WO2024001460A1 - 一种生物合成虾青素的方法及载体 - Google Patents

一种生物合成虾青素的方法及载体 Download PDF

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WO2024001460A1
WO2024001460A1 PCT/CN2023/089919 CN2023089919W WO2024001460A1 WO 2024001460 A1 WO2024001460 A1 WO 2024001460A1 CN 2023089919 W CN2023089919 W CN 2023089919W WO 2024001460 A1 WO2024001460 A1 WO 2024001460A1
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carotene
pdz
astaxanthin
seq
carotene hydroxylase
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叶紫玲
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武汉合生科技有限公司
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
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    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13129—Beta-carotene 3-hydroxylase (1.14.13.129)
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    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
    • C12Y114/15802—Spheroidene monooxygenase (1.14.15.B2), i.e. acyclic carotenoid 2-ketolase
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast

Definitions

  • the present application belongs to the field of astaxanthin biosynthesis, and specifically relates to a method and carrier for biosynthesizing astaxanthin.
  • astaxanthin is 3,3'-dihydroxy-4,4'-diketo- ⁇ , ⁇ '-carotene. It is a terpene unsaturated compound and a ketone secondary compound.
  • Carotenoids the molecular formula is C 40 H 52 O 4 .
  • Astaxanthin is one of the carotenoids with the strongest antioxidant activity in nature. It has been used in food, medicine, health products, cosmetics, feed and other fields, and has extremely high economic and application value. Since there are few sources of natural astaxanthin, it is generally necessary to obtain a large amount of astaxanthin artificially. However, chemically synthesized astaxanthin is a mixture of three configurations, with poor biological activity and low utilization.
  • Haematococcus pluvialis can naturally synthesize 3S-3S'-astaxanthin with the highest biological activity.
  • the astaxanthin thus obtained can be used in the fields of food, cosmetics and medicine. It is the only 3S-3S currently available on the market. '-Main source of astaxanthin.
  • Bacillus, Saccharomyces cerevisiae, etc. were modified to construct a microbial cell factory with high yield of 3S-3S'-astaxanthin.
  • Yarrowia lipolytica is a GRAS (generally recognized as safe) unconventional oil-producing yeast certified by the US FDA. Its genetic background is clear, and relatively complete metabolic modification tools have been developed in recent years. In 2017, researchers reported for the first time the de novo synthesis of astaxanthin using Yarrowia lipolytica. By introducing exogenous lycopene cyclase (CrtYB) and phytene desaturase (CrtI), an engineering strain of Yarrowia lipolytica that can synthesize ⁇ -carotene was constructed, and the mevalonate pathway was optimized on this basis.
  • CeYB exogenous lycopene cyclase
  • CrtI phytene desaturase
  • HpCrtZ derived from Haematococcus pluvialis was simultaneously targeted to liposomes, endoplasmic reticulum and peroxisomes, and the lipolytic enzyme obtained
  • the engineered strain of Saccharomyces cerevisiae can synthesize 858 mg/L of astaxanthin through continuous fed-batch fermentation.
  • HpCrtW and HpCrtZ derived from Haematococcus pluvialis as the research object, and carried out research on directed evolution, protein scaffold construction, and protein ratio optimization, in order to provide ideas for further improving the astaxanthin production of the Yarrowia lipolytica engineered strain. .
  • the purpose of this application is to provide a method and carrier for biosynthesizing astaxanthin, so as to increase the yield of biosynthesizing astaxanthin.
  • the first aspect of the present application provides a method for biosynthesizing astaxanthin, which includes using engineering bacteria capable of expressing ⁇ -carotene ketolase and ⁇ -carotene hydroxylase derived from Haematococcus pluvialis in shrimps. Biosynthesis of cyanins, wherein the ⁇ -carotene hydroxylase contains a G135L point mutation; preferably, the engineering bacterium is Yarrowia lipolytica; preferably, the engineering bacterium is capable of synthesizing ⁇ -carotene.
  • the second aspect of the present application provides a method for biosynthesizing astaxanthin, which includes using an engineered bacterium capable of expressing ⁇ -carotene ketolase and ⁇ -carotene hydroxylase to achieve the biosynthesis of astaxanthin, wherein , the ⁇ -carotene ketolase and ⁇ -carotene hydroxylase are co-localized through scaffolding proteins; preferably, the ⁇ -carotene ketolase and ⁇ -carotene hydroxylase are R.
  • pluvialis Source of algae preferably, the engineering bacterium is Yarrowia lipolytica; preferably, the engineering bacterium can synthesize ⁇ -carotene.
  • the third aspect of the present application provides a ⁇ -carotene hydroxylase containing the G135L point mutation, which has the amino acid sequence shown in SEQ ID NO. 10.
  • the fourth aspect of the present application provides a ⁇ -carotene hydroxylase-PDZ ligand fusion protein containing the G135L point mutation, which has the amino acid sequence shown in SEQ ID NO. 12.
  • the fifth aspect of this application provides a ⁇ -carotene ketolase-PDZ fusion protein, which has the amino acid sequence shown in SEQ ID NO. 14.
  • the sixth aspect of the present application provides a polynucleotide molecule encoding the ⁇ -carotene hydroxylase containing the G135L point mutation of the third aspect of the present application and the fusion protein of the fourth or fifth aspect of the present application; preferably, the The polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO.9, SEQ ID NO.11 or SEQ ID NO.13.
  • 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.
  • the eighth aspect of the present application provides an engineering bacterium for producing astaxanthin, which contains the polynucleotide component of the sixth aspect of the present application. or at least one of the nucleic acid constructs of the seventh aspect of the application; preferably, it is capable of expressing the ⁇ -carotene hydroxylase comprising the G135L point mutation of the third aspect of the application, or the fourth or fifth aspect of the application.
  • At least one fusion protein of the aspect Preferably, the engineered bacterium expresses the fusion protein of the fourth and fifth aspects of the present application; Preferably, the engineered bacterium is capable of synthesizing ⁇ -carotene; Preferably, the engineered bacterium The bacteria is Yarrowia lipolytica.
  • the inventor introduced point mutations in ⁇ -carotene hydroxylase to obtain engineered bacteria with improved astaxanthin production. After introducing point mutations, compared with wild-type ⁇ -carotene hydroxylase, , astaxanthin production increased by 2.8 times.
  • Figure 2 is a comparison of the production of canthaxanthin (Canthaxanthin) expressed and synthesized by wild-type HpCrtW (W*) and five HpCrtW mutants in ⁇ -carotene-producing Yarrowia lipolytica engineered strain BC;
  • Figure 3 shows the comparison of astaxanthin content after combined expression of wild-type HpCrtW and five HpCrtZ mutants in ⁇ -carotene-producing Yarrowia lipolytica engineered strain BC;
  • Figure 4 shows the comparison of astaxanthin production after using PDZ-PL protein scaffold to connect different proportions of HpCrtW and HpCrtZ and expressing them in ⁇ -carotene-producing Yarrowia lipolytica engineered strain BC.
