A method and carrier for biosynthesizing astaxanthin

Abstract

This application discloses a method for biosynthesizing astaxanthin, which involves using an engineered bacterium capable of expressing β-carotene ketolase and β-carotene hydroxylase from Haematococcus pluvialis, wherein the β-carotene hydroxylase contains a G135L point mutation. After introducing the point mutation, astaxanthin production increased by 2.8 times.

Description

Technical Field

[0001] The present application belongs to the field of astaxanthin biosynthesis, and specifically relates to a method and carrier for biosynthesizing astaxanthin. Background Art

[0002] Astaxanthin is chemically known as 3,3'-dihydroxy-4,4'-diketo-β,β'-carotene. It is a terpene unsaturated compound and a keto secondary carotenoid. Its molecular formula is C 40 H 52 O4. Astaxanthin is one of the carotenoids with the strongest antioxidant activity in nature. It has been used in food, medicine, health products, cosmetics and feed, and has extremely high economic and application value. Due to the limited sources of natural astaxanthin, artificial synthesis is generally required to obtain a large amount of astaxanthin. However, chemically synthesized astaxanthin is a mixture of three configurations, with poor biological activity and low utilization. At the same time, the process of chemical synthesis of astaxanthin is complicated, and other impurities will inevitably be introduced during the synthesis process, and its biosafety is questioned. Therefore, chemically synthesized astaxanthin is only used as a feed additive in aquaculture and other breeding industries, and is prohibited from being used in food, cosmetics and pharmaceutical applications. Biosynthesized 3S-3S'-astaxanthin can not only be used in the breeding industry, but is also allowed to be used in food, cosmetics and pharmaceutical fields. Therefore, biosynthesized 3S-3S'-astaxanthin has attracted much attention.

[0003] Haematococcus pluvialis can naturally synthesize the highly bioactive 3S-3S'-astaxanthin, a single substance. The resulting astaxanthin is currently the primary source of 3S-3S'-astaxanthin for use in food, cosmetics, and pharmaceuticals. While commercial production has been achieved, long production cycles and high costs hinder the large-scale application of natural astaxanthin. Consequently, a growing number of researchers are identifying key genes for astaxanthin synthesis in natural astaxanthin-producing hosts like Haematococcus pluvialis. Using metabolic engineering approaches, they are modifying model microorganisms with well-established genetic backgrounds, such as Escherichia coli and Saccharomyces cerevisiae, to construct high-yield 3S-3S'-astaxanthin microbial cell factories.

[0004] Yarrowia lipolytica is a GRAS (generally recognized as safe) unconventional oil-producing yeast certified by the US FDA. Its genetic background is well-established, and relatively comprehensive metabolic engineering tools have been developed in recent years. In 2017, researchers first reported the de novo synthesis of astaxanthin using Yarrowia lipolytica. By introducing exogenous lycopene cyclase (CrtYB) and phytoene desaturase (CrtI), an engineered Yarrowia lipolytica strain capable of synthesizing β-carotene was constructed. Furthermore, by optimizing the mevalonate pathway and downregulating the competing pathway for squalene synthesis, the genes encoding β-carotene ketolase (PaCrtW) and β-carotene hydroxylase (PaCrtZ) from Pantoea ananatis were introduced and their copy numbers optimized, resulting in an engineered Yarrowia lipolytica strain producing astaxanthin with a yield of 54.6 mg / L. In 2019, it was reported that the β-carotene ketolase (HpCrtW) and β-carotene hydroxylase (HpCrtZ) from Haematococcus pluvialis were introduced into the β-carotene-producing Yarrowia lipolytica strain and the copy number was adjusted. The astaxanthin production of the constructed astaxanthin engineered bacteria reached 285±19 mg / L after 7 days of fermentation. It was subsequently discovered that the fusion protein of PsCrtW from Paracoccus sp. and HpCrtZ from Haematococcus pluvialis was simultaneously targeted to liposomes, endoplasmic reticulum and peroxisomes. The resulting Yarrowia lipolytica engineered bacteria could synthesize 858 mg / L of astaxanthin through continuous fed-batch fermentation.

[0005] Consistent with previously reported experimental results, we also found that the PsCrtW and HpCrtZ combination produced the highest astaxanthin concentration (mg / L) per unit volume compared to the HpCrtW and HpCrtZ combination. However, the astaxanthin content (mg / g dry cell weight) was lower than that of the HpCrtW and HpCrtZ combination, and the PsCrtW and HpCrtZ combination also produced more intermediates. Therefore, this study selected HpCrtW and HpCrtZ from Haematococcus pluvialis as research objects, conducting studies such as directed evolution, protein scaffold construction, and protein ratio optimization, in order to provide ideas for further improving astaxanthin production in engineered Yarrowia lipolytica strains. Summary of the Invention

[0006] The purpose of this application is to provide a method and carrier for biosynthesizing astaxanthin to increase the yield of biosynthesized astaxanthin.

[0007] In order to solve the above technical problems, this application proposes the following technical solutions:

[0008] In a first aspect, the present application provides a method for biosynthesizing astaxanthin, comprising achieving the biosynthesis of astaxanthin in an engineered bacterium capable of expressing β-carotene ketolase and β-carotene hydroxylase derived from Haematococcus pluvialis, wherein the β-carotene hydroxylase comprises a G135L point mutation; preferably, the engineered bacterium is Yarrowia lipolytica; preferably, the engineered bacterium is capable of synthesizing β-carotene.

[0009] A second aspect of the present application provides a method for biosynthesizing astaxanthin, which comprises achieving the biosynthesis of astaxanthin in an engineered bacterium capable of expressing β-carotene ketolase and β-carotene hydroxylase, wherein the β-carotene ketolase and β-carotene hydroxylase are co-localized through a scaffold protein; preferably, the β-carotene ketolase and β-carotene hydroxylase are derived from Haematococcus pluvialis; preferably, the engineered bacterium is Yarrowia lipolytica; preferably, the engineered bacterium is capable of synthesizing β-carotene.

[0010] In a third aspect, the present application provides a β-carotene hydroxylase comprising a G135L point mutation, which has an amino acid sequence shown in SEQ ID NO.10.

[0011] In a fourth aspect, the present application provides a β-carotene hydroxylase-PDZ ligand fusion protein comprising a G135L point mutation, which has an amino acid sequence shown in SEQ ID NO.12.

[0012] In a fifth aspect, the present application provides a β-carotene ketolase-PDZ fusion protein having an amino acid sequence shown in SEQ ID NO.14.

[0013] The sixth aspect of the present application provides a polynucleotide molecule encoding the β-carotene hydroxylase containing the G135L point mutation of the third aspect of the present application, or the fusion protein of the fourth aspect or the fifth aspect of the present application; preferably, the polynucleotide molecule has a nucleotide sequence shown in SEQ ID NO.9, SEQ ID NO.11 or SEQ ID NO.13.

[0014] The seventh aspect of the present application provides a nucleic acid construct, which includes the polynucleotide molecule of the sixth aspect of the present application.

[0015] In an eighth aspect, the present application provides an engineered bacterium for producing astaxanthin, comprising at least one of the polynucleotide molecule of the sixth aspect of the present application or the nucleic acid construct of the seventh aspect of the present application; preferably, it is capable of expressing the β-carotene hydroxylase comprising the G135L point mutation of the third aspect of the present application, or at least one of the fusion proteins of the fourth aspect or the fifth aspect of the present application; preferably, the engineered bacterium expresses the fusion protein of the fourth aspect and the fifth aspect of the present application; preferably, the engineered bacterium is capable of synthesizing β-carotene; preferably, the engineered bacterium is Yarrowia lipolytica.

