Intelligent design and optimization method of beta-carotene hydroxylase and its application in astaxanthin and shikonin biosynthesis

Abstract

The application discloses an intelligent design and optimization method of beta-carotene hydroxylase and application of the beta-carotene hydroxylase in astaxanthin and violaxanthin biosynthesis. Pantoea ananatis The beta-carotene hydroxylase is obtained by mutating CrtZ from The application establishes an enzyme-substrate complex model based on a consensus result of molecular docking and co-folding prediction, and uses a cross-species motif mode to guide the modification of key sites of CrtZ, thereby obtaining a series of mutants with high activity and low promiscuity. The mutants can improve the beta-carotene hydroxylation efficiency, reduce the generation of by-products, and promote the accumulation of target products in a yeast bottom astaxanthin synthesis system. Part of the combined mutants can reduce the by-product level by about 50% while maintaining the astaxanthin yield basically unchanged; in the violaxanthin biosynthesis pathway, the by-product level is reduced by about 70% while the main product yield is maintained. The application can be applied to the biosynthesis of astaxanthin, violaxanthin and other hydroxylated carotenoids.

Description

Technical Field

[0001] This invention relates to the fields of microbial technology and molecular biology, specifically to a method for the intelligent design and optimization of β-carotene hydroxylase and its application in the biosynthesis of astaxanthin and violetin. Pantoea ananatis The design method of CrtZ-related mutants of β-carotene hydroxylase and their application in the synthesis of 3,3'-hydroxylated carotenoids and downstream products astaxanthin and violetin in yeast. Background Technology

[0002] Carotenoids are a class of natural fat-soluble pigments widely found in plants, algae, fungi, and bacteria, possessing important physiological functions and broad application value. Among them, xanthophylls have attracted widespread attention due to their excellent antioxidant activity, biocompatibility, and nutritional and health benefits. Astaxanthin, violaxanthin, and zeaxanthin are the most representative high-value-added products, with broad market demand in food colorings, dietary supplements, animal feed, pharmaceuticals, and cosmetics.

[0003] β-Carotene hydroxylase (CrtZ) is a key enzyme in the biosynthetic pathway of carotenoids such as astaxanthin. It primarily catalyzes the hydroxylation of the 3' and 3' carbon atoms at the two ends of the β-carotene molecule, thereby generating hydroxylated carotenoid products such as zeaxanthin. Further synergistic action with β-carotene ketolase or zeaxanthin cyclooxygenase can achieve the biosynthesis of high-value carotenoids such as astaxanthin and violetin. Therefore, the catalytic performance of CrtZ directly affects the metabolic flux distribution and final product accumulation level of the entire carotenoid biosynthetic pathway.

[0004] Currently, industrially produced astaxanthin mainly comes from natural biological resources such as Haematococcus pluvialis, while zeaxanthin is primarily obtained through extraction from marigold petals. Violaxanthin, due to its role as a photosynthetic regulator in plants, suffers from instability and low content, and therefore lacks a stable commercial source. These lutein compounds generally suffer from long cultivation cycles, high production costs, significant susceptibility to environmental factors, and poor product quality stability, severely limiting their commercial production. With the development of synthetic biology and metabolic engineering technologies, the green biomanufacturing of carotenoids using engineered microorganisms, especially yeast cell factories, has become an important development direction. Compared to natural extraction methods, microbial fermentation methods offer advantages such as shorter production cycles, stable raw material sources, ease of large-scale scaling, and environmental friendliness.

[0005] However, in existing carotenoid production systems, CrtZ remains a significant factor limiting the accumulation of the target product. On the one hand, some CrtZ sources have limited catalytic activity, leading to insufficient conversion of β-carotene and its subsequent accumulation within cells. On the other hand, CrtZ exhibits a degree of catalytic heterogeneity; existing reports indicate that PaCrtZ can further over-oxidize intermediates that have already undergone hydroxylation, generating 3-(,3'-)ketoylation byproducts such as rhodoxanthin. These side reactions not only consume metabolic flux but also reduce the purity of the target product, increase subsequent separation and purification costs, and ultimately affect the economics of the entire biomanufacturing process.