  • the terms “about,” “substantially,” and “similar to” mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range may depend in part on the measurement of the value Either the way it is determined, or it depends on the limitations of the measurement system.
  • the first aspect of the present application provides a method for biosynthesizing astaxanthin, which includes using engineering bacteria capable of expressing ⁇ -carotene ketolase and ⁇ -carotene hydroxylase derived from Haematococcus pluvialis in shrimps. Biosynthesis of cyanins, wherein the ⁇ -carotene hydroxylase contains a G135L point mutation. The inventors found that the point mutation increased the production of astaxanthin by 2.8 times.
  • the ⁇ -carotene hydroxylase comprising the G135L point mutation has the amino acid sequence shown in SEQ ID NO. 10.
  • the engineered bacteria are capable of synthesizing beta-carotene.
  • they are commonly used engineering bacteria in this field.
  • lycopene cyclase (CrtYB) and phytene desaturase (CrtI) are introduced into the host bacteria to construct a synthetic ⁇ -carotene.
  • This application does not limit the genetic engineering strain.
  • the species of the engineered bacteria The class is not limited, for example, it can be Escherichia coli, Saccharomyces cerevisiae, Yarrowia lipolytica, etc.; in some embodiments, the engineering bacterium is Yarrowia lipolytica.
  • the method further includes using a scaffold protein to colocalize ⁇ -carotene ketolase and ⁇ -carotene hydroxylase comprising the G135L point mutation;
  • the "Scaffolding protein” refers to a protein that can interact or bind with multiple proteins to form a complex, thereby localizing the protein (in the form of a complex) in a specific region (colocalization), which can include forming a complex.
  • Protein scaffolds constructed from pairs of interacting proteins, protein domains or polypeptide chains, protein scaffolds constructed through self-assembled proteins or protein scaffolds constructed through chemically modified proteins, etc.
  • Some non-limiting examples of the scaffold protein include a scaffold protein composed of GBD and its ligands, a scaffold protein composed of SH3 and its ligands, a scaffold protein composed of PDZ and its ligands, and the like.
  • the scaffold protein includes a murine PSD95/DlgA/Zo-1 domain (PDZ) and a PDZ ligand (PL); preferably, the murine PSD95/DlgA/Zo-1 domain (PDZ) It 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.
  • PDZ murine PSD95/DlgA/Zo-1 domain
  • PL PDZ ligand
  • the ratio of the ⁇ -carotene ketolase connected to the scaffold protein and the ⁇ -carotene hydroxylase comprising the G135L point mutation is (1-4):1; it is understood that the The ratio is the ratio of the number of molecules, for example, it can be 1:1, 2:1, 3:1 or 4:1.
  • the ⁇ -carotene ketolase is fused and expressed with the PSD95/DlgA/Zo-1 region (PDZ); the ⁇ -carotene hydroxylase comprising the G135L point mutation is fused and expressed with a PDZ ligand ; Preferably, the ⁇ -carotene hydroxylase containing the G135L point mutation is fused and expressed with 1-4 of the PDZ ligand molecules; Preferably, the ⁇ -carotene hydroxylase containing the G135L point mutation is expressed with Three of the PDZ ligand molecules are fused and expressed.
  • PDZ PSD95/DlgA/Zo-1 region
  • the scaffold protein composed of the PDZ and its ligands of the present application can spatially recruit specific proteins (such as the ⁇ -carotene ketolase of the present application and ⁇ -carotene ketolase containing the G135L point mutation) in a designable manner.
  • specific proteins such as the ⁇ -carotene ketolase of the present application and ⁇ -carotene ketolase containing the G135L point mutation
  • -carotene hydroxylase the two proteins HpCrtW and HpCrtZ3 ( ⁇ -carotene hydroxylase containing the G135L point mutation) are elastically "bundled" in different proportions; the design idea is shown in Figure 1.
  • Using the protein scaffold to connect HpCrtW and HpCrtZ3 can further improve the synergistic catalytic efficiency of HpCrtW and HpCrtZ3.
  • the ⁇ -carotene ketolase and the PDZ can be directly connected or connected through a linker (for example: (GGGGS)*3 (SEQ ID NO. 42)).
  • a linker for example: (GGGGS)*3 (SEQ ID NO. 42)
  • the protein expressed by fusion of the ⁇ -carotene ketolase and PDZ has the amino acid sequence shown in SEQ ID NO. 14.
  • the ⁇ -carotene hydroxylase and the PDZ ligand molecule are connected through a linker; preferably, the linker is (GGGS)n, and n is an integer from 1 to 4.
  • the ⁇ -carotene hydroxylase containing the G135L point mutation is connected to multiple PDZ ligand molecules in sequence through a linker; preferably, it is connected to three PDZ ligand molecules.
  • the protein expressed by fusion of the ⁇ -carotene hydroxylase containing the G135L point mutation and the PDZ ligand has the amino acid sequence shown in SEQ ID NO. 12.
  • the second aspect of the present application provides a method for biosynthesizing astaxanthin, which includes using a method capable of expressing ⁇ -carotene ketone
  • the biosynthesis of astaxanthin is achieved in engineered bacteria using alcoholase and ⁇ -carotene hydroxylase, wherein the ⁇ -carotene ketolase and ⁇ -carotene hydroxylase are colocalized through scaffold proteins.
  • the scaffold protein includes a murine PSD95/DlgA/Zo-1 domain (PDZ) and a PDZ ligand (PL); preferably, the murine PSD95/DlgA/Zo-1 domain (PDZ) It 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.
  • PDZ murine PSD95/DlgA/Zo-1 domain
  • PL PDZ ligand
  • the ⁇ -carotene ketolase is fused and expressed with the PSD95/DlgA/Zo-1 region (PDZ); the ⁇ -carotene hydroxylase is fused and expressed with a PDZ ligand; preferably, the The ⁇ -carotene hydroxylase is fused and expressed with 1-4 PDZ ligand molecules.
  • PDZ PSD95/DlgA/Zo-1 region
  • the ⁇ -carotene hydroxylase and the PDZ ligand are connected through a linker; the ⁇ -carotene hydroxylase and the PDZ ligand molecule are connected through a linker; preferably, the linker is (GGGS)n, n is an integer from 1 to 4.
  • the ⁇ -carotene hydroxylase is sequentially connected to multiple PDZ ligand molecules through a linker, preferably three PDZ ligand molecules.
