pYES2-Kan-DGA1-Cat2-PAH1.

[0013] Application of a lycopene cyclase mutant in the construction of high-yield α-carotene bacteria.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The mutant enzyme has a significantly stronger ε-cyclization ability than β-cyclization ability, and can improve the efficiency of catalytic production of α-carotene when lycopene is used as a substrate.

[0015] (2) The α-carotene production of the mutant E321K was 4.73 times that of the α-carotene production of the unmutated lycopene cyclase.

[0016] (3) Overexpression of TAG synthesis-related genes DGA1, Cat2 and PAH1 can effectively promote the accumulation of lycopene, with a yield 3.52 times that of the original strain. Attached Figure Description

[0017] Figure 1 This is the α-carotene synthesis pathway in Saccharomyces cerevisiae; Figure 2 The images show plate images of lycopene production chassis strains; a is Saccharomyces cerevisiae BY4741; b is ZA1, a lycopene production chassis constructed by integrating BTS1, CrtB, and CrtI; Saccharomyces cerevisiae BY4741 colonies are white, while strain ZA1 colonies show a distinct orange-yellow color after the co-expression of the three genes, indicating that strain ZA1 can synthesize lycopene. Figure 3 This is the LC-MS detection result of α-carotene; Figure 4 The effects of AtLCYE and its mutants on the production of α-carotene and β-carotene are investigated. Detailed Implementation

[0018] Example 1: Step 1: Construction of Lycopene Strains (1) Construction of Left-ty4-URA3 fragment ① Cloning of HOleft and URA3 Cloning and construction of the HOleft-URA3 fragment included the left homologous arm of the integration site and the URA3 selection tag. The selection tag had LoxP sequences at both ends to facilitate subsequent removal of the selection tag. Specific primers were designed, and the entire URA3 selection tag fragment was cloned using primers URA3-F and URA3-R with the laboratory's pESC-LEU plasmid as a template. The entire URA3 selection tag fragment included the URA3 gene promoter, URA3 tag, and URA3 gene terminator. The genome of Saccharomyces cerevisiae BY4741 was extracted, and the left homologous arm of the integration site, Left-ty4, was cloned using primers Left-ty4-F and Left-ty4-R with the BY4741 genome as a template. After PCR amplification, the target fragment was recovered by gel electrophoresis and gel excision.

[0019] ② Fragment HOleft-URA3 recombination and identification The pYES2-Kan-URA3 vector was digested with the restriction enzyme BamHI. After digestion, the vector was run on an agarose gel and the gel was cut to recover the target fragment. The Left-ty4, URA3 and the digested vector gel product were recombined and ligated for transformation. After successful transformation, plasmids were extracted, digested, and verified before sequencing.

[0020] (2) Construction of PPGK1-BTS1-TPGK1 fragment ① Cloning of PPGK1, BTS1 and TPGK1 The cloning and construction of the PPGK1-BTS1-TPGK1 fragment included the promoter PGK1, BTS1, and terminator PGK1. Specific primers were designed, and using the laboratory plasmid as a template, primers PPGK1-F and PPGK1-R were used to clone the promoter PGK1, primers TPGK1-F and TPGK1-R were used to clone the terminator PGK1, and primers BTS1-F and BTS1-R were used to clone BTS1. After PCR amplification, the fragment was recovered by agarose gel electrophoresis and gel excision.

[0021] ② Recombination and identification of fragment PPGK1-BTS1-TPGK1 The pYES2-Kan-URA3 vector was digested with the restriction enzyme Sal I. After digestion, the target fragment was recovered by agarose gel electrophoresis and gel excision. PPGK1, BTS1, TPGK1 and the digested vector gel recovery product were recombined and ligated for transformation. After successful transformation, plasmid was extracted, digested and verified, and then sent for sequencing.

[0022] (3) Construction of PTDH3-CrtB-TPRM9 fragment ① Cloning of PTDH3, CrtB and TPRM9 The cloning and construction of the PTDH3-CrtB-TPRM9 fragment included the promoter TDH3, CrtB, and the terminator PRM9. Specific primers were designed, and using the laboratory plasmid as a template, primers PTDH3-F and PTDH3-R were used to clone the promoter TDH3, primers TPRM9-F and TPRM9-R were used to clone the terminator PRM9, and primers CrtB-F and CrtB-R were used to clone CrtB. After PCR amplification, the fragment was recovered by agarose gel electrophoresis and gel excision.

[0023] ② Recombination and identification of fragment PTDH3-CrtB-TPRM9 The pYES2-Kan-URA3 vector was digested with the restriction enzyme Sal I. After digestion, the target fragment was recovered by agarose gel electrophoresis and gel excision. PTDH3, CrtB, TPRM9 and the digested vector gel recovery product were recombined and ligated for transformation. After successful transformation, plasmid was extracted, digested and verified, and then sent for sequencing.