[0016] Beneficial effects of the present application: The inventors introduced a point mutation into β-carotene hydroxylase to obtain an engineered bacterium with improved astaxanthin production. After the point mutation was introduced, the astaxanthin production was increased by 2.8 times compared with the wild-type β-carotene hydroxylase. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the protein scaffold linking proteins HpCrtW and HpCrtZ3 at different ratios (n=HpCrtW:HpCrtZ3);

[0018] Figure 2 Comparison of canthaxanthin production by wild-type HpCrtW (W*) and five HpCrtW mutants expressed in the β-carotene-producing Yarrowia lipolytica engineered strain BC;

[0019] Figure 3 Comparison of astaxanthin content after combined expression of wild-type HpCrtW and five HpCrtZ mutants in the β-carotene-producing Yarrowia lipolytica engineered strain BC;

[0020] Figure 4 To compare the astaxanthin production after different ratios of HpCrtW and HpCrtZ were linked using the PDZ-PL protein scaffold and expressed in the β-carotene-producing Yarrowia lipolytica engineered strain BC. DETAILED DESCRIPTION

[0021] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only one embodiment of the present application. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0022] definition

[0023] As used herein, the terms "a," "an," "the," and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0024] As used herein, the terms "about," "substantially," and "similar to" mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which error range may depend in part on how the value is measured or determined or on the limitations of the measurement system.

[0025] In a first aspect, the present application provides a method for biosynthesizing astaxanthin, comprising: utilizing an engineered bacterium capable of expressing β-carotene ketolase and β-carotene hydroxylase derived from Haematococcus pluvialis; wherein the β-carotene hydroxylase comprises a G135L point mutation. The inventors have discovered that this point mutation increases astaxanthin production by 2.8-fold.

[0026] In some embodiments, the β-carotene hydroxylase comprising the G135L point mutation has an amino acid sequence as shown in SEQ ID NO.10.

[0027] In some embodiments, the engineered bacteria are capable of synthesizing β-carotene. These bacteria are commonly used in the art, for example, by introducing lycopene cyclase (CrtYB) and phytoene desaturase (CrtI) into a host bacterium to construct an engineered strain capable of synthesizing β-carotene. This application does not limit this. This application does not limit the type of engineered bacteria; for example, the bacteria may be Escherichia coli, Saccharomyces cerevisiae, or Yarrowia lipolytica. In some embodiments, the engineered bacteria is Yarrowia lipolytica.

[0028] In some embodiments, the method further comprises colocalizing β-carotene ketolase and β-carotene hydroxylase comprising a G135L point mutation using a scaffold protein; it is understood that, in this application, the "scaffold protein" refers to a protein that can interact or bind with multiple proteins to form a complex, thereby localizing the proteins (in the form of a complex) in a specific region (colocalization), which may include protein scaffolds constructed by paired interacting proteins, protein domains, or polypeptide chains, protein scaffolds constructed by self-assembling proteins, or protein scaffolds constructed by chemically modified proteins, etc. Some non-limiting examples of the scaffold protein include scaffold proteins composed of GBD and its ligands, scaffold proteins composed of SH3 and its ligands, scaffold proteins composed of PDZ and its ligands, etc.

[0029] In some embodiments, the scaffold protein includes a mouse PSD95 / DlgA / Zo-1 region (PDZ) and a PDZ ligand (PL); preferably, the mouse PSD95 / DlgA / Zo-1 region (PDZ) has the amino acid sequence shown in SEQ ID NO.6; the PDZ ligand has the amino acid sequence shown in SEQ ID NO.8.

[0030] In some embodiments, the ratio of the β-carotene ketolase linked to the scaffold protein and the β-carotene hydroxylase containing the G135L point mutation is (1-4):1; it is understood that the ratio is the ratio of the number of molecules, for example, it can be 1:1, 2:1, 3:1 or 4:1.

[0031] In some embodiments, the β-carotene ketolase is expressed in fusion with the PSD95 / DlgA / Zo-1 region (PDZ); the β-carotene hydroxylase comprising the G135L point mutation is expressed in fusion with a PDZ ligand; preferably, the β-carotene hydroxylase comprising the G135L point mutation is expressed in fusion with 1-4 molecules of the PDZ ligand; preferably, the β-carotene hydroxylase comprising the G135L point mutation is expressed in fusion with 3 molecules of the PDZ ligand.

[0032] The inventors discovered that the scaffold protein composed of the PDZ and its ligand of the present application can spatially recruit specific proteins (such as the β-carotene ketolase and β-carotene hydroxylase containing the G135L point mutation of the present application) in a designable manner, and flexibly "bundle" the two proteins HpCrtW and HpCrtZ3 (β-carotene hydroxylase containing the G135L point mutation) in different proportions; the design idea is as follows Figure 1 As shown. Using the protein scaffold to connect HpCrtW and HpCrtZ3 can further improve the synergistic catalytic efficiency of HpCrtW and HpCrtZ3. Furthermore, the inventors found that when the ratio of HpCrtW and HpCrtZ3 connected by the scaffold protein was 1:1, 3:1, and 4:1, the astaxanthin production was improved compared to when no scaffold protein was used for connection. In particular, when the ratio of HpCrtW and HpCrtZ3 connected by the scaffold protein was 3:1, the astaxanthin content was the highest, and the astaxanthin content was increased by 3.0 times compared to the control without the protein scaffold.

[0033] In some embodiments, the β-carotene ketolase and the PDZ can be directly linked or linked via a linker (eg, (GGGGS)*3 (SEQ ID NO.42)).

[0034] In some embodiments, the protein expressed by fusion of β-carotene ketolase and PDZ has an amino acid sequence shown in SEQ ID NO.14.

[0035] In some embodiments, the β-carotene hydroxylase is connected to the PDZ ligand molecule via a linker; preferably, the linker is (GGGS)n, where n is an integer of 1-4.

[0036] In some embodiments, the β-carotene hydroxylase comprising the G135L point mutation is sequentially linked to multiple PDZ ligand molecules via a linker; preferably, it is linked to three PDZ ligand molecules.

[0037] In some embodiments, the protein expressed by fusion of the β-carotene hydroxylase containing the G135L point mutation and the PDZ ligand has an amino acid sequence shown in SEQ ID NO.12.

[0038] The second aspect of the present application provides a method for biosynthesizing astaxanthin, which comprises realizing the biosynthesis of astaxanthin in an engineered bacterium capable of expressing β-carotene ketolase and β-carotene hydroxylase, wherein the β-carotene ketolase and β-carotene hydroxylase are co-localized through a scaffold protein.

[0039] In some embodiments, the scaffold protein includes a mouse PSD95 / DlgA / Zo-1 region (PDZ) and a PDZ ligand (PL); preferably, the mouse PSD95 / DlgA / Zo-1 region (PDZ) has the amino acid sequence shown in SEQ ID NO.6; the PDZ ligand has the amino acid sequence shown in SEQ ID NO.8.

[0040] In some embodiments, the β-carotene ketolase is expressed in fusion with the PSD95 / DlgA / Zo-1 region (PDZ); the β-carotene hydroxylase is expressed in fusion with a PDZ ligand; preferably, the β-carotene hydroxylase is expressed in fusion with 1-4 molecules of the PDZ ligand.

[0041] In some embodiments, the β-carotene hydroxylase is connected to the PDZ ligand via a linker; the β-carotene hydroxylase is connected to the PDZ ligand molecule via a linker; preferably, the linker is (GGGS)n, where n is an integer of 1-4.

[0042] In some embodiments, the β-carotene hydroxylase is sequentially linked to multiple PDZ ligand molecules via a linker, preferably three PDZ ligand molecules.

[0043] In the present application, the linker can be understood as a linker sequence, that is, an amino acid or polynucleotide fragment used to connect two proteins and / or nucleic acid molecules. The presence of a linker is usually used to reduce the impact of steric hindrance on the functions of the two molecules it connects.