  • 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 existence of the linker is usually used to reduce steric hindrance between the two connected proteins. Effects on molecular function.
  • the ⁇ -carotene ketolase and ⁇ -carotene hydroxylase may be ⁇ -carotene ketolase and ⁇ -carotene hydroxylase from any source used to prepare astaxanthin , for example, it can be derived from Haematococcus pluvialis, Chromchloris zofingiensis, Paracoccus sp., Pantoea ananatis, Sphingomonas sp. ) source, Brevundimonas sp. source, etc.; preferably, it is a Haematococcus pluvialis source, which synthesizes astaxanthin with a high yield and produces less intermediate metabolites.
  • the ⁇ -carotene ketolase and ⁇ -carotene hydroxylase can be optimized, for example, through codon optimization, point mutation and other technologies, so that they have higher catalytic activity and stability. Sex etc.
  • the engineered bacteria are capable of synthesizing beta-carotene.
  • the engineered bacterium is Yarrowia lipolytica.
  • the third aspect of the present application provides a ⁇ -carotene hydroxylase containing the G135L point mutation, which has the amino acid sequence shown in SEQ ID NO. 10.
  • the fourth aspect of the present application provides a ⁇ -carotene hydroxylase-PDZ ligand fusion protein containing the G135L point mutation, which has the amino acid sequence shown in SEQ ID NO. 12.
  • the fifth aspect of this application provides a ⁇ -carotene ketolase-PDZ fusion protein, which has the amino acid sequence shown in SEQ ID NO. 14.
  • the sixth aspect of the present application provides a polynucleotide molecule encoding the ⁇ -carotene hydroxylase containing the G135L point mutation of the third aspect of the present application and the fusion protein of the fourth or fifth aspect of the present application.
  • a polynucleotide molecule encoding a ⁇ -carotene hydroxylase comprising a G135L point mutation has the nucleotide sequence shown in SEQ ID NO. 9.
  • the polynucleotide molecule encoding the ⁇ -carotene hydroxylase-PDZ ligand fusion protein comprising the G135L point mutation has the nucleotide sequence shown in SEQ ID NO. 11.
  • the polynucleotide molecule encoding ⁇ -carotene ketolase-PDZ fusion protein has SEQ ID The nucleotide sequence shown in NO.13.
  • 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.
  • the eighth aspect of this application provides an engineering bacterium that produces astaxanthin, which contains at least one of the polynucleotide molecule of the sixth aspect of this application or the nucleic acid construct of the seventh aspect of this application; preferably, it can express The ⁇ -carotene hydroxylase containing the G135L point mutation of the third aspect of the application, or at least one of the fusion proteins of the fourth aspect or the fifth aspect of the application; preferably, the engineering bacteria express the fourth aspect of the application and the fusion protein of the fifth aspect; preferably, the engineering bacterium is capable of synthesizing ⁇ -carotene; preferably, the engineering bacterium is Yarrowia lipolytica.
  • the engineering bacterium is Yarrowia lipolytica with deposit number CCTCC M 2022882 or CCTCC M 2022883.
  • the top five mutant proteins were selected, which are HpCrtW D130L , HpCrtW A137F , HpCrtW G148L , HpCrtW K185F , HpCrtW K185L , and HpCrtZ G183L , HpCrtZ G123L , HpCrtZ G135L , HpCrtZ G13. 5F and HpCrtZ G135I .
  • HpCrtW and HpCrtZ are simultaneously expressed in the ⁇ -carotene-producing Yarrowia lipolytica engineering strain BC, HpCrtW preferentially uses ⁇ -carotene to synthesize echinenone and canthaxanthin, while echinenone and canthaxanthin respectively form hydroxyechinenone (hydroxyechinenone) and phoenicoxanthin (phoenicoxanthin) under the action of HpCrtZ, and finally are catalyzed by HpCrtW and HpCrtZ respectively to form astaxanthin (Zhu, H.-Z., et al. al., 2022).
  • the amplification products were recovered and constructed into pYLXP' using the Gibson Assembly kit (Thermo Fisher) 2 (Lv, YK, et al., Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica, ACS synthetic Biology, 2019.8: p.568-576) vector. Finally, CX-F/CX-R was used as the universal sequencing primer. Correct sequencing indicates that the point mutation vector pYLXP'2::HpCrtW D130L was successfully obtained. The other point mutation processes and naming methods of HpCrtW and HpCrtZ proteins are the same as above.
  • Table 1 Primer information used in the point mutation process of HpCrtW and HpCrtZ proteins:
  • Table 2 PCR amplification system of HpCrtW and HpCrtZ protein point mutation process
  • Carotenoids were quantitatively detected using high-performance liquid chromatography (HPLC, Agilent Technologies, USA).
  • HPLC high-performance liquid chromatography
  • An Agilent 1200 series LC system was used, equipped with a YMC carotenoid C30 column (250mm ⁇ 4.6mm i.d. ⁇ 5 ⁇ m thickness).
  • the mobile phases and gradients used were as follows. Initially, use eluent A (81% methanol, 15% MTBE, 4% water) at a flow rate of 1 mL/min for 50-70 min and gradually decrease from 100 to 60%. At the same time, eluent B (7% methanol, 90% MTBE and 3% water) was increased from 0 to 40%.
  • HpCrtZ (the nucleotide sequence is shown in SEQ ID NO.3, the amino acid sequence is shown in SEQ ID NO.4) and 5 mutations.
  • HpCrtZ was co-transformed with wild-type HpCrtW and point mutation HpCrtW K185F and co-expressed in Yarrowia lipolytica engineering strain BC, and the production of astaxanthin (Ast) in the engineering strain was detected using the method of 1.2. The results are shown in Figure 3 Show.
  • HpCrtZ G135L and wild-type HpCrtW can significantly increase the production of astaxanthin in the engineered strain , the maximum improvement is about 2.8 times.
  • the mutant HpCrtZ G135L is named HpCrtZ3 in this application. In the following, HpCrtZ G135L and HpCrtZ3 can be replaced.
  • the strain with the highest astaxanthin yield was named pHpWZ3 and was deposited in China on June 14, 2022.
  • Type Culture Collection Center (CCTCC for short, address: Wuhan University, Wuhan, China), the collection number is CCTCC M 2022882.
  • Example 2 Using scaffold protein to increase astaxanthin production
  • the five designed sequences were sent to General 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 and pYLXP'2. ::HpCrtZ3-PL*1, pYLXP'2::HpCrtZ3-PL*2, pYLXP'2-HpCrtZ3-PL*3 and pYLXP'2-HpCrtZ3-PL*4.