[0006] Therefore, developing novel CrtZ mutants with both high catalytic activity and high substrate specificity to inhibit excessive oxidation side reactions while ensuring efficient conversion of β-carotene is of great significance for improving the biomanufacturing efficiency of carotenoid products such as astaxanthin and violetin. Summary of the Invention

[0007] To address the problems of insufficient catalytic efficiency, poor substrate specificity, and excessive byproduct formation in existing β-carotene hydroxylase technologies, this invention provides a smart design and optimization method for β-carotene hydroxylases and its application in the biosynthesis of astaxanthin and violetin. Based on this method, highly active β-carotene hydroxylase mutants with low heterogeneity can be obtained. Through further research... Pantoea ananatis By intelligently designing and directionally modifying the source CrtZ, a series of engineered enzyme mutants with both high catalytic efficiency and high reaction specificity were obtained, thereby achieving optimized regulation of the synthesis pathway of carotenoids such as astaxanthin.

[0008] This invention first utilizes protein structure prediction and molecular docking analysis techniques to study the interaction pattern between β-carotene and the CrtZ active site, identifying key amino acid residues in β-carotene hydroxylase that affect substrate binding and catalytic reactions. Subsequently, using a cross-species motif model (combined with evolutionary conservation analysis of β-carotene hydroxylases from different species), several key sites potentially affecting enzyme activity and substrate specificity were screened, and single-point mutations, fragment substitutions, and multi-site combination mutants were constructed targeting these sites. Through functional validation in astaxanthin and violetin production systems, the effects of different β-carotene hydroxylase mutants on β-carotene conversion efficiency, astaxanthin and violetin accumulation levels, and byproduct formation were systematically evaluated.

[0009] In a first aspect, the present invention provides a series of β-carotene hydroxylase mutants. The mutants are based on… Pantoea ananatisThe source CrtZ is the parent, and its amino acid sequence contains at least one or more single or multiple amino acid substitutions selected from Table 1 below: Table 1

[0010] Furthermore, the amino acid substitutions specifically refer to amino acid substitutions selected from the following key sites: A47P, V50L, V50M, Y54F, M83L, Y86F, and R121Q.

[0011] In some implementations, the mutant comprises a superposition combination of the above amino acid mutations, as shown in Table 2 below: Table 2

[0012] In a preferred embodiment, the mutant comprises the following core superposition combination: M83I+Y54F+V50L or M83I+V50L+A47P.

[0013] Furthermore, the β-carotene hydroxylase CrtZ encoding gene has undergone codon optimization, and its sequence is shown in SEQ ID NO. 1. All mutants in this application are obtained by mutation based on this sequence. The starting strains are a yeast strain that has been introduced with the complete astaxanthin biosynthetic pathway other than CrtZ, and a yeast strain that has been introduced with the complete violetin biosynthetic pathway other than CrtZ.

[0014] Secondly, the present invention provides a nucleic acid that encodes the aforementioned mutant.

[0015] Thirdly, the present invention provides a carrier for use in yeast cell factories, the carrier comprising the aforementioned nucleic acids.

[0016] Fourthly, the present invention provides a host cell that can functionally express the above-mentioned nucleic acid, or can be transformed and introduced into the above-mentioned vector to ultimately achieve functional expression.

[0017] Fifthly, the present invention provides a method for preparing astaxanthin, the method comprising using the above-mentioned nucleic acid, vector and host cell.

[0018] In a sixth aspect, the present invention provides a method for preparing violetin, the method comprising using the above-mentioned nucleic acid, vector and host cell.

[0019] In a seventh aspect, the present invention provides a smart design and optimization method for β-carotene hydroxylation with high activity and low heterogeneity, the method comprising the following steps: (1) Structural prediction and molecular docking: Obtain the three-dimensional structural model of β-carotene hydroxylase, and study the interaction mode between β-carotene and CrtZ active center through molecular docking and co-folding prediction, and identify the important amino acid residues in β-carotene hydroxylase that affect substrate binding and catalytic reaction. (2) Conservation analysis: Multiple sequence alignment was performed to analyze conserved and variable regions and screen candidate mutation sites by combining the evolutionary conservation of β-carotene hydroxylase from different species. (3) Rosetta screening: Mutation strategies were constructed for the candidate mutation sites, and the effects of each mutation strategy on the stability of the protein itself were evaluated. Mutation strategies with free energy change ΔΔG < 2 REU were screened to obtain candidate mutants. (4) Yeast validation: The candidate mutant was expressed in yeast strains (yeast strains that have been introduced into the complete biosynthetic pathway of astaxanthin except CrtZ and yeast strains that have been introduced into the complete biosynthetic pathway of violetin except CrtZ), and the accumulation levels of the target product and by-products were measured to screen out the optimal mutant, which is the optimized β-carotene hydroxylase.

