Method for producing esterified astaxanthin and application of esterification gene

Abstract

The present disclosure provides a method for producing esterified astaxanthin and the application of esterification genes, belonging to the field of bioengineering technology. The production method comprises: obtaining an esterification gene, and the protein formed by the esterification gene has a homology with the CzDGAT2C protein or the CzDGAT2D protein higher than 90%, the CzDGAT2C protein is formed by the diacylglycerol acyltransferase gene Czdgat2c of Zofingen Chromococcus algae, and the CzDGAT2D protein is formed by the diacylglycerol acyltransferase gene Czdgat2d of Zofingen Chromococcus algae; using the esterification gene, constructing an expression vector of esterified astaxanthin; transforming the expression vector into a strain producing free astaxanthin to obtain esterified astaxanthin. The present disclosure can produce esterified astaxanthin through the production method of esterified astaxanthin.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a method for producing esterified astaxanthin and the application of esterification genes. Background Art

[0002] Astaxanthin is a natural red keto-carotenoid. The natural forms of astaxanthin include free astaxanthin, esterified astaxanthin, glycosylated astaxanthin, etc. Compared with free astaxanthin, esterified astaxanthin has better stability, and there are documents showing that the two have different biological activities. At present, esterified astaxanthin is mainly obtained by fermentation of Haematococcus pluvialis. Haematococcus pluvialis is placed in a culture medium, and the culture medium is placed under light conditions. After accumulating a certain amount of biomass, it is converted to a pressure culture method such as nitrogen deficiency, so that the astaxanthin in the cells of Haematococcus pluvialis accumulates rapidly and most of it accumulates in the form of esterified astaxanthin, and then the esterified astaxanthin is extracted by leaching, filtration, etc. However, when esterified astaxanthin is formed by the above method, the synthesis cycle is long and the efficiency is low. The high fermentation cost of esterified astaxanthin limits its wide application. In addition, although Haematococcus pluvialis is the main source of esterified astaxanthin, the genes responsible for the synthesis of esterified astaxanthin in Haematococcus pluvialis have not been elucidated to date.

[0003] As early as 1994, Chromochloris zofingiensis was found to produce astaxanthin. After that, Liu et al. proposed that Chromochloris zofingiensis has the potential to replace Haematococcus pluvialis. Similar to Haematococcus pluvialis, the astaxanthin of Chromochloris zofingiensis mainly exists in the form of monoesters and diesters, indicating that it contains genes that can efficiently catalyze the conversion of free astaxanthin into esterified astaxanthin. Chromochloris zofingiensis and Haematococcus pluvialis are closely related green algae. Compared with the latter, Chromochloris zofingiensis can not only use light energy and CO2 for photoautotrophic growth, but also use organic carbon sources for growth. Therefore, it can be cultured under heterotrophic conditions. The dry cell weight of Chromochloris zofingiensis during the fermentation process can reach 98.4g L -1 , which is much higher than the weight of stem cells under photoautotrophic conditions. In the latest study, a two-stage heterotrophic culture process was used to achieve an astaxanthin production of 0.318 g L -1 , of which the biomass yield was 235.4 g L -1 , astaxanthin content was 0.144% of dry weight. At the same time, the proportion of total fatty acid content in dry weight increased from 23% to 42%.

[0004] β-Carotene hydroxylase (CrtZ) and β-carotene ketolase (CrtW) are two key proteins for the synthesis of free astaxanthin in Haematococcus pluvialis and Chlorococcus zofingensis, but CrtZ and CrtW from different sources have different substrate preferences. In Haematococcus pluvialis, CrtW starts the catalytic reaction with β-carotene as the substrate, while in Chlorococcus zofingensis, CrtZ starts the catalytic reaction with β-carotene as the substrate. Although both of them eventually accumulate esterified astaxanthin, the gene that catalyzes the synthesis of esterified astaxanthin from free astaxanthin has not been resolved.

[0005] The exploration of esterification genes has been ongoing. In Haematococcus pluvialis, Chen et al. proposed that the diacylglycerol acyltransferase gene (DGAT) may be the key gene for esterification of astaxanthin, and extracted the endoplasmic reticulum region of Haematococcus pluvialis for in vitro enzyme activity analysis, but no accurate results were obtained. DGAT inhibitors cannot completely inhibit the production of astaxanthin esters. In Zofingensis, Liu et al. also studied the esterification genes of Zofingensis, and also found in transcriptome analysis that the expression of DGAT was upregulated when astaxanthin increased. However, after heterologously expressing different DGATs from Zofingensis in Escherichia coli engineered bacteria that can synthesize free astaxanthin, no esterified astaxanthin was detected, so it is believed that other undiscovered acyltransferases may catalyze this process. Summary of the invention

[0006] The disclosed embodiment provides a method for producing esterified astaxanthin and the application of esterification genes, which can assist in producing free astaxanthin-producing engineered strains to obtain esterified astaxanthin. The technical solution is as follows:

[0007] The disclosed embodiment provides a method for producing esterified astaxanthin, the method comprising: obtaining an esterification gene, wherein the protein formed by the esterification gene has a homology with the CzDGAT2C protein or the CzDGAT2D protein higher than 90%, the CzDGAT2C protein is formed by the diacylglycerol acyltransferase gene Czdgat2c of the zofingensis chromochlorococcus algae, and the CzDGAT2D protein is formed by the diacylglycerol acyltransferase gene Czdgat2d of the zofingensis chromochlorococcus algae; using the esterification gene, constructing an expression vector for esterified astaxanthin; and transforming the expression vector into a strain producing free astaxanthin to obtain esterified astaxanthin.