  • pYLXP'2::HpCrtW and pYLXP'2::HpCrtW-PDZ were linearized with restriction enzymes ClaI and NheI
  • pYLXP'2::HpCrtZ3 was linearized with restriction enzymes Avr II and ClaI (i.e. Example 1 pYLXP'2::HpCrtZ G135L ), pYLXP'2::HpCrtZ3-PL*1, pYLXP'2::HpCrtZ3-PL*2, pYLXP'2-HpCrtZ3-PL*3 and pYLXP'2-HpCrtZ3-PL* 4.
  • the strain with a ratio of HpCrtW and HpCrtZ3 of 3:1 obtained in this example was named pHpW PDZ Z3 PL *3 and was deposited at the China Type Culture Collection Center (CCTCC for short) on June 14, 2022. The address is Wuhan, China. Wuhan University), the accession number is CCTCC M 2022883.

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Abstract

本申请公开了一种生物合成虾青素的方法及载体，该方法包括采用能够表达雨生红球藻来源的β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶的工程菌中实现虾青素的生物合成，其中，所述β-胡萝卜素羟化酶包含G135L点突变。引入点突变后，虾青素产量提高了2.8倍。

Description

一种生物合成虾青素的方法及载体
相关申请的交叉引用
本申请要求于2022年6月30日提交至中国知识产权局的发明名称为“一种生物合成虾青素的方法及载体”的中国专利申请CN202210778671.5的优先权权益。在此通过引用方式将其全部内容整体并入本申请。
技术领域
本申请属于虾青素生物合成领域，具体涉及一种生物合成虾青素的方法及载体。
背景技术
虾青素(astaxanthin)化学名称为3,3’-二羟基-4,4’-二酮基-β,β’-胡萝卜素，为萜烯类不饱和化合物，是一种酮式次生类胡萝卜素(Carotenoids)，分子式为C40H52O4。虾青素是自然界中抗氧化活性最强的类胡萝卜素之一，已经被应用于食品、药品、保健品、化妆品和饲料等领域，具有极高的经济与应用价值。由于天然虾青素的来源少，一般需要人工合成获取大量的虾青素。但化学合成的虾青素为三种构型的混合物，生物活性差且利用度不高，同时化学合成虾青素过程复杂，合成过程中不可避免地会引入其他杂质，生物安全性受到质疑。所以，化学合成的虾青素仅作为饲料添加剂应用于水产等养殖行业，被禁止用于食品、化妆品和药品应用等领域。而生物合成的3S-3S’-虾青素不仅可用于养殖行业，还被允许用于食品、化妆品和医药领域，因此生物合成3S-3S’-虾青素备受关注。
雨生红球藻(Haematococcus pluvialis)可天然单一合成生物活性最高的3S-3S’-虾青素，由此获得的虾青素可应用于食品、化妆品和医药领域，是目前市场获取3S-3S’-虾青素主要来源。虽已实现了商业化生产，但存在生产周期长、生产成本高等问题，阻碍了天然虾青素的大规模应用。因此越来越多的研究者将雨生红球藻等天然虾青素合成宿主中合成虾青素的关键基因挖掘出来，通过代谢工程手段对遗传背景清楚和基因操作成熟的一些模式微生物如大肠杆菌、酿酒酵母等进行改造，构建高产3S-3S’-虾青素的微生物细胞工厂。
解脂耶氏酵母(Yarrowia lipolytica)是美国FDA认证的GRAS(generally recognized as safe)非常规产油酵母，其遗传背景清楚，近些年来开发了较为完善的代谢改造工具。2017年研究人员首次报道了利用解脂耶氏酵母从头合成虾青素。通过引入外源番茄红素环化酶(CrtYB)和植物烯去饱和酶(CrtI)，构建了可合成β-胡萝卜素的解脂耶氏酵母工程菌株，在此基础上优化甲羟戊酸路径、下调角鲨烯合成竞争路径，再引入菠萝泛菌(Pantoea ananatis)来源的β-胡萝卜素酮醇酶(PaCrtW)和β-胡萝卜素羟化酶(PaCrtZ)编码基因并优化二者的拷贝数，获得产虾青素的解脂耶氏酵母工程菌，产量为54.6mg/L。2019年报道，在产β-胡萝卜素的解脂 耶氏酵母工程菌株基础上引入雨生红球藻来源的β-胡萝卜素酮醇酶(HpCrtW)和β-胡萝卜素羟化酶(HpCrtZ)并调节拷贝数，所构建的虾青素工程菌发酵7天后合成的虾青素产量可达285±19mg/L。随后发现，将副球菌(Paracoccus sp.)来源的PsCrtW和雨生红球藻来源的HpCrtZ融合蛋白，同时靶向于脂质体、内质网和过氧化物酶体，所获得的解脂耶氏酵母工程菌经连续补料分批发酵中可合成858mg/L的虾青素。
和前期报道实验结果一致，我们也发现相较于HpCrtW和HpCrtZ组合，PsCrtW和HpCrtZ组合在单位体积合成的虾青素浓度(mg/L)最高，但是虾青素占细胞干重的含量(mg/g dry cell weight)低于HpCrtW和HpCrtZ组合，并且PsCrtW和HpCrtZ组合产生的中间产物也更多。因此，本研究选择雨生红球藻来源的HpCrtW和HpCrtZ为研究对象，开展定向进化、蛋白支架构建、蛋白比例优化等研究，以期为进一步提高解脂耶氏酵母工程菌株虾青素产量提供思路。
发明内容
本申请的目的在于提供一种生物合成虾青素的方法及载体，以提高生物合成虾青素的产量。
本申请为解决上述技术问题，提出了如下技术方案：
本申请第一方面提供了一种生物合成虾青素的方法，其包括采用能够表达雨生红球藻来源的β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶的工程菌中实现虾青素的生物合成，其中，所述β-胡萝卜素羟化酶包含G135L点突变；优选地，所述工程菌为解脂耶氏酵母；优选地，所述工程菌能够合成β-胡萝卜素。
本申请第二方面提供了一种生物合成虾青素的方法，其包括采用能够表达β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶的工程菌中实现虾青素的生物合成，其中，所述β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶通过支架蛋白共区域化；优选地，所述β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶为雨生红球藻来源；优选地，所述工程菌为解脂耶氏酵母；优选地，所述工程菌能够合成β-胡萝卜素。