[0044] In some embodiments, the β-carotene ketolase and β-carotene hydroxylase can be β-carotene ketolase and β-carotene hydroxylase from any source used to prepare astaxanthin, for example, they can be derived from Haematococcus pluvialis, Chromchloris zofingiensis, Paracoccus sp., Pantoea ananatis, Sphingomonas sp., Brevundimonas sp., etc.; preferably, they are derived from Haematococcus pluvialis, which has a high yield of synthesized astaxanthin and produces fewer intermediate metabolites.

[0045] In some embodiments, the β-carotene ketolase and β-carotene hydroxylase may be optimized, for example, by codon optimization, point mutation and other techniques, so as to have higher catalytic activity, stability and the like.

[0046] In some embodiments, the engineered bacteria is capable of synthesizing β-carotene.

[0047] In some embodiments, the engineered bacteria is Yarrowia lipolytica.

[0048] In a third aspect, the present application provides a β-carotene hydroxylase comprising a G135L point mutation, which has an amino acid sequence shown as SEQ ID NO.10.

[0049] In a fourth aspect, the present application provides a β-carotene hydroxylase-PDZ ligand fusion protein comprising a G135L point mutation, which has an amino acid sequence shown in SEQ ID NO.12.

[0050] In a fifth aspect, the present application provides a β-carotene ketolase-PDZ fusion protein having an amino acid sequence shown in SEQ ID NO.14.

[0051] In a sixth aspect, the present application provides a polynucleotide molecule encoding the β-carotene hydroxylase comprising the G135L point mutation according to the third aspect of the present application, or the fusion protein according to the fourth aspect or the fifth aspect of the present application.

[0052] In some embodiments, the polynucleotide molecule encoding the β-carotene hydroxylase comprising the G135L point mutation has a nucleotide sequence shown in SEQ ID NO.9.

[0053] In some embodiments, the polynucleotide molecule encoding the β-carotene hydroxylase-PDZ ligand fusion protein comprising the G135L point mutation has a nucleotide sequence shown in SEQ ID NO.11.

[0054] In some embodiments, the polynucleotide encoding the β-carotene ketolase-PDZ fusion protein has a nucleotide sequence shown in SEQ ID NO.13.

[0055] The seventh aspect of the present application provides a nucleic acid construct, which includes the polynucleotide molecule of the sixth aspect of the present application.

[0056] In an eighth aspect, the present application provides an engineered bacterium for producing astaxanthin, comprising at least one of the polynucleotide molecule of the sixth aspect of the present application or the nucleic acid construct of the seventh aspect of the present application; preferably, it is capable of expressing the β-carotene hydroxylase comprising the G135L point mutation of the third aspect of the present application, or at least one of the fusion proteins of the fourth aspect or the fifth aspect of the present application; preferably, the engineered bacterium expresses the fusion protein of the fourth aspect and the fifth aspect of the present application; preferably, the engineered bacterium is capable of synthesizing β-carotene; preferably, the engineered bacterium is Yarrowia lipolytica.

[0057] In some embodiments, the engineered bacteria is Yarrowia lipolytica with a deposit number of CCTCC M 2022882 or CCTCC M2022883.

[0058] The following specific examples illustrate the method and carrier for biosynthesizing astaxanthin of the present application. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The plasmids involved in the following examples are all plasmids well known to those skilled in the art. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in accordance with the product instructions are used. Reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0059] Example 1 Screening of point mutations

[0060] 1.1 Construction of point mutation vector

[0061] Protein structure prediction was performed for HpCrtW and HpCrtZ (https: / / zhanggroup.org / I-TASSER / ). Based on the protein model with the highest score, the stability changes of HpCrtW and HpCrtZ proteins after various point mutations were predicted on the DeepDDG server (http: / / protein.org.cn / ddg.html). Based on the stability scores of the mutants, the top five mutant proteins were selected, namely HpCrtWD130L 、HpCrtW A137F 、HpCrtW G148L 、HpCrtW K185F 、HpCrtW K185L , and HpCrtZ G183L 、HpCrtZ G123L 、HpCrtZ G135L 、HpCrtZ G135F and HpCrtZ G135I Previous studies have shown that when HpCrtW and HpCrtZ are simultaneously expressed in an engineered β-carotene-producing Yarrowia lipolytica strain BC, HpCrtW preferentially uses β-carotene to synthesize echinenone and canthaxanthin. Echinenone and canthaxanthin are then converted to hydroxyechinenone and phoenicoxanthin, respectively, under the action of HpCrtZ, and finally catalyzed by HpCrtW and HpCrtZ to form astaxanthin (Zhu, H.-Z., et al., 2022).

[0062] According to the prediction results, amino acids that may improve protein stability were selected for point mutation. First, the plasmid pYLXP'2::HpCrtW (Zhu, H.-Z., et al., Production of High Levels of 3S, 3′S-Astaxanthin in Yarrowia lipolytica via Iterative Metabolic Engineering. Journal of Agricultural and Food Chemistry, 2022.70(8): p.2673-2683.) containing the gene sequence encoding the wild-type HpCrtW protein (SEQ ID NO.1) was used as a template. D130L -R1 and HpCrtW D130L-F2 / HpCrtW-R two pairs of primers were used for PCR amplification. The amplified products were then mixed in a 1:1 molar ratio, and the mixture was used as a template to amplify again with primers HpCrtW-F / HpCrtW-R. The amplified products were recovered and constructed into the pYLXP'2 (Lv, YK, et al., Combining 26s rDNA and the Cre-loxP Systemfor Iterative Gene Integration and Efficient Marker Curation in Yarrowialipolytica, ACS synthetic Biology, 2019.8: p.568-576) vector using the Gibson Assembly kit (Thermo Fisher). Finally, CX-F / CX-R was used as a universal sequencing primer. Correct sequencing indicated that the point mutation vector pYLXP'2::HpCrtW was successfully obtained. D130L The process and nomenclature of other point mutations in HpCrtW and HpCrtZ proteins are the same as above.

[0063] Table 1: Primers used in the process of HpCrtW and HpCrtZ protein point mutations:

[0064]

[0065]

[0066] Table 2: PCR amplification system for HpCrtW and HpCrtZ protein point mutation process

[0067]

[0068] Table 3: PCR amplification procedures for point mutations in HpCrtW and HpCrtZ proteins

[0069]

[0070]

[0071] 1.2 HpCrtW point mutation screening

[0072] The above-mentioned vectors containing the correct sequenced mutant proteins were transformed into the Yarrowia lipolytica engineered strain BC (ZHU Hang-zhi, J Set al., Improving the Biosynthesis of β-Carotene in Yarrowia lipolyticaby Introducing anArtificial Isopentenol Utilization Pathway.China Biotechnology,2021.41(4):p.37-46.). Carotenoids such as canthaxanthin, zeaxanthin, and astaxanthin were detected by high performance liquid chromatography.

[0073] The yeast transformation steps are as follows:

[0074] (1) Streak the activated β-carotene-producing Yarrowia lipolytica engineered strain BC on a YPD plate and culture at 28°C for 16-22 h.

[0075] (2) Prepare 100 μL of yeast transformation buffer: 90 μL of sterile 50% PEG 6000, 5 μL of 2 M lithium acetate, and 5 μL of salmon sperm DNA. Preheat the salmon sperm DNA to 100°C for 5 minutes and allow it to cool before adding it to the transformation buffer.

[0076] (3) Use a 200 μL sterile pipette tip to scrape an appropriate amount of "yeast moss" from the overnight YPD plate and transfer it to a 1.5 mL sterile EP tube containing yeast transformation buffer and vortex mix for at least 10 seconds.

[0077] (4) The above-mentioned vector plasmid containing the HpCrtW point mutation (0.2-0.5 μg DNA) was added separately, and each sample was vortexed for 10 seconds to mix thoroughly.