[0008] In another implementation of the present disclosure, the step of obtaining the esterification gene includes:

[0009] Obtain multiple dgat2 genes of the Zofingen Chlorochlorococcum algae; and obtain at least one of the Czdgat2c gene or the Czdgat2d gene from the dgat2 gene as the esterification gene.

[0010] In another implementation of the present disclosure, the esterification gene is used to construct an expression vector for esterified astaxanthin, comprising:

[0011] The esterification gene is codon optimized; the optimized esterification gene is cloned into the pYLXP'2 linear vector to obtain an expression vector, which is a pYLXP'2::Czdgat2c vector or a pYLXP'2::Czdgat2d vector.

[0012] In another implementation of the present disclosure, cloning the optimized esterification gene into the pYLXP'2 linear vector comprises:

[0013] The pYLXP'2 vector is double-digested to obtain a plurality of linearized pYLXP'2 vector fragments, each of which has a sticky end; the plurality of linearized pYLXP'2 vector fragments are separated by gel electrophoresis, and the target vector fragments in the plurality of linearized pYLXP'2 vector fragments are recovered by a DNA gel recovery test kit to obtain pYLXP'2 linearized vector fragments; the pYLXP'2 linearized vector fragments and the optimized esterification gene are connected by ligase, so that the optimized esterification gene is cloned into the pYLXP'2 linearized vector fragment.

[0014] In another implementation of the present disclosure, the expression vector is transformed into an engineered strain that produces free astaxanthin to obtain esterified astaxanthin, comprising:

[0015] The free astaxanthin-producing strain is activated; the expression vector is transferred into the activated free astaxanthin-producing strain for cultivation to obtain a culture solution; and the culture solution is treated to obtain the esterified astaxanthin.

[0016] In another implementation of the present disclosure, the expression vector is transferred into the activated free astaxanthin-producing strain for culturing to obtain a culture solution, comprising:

[0017] The bacterial liquid of the activated free astaxanthin-producing strain is smeared on a culture medium plate for cultivation to obtain a chassis strain; the expression vector is transferred into the chassis strain for cultivation to obtain the culture liquid.

[0018] In another implementation of the present disclosure, the bacterial liquid of the activated free astaxanthin-producing strain is smeared on a culture medium plate for culturing to obtain a chassis strain, comprising:

[0019] The bacterial liquid of the free astaxanthin-producing strain after activation for 24-36 hours is applied to a solid culture medium and cultured at 28-30° C. for 48 hours; the strain with the highest free astaxanthin production content is picked out from the solid culture medium plate as the chassis strain.

[0020] In another implementation of the present disclosure, the processing of the culture solution to obtain the esterified astaxanthin comprises:

[0021] The culture solution is separated to obtain an extract; the extract is separated by thin layer chromatography and detected by liquid chromatography to obtain the esterified astaxanthin.

[0022] In another implementation of the present disclosure, the free astaxanthin-producing strain is one of an engineered strain of Yarrowia lipolytica, an engineered bacterial strain, an engineered yeast strain, and an engineered microalgae strain.

[0023] In another implementation of the present disclosure, an application of an esterification gene is also provided, wherein the esterification gene is used to esterify free astaxanthin, and the protein formed by the esterification gene has a homology with the CzDGAT2C protein or the CzDGAT2D protein greater than 90%, wherein the CzDGAT2C protein is formed by the diacylglycerol acyltransferase gene Czdgat2c of Chromococcus zofingensis, and the CzDGAT2D protein is formed by the diacylglycerol acyltransferase gene Czdgat2d of Chromococcus zofingensis.

[0024] The technical solution provided by the embodiments of the present disclosure brings the following beneficial effects:

[0025] By simply expressing Czdgat2c and / or Czdgat2d genes in strains (engineered strains or wild strains) capable of synthesizing free astaxanthin and synthesizing the corresponding proteins, free astaxanthin can be directed to be catalyzed into esterified astaxanthin. The gene can be used to efficiently prepare esterified astaxanthin and improve the stability of astaxanthin. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 is a flow chart of a method for producing esterified astaxanthin provided in an embodiment of the present disclosure;

[0028] Figure 2 is a flow chart of another method for producing esterified astaxanthin provided in an embodiment of the present disclosure;

[0029] Figure 3 This is a thin layer chromatography separation diagram of the UHpDA*16-pYLXP'2::Czdgat2 transformant provided in the examples of the present disclosure;

[0030] Figure 4 This is the LC / MS analysis chart of the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c recovery provided by the embodiment of the present disclosure;

[0031] Figure 5 This is an analysis diagram of AST-C18:1 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c recovery provided in the embodiments of the present disclosure;

[0032] Figure 6 This is an analysis diagram of AST-C18:2 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c recovery provided by the embodiments of the present disclosure;

[0033] Figure 7 This is an analysis diagram of AST-C18:0 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c recovery provided in the embodiments of the present disclosure;

[0034] Figure 8 This is an analysis diagram of AST-C16:0 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c recovery provided in the embodiments of the present disclosure;

[0035] Fig. 9 This is an analysis diagram of AST-C16:1 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c recovery provided by the embodiments of the present disclosure;

[0036] Fig.10 This is an LC-MS analysis chart of a new TLC band of UHpDA*16-pYLXP'2::Czdgat2d recovered according to an embodiment of the present disclosure;