本申请第三方面提供了一种包含G135L点突变的β-胡萝卜素羟化酶，其具有SEQ ID NO.10所示的氨基酸序列。
本申请第四方面提供了一种包含G135L点突变的β-胡萝卜素羟化酶-PDZ配体融合蛋白，其具有SEQ ID NO.12所示的氨基酸序列。
本申请第五方面提供了一种β-胡萝卜素酮醇酶-PDZ融合蛋白，其具有SEQ ID NO.14所示的氨基酸序列。
本申请第六方面提供了编码本申请第三方面的包含G135L点突变的β-胡萝卜素羟化酶、本申请第四方面或第五方面的融合蛋白的多核苷酸分子；优选地，所述多核苷酸分子具有SEQ ID NO.9、SEQ ID NO.11或SEQ ID NO.13所示的核苷酸序列。
本申请第七方面提供了一种核酸构建体，其包括本申请第六方面的多核苷酸分子。
本申请第八方面提供了一种生产虾青素的工程菌，其包含本申请第六方面的多核苷酸分 子或本申请第七方面的核酸构建体的至少一种；优选的，其能够表达本申请第三方面的包含G135L点突变的β-胡萝卜素羟化酶、或本申请第四方面或第五方面的融合蛋白的至少一种；优选地，所述工程菌表达本申请第四方面和第五方面的融合蛋白；优选地，所述工程菌能够合成β-胡萝卜素；优选的，所述工程菌为解脂耶氏酵母。
本申请的有益效果：发明人通过在β-胡萝卜素羟化酶中引入点突变，获得的虾青素产量提高的工程菌，引入点突变后，相比于野生型β-胡萝卜素羟化酶，虾青素产量提高了2.8倍。
附图说明
图1为蛋白支架按不同比例链接蛋白HpCrtW和HpCrtZ3示意图(n＝HpCrtW:HpCrtZ3)；
图2为野生型HpCrtW(W*)和5个HpCrtW突变体分别在产β-胡萝卜素的解脂耶氏酵母工程菌株BC中表达合成角黄素(Canthaxanthin)产量的比较；
图3为野生型HpCrtW分别和5个HpCrtZ突变体在产β-胡萝卜素的解脂耶氏酵母工程菌株BC中组合表达后虾青素含量的比较；
图4为利用PDZ-PL蛋白支架连接不同比例的HpCrtW和HpCrtZ，并在产β-胡萝卜素的解脂耶氏酵母工程菌株BC中表达后虾青素产量的比较。
具体实施方式
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一种实施方式，对于本领域普通技术人员来讲，还可以根据这些附图获得其他的实施方式。
定义
如本文所用，术语“一个”和“一种”以及“所述”和类似的指代物指示单数和复数，除非本文另外指明或上下文明显矛盾。
如本文所用，术语“约”、“基本上”和“类似于”是指在本领域普通技术人员所确定的特定值的可接受误差范围内，所述误差范围可部分取决于该值的测量或确定方式，或取决于测量系统的局限性。
本申请第一方面提供了一种生物合成虾青素的方法，其包括采用能够表达雨生红球藻来源的β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶的工程菌中实现虾青素的生物合成，其中，所述β-胡萝卜素羟化酶包含G135L点突变。发明人发现，所述点突变使得虾青素的产量提高2.8倍。
在一些实施方式中，所述包含G135L点突变的β-胡萝卜素羟化酶具有如SEQ ID NO.10所示的氨基酸序列。
在一些实施方式，所述工程菌能够合成β-胡萝卜素。对于能够合成β-胡萝卜素的工程菌，为本领域的常用工程菌，例如在宿主菌中引入番茄红素环化酶(CrtYB)和植物烯去饱和酶(CrtI)，构建可合成β-胡萝卜素的工程菌株，本申请对此不做限定。本申请对所述工程菌的种 类不做限定，例如可以是大肠杆菌、酿酒酵母、解脂耶氏酵母等；在一些实施方式，所述工程菌为解脂耶氏酵母。
在一些实施方式中，所述方法还包括利用支架蛋白将β-胡萝卜素酮醇酶和包含G135L点突变的β-胡萝卜素羟化酶共区域化；可以理解的是，本申请中，所述“支架蛋白”指的是能够与多个蛋白互相作用或结合，形成复合体，从而将所述蛋白(以复合体的形式)定位在特定区域(共区域化)的蛋白，其可以包括通过成对的相互作用的蛋白质、蛋白结构域或多肽链构建的蛋白支架，通过自组装蛋白质构建的蛋白支架或通过借助化学修饰蛋白构建的蛋白支架等。所述支架蛋白的一些示例非限制性举例包括由GBD及其配体组成的支架蛋白、由SH3及其配体组成的支架蛋白、由PDZ及其配体组成的支架蛋白等。
在一些实施方式中，所述支架蛋白包括鼠源PSD95/DlgA/Zo-1区域(PDZ)和PDZ配体(PL)；优选地，所述鼠源PSD95/DlgA/Zo-1区域(PDZ)具有SEQ ID NO.6所示的氨基酸序列；所述PDZ配体具有SEQ ID NO.8所示的氨基酸序列。
在一些实施方式中，所述支架蛋白连接的β-胡萝卜素酮醇酶和包含G135L点突变的β-胡萝卜素羟化酶的比例为(1-4):1；可以理解的是，所述比例为分子个数之比，例如可以是1:1、2:1、3:1或4:1。
在一些实施方式中，所述β-胡萝卜素酮醇酶与PSD95/DlgA/Zo-1区域(PDZ)融合表达；所述包含G135L点突变的β-胡萝卜素羟化酶与PDZ配体融合表达；优选地，所述包含G135L点突变的β-胡萝卜素羟化酶与1-4个所述PDZ配体分子融合表达；优选地，所述包含G135L点突变的β-胡萝卜素羟化酶与3个所述PDZ配体分子融合表达。
发明人发现，采用本申请的PDZ及其配体构成的支架蛋白，能够以可设计的方式在空间上招募特定的蛋白(如本申请的β-胡萝卜素酮醇酶和包含G135L点突变的β-胡萝卜素羟化酶)，将HpCrtW和HpCrtZ3(包含G135L点突变的β-胡萝卜素羟化酶)两个蛋白按不同比例进行弹性“捆绑”；设计思路如图1所示。利用所述蛋白支架连接HpCrtW和HpCrtZ3能够进一步提高HpCrtW和HpCrtZ3的协同催化效率。进一步地，发明人发现，当支架蛋白连接的HpCrtW和HpCrtZ3比例为1:1、3:1、4:1时，虾青素产量相比于未采用支架蛋白连接时均有所提高，尤其当支架蛋白连接的HpCrtW和HpCrtZ3比例为3:1时，虾青素含量最高，相较于无蛋白支架的对照，虾青素含量提高了3.0倍。
在一些实施方式中，所述β-胡萝卜素酮醇酶与所述PDZ可以直接连接或通过linker(例如:(GGGGS)*3(SEQ ID NO.42))连接。
在一些实施方式中，所述β-胡萝卜素酮醇酶与PDZ融合表达的蛋白具有SEQ ID NO.14所示的氨基酸序列。
在一些实施方式中，所述β-胡萝卜素羟化酶与所述PDZ配体分子通过linker连接；优选地，所述linker为(GGGS)n，n为1-4的整数。