[0020] Compared with the prior art, the present invention has the following beneficial effects: First, this invention focuses on the relatively low heterogeneity and high stability of β-carotene hydroxylase in plants. Through structure-guided protein engineering combined with sequence alignment, a series of novel and rational design schemes were proposed, resulting in a series of CrtZ mutants with excellent catalytic performance. These mutants provide new key functional elements for the metabolic engineering of carotenoids such as astaxanthin and violetin. Second, the mutants obtained in this invention significantly improve the conversion efficiency of β-carotene to hydroxylated products, effectively alleviating substrate accumulation problems and improving the overall throughput efficiency of metabolic pathways. Third, the mutants obtained in this invention reduce further oxidation of hydroxylated products, significantly reducing the formation of 3,3'-ketoylation byproducts and improving the purity of astaxanthin and violetin. Furthermore, through a multi-site synergistic optimization strategy, a balanced improvement in enzyme activity and reaction specificity was achieved, overcoming the problem that increased activity in traditional protein engineering is often accompanied by increased side reactions. Finally, the mutants obtained by this invention can be directly applied to the bio-manufacturing process of carotenoids in yeast and other industrial microbial chassis, and have important application value and industrialization prospects for the green and efficient production of astaxanthin, violetin, zeaxanthin and other 3,3'-hydroxylated carotenoids. Attached Figure Description

[0021] Figure 1 Conformation diagram of the CrtZ-β-carotene complex obtained by Autodock Vina docking.

[0022] Figure 2 Protenix predicts the conformation of the CrtZ co-folded complex with β-carotene.

[0023] Figure 3 Biosynthetic pathway of astaxanthin.

[0024] Figure 4 Figure showing the test results of the single-mutation CrtZ mutant in the astaxanthin synthesis chassis.

[0025] Figure 5 Figure showing the test results of the superimposed mutant in the astaxanthin synthesis chassis.

[0026] Figure 6 Biosynthetic pathway of violetin.

[0027] Figure 7 Figure showing the test results of the mutant in the violetin synthesis chassis. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] The β-carotene hydroxylase (CrtZ) described in this application belongs to the non-heme ferrous hydroxylase family. Encoded by the β-carotene hydroxylase gene, it belongs to the fatty acid desaturase superfamily and is widely found in plants, bacteria, and other organisms. In different species, β-carotene hydroxylase has different catalytic preferences and catalytic activities, and at the same time, it has high homology, providing space for trying mutation strategies for protein engineering.

[0030] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0031] Example 1: Experimental Materials and Methods Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, and instruments used in the following examples, unless otherwise specified, are all conventional materials, reagents, and instruments in the art and are commercially available.

[0032] The original yeast strain involved in this invention was preserved in our laboratory. (Genotype) HIS4 :: Cas9 pGAP- CrtE pGAP- CrtI pTEF- CrtYBpGAP- CrtW ; HIS4 :: Cas9 pGAP- CrtE pGAP- CrtI pTEF- CrtYB pGAP- ZEP ) The YPD medium used in this invention comprises: 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose. The LB medium used in this invention comprises: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L sodium chloride. An additional 20 g / L agar powder is added to the solid medium. Fermentation of the yeast strain is carried out in YPD medium, supplemented with 2% glucose every 24 h.

[0033] The yeast transformation method involved in this invention employs electroconversion, and during unit site integration, the amounts of gRNA and donor fragments added are 500 ng and 1000 ng, respectively.

[0034] In this embodiment, the extraction and quantitative analysis steps of carotenoids are as follows: 1. Extraction of carotenoids (1) Take 500 μL of the mixed fermentation broth, centrifuge at 12000 rpm for 1 min, and wash once with pure water.

[0035] (2) After removing the supernatant and draining the water, add 500 μL of acetone and about 40 μL of zirconium oxide grinding beads, and use a grinder to break the cells.

[0036] (3) Centrifuge the crushed sample at 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube for storage in the dark. Mix the supernatant with an equal volume of acetone, filter through a 0.22 μm nylon filter, and transfer to a liquid chromatography vial for high performance liquid chromatography (HPLC) analysis.

[0037] 2. Detection of carotenoid composition by high performance liquid chromatography The liquid chromatography system used in this invention is an Agilent Technologies 1260 Infinity II, with an Agilent Poroshell EC-C18 column, an ultraviolet absorption wavelength of 472 nm, and a flow rate of 0.4 mL / min. The mobile phase A is acetonitrile:water = 9:1 (v / v), and the mobile phase B is isopropanol:acetonitrile = 3:1 (v / v). The gradient elution program is as follows: 0 min, 100% A, 0% B; 15 min, 0% A, 100%, B; 26 min, 0% A, 100% B; 28 min, 100% A, 0% B; 35 min, 100% A, 0% B.