[0078] (5) Incubate the transformation mixture at 30°C for 30-45 minutes, vortex the transformation mixture for 15 seconds every 10 minutes, and continue heat shock at 39°C for an additional 10 minutes to improve transformation efficiency.

[0079] (6) Add 200 μL of sterile deionized water for dilution.

[0080] (7) Inoculate 100 μL of the diluted cells onto yeast selective medium CSM-Ura and culture at 28-30°C for 48 h.

[0081] Carotenoid extraction and detection:

[0082] After fermentation of yeast transformants in shake flasks at 28°C for 48 hours, 10 mL of fermentation broth was collected into a 10 mL EP tube and centrifuged at 4000 rpm for 10 minutes. The supernatant was removed and 2.5 g of 3-4 mm diameter glass beads and 5 mL of ethyl acetate containing 0.02% 3,5-di-tert-4-butylhydroxytoluene (BHT) were added. BHT was used to prevent carotenoid oxidation. Cells were vortexed at 2500 rpm for 30-50 minutes to disrupt the cells and release the carotenoids. The carotenoids were then dissolved in ethyl acetate and centrifuged at 4000 rpm for 5 minutes. After filtration twice through a 0.2 μm hydrophobic filter, the solution was transferred to a brown liquid phase vial with an inner cannula.

[0083] Carotenoids were quantitatively detected by high performance liquid chromatography (HPLC, Agilent Technologies, USA). An Agilent 1200 series LC system was used, equipped with a YMC carotenoid C30 column (250 mm × 4.6 mm id. × 5 μm thickness). The mobile phase and gradient used were as follows. Initially, eluent A (81% methanol, 15% MTBE, 4% water) was used at a flow rate of 1 mL / min for 50-70 min and gradually decreased from 100 to 60%. At the same time, eluent B (7% methanol, 90% MTBE and 3% water) was increased from 0 to 40%. The entire process was completed within 50-70 min, and the absorbance of all carotenoids was detected at 450 nm. Standard curves for β-carotene, astaxanthin, zeaxanthin and zeaxanthin were drawn under the same conditions as above. The actual carotenoid content in the sample was calculated based on the standard curve.

[0084] Among them, the canthaxanthin (Can) content results of the engineered bacteria containing wild-type HpCrtW (amino acid sequence as shown in SEQ ID NO.2) and HpCrtW with different point mutations are as follows: Figure 2 As shown (W* represents wild type, multiple clones were selected from each plate), the results show that compared with the original HpCrtW, the five designed HpCrtW mutation sites all resulted in a decrease in canthaxanthin content, indicating that the mutated HpCrtW activity was weakened or lost. This result also shows that the D130, A137, and G148 sites are very important for HpCrtW activity.

[0085] 1.3 HpCrtZ point mutation screening

[0086] Furthermore, the same yeast transformation method as in 1.2 was used to transform wild-type HpCrtZ (Z*) (nucleotide sequence shown in SEQ ID NO.3, amino acid sequence shown in SEQ ID NO.4) and five mutant HpCrtZs into wild-type HpCrtW and point mutant HpCrtW. K185FThe protein was co-transformed and co-expressed in the Yarrowia lipolytica engineered strain BC, and the production of astaxanthin (Ast) in the engineered strain was detected using the method 1.2. The results were as follows: Figure 3 The results showed that compared with the combination of wild-type HpCrtZ and wild-type HpCrtW (W*Z*), HpCrtZ G135L The combined expression of HpCrtW and wild type HpCrtW (W*Z-G135L) can significantly increase the astaxanthin production in the engineered strain, with the highest increase of about 2.8 times. G135L Named HpCrtZ3, hereinafter referred to as HpCrtZ G135L and HpCrtZ3 can be replaced. G135L Among the strains expressed in combination with wild-type HpCrtW (W*Z-G135L), the strain with the highest astaxanthin production was named pHpWZ3 and was deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) on June 14, 2022, with the accession number CCTCC M 2022882.

[0087] Example 2 Using scaffold protein to increase astaxanthin production

[0088] First, the nucleic acid sequences of HpCrtW (SEQ ID NO.1) and PDZ (SEQ ID NO.5) were connected with a linker sequence (SEQ ID NO.41). The nucleic acid sequence of HpCrtZ3 (SEQ ID NO.9) was connected with 1 to 4 PL nucleic acid sequences (SEQ ID NO.7) using the same linker sequence as above. The nucleic acid sequence connected with 3 PLs is shown in SEQ ID NO.11. Homology arms were designed at both ends of all fragments. Exemplary nucleotide sequences of the upstream and downstream homology arms are shown in SEQ ID NO.43 and SEQ ID NO.44, respectively, to facilitate subsequent connection with the vector pYLXP'2 by homologous recombination. The five designed sequences were sent to Universal Biosystems (Anhui) Co., Ltd. for synthesis. The synthetic fragments were constructed into the pYLXP'2 vector using the Gibson Assembly kit. The obtained vectors were named pYLXP'2::HpCrtW-PDZ, pYLXP'2::HpCrtZ3-PL*1, pYLXP'2::HpCrtZ3-PL*2, pYLXP'2-HpCrtZ3-PL*3, and pYLXP'2-HpCrtZ3-PL*4, respectively. pYLXP'2::HpCrtW and pYLXP'2::HpCrtW-PDZ were linearized with restriction endonucleases ClaI and NheI, and pYLXP'2::HpCrtZ3 (i.e., pYLXP'2::HpCrtZ3 in Example 1) was linearized with restriction endonucleases Avr II and ClaI. G135L ), pYLXP'2::HpCrtZ3-PL*1, pYLXP'2::HpCrtZ3-PL*2, pYLXP'2-HpCrtZ3-PL*3 and pYLXP'2-HpCrtZ3-PL*4. Then, the pYLXP'2 vector and the different fragments obtained above were ligated by T4 ligase to construct the following expression vectors: pYLXP'2::HpCrtW::HpCrtZ3(n0), pYLXP'2::HpCrtW-PDZ::HpCrtZ3(n0(W-PDZ)), pYLXP'2::HpCrtW::HpCrtZ3-PL*1(n0(Z-PL)), p YLXP'2::HpCrtW-PDZ::HpCrtZ3-PL*1(n1), pYLXP'2::HpCrtW-PDZ::HpCrtZ3-PL*2(n2), pYLXP'2::HpCrtW-PDZ::HpCrtZ3-PL*3(n3) and pYLXP'2::HpCrtW-PDZ::HpCrtZ3-PL*4(n4).

[0089] After enzyme digestion verification was correct, the same yeast transformation method as in 1.2 of Example 1 was used to transform the above expression vector into the β-carotene-producing Yarrowia lipolytica engineered strain BC, and the same carotenoid extraction and detection method as in 1.2 of Example 1 was used to detect the astaxanthin production in the obtained strain. The results are as follows: Figure 4 shown.

[0090] The results showed that when the ratio of HpCrtW and HpCrtZ3 connected by the scaffold protein was 1:1 (n1), 3:1 (n3) and 4:1 (n4), the astaxanthin content synthesized by the Yarrowia lipolytica engineered strain was increased, especially when the ratio of HpCrtW and HpCrtZ3 was 3:1, the astaxanthin content was increased by 3.0 times compared with the control without protein scaffold (n0). The strain with a ratio of HpCrtW and HpCrtZ3 of 3:1 obtained in this example was named pHpW PDZ Z3 PL *3, deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) on June 14, 2022, with the accession number CCTCC M 2022883.