在一些实施方式中，所述包含G135L点突变的β-胡萝卜素羟化酶通过linker依次与多个PDZ配体分子连接；优选与三个PDZ配体分子连接。
在一些实施方式中，所述包含G135L点突变的β-胡萝卜素羟化酶与PDZ配体融合表达的蛋白具有SEQ ID NO.12所示的氨基酸序列。
本申请第二方面提供了一种生物合成虾青素的方法，其包括采用能够表达β-胡萝卜素酮 醇酶和β-胡萝卜素羟化酶的工程菌中实现虾青素的生物合成，其中，所述β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶通过支架蛋白共区域化。
在一些实施方式中，所述支架蛋白包括鼠源PSD95/DlgA/Zo-1区域(PDZ)和PDZ配体(PL)；优选地，所述鼠源PSD95/DlgA/Zo-1区域(PDZ)具有SEQ ID NO.6所示的氨基酸序列；所述PDZ配体具有SEQ ID NO.8所示的氨基酸序列。
在一些实施方式中，所述β-胡萝卜素酮醇酶与PSD95/DlgA/Zo-1区域(PDZ)融合表达；所述β-胡萝卜素羟化酶与PDZ配体融合表达；优选地，所述β-胡萝卜素羟化酶与1-4个所述PDZ配体分子融合表达。
在一些实施方式中，所述β-胡萝卜素羟化酶与PDZ配体通过linker连接；所述β-胡萝卜素羟化酶与所述PDZ配体分子通过linker连接；优选地，所述linker为(GGGS)n，n为1-4的整数。
在一些实施方式中，所述β-胡萝卜素羟化酶通过linker依次与多个PDZ配体分子连接，优选为3个PDZ配体分子连接。
本申请中，所述linker可以理解为接头序列，即用于连接两个蛋白和/或核酸分子的一段氨基酸或多核苷酸片段，linker的存在通常用于减少空间位阻对其连接的两个分子功能的影响。
在一些实施方式中，所述β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶可以是用于制备虾青素的任何来源的β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶，例如可以是雨生红球藻来源、佐夫色绿藻(Chromchloris zofingiensis)来源、副球菌(Paracoccus sp.)来源、菠萝泛菌(Pantoea ananatis)来源、鞘氨醇单胞菌(Sphingomonas sp.)来源、短波单胞菌(Brevundimonas sp.)来源等；优选地，是雨生红球藻来源，其合成虾青素产量高、产生更少的中间代谢物。
在一些实施方式中，所述β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶可以是经过优化的，例如通过密码子优化、点突变等技术，使其具有更高的催化活性、稳定性等。
在一些实施方式中，所述工程菌能够合成β-胡萝卜素。
在一些实施方式中，所述工程菌为解脂耶氏酵母。
本申请第三方面提供了一种包含G135L点突变的β-胡萝卜素羟化酶，其具有SEQ ID NO.10示的氨基酸序列。
本申请第四方面提供了包含G135L点突变的β-胡萝卜素羟化酶-PDZ配体融合蛋白，其具有SEQ ID NO.12所示的氨基酸序列。
本申请第五方面提供了β-胡萝卜素酮醇酶-PDZ融合蛋白，其具有SEQ ID NO.14所示的氨基酸序列。
本申请第六方面提供了编码本申请第三方面的包含G135L点突变的β-胡萝卜素羟化酶、本申请第四方面或第五方面的融合蛋白的多核苷酸分子。
在一些实施方式中，编码包含G135L点突变的β-胡萝卜素羟化酶的多核苷酸分子具有SEQ ID NO.9所示的核苷酸序列。
在一些实施方式中，编码包含G135L点突变的β-胡萝卜素羟化酶-PDZ配体融合蛋白的多核苷酸分子具有SEQ ID NO.11所示的核苷酸序列。
在一些实施方式中，编码β-胡萝卜素酮醇酶-PDZ融合蛋白的多核苷酸分子具有SEQ ID  NO.13所示的核苷酸序列。
本申请第七方面提供了一种核酸构建体，其包括本申请第六方面的多核苷酸分子。
本申请第八方面提供了一种生产虾青素的工程菌，其包含本申请第六方面的多核苷酸分子或本申请第七方面的核酸构建体的至少一种；优选的，其能够表达本申请第三方面的包含G135L点突变的β-胡萝卜素羟化酶、或本申请第四方面或第五方面的融合蛋白的至少一种；优选地，所述工程菌表达本申请第四方面和第五方面的融合蛋白；优选地，所述工程菌能够合成β-胡萝卜素；优选的，所述工程菌为解脂耶氏酵母。
在一些实施方式中，所述工程菌为保藏编号为CCTCC M 2022882或CCTCC M 2022883的解脂耶氏酵母。
下面通过具体实施例来说明本申请的生物合成虾青素的方法及载体。下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。以下实施例中所涉及的质粒均为本领域技术人员公知质粒。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1点突变的筛选
1.1点突变载体构建
分别对HpCrtW和HpCrtZ进行蛋白质结构预测(https://zhanggroup.org/I-TASSER/)。以获得得分最高的蛋白模型为基础，在DeepDDG服务器(http://protein.org.cn/ddg.html)预测HpCrtW和HpCrtZ蛋白各种点突变后稳定性的变化。依据突变体稳定性的评分，各选择评分前五的突变蛋白，分别是HpCrtWD130L、HpCrtWA137F、HpCrtWG148L、HpCrtWK185F、HpCrtWK185L，以及HpCrtZG183L、HpCrtZG123L、HpCrtZG135L、HpCrtZG135F和HpCrtZG135I。前期研究表明，HpCrtW和HpCrtZ同时在产β-胡萝卜素的解脂耶氏酵母工程菌株BC中表达时，HpCrtW优先利用β-胡萝卜素合成海胆烯酮(echinenone)以及角黄素，而海胆烯酮和角黄素再在HpCrtZ的作用下分别形成羟化海胆酮(hydroxyechinenone)和绿蝇黄质(phoenicoxanthin)，最后再分别被HpCrtW和HpCrtZ催化形成虾青素(Zhu,H.-Z.,et al.,2022)。