[0038] Example 2: CrtZ Mutation Targets and Construction of Mutation Strategy Library To obtain a β-carotene hydroxylase with higher catalytic activity and lower heterogeneity, this invention uses... Pantoea ananatis The study used CrtZ as the research subject for rational design. First, a three-dimensional structural model of CrtZ was obtained using protein structure prediction tools. Then, molecular docking analysis was performed using AutoDock Vina software with β-carotene as the ligand. Figure 1 Meanwhile, the advanced Protenix was used to predict the cofolding of the CrtZ and β-carotene complex. Figure 2 The binding mode of β-carotene in the enzyme active site was identified using a dual-model cross-validation approach. By analyzing the interaction between substrate and enzyme active site residues in different docking conformations, several key amino acid sites that may affect substrate recognition, binding stability, and catalytic reaction efficiency were identified.

[0039] Based on this, further collection of plant-derived β-carotene hydroxylase sequences, including those from grapes ( Vitis vinifera ),corn( Zea mays ),tomato( Solanum lycopersicum ) and honeysuckle ( Lonicera japonica ) and other species, and their relationship with Pantoea ananatis Multiple sequence alignment analysis was performed using CrtZ. By comparing conserved and variable regions of hydroxylases from different sources, the study focused on differences in amino acid residues located near substrate-binding pockets and catalytic channels, and used molecular docking results to screen for potential functional sites.

[0040] Based on the combined results of structural and sequence evolution analysis, several key sites, including Y54, R121, V50, M83, Y86, and A47, were identified as key targets for modification. Simultaneously, two sets of amino acid fragment substitution schemes were designed for regions that might affect substrate binding conformation and catalytic channel spatial structure. A series of single-point mutants and fragment substitution mutants were constructed using these strategies, as shown in Table 1, laying the foundation for further screening of high-performance CrtZ mutants.

[0041] Table 1. CrtZ Single-Point Mutants and Fragment Substitution Mutation Strategy Library

[0042] Example 3: Screening and Validation of Effective Targets for CrtZ Mutation Based on the mutation strategy selected in Example 2, to further improve screening efficiency, the RosettaCartesian_ddG function was used to evaluate the impact of mutations on protein stability. The change in free energy (ΔΔG) before and after mutation was calculated with the wild-type structure as a reference. Mutations with less impact on stability were preferentially selected for experimental verification. The results are shown in Table 2.

[0043] Table 2 Calculated values ​​of free energy changes before and after CrtZ mutation

[0044] A larger ΔΔG value indicates that the mutation makes the protein structure more unstable. Candidate mutation strategies with ΔΔG < 2 REU and two fragment substitution strategies were selected to construct mutants for functional verification. Each mutant was used to construct an expression vector using standard molecular cloning techniques and introduced into the aforementioned yeast host strain. This host strain already carries the complete astaxanthin biosynthetic pathway (…). Figure 3 However, the CrtZ step was not introduced to ensure that the experimental results accurately reflect the effects of each mutation on hydroxylation efficiency (target product generation) and byproduct generation.

[0045] After in vivo expression, the contents of astaxanthin, β-carotene, and 3-ketoylation byproducts were measured. The peak areas of the main product and byproducts in wild-type CrtZ were 817.75 and 122.55, respectively. The results showed that different mutation sites had significant differences in substrate transformation efficiency and byproduct formation. Results for other mutation types are as follows... Figure 4Different mutation sites significantly affected the hydroxylation efficiency of β-carotene and the level of byproduct formation. Mutants such as LLYFM88-92MAYMF, M83I, Y86F, and Y54F showed a slight decrease in main product yield compared to the control system, but a significant decrease in byproduct levels, exhibiting a clear side reaction inhibition effect. This indicates that these mutations primarily optimize product profile distribution by reducing the flux of non-specific oxidation reactions. Mutants such as V50L, A47P, V50M, and R121Q showed increased main product yield, but were accompanied by a significant increase in byproducts. In some mutants, the byproduct level even exceeded the main product accumulation, indicating that while enhancing catalytic activity, these mutations reduced catalytic specificity and introduced a strong side reaction flux. Overall, the M83I mutant exhibited the best comprehensive performance, significantly reducing byproduct formation levels without affecting the main product.