[0024] (4) Construction of PTEF1-CrtI-TCPS1-Right-ty4 fragment ① Cloning of PTEF1, CrtI, TCPS1 and Right-ty4 The cloning and construction of the fragment PTEF1-CrtI-TCPS1-Right-ty4 included the promoter TEF1, CrtI, terminator CPS1, and the right homologous arm Right-ty4. Specific primers were designed, and genomic DNA of Saccharomyces cerevisiae BY4741 was extracted as a template. The promoter TEF1 was cloned using primers PTEF1-F and PTEF1-R, the terminator CPS1 was cloned using primers TCPS1-F and TCPS1-R, and CrtI was cloned using primers CrtI-F and CrtI-R. Genomic DNA of Saccharomyces cerevisiae BY4741 was extracted as a template, and the right homologous arm Right-ty4, the integration site, was cloned using primers Right-ty4-F and Right-ty4-R. After PCR amplification, the fragment was recovered by agarose gel electrophoresis and gel excision.

[0025] ② Reassembly and identification of fragment PTEF1-CrtI-TCPS1-Right-ty4 The pYES2-Kan-URA3 vector was digested with the restriction enzyme BamHI. After digestion, the target fragment was recovered by agarose gel electrophoresis and gel excision. PTEF1, CrtI, TCPS1, Right-ty4, and the recovered digested vector fragments were recombined and ligated for transformation. After successful transformation, plasmids were extracted, digested, and verified before sequencing.

[0026] Step 2: Construction of AtLCYE and its mutant expression plasmids (1) Construction of pYES2-Kan-AtLCYE plasmid The pYES2-Kan plasmid was digested with the restriction endonuclease BamHI to obtain a linearized vector. The target fragment was then obtained using AtLCYE-F / R primers and constructed into the pYES2-Kan plasmid to obtain the pYES2-Kan-AtLCYE expression plasmid.

[0027] (2) Constructing the pYES2-Kan-AtLCYE-E321K plasmid The pYES2-Kan plasmid was digested with restriction endonuclease BamHI to obtain a linearized vector. The target fragment was then obtained by overlapping PCR using primers AtLCYE-F / E321K-Mut-DO and E321K-Mut-UP / AtLCYE-R. The target fragment was then constructed into the pYES2-Kan plasmid to obtain the pYES2-Kan-AtLCYE-E321K plasmid.

[0028] (3) Constructing the pYES2-Kan-AtLCYE-F319L plasmid The pYES2-Kan plasmid was digested with the restriction endonuclease BamHI to obtain a linearized vector. The target fragment was then obtained by overlapping PCR using primers AtLCYE-F / F319L-Mut-DO and F319L-Mut-UP / AtLCYE-R. The target fragment was then constructed into the pYES2-Kan plasmid to obtain the pYES2-Kan-AtLCYE-F319L plasmid.

[0029] The construction methods for the remaining mutants are the same as those for constructing the pYES2-Kan-AtLCYE-E321K plasmid and the pYES2-Kan-AtLCYE-F319L plasmid.

[0030] 2. Functional verification of mutants The constructed plasmids pYES2-Kan-AtLCYE, pYES2-Kan-AtLCYE-E321A, pYES2-Kan-AtLCYE-E321K, pYES2-Kan-AtLCYE-E321R, pYES2-Kan-AtLCYE-F319L, pYES2-Kan-AtLCYE-C323A, and pYES2-Kan-AtLCYE-S391M were transformed into the chassis strain ZA1, and the strains were screened using defective solid medium. After colony verification, the correct positive clones of engineered strains were selected, and strains producing α-carotene and β-carotene were obtained.

[0031] like Figure 4 As shown, the α-carotene production of mutant strain E321K was 4.73 times that of the non-mutated enzyme, the α-carotene production of mutant strain F319L was 2.56 times that of the non-mutated enzyme, and the α-carotene production of mutant strain C323A was 2.16 times that of the non-mutated enzyme.

[0032] Step 3: Construction of recombinant plasmids for lipid metabolism-related genes and perilipoproteins ① Construction of the pYES2-Kan-DGA1-Cat2-PAH1 recombinant plasmid Using *Saccharomyces cerevisiae* BY4741 genomic DNA as a template, the DGA1 gene was cloned using primers DGA1-BamH IF / R. The pYES2-Kan vector was digested with BamH I, and the DGA1 gene was ligated into the pYES2-Kan vector to obtain pYES2-Kan-DGA1. Using *Saccharomyces cerevisiae* BY4741 genomic DNA as a template, the Cat2 gene was cloned using primers Cat2-Sac IF / R. pYES2-Kan-DGA1 was digested with Sac I to obtain a linearized plasmid. The Cat2 gene was ligated into the YES2-Kan-DGA1 vector to construct the recombinant plasmid pYES2-Kan-DGA1-Cat2. Using the expression vector pYES2-Kan and Saccharomyces cerevisiae BY4741 genomic DNA as templates, the PPGK1, PAH1, and TPGK1 genes were cloned using primers PPGK1-Xba IF / R, PAH1-Xba IF / R, and TPGK1-Xba IF / R, respectively. The pYES2-Kan-DGA1-Cat2 vector was digested with Xba I to obtain a linearized plasmid. The PAH1 gene was ligated into the YES2-Kan-DGA1-Cat2 vector to obtain the recombinant plasmid pYES2-Kan-DGA1-Cat2-PAH1.