[0037] Fig.11 This is an analysis diagram of AST-C18:1 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2d recovery provided in the embodiments of the present disclosure;

[0038] Fig.12 This is an analysis diagram of AST-C18:2 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2d recovered provided by the embodiments of the present disclosure;

[0039] Fig.13 This is an analysis diagram of AST-C18:0 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2d recovered provided by the embodiments of the present disclosure;

[0040] Fig.14 This is an analysis diagram of AST-C16:0 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2d recovered provided by the embodiments of the present disclosure;

[0041] Fig.15 This is an analysis diagram of AST-C16:1 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2d recovered provided by the embodiments of the present disclosure;

[0042] Fig.16 is an LC-MS analysis chart of the TLC astaxanthin ester band recovered by UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2d provided in the embodiments of the present disclosure;

[0043] Fig.17 This is an analysis diagram of AST-C18:1 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2d recovery provided in the embodiments of the present disclosure;

[0044] Fig.18 This is an analysis diagram of AST-C18:2 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2d recovered provided by the embodiments of the present disclosure;

[0045] Fig.19 This is an analysis diagram of AST-C18:0 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2d recovered provided by the embodiments of the present disclosure;

[0046] Fig. 20 This is an analysis diagram of AST-C16:0 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2d recovered provided by the embodiments of the present disclosure;

[0047] Fig.21 This is an analysis diagram of AST-C16:1 in the new TLC band of UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2d recovered provided by the embodiments of the present disclosure;

[0048] Fig. 22 is a protein sequence alignment diagram of CzDGAT2c and ScDGAT2a provided in the embodiments of the present disclosure;

[0049] Fig.23 is a protein sequence alignment diagram of CzDGAT2d and ScDGAT2a provided in the embodiments of the present disclosure;

[0050] Fig.24 It is a schematic diagram of the nucleic acid evolution tree of the DGAT protein provided in the embodiments of the present disclosure. DETAILED DESCRIPTION

[0051] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0052] The chemical name of astaxanthin is 3,3'-dihydroxy-4,4'-diketo-β,β'-carotene, which is a terpene unsaturated compound with a molecular formula of C 40 H 52 O4. Astaxanthin is easily oxidized. Astaxanthin is a natural red keto-carotenoid. Only a few types of bacteria, microalgae and a type of plant marigold can synthesize natural astaxanthin from scratch. Although marine animals (such as fish and crustaceans) cannot synthesize astaxanthin from scratch, they can ingest astaxanthin by ingesting zooplankton or phytoplankton, or convert other ingested carotenoids into astaxanthin.

[0053] The present disclosure also provides a method for producing esterified astaxanthin, such as Figure 1 As shown, the manufacturing method includes:

[0054] S101: Obtain an esterification gene, wherein the protein formed by the esterification gene has a homology higher than 90% with the CzDGAT2C protein or the CzDGAT2D protein.

[0055] The CzDGAT2C protein is formed by the diacylglycerol acyltransferase gene Czdgat2c of Chromococcus zofingensis, and the CzDGAT2D protein is formed by the diacylglycerol acyltransferase gene Czdgat2d of Chromococcus zofingensis.

[0056] The esterification gene comes from Chromochloris zofingiensis.

[0057] Zofingensis contains at least 8 DGAT proteins. Although studies have shown that none of these discovered DGAT proteins have the ability to catalyze the synthesis of esterified astaxanthin, the inventors still re-analyzed the functions of these proteins, and the results showed that overexpressing at least one of the Czdgat2c or Czdgat2d genes in the engineered strain UHpDA*16 of Yarrowia lipolytica that synthesizes free astaxanthin can convert part of the free astaxanthin into esterified astaxanthin.

[0058] Mass spectrometry analysis of the product confirmed the production of esterified astaxanthin, and five esterified astaxanthins were detected, namely C16:0, C16:1, C18:0, C18:1, and C18:2. These five esterified astaxanthins have similar fatty acid compositions to those of Yarrowia lipolytica, indicating that the two proteins CzDGAT2C and CzDGAT2D have a wide range of substrate preferences.

[0059] S102: Use the esterification gene to construct an expression vector for esterified astaxanthin.

[0060] An expression vector is a vector that adds expression elements (such as promoters, terminators, etc.) to the basic skeleton of a cloning vector to enable the expression of the target gene.

[0061] In the embodiments disclosed herein, the target gene refers to the esterification gene mentioned above. That is, the expression vector is a cloning vector with the esterification gene added thereto.

[0062] In this embodiment, the cloning vector is a plasmid.

[0063] S103: Transform the expression vector into a strain that produces free astaxanthin to obtain esterified astaxanthin.

[0064] In this embodiment, the strain producing free astaxanthin can be the engineered strain UHpDA*16 of Yarrowia lipolytica. Of course, it can also be other strains producing free astaxanthin, such as wild strains, including bacterial engineered strains, yeast engineered strains, microalgae engineered strains, and the like.

[0065] By simply expressing Czdgat2c and / or Czdgat2d genes in strains (engineered strains or wild strains) capable of synthesizing free astaxanthin and synthesizing the corresponding proteins, free astaxanthin can be directed to be catalyzed into esterified astaxanthin. The gene can be used to efficiently prepare esterified astaxanthin and improve the stability of astaxanthin.

[0066] In other words, the above method can be combined with biosynthesis to obtain esterified astaxanthin from non-natural sources, greatly improving the stability and efficiency of synthetic esterified astaxanthin.