根据预测结果选择可能提高蛋白稳定性的氨基酸进行点突变。首先以含有编码HpCrtW野生型蛋白基因序列(SEQ ID NO.1)的质粒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.)为模板，分别用HpCrtW-F/HpCrtWD130L-R1和HpCrtWD130L-F2/HpCrtW-R两对引物进行PCR扩增。然后扩增产物按1：1的摩尔比混合，以该混合物作为模板用引物HpCrtW-F/HpCrtW-R再次扩增，回收扩增产物，通过Gibson Assembly试剂盒(赛默飞)构建到pYLXP'2(Lv,Y.K.,et al.,Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica,ACS synthetic Biology,2019.8:p.568-576)载体上。最后以CX-F/CX-R为通用测序引物，测序正确则表明成功获得点突变载体pYLXP'2::HpCrtWD130L。HpCrtW和HpCrtZ蛋白的其他点突变过程和命名方式同上。
表1：HpCrtW和HpCrtZ蛋白点突变过程中使用的引物信息：
表2：HpCrtW和HpCrtZ蛋白点突变过程PCR扩增体系
表3：HpCrtW和HpCrtZ蛋白点突变过程PCR扩增程序
1.2 HpCrtW点突变筛选
将测序正确的、含有突变蛋白的上述载体，通过酵母转化分别转入可以合成β-胡萝卜素(β-carotene)的解脂耶氏酵母工程菌株BC(ZHU Hang-zhi,J.S.et al.,Improving the Biosynthesis of β-Carotene in<i>Yarrowia lipolytica</i>by Introducing an Artificial Isopentenol Utilization Pathway.China Biotechnology,2021.41(4):p.37-46.)中。通过高效液相色谱检测角黄素(canthaxanthin)、玉米黄质(zeaxanthin)、虾青素等类胡萝卜素。
酵母转化步骤如下：
(1)在YPD平板上划线活化产β-胡萝卜素的解脂耶氏酵母工程菌株BC，并在28℃下培养16-22h。
(2)准备酵母转化缓冲体系100μL：无菌50％PEG6000，90μL；2M醋酸锂，5μL；鲑鱼精DNA，5μL。鲑鱼精DNA应100℃预热5分钟并冷却后再加入转化缓冲液中。鲑鱼精DNA应100℃预热5分钟并冷却后再加入转化缓冲液中。
(3)使用200μL无菌移液器吸头，从过夜的YPD平板上刮取适量“酵母菌苔”并转移到含有酵母转化缓冲液的1.5mL无菌EP管中，涡旋混合至少10s。
(4)分别加入上述包含HpCrtW点突变的载体质粒(0.2～0.5μg DNA)，每个样品涡旋10s充分混合。
(5)将转化混合物在30℃下孵育30-45分钟，每10分钟涡旋转化混合物15s，继续在39℃下额外热激10min以提高转化效率。
(6)加入200μL无菌去离子水稀释。
(7)将100μL稀释的细胞接种到酵母选择性培养基CSM-Ura上，并置于28-30℃培养48h。
类胡萝卜素提取与检测：
经酵母转化获得的转化子经28℃摇瓶发酵48h后，收集10mL发酵液至10mL EP管中，4000rpm离心10min后去上清，并加入2.5g直径为3-4mm的玻璃珠和5mL含0.02％3,5-二叔-4-丁基羟基甲苯(BHT)的乙酸乙酯。BHT用于预防类胡萝卜素氧化。用涡旋振荡仪2500rpm震荡30-50min，使细胞破碎释放类胡萝卜素，此时类胡萝卜素溶于乙酸乙酯中，4000rpm离心5min后用0.2μm疏水过滤器过滤两次转移至带内插管的棕色液相小瓶。
类胡萝卜素采用高效液相色谱法(HPLC，安捷伦技术公司，美国)进行定量检测。采用了安捷伦1200系列LC系统，配备了YMC类胡萝卜素C30柱(250mm×4.6mm i.d。×5μm厚度)。所使用的流动相位和梯度如下。最初，使用洗脱液A(81％甲醇，15％MTBE，4％水)在1mL/min的流速下使用50-70min，并逐渐从100降至60％。同时，洗脱液B(7％甲醇，90％MTBE和3％水)从0增加到40％。整个过程在50-70min内完成，在450nm处检测所有类胡萝卜素的吸光度。在上述相同条件下完成β-胡萝卜素、虾青素、玉米虾黄质和玉米黄质标准曲线绘制。基于标准曲线计算样品中真实类胡萝卜素含量。
其中，包含野生型HpCrtW(氨基酸序列如SEQ ID NO.2所示)和具有不同点突变的HpCrtW的工程菌中，角黄素(Can)含量结果如图2所示(W*代表野生型，每个平板挑取多个克隆)，结果表明，与原始HpCrtW相比，5个设计的HpCrtW突变位点均造成角黄素含量的下降，说明突变后的HpCrtW活性减弱或丧失。该结果也说明了D130、A137、G148这几个位点对HpCrtW活性十分重要。
1.3 HpCrtZ点突变筛选
进一步的，采用与1.2相同的酵母转化的方法，将野生型HpCrtZ(Z*)(核苷酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO.4所示)以及5个突变的HpCrtZ分别与野生型HpCrtW以及点突变HpCrtWK185F共转化并在解脂耶氏酵母工程菌株BC中共表达，并采用1.2的方法检测工程菌中虾清素(Ast)的产量，结果如图3所示。结果显示，相较于野生型HpCrtZ和野生型HpCrtW的组合(W*Z*)，HpCrtZG135L与野生型HpCrtW的组合表达(W*Z-G135L)，可明显提高工程菌株中虾青素的产量，最高提高了约2.8倍。为表述方便，本申请中将突变体HpCrtZG135L命名为HpCrtZ3，下文中，HpCrtZG135L和HpCrtZ3可以替换。其中，本实施例中获得的HpCrtZG135L与野生型HpCrtW的组合表达(W*Z-G135L)的菌株中，虾青素的产量最高的菌株命名为pHpWZ3，于2022年6月14日保藏于中国典型培养物保藏中心(简称CCTCC，地址为中国，武汉，武汉大学)，保藏号为CCTCC M 2022882。
实施例2采用支架蛋白提高虾青素产量
首先设计将HpCrtW(SEQ ID NO.1)和PDZ(SEQ ID NO.5)的核酸序列用一段接头序列(SEQ ID NO.41)连接起来，HpCrtZ3的核酸序列(SEQ ID NO.9)则利用与上相同的接头序列分别与1至4个PL核酸序列(SEQ ID NO.7)连接，其中，与3个PL连接的核酸序列见SEQ ID NO.11，所有片段的两端设计一段同源臂，其中，上下游同源臂的示例性核苷酸序列分别如SEQ ID NO.43和SEQ ID NO.44所示，方便后期与载体pYLXP'2利用同源重组的方式连接。五个设计好的序列送通用生物系统(安徽)有限公司合成，合成片段通过Gibson Assembly试剂盒构建到pYLXP'2载体上，获得的载体依次命名为pYLXP'2::HpCrtW-PDZ、pYLXP'2::HpCrtZ3-PL*1、pYLXP'2::HpCrtZ3-PL*2、pYLXP'2-HpCrtZ3-PL*3和pYLXP'2-HpCrtZ3-PL*4。