[0033] ② Construction of pYES2-Kan-PET10 recombinant plasmid Using the genomic DNA of Saccharomyces cerevisiae BY4741 as a template, the PET10 gene was cloned using primers PET10-BamH IF / R, and pYES2-Kan was digested with BamH I. The PET10 gene was then ligated into the pYES2-Kan vector to obtain the recombinant plasmid pYES2-Kan-PET10.

[0034] Step 4: Shake fermentation of brewing yeast Positive monoclonal strains were selected and cultured in SD-URA medium at 28 ℃ and 180 rpm to prepare fermentation seed culture. The seed culture was then transferred to 100 mL of YPD medium at a 5% inoculum and cultured at 28 ℃ and 180 rpm for 2-3 days.

[0035] Step 5: Extraction and detection of α-carotene and β-carotene (1) Extraction of α-carotene and β-carotene Centrifuge at 12000 rpm for 3 min to enrich 1 mL of fermentation broth. Resuspend the bacterial cells in 1 mL ddH2O, centrifuge at 12000 rpm for 3 min, and repeat once. Resuspend the bacterial cells in 1 mL 3 M HCl, boil in water for 3 min, incubate on ice for 3 min to return to room temperature, and centrifuge at 12000 rpm for 3 min. Resuspend the bacterial cells in 1 mL ddH2O, centrifuge at 12000 rpm for 3 min, and repeat once. Resuspend the bacterial cells in 1 mL acetone, sonicate for 10 min, and centrifuge at 12000 rpm for 5 min until the bacterial cells turn white. The supernatant is the sample to be tested. After filtering the sample through a membrane, perform high-performance liquid chromatography (HPLC) analysis.

[0036] (2) Detection of α-carotene and β-carotene An MD-2015 detector, a Unitary C18 column (4.6 × 250 mm, 5 μm), a mobile phase of acetonitrile:methanol:isopropanol (50:30:20), a flow rate of 1.0 mL / min, an injection volume of 10 µL, a column temperature of 28 ℃, and a detection wavelength of 450 nm were used. Each sample was tested in triplicate. α-carotene and β-carotene standards were dissolved in acetone for quantitative analysis; the content of α-carotene and β-carotene was calculated using peak area.

[0037] like Figure 3 As shown, the fermentation sample was analyzed by LC-MS after membrane filtration to determine the molecular weight of the sample. The precise molecular weight of α-carotene was 536. The mass-to-charge ratio of the main peak detected by LC-MS analysis was 536, which is consistent with the mass-to-charge ratio of α-carotene.

[0038] Original amino acid sequence SEQ ID NO: 1: MECVGARNFAAMAVSTFPSWSCRRKFPVVKRYSYRNIRFGLCSVRASGGGSSGSESCVAVREDFADEEDFVKAGGSEILFVQMQQNKDMDEQSKLVDKLPPISIGDGALDLVVIGCGPAGLALAAESAKLGLKVGLIGPDLPFTNNYGVWEDEFNDLGLQKCIEHVWRETIVYLDDDKPITIGRAYGRVSRRLLHEELLRRCVESGVSYLSSKVDSITEASDGLRLVACDDNNVIPCRLATVASGAASGKLLQYEVGGPRVCVQTAYGVEVEVENSPYDPDQMVFMDYRDYTNEKVRSLEAEYPTFLYAMPMTKSRLFFEETCLASKDVMPFDLLKTKLMLRLDTLGIRILKTYEEEWSYIPVGGSLPNTEQKNLAFGAAASMVHPATGYSVVRSLSEAPKYASVIAEILREETTKQINSNISRQAWDTLWPPERKRQRAFFLFGLALIVQFDTEGIRSFFRTFFRLPKWMWQGFLGSTLTSGDLVLFALYMFVISPNNLRKGLINHLISDPTGATMIKTYLKV。

[0039] Mutant sequence SEQ ID NO.2: .

[0040] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A lycopene cyclase mutant, characterized in that, The glutamic acid at position 321 of wild-type lycopene cyclase, derived from Arabidopsis thaliana and with the amino acid sequence shown in SEQ ID NO. 1, is mutated to lysine, or the phenylalanine at position 319 is mutated to leucine, or the cysteine ​​at position 323 is mutated to alanine.

2. The application of the lycopene cyclase mutant as described in claim 1 in the construction of high-yield α-carotene bacteria.

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