[0046] Example 4: Mutation Target Stacking Test The properties of each mutation were determined in Example 3. Mutations that significantly increased β-carotene accumulation were removed, and the remaining mutations with the best effects were selected as beneficial mutations. These beneficial mutations were divided into two categories: mutations that reduced heterogeneity, accompanied by an increase in β-carotene accumulation within the acceptable range; and mutations that increased activity, resulting in a decrease in β-carotene accumulation, accompanied by an increase in byproducts. To reduce side reaction activity while maintaining high catalytic activity, various combinations of these two mutations were constructed to create related superimposed mutants. These mutants were introduced into the aforementioned yeast starting strain for testing and shake-flask fermentation. In this round of testing, the peak areas of the main product (astaxanthin) and its over-oxidized byproducts produced by wild-type CrtZ were 675.50 and 109.73, respectively. Other combination screening results are as follows: Figure 5 .

[0047] Through superposition testing, mutants that significantly reduced excessive oxidation byproducts while maintaining no significant decrease in astaxanthin content were obtained. In combination tests, the mutants M83I+A47P, M83I+V50M, M83I+V50L, M83I+R121Q, M83I+V50L+Y54F, and M83I+V50L+A47P showed superior effects, further improving byproduct distribution while maintaining high product levels. Among them, the optimal combination M83I+Y54F+V50L mutant reduced byproduct levels by approximately 50% compared to the control system, demonstrating significant potential for engineering applications.

[0048] Example 5: Testing of mutants in violetin biosynthesis Several mutants with low heterobiological activity were obtained in Example 4. Since violetin can also be over-oxidized by CrtZ, the above mutants were attempted for the biosynthesis of violetin. The above mutants (M83I+V50L+Y54F, M83I+V50L+A47P) were introduced into the violetin completion pathway (excluding CrtZ) that had been introduced. Figure 6 A yeast strain was selected and shake-flask fermentation tests were conducted. For example... Figure 7 As shown, compared with the control strain expressing wild-type CrtZ, the reduction in violetin yield (expressed as peak area) of all mutants was no more than 20%, while the content of excessive oxidation byproducts was reduced by about 70%. The content of violetin as the main product was effectively accumulated, indicating that the combined mutants can significantly inhibit excessive oxidation side reactions while maintaining a high level of violetin accumulation, showing application potential in the biosynthesis of different lutein compounds.

[0049] The scope of this invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this invention are also within the scope of this invention. All equivalent transformations and modifications made without departing from the conceptual framework of this invention are covered within its scope.

Claims

1. A β-carotene hydroxylase, characterized in that, From Pantoea ananatis The β-carotene hydroxylase was used as the parent, and the parent was mutated to obtain the product. The mutation includes at least one amino acid substitution selected from the following sites: Y54, V50, M83, Y86, R121 or A47, or includes at least one amino acid segment substitution: AGMTAY81-86 and LLYFM88-92.

2. The β-carotene hydroxylase according to claim 1, characterized in that, The specific amino acid substitutions are: Y54F, V50L, V50M, M83I, Y86F, R121Q, or A47P; the specific amino acid fragment substitutions are: AGMTAY81-86LGITVF or LLYFM88-92MAYMF.

3. The β-carotene hydroxylase according to claim 2, characterized in that, The mutation is a combination of two amino acid substitutions: M83I+A47P, M83I+V50M, M83I+V50L, and M83I+R121Q.

4. The β-carotene hydroxylase according to claim 2, characterized in that, The mutation includes any of the following combinations of amino acid mutations: 1) M83I + Y54F + V50L; 2) M83I + V50L + A47P.

5. A nucleic acid encoding the β-carotene hydroxylase as described in any one of claims 1-4.

6. A vector comprising the nucleic acid as described in claim 5.

7. A host cell capable of functionally expressing the nucleic acid as described in claim 5, or capable of functionally expressing it by conversion into the vector as described in claim 6.

8. The application of the β-carotene hydroxylase as described in any one of claims 1-4 in the biosynthesis of astaxanthin and violetin.

9. A method for intelligent design and optimization of β-carotene hydroxylase, characterized in that, Includes the following steps: 1) Structural prediction and molecular docking: Obtain a three-dimensional structural model of β-carotene hydroxylase, and identify key amino acid sites in β-carotene hydroxylase that affect substrate recognition, binding stability and catalytic reaction efficiency through molecular docking and cofolding prediction. 2) Conservation analysis: Multiple sequence alignment was performed on β-carotene hydroxylase sequences from different species to analyze conserved and variable regions and identify candidate mutation targets. 3) Rosetta screening: The effect of candidate mutation strategies on the stability of β-carotene hydroxylase was evaluated, and mutation strategies with free energy change ΔΔG < 2 REU were screened to obtain candidate mutants; 4) Yeast validation: The candidate mutants selected in step 3) are introduced into yeast carrying the biosynthetic pathways of astaxanthin and violetin, and the accumulation of target products and by-products is measured for validation, and better mutants are screened out.

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