用限制性内切酶ClaI和NheI线性化pYLXP'2::HpCrtW和pYLXP'2::HpCrtW-PDZ，用限制性内切酶Avr II和ClaI线性化pYLXP'2::HpCrtZ3(即实施例1的pYLXP'2::HpCrtZG135L)、pYLXP'2::HpCrtZ3-PL*1、pYLXP'2::HpCrtZ3-PL*2、pYLXP'2-HpCrtZ3-PL*3和pYLXP'2-HpCrtZ3-PL*4。再通过T4连接酶连接pYLXP'2载体和上述获得的不同片段，分别构建以下表达载体：pYLXP'2::HpCrtW::HpCrtZ3(n0)、 pYLXP'2::HpCrtW-PDZ::HpCrtZ3(n0(W-PDZ))、pYLXP'2::HpCrtW::HpCrtZ3-PL*1(n0(Z-PL))、pYLXP'2::HpCrtW-PDZ::HpCrtZ3-PL*1(n1)、pYLXP'2::HpCrtW-PDZ::HpCrtZ3-PL*2(n2)、pYLXP'2::HpCrtW-PDZ::HpCrtZ3-PL*3(n3)和pYLXP'2::HpCrtW-PDZ::HpCrtZ3-PL*4(n4)。
酶切验证正确后，采用与实施例1的1.2中相同的酵母转化方法，将上述表达载体转化入产β-胡萝卜素的解脂耶氏酵母工程菌株BC中，并采用与实施例1的1.2中相同类胡萝卜素提取与检测方法检测获得的菌株中虾青素的产量，结果如图4所示。
结果显示，当支架蛋白连接的HpCrtW和HpCrtZ3比例为1:1(n1)、3:1(n3)和4:1(n4)时，解脂耶氏酵母工程菌株合成的虾青素含量均有所提高，尤其是HpCrtW和HpCrtZ3比例为3:1时，相较于无蛋白支架的对照(n0)，虾青素含量提高了3.0倍。本实施例中获得的HpCrtW和HpCrtZ3比例为3:1的菌株命名为pHpWPDZZ3PL*3，于2022年6月14日保藏于中国典型培养物保藏中心(简称CCTCC，地址为中国，武汉，武汉大学)，保藏号为CCTCC M 2022883。
以上所述仅为本申请的较佳实施例，并不用以限制本申请，凡在本申请的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本申请保护的范围之内。

Claims (14)

  1. 一种生物合成虾青素的方法，其包括采用能够表达雨生红球藻来源的β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶的工程菌中实现虾青素的生物合成，其中，所述β-胡萝卜素羟化酶包含G135L点突变；优选地，所述工程菌为解脂耶氏酵母；优选地，所述工程菌能够合成β-胡萝卜素。
  2. 根据权利要求1所述的方法，其包括利用支架蛋白将β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶共区域化；优选地，所述支架蛋白包括鼠源PSD95/DlgA/Zo-1区域(PDZ)和PDZ配体；优选地，所述鼠源PSD95/DlgA/Zo-1区域(PDZ)具有SEQ ID NO.6所示的氨基酸序列；所述PDZ配体具有SEQ ID NO.8所示的氨基酸序列。
  3. 根据权利要求2所述的方法，其中，所述支架蛋白连接的β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶的比例为(1-4):1。
  4. 根据权利要求1所述的方法，其中，所述β-胡萝卜素酮醇酶与PSD95/DlgA/Zo-1区域(PDZ)融合表达；所述β-胡萝卜素羟化酶与PDZ配体融合表达；优选地，所述β-胡萝卜素羟化酶与1-4个所述PDZ配体分子融合表达；优选地，所述β-胡萝卜素羟化酶与PDZ配体通过linker连接；所述β-胡萝卜素羟化酶与所述PDZ配体分子通过linker连接；优选地，所述linker为(GGGS)n，n为1-4的整数。
  5. 一种生物合成虾青素的方法，其包括采用能够表达β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶的工程菌中实现虾青素的生物合成，其中，所述β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶通过支架蛋白共区域化；优选地，所述β-胡萝卜素酮醇酶和β-胡萝卜素羟化酶为雨生红球藻来源；优选地，所述工程菌为解脂耶氏酵母；优选地，所述工程菌能够合成β-胡萝卜素。
  6. 根据权利要求5所述的方法，其中，所述支架蛋白包括鼠源PSD95/DlgA/Zo-1区域(PDZ)和PDZ配体；优选地，所述鼠源PSD95/DlgA/Zo-1区域(PDZ)具有SEQ ID NO.6所示的氨基酸序列；所述PDZ配体具有SEQ ID NO.8所示的氨基酸序列。
  7. 根据权利要求6所述的方法，其中，所述β-胡萝卜素酮醇酶与PSD95/DlgA/Zo-1区域(PDZ)融合表达；所述β-胡萝卜素羟化酶与PDZ配体融合表达；优选地，所述β-胡萝卜素羟化酶与1-4个所述PDZ配体分子融合表达；优选地，所述β-胡萝卜素羟化酶与PDZ配体通过linker连接；所述β-胡萝卜素羟化酶与所述PDZ配体分子通过linker连接；优选地，所述linker为(GGGS)n，n为1-4的整数。
  8. 包含G135L点突变的β-胡萝卜素羟化酶，其具有SEQ ID NO.10所示的氨基酸序列。
  9. 包含G135L点突变的β-胡萝卜素羟化酶-PDZ配体融合蛋白，其具有SEQ ID NO.12所示的氨基酸序列。
  10. β-胡萝卜素酮醇酶-PDZ融合蛋白，其具有SEQ ID NO.14所示的氨基酸序列。
  11. 编码权利要求8-10中任一项所述的酶或融合蛋白的多核苷酸分子；优选地，所述多核苷酸分子具有SEQ ID NO.9、SEQ ID NO.11或SEQ ID NO.13所示的核苷酸序列。
  12. 一种核酸构建体，其包括权利要求11所述的多核苷酸分子。
  13. 一种生产虾青素的工程菌，其包含权利要求11所述的多核苷酸分子或权利要求12所 示的核酸构建体的至少一种；优选的，其能够表达权利要求8所述的包含G135L点突变的β-胡萝卜素羟化酶、权利要求9或10所述的融合蛋白的至少一种；优选地，所述工程菌表达权利要求9和10所述的融合蛋白；优选地，所述工程菌能够合成β-胡萝卜素；优选的，所述工程菌为解脂耶氏酵母。
  14. 根据权利要求13所述的工程菌，其为保藏编号为CCTCC M 2022882或CCTCC M 2022883的解脂耶氏酵母。
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