[0091] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application. Sequence Listing <110> Wuhan Hesheng Technology Co., Ltd. <120> A method and carrier for biosynthesizing astaxanthin <130> MTI220265 <160> 44 <170> SIPOSequenceListing 1.0 <210> 1 <211> 960 <212> DNA <213> Haematococcus pluvialis <400> 1 cacgtggcct ctgctctgat ggtcgagcag aagggctctg aggccgctgc ctcttctccc 60 gacgtgctgc gagcctgggc tacccagtac cacatgcctt ccgagtcctc tgacgccgct 120 cgacccgctc tgaagcacgc ctacaagcct ccagcctccg acgccaaggg catcaccatg 180 gctctgacca tcatcggaac ctggaccgcc gtgttcctgc acgccatctt ccagattcga 240 ctgcccacct ctatggacca gctgcactgg ctgcccgtgt ctgaggccac cgctcagctg 300 ctcggcggat cttcttctct gctgcacatt gccgccgtct ttatcgtgct cgagttcctg 360 tacaccggcc tgttcatcac cactcacgac gccatgcacg gcaccattgc tctgcgacac 420 cgacagctga acgacctgct gggcaacatc tgcatctccc tgtacgcctg gttcgactac 480 tctatgctgc accgaaagca ctgggagcac cacaaccaca ccggcgaggt cggcaaggac 540 cccgacttcc acaagggcaa ccccggactg gtgccctggt tcgcctcttt catgtcctct 600 tacatgtctc tgtggcagtt cgcccgactg gcctggtggg ccgtcgtcat gcagatgctg 660 ggcgctccca tggccaacct gctggtgttc atggccgctg ctcccatcct gtccgccttc 720 cgactgttct acttcggcac ctacctgcct cacaagcccg agcctggacc tgccgccgga 780 tctcaggtga tggcctggtt ccgagccaag acctctgagg cttctgacgt gatgtctttc 840 ctgacctgct accacttcga cctgcattgg gagcatcaca gatggccctt cgctccctgg 900 tggcagctcc ctcactgccg acgactgtct ggccgaggac tggtccccgc tctggcctaa 960 <210> 2 <211> 319 <212> PRT <213> Haematococcus pluvialis <400> 2 His Val Ala Ser Ala Leu Met Val Glu Gln Lys Gly Ser Glu Ala Ala 1 5 10 15 Ala Ser Ser Pro Asp Val Leu Arg Ala Trp Ala Thr Gln Tyr His Met 20 25 30 Pro Ser Glu Ser Ser Asp Ala Ala Arg Pro Ala Leu Lys His Ala Tyr 35 40 45 Lys Pro Pro Ala Ser Asp Ala Lys Gly Ile Thr Met Ala Leu Thr Ile 50 55 60 Ile Gly Thr Trp Thr Ala Val Phe Leu His Ala Ile Phe Gln Ile Arg 65 70 75 80 Leu Pro Thr Ser Met Asp Gln Leu His Trp Leu Pro Val Ser Glu Ala 85 90 95 Thr Ala Gln Leu Leu Gly Gly Ser Ser Ser Leu Leu His Ile Ala Ala 100 105 110 Val Phe Ile Val Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr 115 120 125 His Asp Ala Met His Gly Thr Ile Ala Leu Arg His Arg Gln Leu Asn 130 135 140 Asp Leu Leu Gly Asn Ile Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr 145 150 155 160 Ser Met Leu His Arg Lys His Trp Glu His His Asn His Thr Gly Glu 165 170 175 Val Gly Lys Asp Pro Asp Phe His Lys Gly Asn Pro Gly Leu Val Pro 180 185 190 Trp Phe Ala Ser Phe Met Ser Ser Tyr Met Ser Leu Trp Gln Phe Ala 195 200 205 Arg Leu Ala Trp Trp Ala Val Val Met Gln Met Leu Gly Ala Pro Met 210 215 220 Ala Asn Leu Leu Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe 225 230 235 240 Arg Leu Phe Tyr Phe Gly Thr Tyr Leu Pro His Lys Pro Glu Pro Gly 245 250 255 Pro Ala Ala Gly Ser Gln Val Met Ala Trp Phe Arg Ala Lys Thr Ser 260 265 270 Glu Ala Ser Asp Val Met Ser Phe Leu Thr Cys Tyr His Phe Asp Leu 275 280 285 His Trp Glu His His Arg Trp Pro Phe Ala Pro Trp Trp Gln Leu Pro 290 295 300 His Cys Arg Arg Leu Ser Gly Arg Gly Leu Val Pro Ala Leu Ala 305 310 315 <210> 3 <211> 879 <212> DNA <213> Haematococcus pluvialis <400> 3 ctgtctaagc tgcagctat ctctgtgaag gcccgacgag tcgagctggc ccgagacatc 60 acccgaccta aggtgtgcct gcacgcccag agatgttctc tggtgcgact gcgagtggct 120 gctccccaga ccgaagaggc cctgggcacc gtgcaggccg ctggcgctgg cgacgagcac 180 tctgccgacg tggccctgca gcagctggac cgagccattg ccgagcgacg agcccgacga 240 aagcgagagc agctgtctta ccaggctgcc gctatcgccg cctctatcgg cgtgtctgga 300 atcgccatct tcgccaccta cctgcgattc gccatgcaca tgaccgtcgg cggagctgtg 360 ccctggggcg aagtggctgg caccctgctg ctggtggtcg gaggcgccct cggcatggaa 420 atgtacgccc gatacgccca caaggccatc tggcacgagt cgcccctcgg ctggctgctg 480 cacaagtctc atcacacccc tcgaaccgga cctttcgagg ccaacgacct gttcgccatc 540 atcaacggac tgcccgccat gctgctgtgt accttcggct tctggctgcc caacgtgctg 600 ggagccgcct gcttcggagc cggcctgggc atcaccctgt acggcatggc ctacatgttc 660 gtccacgacg gcctggtgca ccgacgattc cccaccggac ctatcgctgg actgccctac 720 atgaagcgac tgaccgtggc tcaccagctg caccactctg gcaagtacgg cggagcccct 780 tggggcatgt tcctgggacc tcaagagctg cagcacatcc ccggtgccgc cgaagaggtc 840 gagcgactgg tgctggaact ggactggtct aagcgataa 879 <210> 4 <211> 292 <212> PRT <213> Haematococcus pluvialis <400> 4 Leu Ser Lys Leu Gln Ser Ile Ser Val Lys Ala Arg Arg Val Glu Leu 1 5 10 15 Ala Arg Asp Ile Thr Arg Pro Lys Val Cys Leu His Ala Gln Arg Cys 20 25 30 Ser Leu Val Arg Leu Arg Val Ala Ala Pro Gln Thr Glu Glu Ala Leu 35 40 45 Gly Thr Val Gln Ala Ala Gly Ala Gly Asp Glu His Ser Ala Asp Val 50 55 60 Ala Leu Gln Gln Leu Asp Arg Ala Ile Ala Glu Arg Arg Ala Arg Arg 65 70 75 80 Lys Arg Glu Gln Leu Ser Tyr Gln Ala Ala Ala Ile Ala Ala Ser Ile 85 90 95 Gly Val Ser Gly Ile Ala Ile Phe Ala Thr Tyr Leu Arg Phe Ala Met 100 105 110 His Met Thr Val Gly Gly Ala Val Pro Trp Gly Glu Val Ala Gly Thr 115 120 125 Leu Leu Leu Val Val Gly Gly Ala Leu Gly Met Glu Met Tyr Ala Arg 130 135 140 Tyr Ala His Lys Ala Ile Trp His Glu Ser Pro Leu Gly Trp Leu Leu 145 150 155 160 His Lys Ser His His Thr Pro Arg Thr Gly Pro Phe Glu Ala Asn Asp 165 170 175 Leu Phe Ala Ile Ile Asn Gly Leu Pro Ala Met Leu Leu Cys Thr Phe 180 185 190 Gly Phe Trp Leu Pro Asn Val Leu Gly Ala Ala Cys Phe Gly Ala Gly 195 200 205 Leu Gly Ile Thr Leu Tyr Gly Met Ala Tyr Met Phe Val His Asp Gly 210 215 220 Leu Val His Arg Arg Phe Pro Thr Gly Pro Ile Ala Gly Leu Pro Tyr 225 230 235 240 Met Lys Arg Leu Thr Val Ala His Gln Leu His His Ser Gly Lys Tyr 245 250 255 Gly Gly Ala Pro Trp Gly Met Phe Leu Gly Pro Gln Glu Leu Gln His 260 265 270 Ile Pro Gly Ala Ala Glu Glu Val Glu Arg Leu Val Leu Glu Leu Asp 275 280 285 Trp Ser Lys Arg 290 <210> 5 <211> 285 <212> DNA <213> Mus musculus <400> 5 ctgcagcgac gacgagtgac cgtgcgaaag gccgacgctg gcggcctggg catctctatc 60 aagggcggac gagagaacaa gatgcccatc ctgatctcta agatcttcaa gggcctcgcc 120 gccgaccaga ccgaggctct gttcgtgggc gacgccatcc tgtctgtgaa cggcgaggac 180 ctgtccctctg ctacccacga cgaggccgtg caggccctga agagaccgg caggaagtc 240 gtgctcgagg tcaagtacat gaggaagtg tctcctact tcaag 285 <210> 6 <211> 95 <212> PRT <213> Muscles <400> 6 Leu Gln Arg Arg Arg Val Thr Val Arg Lys Ala Asp Ala Gly Gly Leu 1 5 10 15 Gly Ser Isolation Gly Gly Arg Glu Asn Lys Met Pro Isolation 20 25 30 Ser Lys Ile Phe Lys Gly Leu Ala Ala Asp Gln Thr Glu Ala Leu Phe 35 40 45 Val Gly Asp Ala Ile Leu Ser Val Asn Gly Glu Asp Ala Leu Ser Ser Ala 50 55 60 Thr His Asp Glu Ala Val Gln Ala Leu Lys