[0067] The disclosed embodiments also provide another method for producing esterified astaxanthin, such as Figure 2 As shown, the manufacturing method includes:

[0068] S201: Obtain an esterification gene, wherein the protein formed by the esterification gene has a homology higher than 90% with the CzDGAT2C protein or the CzDGAT2D protein.

[0069] The CzDGAT2C protein is formed by the diacylglycerol acyltransferase gene Czdgat2c of Chromococcus zofingensis, and the CzDGAT2D protein is formed by the diacylglycerol acyltransferase gene Czdgat2d of Chromococcus zofingensis.

[0070] Step S201 includes:

[0071] 2011: Obtaining multiple DGAT2 genes from Chlorochlorozoanthellate.

[0072] Multiple dgat2 genes of Chlorochlorococcus zofingensis are available from gene banks.

[0073] 2012: From the dgat2 gene, obtain at least one of the Czdgat2c gene or the Czdgat2d gene as an esterification gene.

[0074] Zofingensis contains at least 8 DGAT proteins. Although studies have shown that none of these discovered DGAT proteins have the ability to catalyze the synthesis of esterified astaxanthin, the inventors of the present application have re-analyzed the functions of these proteins. The results show that overexpressing at least one of the Czdgat2c or Czdgat2d genes in the engineered strain UHpDA*16 of Yarrowia lipolytica that synthesizes free astaxanthin can convert part of the free astaxanthin into esterified astaxanthin.

[0075] The mass spectrometry analysis product once again confirmed the production of esterified astaxanthin, and five esterified astaxanthins, namely C16:0, C16:1, C18:0, C18:1, and C18:2, could be detected. These five esterified astaxanthins have similar fatty acid compositions to those of Yarrowia lipolytica, indicating that the two proteins CzDGAT2C and CzDGAT2D have a wide substrate preference. However, overexpression of Czdgat genes from other Zofingen Chlorella sources failed to detect the formation of esterified astaxanthin. Therefore, at least two genes, Czdgat2c and Czdgat2d, in Zofingen Chlorella have the function of catalyzing the synthesis of esterified astaxanthin from free astaxanthin.

[0076] The sequence of the Czdgat2c gene is shown in SEQ ID NO: 1 in the sequence listing, and the sequence of the Czdgat2d gene is shown in SEQ ID NO: 2 in the sequence listing.

[0077] That is, at least one of the Czdgat2c and Czdgat2d genes is cloned into an expression vector, and then the expression vector is transformed into a strain that produces free astaxanthin and cultured, so that esterified astaxanthin can be obtained. That is, the esterification gene can be at least one of Czdgat2c or Czdgat2d.

[0078] That is, the esterification gene is not limited to one of Czdgat2c and Czdgat2d. For example, it can be Czdgat2c gene, Czdgat2d gene, or a mixed gene of Czdgat2c and Czdgat2d. That is, as long as the homology with Czdgat2c gene or Czdgat2d gene is higher than 90%, it can be used.

[0079] S202: Use the esterification gene to construct an expression vector for esterified astaxanthin.

[0080] Step S202 includes:

[0081] 2021: Codon optimization of esterification genes.

[0082] Codons are important bridges for biological information to flow from genes to proteins. Each codon (containing three bases) will eventually be translated into an amino acid (except for the stop codon). Three codons correspond to one amino acid, but one amino acid may correspond to multiple codons.

[0083] According to the knowledge of permutations and combinations, we know that an amino acid sequence of length N has 3 N If a protein has 100 amino acids, there will be 3 100 Therefore, codon optimization is to select the best base sequence from all possible base sequences corresponding to each protein sequence. In other words, codon optimization is to find the codons suitable for expression in the engineered strain of Yarrowia lipolytica from the various codons corresponding to the esterification gene. The codons suitable for the engineered strain of Yarrowia lipolytica can be determined through experiments.

[0084] To put it simply, after the codon of the esterification gene is optimized, the esterification gene can be efficiently transcribed and expressed when it is expressed in the subsequent free astaxanthin-producing strain.

[0085] 2022: Clone the optimized esterification gene into the pYLXP'2 linear vector to obtain an expression vector, which is a pYLXP'2::Czdgat2c vector, a pYLXP'2::Czdgat2d vector or a pYLXP'2::Czdgat2c-Czdgat2d vector.

[0086] Step 2022 includes:

[0087] (1) The pYLXP'2 vector is double-digested to obtain a plurality of linearized pYLXP'2 vector fragments, each of which has a sticky end.

[0088] For example, the pYLXP'2 vector is from the UMBC Laboratory of Chemistry, Biochemistry and Environmental Engineering.

[0089] The pYLXP'2 vector is a circular plasmid vector, and the pYLXP'2 vector includes a promoter and a terminator.

[0090] Double digestion is to cut with two restriction endonucleases, so that the circular plasmid vector is cut into multiple linearized pYLXP'2 vector fragments, each of which has two gaps at the sticky ends. In other words, the circular plasmid vector will become multiple linearized vector fragments. Then the target vector fragment (that is, the required linearized vector fragment) is recovered, because both ends of the target vector fragment have sticky ends complementary to the target gene (that is, the esterification gene) fragment. In this way, under the action of T4 DNA ligase, the target gene can be connected with the sticky ends of the target vector fragment to obtain a circular expression vector cloned with the esterification gene.

[0091] The linearized pYLXP'2 vector fragment is a gene fragment formed after the pYLXP'2 vector is digested with double enzymes.