Lys Thr Gly Lys Glu Val 65 70 75 80 Val Leu Glu Val Lys Tyr Met Lys Glu Val Ser Pro Tyr Phe Lys 85 90 95 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <400> 7 ggcgtgaagg aatctctggt g 21 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <400> 8 Gly Val Lys Glu Ser Leu Val 1 5 <210> 9 <211> 876 <212> DNA <213> Artificial Sequence <400> 9 ctgtctaagc tgcagtctat ctctgtgaag gcccgacgag tcgagctggc ccgagacatc 60 acccgaccta aggtgtgcct gcacgcccag agatgttctc tggtgcgact gcgagtggct 120 gctccccaga ccgaagaggc cctgggcacc gtgcaggccg ctggcgctgg cgacgagcac 180 tctgccgacg tggccctgca gcagctggac cgagccattg ccgagcgacg agcccgacga 240 aagcgagagc agctgtctta ccaggctgcc gctatcgccg cctctatcgg cgtgtctgga 300 atcgccatct tcgccaccta cctgcgattc gccatgcaca tgaccgtcgg cggagctgtg 360 ccctggggcg aagtggctgg caccctgctg ctggtggtcg gactggccct cggcatggaa 420 atgtacgccc gatacgccca caaggccatc tggcacgagt cgcccctcgg ctggctgctg 480 cacaagtctc atcacacccc tcgaaccgga cctttcgagg ccaacgacct gttcgccatc 540 atcaacggac tgcccgccat gctgctgtgt accttcggct tctggctgcc caacgtgctg 600 ggagccgcct gcttcggagc cggcctgggc atcaccctgt acggcatggc ctacatgttc 660 gtccacgacg gcctggtgca ccgacgattc cccaccggac ctatcgctgg actgccctac 720 atgaagcgac tgaccgtggc tcaccagctg caccactctg gcaagtacgg cggagcccct 780 tggggcatgt tcctgggacc tcaagagctg cagcacatcc ccggtgccgc cgaagaggtc 840 gagcgactgg tgctggaact ggactggtct aagcga 876 <210> 10 <211> 292 <212> PRT <213> Artificial Sequence <400> 10 Leu Ser Lys Leu Gln Ser Ile Ser Val Lys Ala Arg Arg Val Glu Leu 1 5 10 15 Ala Arg Asp Ile Thr Arg Pro Lys Val Cys Leu His Ala Gln Arg Cys 20 25 30 Ser Leu Val Arg Leu Arg Val Ala Ala Pro Gln Thr Glu Glu Ala Leu 35 40 45 Gly Thr Val Gln Ala Ala Gly Ala Gly Asp Glu His Ser Ala Asp Val 50 55 60 Ala Leu Gln Gln Leu Asp Arg Ala Ile Ala Glu Arg Arg Ala Arg Arg 65 70 75 80 Lys Arg Glu Gln Leu Ser Tyr Gln Ala Ala Ala Ile Ala Ala Ser Ile 85 90 95 Gly Val Ser Gly Ile Ala Ile Phe Ala Thr Tyr Leu Arg Phe Ala Met 100 105 110 His Met Thr Val Gly Gly Ala Val Pro Trp Gly Glu Val Ala Gly Thr 115 120 125 Leu Leu Leu Val Val Gly Leu Ala Leu Gly Met Glu Met Tyr Ala Arg 130 135 140 Tyr Ala His Lys Ala Ile Trp His Glu Ser Pro Leu Gly Trp Leu Leu 145 150 155 160 His Lys Ser His His Thr Pro Arg Thr Gly Pro Phe Glu Ala Asn Asp 165 170 175 Leu Phe Ala Ile Ile Asn Gly Leu Pro Ala Met Leu Leu Cys Thr Phe 180 185 190 Gly Phe Trp Leu Pro Asn Val Leu Gly Ala Ala Cys Phe Gly Ala Gly 195 200 205 Leu Gly Ile Thr Leu Tyr Gly Met Ala Tyr Met Phe Val His Asp Gly 210 215 220 Leu Val His Arg Arg Phe Pro Thr Gly Pro Ile Ala Gly Leu Pro Tyr 225 230 235 240 Met Lys Arg Leu Thr Val Ala His Gln Leu His His Ser Gly Lys Tyr 245 250 255 Gly Gly Ala Pro Trp Gly Met Phe Leu Gly Pro Gln Glu Leu Gln His 260 265 270 Ile Pro Gly Ala Ala Glu Glu Val Glu Arg Leu Val Leu Glu Leu Asp 275 280 285 Trp Ser Lys Arg 290 <210> 11 <211> 1074 <212> DNA <213> Artificial Sequence <400> 11 ctgtccaagc tgcaatctat ttccgtgaag gcccgacgag tcgagctggc ccgagacatc 60 acccgaccca aggtgtgcct gcacgcccag cgatgctccc tggtgcgact gcgagtcgcc 120 gcccctcaga ccgaggaggc tctgggtact gtgcaggccg ccggcgctgg agacgagcat 180 tctgccgacg tggccctgca acagctggac cgagccatcg ccgagcgacg agcccgacga 240 aagcgagagc agctgtccta ccaggccgcc gccattgccg cctccattgg tgtgtccggc 300 attgccattt tcgccaccta cctgcgattc gccatgcaca tgaccgtcgg tggcgccgtg 360 ccctggggtg aggttgctgg aaccctgctg ctggtcgtgg gcctggccct gggaatggag 420 atgtacgccc gatacgccca caaggccatt tggcacgagt cccccctggg ctggctgctg 480 cacaagtccc accacacccc ccgaaccggc cccttcgagg ctaacgacct gttcgccatc 540 attaacggtc tgcccgccat gctgctgtgt accttcggtt tctggctgcc caacgtgctg 600 ggcgccgcct gtttcggtgc cggtctggga atcaccctgt acggtatggc ctacatgttc 660 gtccacgacg gtctggtgca ccgacgattc cccaccggac ccatcgccgg cctgccttac 720 atgaagcgac tgaccgtggc ccaccagctg caccactccg gcaagtacgg cggcgcccct 780 tggggtatgt tcctgggtcc ccaggagctg caacacattc ccggtgccgc cgaggaggtc 840 gagcgacttg tgctggagct ggactggtcc aagcgaggtg gcggcggttc cggtggtggt 900 ggatctggtg gtggcggttc cggagtgaag gagtctctgg tcggcggtgg cggctctggt 960 ggtggtggtt ctggcggcgg tggctctggc gtgaaggagt ccctggtggg tggtggcgga 1020 tctggtggcg gcggatctgg cggtggtggt tccggagtca aggagtctct cgtg 1074 <210> 12 <211> 358 <212> PRT <213> Artificial Sequence <400> 12 Leu Ser Lys Leu Gln Ser Ile Ser Val Lys Ala Arg Arg Val Glu Leu 1 5 10 15 Path Arg Asp Ile Thr Arg Pro Lys Val Cys Leu His Path Gln Arg Cys 20 25 30 Ser Leu Val Arg Leu Arg Val Ala Ala Pro Gln Thr Glu Glu Ala Leu 35 40 45 Gly Thr Val Gln Ala Ala Gly Ala Gly Asp Glu His Ser Ala Asp Val 50 55 60 Path Leu Gln Gln Leu Asp Arg Path I Path Glu Arg Arg Path Arg Arg 65 70 75 80 Lys Arg Glu Gln Leu Ser Tyr Gln Ala Ala Ala Ila Ala Ser Ile 85 90 95 Gly Val Ser Gly Ile Ala Ile Phe Ala Thr Tyr Leu Arg Phe Ala Met 100 105 110 His Met Thr Val Gly Gly Ala Val Pro Trp Gly Glu Val Ala Gly Thr 115 120 125 Leu Leu Leu Val Val Gly Leu Ala Leu Gly Met Glu Met Tyr Ala Arg 130 135 140 Tyr Ala His Lys Ala Ile Trp His Glu Ser Pro Leu Gly Trp Leu Leu 145 150 155 160 His Lys Ser His His Thr Pro Arg Thr Gly Pro Phe Glu Ala Asn Asp 165 170 175 Leu Phe Ala Ile Ile Asn Gly Leu Pro Ala Met Leu Leu Cys Thr Phe 180 185 190 Gly Phe Trp Leu Pro Asn Val Leu Gly Ala Ala Cys Phe Gly Ala Gly 195 200 205 Leu Gly Ile Thr Leu Tyr Gly Met Ala Tyr Met Phe Val His Asp Gly 210 215 220 Leu Val His Arg Arg Phe Pro Thr Gly Pro Ile Ala Gly Leu Pro Tyr 225 230 235 240 Met Lys Arg Leu Thr Val Ala His Gln Leu His His Ser Gly Lys Tyr 245 250 255 Gly Gly Ala Pro Trp Gly Met Phe Leu Gly Pro Gln Glu Leu Gln His 260 265 270 Ile Pro Gly Ala Ala Glu Glu Val Glu Arg Leu Val Leu Glu Leu Asp 275 280 285 Trp Ser Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 290 295 300 Gly Gly Ser Gly Val Lys Glu Ser Leu Val Gly Gly Gly Gly Ser Gly 305 310 315 320 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Val Lys Glu Ser Leu Val 325 330 335 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 340 345 350 Val Lys Glu Ser Leu Val 355 <210> 13 <211> 1287 <212> DNA <213> Artificial Sequence <400> 13 cacgtggcct ctgctctgat ggtcgagcag aagggctctg aggccgctgc ctcttctccc 60 gacgtgctgc gagcctgggc tacccagtac cacatgcctt ccgagtcctc tgacgccgct 120 cgacccgctc tgaagcacgc ctacaagcct ccagcctccg acgccaaggg catcaccatg 180 gctctgacca tcatcggaac ctggaccgcc gtgttcctgc acgccatctt ccagattcga 240 ctgcccacct ctatggacca gctgcactgg ctgcccgtgt ctgaggccac cgctcagctg 300 ctcggcggat cttcttctct gctgcacatt gccgccgtct ttatcgtgct cgagttcctg 360 tacaccggcc tgttcatcac cactcacgac gccatgcacg gcaccattgc tctgcgacac 420 cgacagctga acgacctgct gggcaacatc tgcatctccc tgtacgcctg gttcgactac 480 tctatgctgc accgaaagca ctgggagcac cacaaccaca ccggcgaggt cggcaaggac 540 cccgacttcc acaagggcaa ccccggactg gtgccctggt tcgcctcttt catgtcctct 600 tacatgtctc tgtggcagtt cgcccgactg gcctggtggg ccgtcgtcat gcagatgctg 660 ggcgctccca tggccaacct gctggtgttc atggccgctg ctcccatcct gtccgccttc 720 cgactgttct acttcggcac ctacctgcct cacaagcccg agcctggacc tgccgccgga 780 tctcaggtga tggcctggtt ccgagccaag acctctgagg cttctgacgt gatgtctttc 840 ctgacctgct accacttcga cctgcattgg gagcatcaca gatggccctt cgctccctgg 900 tggcagctcc ctcactgccg acgactgtct ggccgaggac tggtccccgc tctggccggc 960 ggaggcggct ctggcggcgg aggatctggc ggtggcggtt ccctgcagcg acgacgagtg 1020 accgtgcgaa aggccgacgc tggcggcctg ggcatctcta tcaagggcgg acgagagaac 1080 aagatgccca tcctgatctc taagatcttc aagggcctcg ccgccgacca gaccgaggct 1140 ctgttcgtgg gcgacgccat cctgtctgtg aacggcgagg acctgtcctc tgctacccac 1200 gacgaggccg tgcaggccct gaagagacc ggcaaggaag tcgtgctcga ggtcaagtac 1260 atgaaggaag tgtctcccta cttcaag 1287 <210> 14 <211> 429 <212> PRT <213> Artificial Sequence <400> 14 His Val Ala Ser Ala Leu Met Val Glu Gln Lys Gly Ser Glu Ala Ala 1 5 10 15 Ala Sir Sir Pro Asp Val Leu Arg Ala Trp Ala Thr Gln Tyr His Met 20 25 30 Pro Sister Glu Sister Sister Asp Ala Ala Arg Pro Ala Leu Lys His Ala Tyr 35 40 45 Lys Pro Pro Ala Ser Asp Ala Lys Gly Ile Thr Met Ala Leu Thr Ile 50 55 60 Ile Gly Thr Trp Thr Ala Val Phe Leu His Ala Ile Phe Gln Ile Arg 65 70 75 80 Leu Pro Thr Ser Met Asp Gln Leu His Trp Leu Pro Val Ser Glu Ala 85 90 95 Thr Ala Gln Leu Leu Gly Gly Ser Ser Ser Leu Leu His Ile Ala Ala 100 105 110 Val Phe Ile Val Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr 115 120 125 His Asp Ala Met His Gly Thr Ile Ala Leu Arg His Arg Gln Leu Asn 130 135 140 Asp Leu Leu Gly Asn Ile Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr 145 150 155 160 Ser Met Leu His Arg Lys His Trp Glu His His Asn His Thr Gly Glu 165 170 175 Val Gly Lys Asp Pro Asp Phe His Lys Gly Asn Pro Gly Leu Val Pro 180 185 190 Trp Phe Ala Ser Phe Met Ser Ser Tyr Met Ser Leu Trp Gln Phe Ala 195 200 205 Arg Leu Ala Trp Trp Ala Val Val Met Gln Met Leu Gly Ala Pro Met 210 215 220 Ala Asn Leu Leu Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe 225 230 235 240 Arg Leu Phe Tyr Phe Gly Thr Tyr Leu Pro His Lys Pro Glu Pro Gly 245 250 255 Pro Ala Ala Gly Ser Gln Val Met Ala Trp Phe Arg Ala Lys Thr Ser 260 265 270 Glu Ala Ser Asp Val Met Ser Phe Leu Thr Cys Tyr His Phe Asp Leu 275 280 285 His Trp Glu His His Arg Trp Pro Phe Ala Pro Trp Trp Gln Leu Pro 290 295 300 His Cys Arg Arg Leu Ser Gly Arg Gly Leu Val Pro Ala Leu Ala Gly 305 310 315 320 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln 325 330 335 Arg Arg Arg Val Thr Val Arg Lys Ala Asp Ala Gly Gly Leu Gly Ile 340 345 350 Ser Ile Lys Gly Gly Arg Glu Asn Lys Met Pro Ile Leu Ile Ser Lys 355 360 365 Ile Phe Lys Gly Leu Ala Ala Asp Gln Thr Glu Ala Leu Phe Val Gly 370 375 380 Asp Leu Serving Val Asn Gly Glu Asp Leu Serving Thr His 385 390 395 400 Asp Glu Ala Val Gln Ala Leu Lys Thr Gly Lys Glu Val Val Val Leu 405 410 415 Glu Val Lys Tyr Met Lys Glu Val Ser Pro Tyr Phe Lys 420 425 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <400> 15 cgccaggtt ttcccagtca cgac 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <400> 16 age 24 <210> 17 <211> 52 <212> DNA <213> Artificial Sequence <400> 17 ccgaccagca cttttgcag tactaccgc agcacgtggc ctctgctctg at 52 <210> 18 <211> 46 <212> DNA <213> Artificial Sequence <400> 18 ggacaggcca tggaactagt cggtacctta ggccagagcg gggacc 46 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <400> 19 gcatggccag gtgagtggtg atgaacagg 29 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <400> 20 ccactcacct ggccatgcac ggcaccattg 30 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <400> 21 ggtgtcgcag gaaaatggtg ccgtgcatgg cg 32 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <400> 22 ccattttcct gcgacaccga cagctg 26 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <400> 23 gcagatgttc agcagcaggt cgttcagctg tc 32 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <400> 24 cctgctgctg aacatctgca tctccctgta cgc 33 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <400> 25 ccggggttgc cgaagtggaa gtcggggtcc ttg 33 <210> 26 <211> 29 <212> DNA <213> Artificial Sequence <400> 26 tccacttcgg caaccccgga ctggtgccc 29 <210> 27 <211> 34 <212> DNA <213> Artificial Sequence <400> 27 ccggggttgc ccaggtggaa gtcggggtcc ttgc 34 <210> 28 <211> 29 <212> DNA <213> Artificial Sequence <400> 28 ccacctgggc aaccccggac tggtgccct 29 <210> 29 <211> 55 <212> DNA <213> Artificial Sequence <400> 29 ccgaccagca ctttttgcag tactaaccgc agctgtctaa gctgcagtct atctc 55 <210> 30 <211> 52 <212> DNA <213> Artificial Sequence <400> 30 ggacaggcca tggaactagt cggtacctta tcgcttagac cagtccagtt cc 52 <210> 31 <211> 37 <212> DNA <213> Artificial Sequence <400> 31 catggcgggc agcaggttga tgatggcgaa caggtcg 37 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <400> 32 caacctgctg cccgccatgc tgctgtg 27 <210> 33 <211> 34 <212> DNA <213> Artificial Sequence <400> 33 gccagccact tccagccagg gcacagctcc gccg 34 <210> 34 <211> 25 <212> DNA <213> Artificial Sequence <400> 34 tggctggaag tggctggcac cctgc 25 <210> 35 <211> 32 <212> DNA <213> Artificial Sequence <400> 35 gccgagggcc agtccgacca ccagcagcag gg 32 <210> 36 <211> 32 <212> DNA <213> Artificial Sequence <400> 36 tggtcggact ggccctcggc atggaaatgt ac 32 <210> 37 <211> 32 <212> DNA <213> Artificial Sequence <400> 37 gccgagggcg aatccgacca ccagcagcag gg 32 <210> 38 <211> 32 <212> DNA <213> Artificial Sequence <400> 38 tggtcggatt cgccctcggc atggaaatgt ac 32 <210> 39 <211> 32 <212> DNA <213> Artificial Sequence <400> 39 gccgagggcg attccgacca ccagcagcag gg 32 <210> 40 <211> 32 <212> DNA <213> Artificial Sequence <400> 40 tggtcggaat cgccctcggc atggaaatgt ac 32 <210> 41 <211> 45 <212> DNA <213> Artificial Sequence <400> 41 ggcggaggcg gctctggcgg cggaggatct ggcggtggcg gttcc 45 <210> 42 <211> 15 <212> PRT <213> Artificial Sequence <400> 42 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 43 <211> 32 <212> DNA <213> Artificial Sequence <400> 43 ccgaccagca ctttttgcag tactaaccgc ag 32 <210> 44 <211> 27 <212> DNA <213> Artificial Sequence <400> 44 ggtaccgact agttccatgg cctgtcc 27