[0092] In this example, the pYLXP'2 vector was double-digested with KpnI (endonuclease 1)-SnaBI (endonuclease 2).

[0093] The relevant reaction system refers to Table 1 (double enzyme digestion reaction system), wherein the reaction conditions are 37° C. and the reaction time is 60 min.

[0094] Table 1: KpnI and SnaBI double restriction digestion of pYLXP'2 vector

[0095] Components Volume (μL) KqI 5 SnaBI 5 Cut Smart (enzyme digestion buffer) 12 PYLXP'2 80 Double distilled water 18 Total volume 120

[0096] In this embodiment, after the double enzyme digestion reaction is completed, gel electrophoresis can be used to separate DNA fragments of different lengths to determine whether the double enzyme digestion reaction is successful.

[0097] (2) Separating multiple linearized pYLXP'2 vector fragments by gel electrophoresis, and recovering the target vector fragment from the linearized pYLXP'2 vector fragments using a DNA gel recovery test kit to obtain pYLXP'2 linearized vector fragments.

[0098] For example, the gel electrophoresis may be agarose gel electrophoresis, which is a method for separating DNA fragments according to their lengths.

[0099] The DNA gel recovery test kit is a kit used to recover the desired gene fragments from DNA agarose gel.

[0100] That is to say, the target vector fragment cut out from the pYLXP'2 vector can be recovered by using the DNA gel recovery test kit.

[0101] (3) The pYLXP'2 linearized vector fragment and at least one of the optimized esterification genes Czdgat2c gene or Czdgat2d gene are connected by homologous recombinase, so that at least one of the optimized esterification genes Czdgat2c gene or Czdgat2d gene is cloned into the pYLXP'2 linearized vector fragment to obtain pYLXP'2::Czdgat2c, pYLXP'2::Czdgat2d or pYLXP'2::Czdgat2c-Czdgat2d.

[0102] The pYLXP'2 linearized vector fragment and the optimized esterification gene Czdgat2c gene, Czdgat2d gene or a mixed gene of Czdgat2c and Czdgat2d are connected by DNA fragments using homologous recombinase, so that the optimized esterification gene Czdgat2c gene or Czdgat2d gene or a mixed gene of Czdgat2c and Czdgat2d can be cloned into the pYLXP'2 linearized vector fragment.

[0103] The specific DNA ligation system is shown in Table 2, wherein the reaction conditions of the ligation system are 37° C. and the reaction time is 60 min.

[0104] Table 2: Optimized esterification gene cloned into pYLXP'2 linearized vector fragment

[0105] Connection system Volume (μL) pYLXP'2 vector fragment 1 Czdgat2c fragment & Czdgat2d fragment 3 Recombinant enzyme cocktail 5 Double distilled water 1 Total volume 10

[0106] In this embodiment, when obtaining pYLXP'2::Czdgat2c-Czdgat2d, it is necessary to first obtain pYLXP'2::Czdgat2c and pYLXP'2::Czdgat2d. For details, please refer to the following steps:

[0107] First, Czdgat2c and Czdgat2d were co-combined into the pYLXP'2 linear vector to obtain the pYLXP'2::Czdgat2c vector or the pYLXP'2::Czdgat2d vector.

[0108] The above steps can be specifically performed as follows:

[0109] (1) The pYLXP'2::Czdgat2c vector and the pYLXP'2::Czdgat2d vector were double-digested to obtain a Czdgat2c linearized vector fragment and a pYLXP'2::Czdgat2d linearized vector fragment. Both the Czdgat2c linearized vector fragment and the pYLXP'2::Czdgat2d linearized vector fragment had sticky ends.

[0110] In this example, the pYLXP'2::Czdgat2c vector was double-digested with ClaI (endonuclease 1)-NheI (endonuclease 2). The relevant reaction system is shown in Table 3 (double-digestion reaction system), wherein the reaction conditions are 37° C. and the reaction time is 60 min.

[0111] Table 3: ClaI and NheI double restriction digestion of pYLXP'2::Czdgat2c vector

[0112] Components Volume (μL) ClaI 1 NeA 1 Cut Smart (enzyme digestion buffer) 3 pYLXP'2::Czdgat2c 15 Double distilled water 10 Total volume 30

[0113] The pYLXP'2::Czdgat2d vector was double-digested with ClaI (endonuclease 1)-AvrII (endonuclease 2). The relevant reaction system is shown in Table 4 (double-digestion reaction system), wherein the reaction conditions are 37° C. and the reaction time is 60 min.

[0114] Table 4: ClaI and AvrII double restriction digestion of pYLXP'2::Czdgat2d vector

[0115] Components Volume (μL) ClaI 1 AvrII 1 Cut Smart (enzyme digestion buffer) 3 pYLXP'2::Czdgat2d 15 Double distilled water 10 Total volume 30

[0116] In this embodiment, after the double enzyme digestion reaction is completed, gel electrophoresis can be used to separate DNA fragments of different lengths to determine whether the double enzyme digestion reaction is successful.

[0117] (2) The enzyme cleavage products of pYLXP'2::Czdgat2c and pYLXP'2::Czdgat2d were separated by gel electrophoresis, and the target vector fragments in the linearized vector fragments of Czdgat2c and pYLXP'2::Czdgat2d were recovered using a DNA gel recovery kit to obtain the linearized vector fragments of Czdgat2c and pYLXP'2::Czdgat2d.

[0118] Exemplarily, the gel electrophoresis may also be the agarose gel electrophoresis mentioned above.