Claims

1. A method for biosynthesizing astaxanthin, comprising achieving the biosynthesis of astaxanthin in an engineered bacterium expressing β-carotene ketolase and β-carotene hydroxylase derived from Haematococcus pluvialis, wherein: The amino acid sequence of the beta-carotene hydroxylase is a wild-type beta-carotene hydroxylase with a G135L point mutation as shown in SEQ ID NO.

4. The engineered bacterium is Yarrowia lipolytica, and the engineered bacterium synthesizes beta-carotene.

2. The method according to claim 1, comprising co-localizing β-carotene ketolase and β-carotene hydroxylase using a scaffold protein, wherein The scaffold protein includes a mouse PSD95 / DlgA / Zo-1 region (PDZ) and a PDZ ligand, the β-carotene ketolase is fused with the PSD95 / DlgA / Zo-1 region (PDZ) and expressed, and the β-carotene hydroxylase is fused with the PDZ ligand and expressed, and the ratio of the β-carotene ketolase and the β-carotene hydroxylase connected by the scaffold protein is 1:1, 3:1 or 4:1; The amino acid sequence of the mouse PSD95 / DlgA / Zo-1 region (PDZ) is SEQ ID NO. 6; the amino acid sequence of the PDZ ligand is SEQ ID NO. 8; The β-carotene hydroxylase is expressed in fusion with 1, 3 or 4 PDZ ligand molecules; The β-carotene ketolase is directly connected to one of the PDZ or connected through a linker; The β-carotene hydroxylase is connected to the PDZ ligand molecule via a linker; The linker is the amino acid sequence shown in SEQ ID NO.

42.

3. A β-carotene hydroxylase comprising a G135L point mutation, the amino acid sequence of which is SEQ ID NO.

10.

4. A β-carotene hydroxylase-PDZ ligand fusion protein comprising a G135L point mutation, the amino acid sequence of which is SEQ ID NO.

12.

5. A polynucleotide molecule encoding the enzyme or fusion protein according to claim 3 or 4. The polynucleotide molecule according to claim 5 , wherein the nucleotide sequence of the polynucleotide molecule is SEQ ID NO. 9 or SEQ ID NO.

11.

7. A nucleic acid construct comprising the polynucleotide molecule according to claim 5 or 6.

8. An engineered bacterium for producing astaxanthin, comprising at least one of the polynucleotide molecule of claim 5 or 6 or the nucleic acid construct of claim 7, wherein the engineered bacterium expresses the fusion protein of claim 4 and the fusion protein with an amino acid sequence as shown in SEQ ID NO. 14, wherein the ratio of β-carotene ketolase and β-carotene hydroxylase linked by the scaffold protein is 3:1, and the engineered bacterium synthesizes β-carotene, wherein the engineered bacterium is Yarrowia lipolytica. 9 . The engineered bacterium according to claim 8 , which is Yarrowia lipolytica with a deposit number of CCTCC M 2022883.

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