[0119] (3) The Czdgat2c linearized fragment and the pYLXP'2::Czdgat2d linearized vector fragment were connected by T4 ligase, so that the esterification gene Czdgat2c gene was cloned into the pYLXP'2::Czdgat2d linearized vector fragment to obtain pYLXP'2::Czdgat2c-Czdgat2d.

[0120] Endonuclease AvrII and endonuclease NheI can cut and produce the same sticky ends, and are homotypic enzymes. Under the action of T4 DNA ligase, the sticky ends produced by endonuclease AvrII and endonuclease NheI can be connected.

[0121] The pYLXP'2::Czdgat2d linearized vector fragment and the Czdgat2c linearized fragment were connected by using T4 DNA ligase, so that the Czdgat2c gene could be cloned into the pYLXP'2::Czdgat2d linearized vector fragment to obtain pYLXP'2::Czdgat2c-Czdgat2d.

[0122] The specific DNA ligation system is shown in Table 5, wherein the reaction conditions of the ligation system are 37° C. and the reaction time is 60 min.

[0123] Table 5: Czdgat2c ligated to pYLXP'2::Czdgat2d linearized vector fragment

[0124] Connection system Volume (μL) pYLXP'2::Czdgat2d vector fragment 1 Czdgat2c linearized fragment 3 Ligase 5 Double distilled water 1 Total volume 10

[0125] Of course, when the optimized esterification gene is cloned into a vector to obtain an expression vector, it can also be cloned into other linear vectors, not limited to the pYLXP'2 linear vector mentioned above, that is, the pYLXP'2 linear vector can also be replaced by other vectors, and the present disclosure does not limit this.

[0126] S203: Activating the free astaxanthin-producing strain.

[0127] Since the strains producing free astaxanthin are kept in a refrigerator before use, they need to be thawed to revive and activate them.

[0128] In this example, the strain UHpDA*16 was activated.

[0129] S204: The expression vector is transferred into the activated free astaxanthin-producing strain for culturing to obtain a culture solution.

[0130] Step S204 includes:

[0131] 2041: Spread the bacterial liquid of the activated free astaxanthin-producing strain on a culture medium plate for cultivation to obtain a chassis strain.

[0132] The bacterial liquid of the engineered strain (UHpDA*16) producing free astaxanthin after activation for 24-36 hours is applied to a solid activation culture medium and cultured at 28-30° C. for 48 hours.

[0133] Then, the strain with the highest free astaxanthin content was picked out from the solid activated culture medium plate and used as the chassis strain.

[0134] 2042: Transform the expression vector into the chassis strain for cultivation to obtain culture fluid.

[0135] Take 100-300 ng of pYLXP'2::Czdgat2c vector, pYLXP'2::Czdgat2d vector or pYLXP'2::Czdgat2c-Czdgat2d vector, 90 μL polyethylene glycol 6000, 2 mol / L lithium acetate, 5 μL ssDNA (single-stranded DNA) and a certain amount of chassis strain, mix well and place in a 30°C incubation bath for 30 min, followed by heat shock for 10 min to obtain a bacterial solution.

[0136] Then, the bacterial solution was spread on a uracil-deficient plate and cultured at 28° C. for 2-3 days to obtain a culture solution.

[0137] S205: Treat the culture solution to obtain esterified astaxanthin.

[0138] Step S205 includes:

[0139] 2051: The culture medium is centrifuged, vortexed and extracted to obtain an extract.

[0140] In this example, 30 mL of culture solution was centrifuged at 4000 rpm for 10 minutes to collect the bacteria, 5 mL of ethyl acetate and 2-3 mL of 2 mm glass beads were added, and the mixture was extracted on a multifunctional vortex mixer at 2500 rpm for 50 min.

[0141] After a short centrifugation, the upper oil phase was collected to obtain the extract.

[0142] 2052: The extract is separated by thin layer chromatography (TLC) and detected by high performance liquid chromatography (HPLC) to obtain esterified astaxanthin and identify it.

[0143] In this embodiment, TLC separation conditions: the extract obtained above is concentrated with nitrogen and re-dissolved with 10 μL of chloroform and then spotted onto a thin layer chromatography plate, and separated in a carrier (ether: n-hexane = 2:3, v / v), and the esterified astaxanthin band is recovered and re-dissolved in chloroform.

[0144] After one run, a new carrier (ether:n-hexane=2:3, v / v) was used to perform a second separation.

[0145] HPLC detection conditions: The chromatographic column is a YMC Carotenoid C 30 (4.6 mm×250 mm, 5 μm) reverse phase liquid chromatography column.

[0146] The mobile phase was AB solution for gradient elution, 0-90min, 0-100% B solution, 100%-0% A solution. The volume flow rate was 1.0mL / min, the column temperature was 30°C, the injection volume was 10μL, and the detection wavelength was 450nm.

[0147] Mobile phase A: methanol / methyl tert-butyl ether / water = 81 / 15 / 4.

[0148] Mobile phase B: methanol / methyl tert-butyl ether / water = 7 / 90 / 3.

[0149] In this embodiment, after the above treatment, what is actually obtained is total astaxanthin, and the so-called total astaxanthin includes esterified astaxanthin and non-esterified astaxanthin.

[0150] S206: Identify esterified astaxanthin.

[0151] The esterified astaxanthin was identified using a Thermo Fisher U3000 HPLC with DAD detector and Q Exactive Orbitrap mass spectrometer.

[0152] Mass spectrometry conditions: APCI ion source, nebulizer gas 45arb.

[0153] Auxiliary gas 15arb; spray voltage 5000V, ion transfer capillary temperature 320℃; auxiliary gas temperature 300℃; scanning mode: positive ion Full MS / dd-MS2, scanning range m / z; 500-1400, positive and negative ion switching.

[0154] After thin layer chromatography separation, 5 transformants were selected from UHpDA*16-pYLXP'2::Czdgat2c and UHpDA*16-pYLXP'2::Czdgat2d for activation fermentation (the control group was UHpDA*16-pYLXP'2). The results are as follows: Figure 3 ,UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2dNo picture. The top of the silica gel plate is the β-carotene band.

[0155] from Figure 3It can be seen that a new band appeared in the carotenoids extracted from the UHpDA*16-pYLXP'2::Czdgat2c and UHpDA*16-pYLXP'2::Czdgat2d engineering strains, such as Figure 3 As indicated by the arrow in .

[0156] In addition, this band also appeared in UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2d, while there was no corresponding band in the carotenoids extracted from the control group UHpDA*16-pYLXP'2 (not shown in the figure).

[0157] The extra bands were recovered and further identified by LC / MS (Liquid Chromatography-Mass Spectrometry), and it was found that the new bands did contain esterified astaxanthin (see Figure 4-5 ).

[0158] Figure 4-9 is the LC-MS analysis chart of the new band of UHpDA*16-pYLXP'2::Czdgat2c recovery TLC, where Figure 4 This is the mass spectrometry ion peak extraction diagram of esterified astaxanthin. Figure 5-9 Comparison of the isotope peaks of the primary mass spectra of AST-C18:1, AST-C18:2, AST-C18:0, AST-C16:0, and AST-C16:1 with those of the expected substances.

[0159] according to Figure 4-Figure 9 It can be seen that the types of esterified astaxanthin synthesized by the UHpDA*16-pYLXP'2::Czdgat2c engineered strain include AST-C18:1, AST-C18:2, AST-C18:0, AST-C16:0, and AST-C16:1.

[0160] Figure 10-15 This is the LC-MS analysis of the new band of UHpDA*16-pYLXP'2::Czdgat2d recovery TLC, where Fig.10 This is the mass spectrometry ion peak extraction diagram of esterified astaxanthin. Figure 11-15 They are the comparison of the isotope peaks of the primary mass spectra of AST-C18:1, AST-C18:2, AST-C18:0, AST-C16:0, and AST-C16:1 with the expected substances. Figure 10-15 It can be seen that in the engineered strain overexpressing Czdgat2d in UHpDA*16, AST-C18:1, AST-C18:2, AST-C18:0, AST-C16:0, and AST-C16:1 can still be detected.

[0161] Figure 16-21 This is the LC-MS analysis diagram of the new band of UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2d recovered by TLC, where Fig.16 This is the mass spectrometry ion peak extraction diagram of esterified astaxanthin. Figure 17-21 They are the comparison of the isotope peaks of the primary mass spectra of AST-C18:1, AST-C18:2, AST-C18:0, AST-C16:0, and AST-C16:1 with the expected substances. Figure 16-21 It can be seen that in the engineered strain overexpressing Czdgat2c-Czdgat2d in UHpDA*16, AST-C18:1, AST-C18:2, AST-C18:0, AST-C16:0, and AST-C16:1 can still be detected.

[0162] Whether overexpressing Czdgat2c or Czdgat2d, the signal intensity of AST-C18:1 and AST-C18:2 esterified astaxanthin was the highest. C18:1 and C18:2 are also the two fatty acids with the highest content in Yarrowia lipolytica, followed by C16:0, C16:1 and C18:0. Therefore, the fatty acids of Yarrowia lipolytica are transferred to the astaxanthin skeleton to form esterified astaxanthin, indicating that the two genes Czdgat2c and Czdgat2d or the mixture of the two genes have no significant preference for fatty acid substrates.

[0163] In other words, through the above spectrum analysis, it can be shown that the UHpDA*16-pYLXP'2::Czdgat2c, UHpDA*16-pYLXP'2::Czdgat2d and UHpDA*16-pYLXP'2::Czdgat2c-Czdgat2d engineering strains can produce a large amount of esterified astaxanthin, which further illustrates that the above method can quickly and effectively synthesize esterified astaxanthin.

[0164] In addition, the inventors of the present application also compared the two genes Czdgat2c and Czdgat2d with the esterification gene Scdgat2a of Schizochytrium discovered earlier to analyze whether there is a genetic relationship between the Czdgat2c gene or Czdgat2d gene of Chlorochlorozoanthellae and the esterification gene Scdgat2a of Schizochytrium.

[0165] First, the amino acid sequence of the Czdgat2c gene or the Czdgat2d gene is compared with the amino acid sequence of the Scdgat2a gene of Schizochytrium. Fig. 22The displayed results, combined with the results of sequence alignment calculation and analysis, show that CzDGAT2c and ScDGAT2a have only 25.25% homology, with a coverage rate of 87%.

[0166] according to Fig.23 The results of the display, combined with the results of sequence alignment calculation and analysis, show that the homology between CzDGAT2d and ScDGAT2a is only 27.04%, with a coverage rate of 70%. The above results show that the amino acid sequence of the Czdgat2c gene or Czdgat2d gene is quite different from the amino acid sequence similarity of the ScDGAT2a gene of Schizochytrium.

[0167] Next, molecular evolution analysis was performed based on the nucleotide sequences of DGAT from different sources. The nucleic acid evolution tree was obtained, see Fig.24 ,according to Fig.24 It can be seen that CzDGAT2c and CzDGAT2d are far away from the corresponding branches of ScDGAT2a, respectively, indicating that CzDGAT2c and CzDGAT2d are distantly related to ScDGAT2a.

[0168] The disclosed embodiments also provide an application of an esterification gene, wherein the esterification gene is used to esterify free astaxanthin, and the protein formed by the esterification gene has a homology with the CzDGAT2C protein or the CzDGAT2D protein greater than 90%, the CzDGAT2C protein is formed by the diacylglycerol acyltransferase gene Czdgat2c of the zofingensis chromochlorococcus algae, and the CzDGAT2D protein is formed by the diacylglycerol acyltransferase gene Czdgat2c of the zofingensis chromochlorococcus algae.

[0169] In this embodiment, the esterification gene is the Czdgat2c gene or the Czdgat2d gene in Chlorochlorococcus zofingensis.

[0170] The astaxanthin esterification gene Czdgat2c or Czdgat2d provided by the embodiments of the present disclosure can be used to biosynthesize esterified astaxanthin, prevent oxidation of free astaxanthin, and increase the yield of total astaxanthin (including free and esterified astaxanthin) synthesized by the strain.

[0171] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. A method for producing esterified astaxanthin, characterized in that: The manufacturing method comprises: Obtaining multiple dgat2 genes of the Zofingensis chlorophyll; From the dgat2 gene, at least one of the Czdgat2c gene and the Czdgat2d gene is obtained as an esterification gene, and the Czdgat2c gene is used to form a CzDGAT2C protein, and the Czdgat2d gene is used to form a CzDGAT2D protein, the sequence of the Czdgat2c gene is shown in SEQ ID NO: 1 in the sequence listing, and the sequence of the Czdgat2d gene is shown in SEQ ID NO: 2 in the sequence listing; Using the esterification gene, constructing an expression vector for esterified astaxanthin; The expression vector is transformed into a strain producing free astaxanthin to obtain esterified astaxanthin.

2. The manufacturing method according to claim 1, characterized in that: The esterification gene is used to construct an expression vector for esterified astaxanthin, comprising: performing codon optimization on the esterification gene; The optimized esterification gene is cloned into the pYLXP'2 linear vector to obtain an expression vector, which is a pYLXP'2::Czdgat2c vector, a pYLXP'2::Czdgat2d vector or a pYLXP'2::Czdgat2c-Czdgat2d vector.

3. The manufacturing method according to claim 2, characterized in that: The method of cloning the optimized esterification gene into the pYLXP'2 linear vector comprises: Performing double enzyme digestion on the pYLXP'2 vector to obtain a plurality of linearized pYLXP'2 vector fragments, each of which has a sticky end; Separating the plurality of linearized pYLXP'2 vector fragments by gel electrophoresis, and recovering the target vector fragment from the plurality of linearized pYLXP'2 vector fragments using a DNA gel recovery test kit to obtain pYLXP'2 linearized vector fragments; The pYLXP'2 linearized vector fragment and the optimized esterification gene are connected by ligase, so that the optimized esterification gene is cloned into the pYLXP'2 linearized vector fragment.

4. The manufacturing method according to claim 1, characterized in that: The step of transforming the expression vector into a strain producing free astaxanthin to obtain esterified astaxanthin comprises: activating the free astaxanthin-producing strain; The expression vector is transferred into the activated free astaxanthin-producing strain for culturing to obtain a culture solution; The culture solution is treated to obtain the esterified astaxanthin.

5. The manufacturing method according to claim 4, characterized in that: The step of transferring the expression vector into the activated free astaxanthin-producing strain for culturing to obtain a culture solution comprises: Spreading the activated bacterial liquid of the free astaxanthin-producing strain on a culture medium plate for culturing to obtain a base strain; The expression vector is transferred into the chassis strain for culturing to obtain the culture fluid.

6. The manufacturing method according to claim 5, characterized in that: The method comprises applying the bacterial liquid of the activated free astaxanthin-producing strain on a culture medium plate for culturing to obtain a base strain, comprising: Applying the bacterial liquid of the free astaxanthin-producing strain after activation for 24-36 hours to a solid culture medium, and culturing it statically at 28° C. for 48 hours; The strain with the highest free astaxanthin production content was selected from the solid culture medium plate as the chassis strain.

7. The manufacturing method according to claim 4, characterized in that: The step of treating the culture solution to obtain the esterified astaxanthin comprises: separating the culture solution to obtain an extract; The extract is subjected to thin layer chromatography separation and liquid chromatography detection to obtain the esterified astaxanthin.

8. The manufacturing method according to any one of claims 1 to 7, characterized in that: The free astaxanthin-producing strain is one of an engineered strain of Yarrowia lipolytica, an engineered bacterial strain, and an engineered microalgae strain.

9. An application of an esterification gene, characterized in that: The esterification gene is used to esterify free astaxanthin, and the esterification gene is the diacylglycerol acyltransferase gene Czdgat2c or Czdgat2d in Zofingen Chlorella, the Czdgat2c gene is used to form CzDGAT2C protein, and the Czdgat2d gene is used to form CzDGAT2D protein; The sequence of the Czdgat2c gene is shown in SEQ ID NO: 1 in the sequence listing, and the sequence of the Czdgat2d gene is shown in SEQ ID NO: 2 in the sequence listing.

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