Flavone 3beta-hydroxylase mutant and coenzyme mutant thereof and application thereof

Abstract

The application provides flavone 3beta-hydroxylase mutants and coenzyme mutants thereof, and is used for catalyzing synthesis of eriodictyol. Specifically, the application obtains flavone 3beta-hydroxylase mutants and coenzyme mutants thereof with an overall 10-fold performance improvement through artificial intelligence and high-throughput screening, and the mutants can be used to catalyze naringenin to produce 30g / L of eriodictyol with a yield of more than 95%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of synthetic biology and protein engineering, and particularly relates to flavone 3β-hydroxylase mutants and coenzyme mutants thereof and application thereof. BACKGROUND

[0002] Eriodictyol is a polyphenolic flavonoid compound widely distributed in fruits and vegetables, especially lemons and citrus, and has the effects of antioxidant, anti-inflammatory, analgesic, improvement of diabetes and diabetic complications. At present, eriodictyol is mainly obtained by extraction. Due to the too low content of plants, the extraction cost is high. For example, Chinese invention patent CN201410833103.6 provides a technology for extracting eriodictyol from eleocharis tuberosa peel, but only 20 mg of eriodictyol can be extracted from 8 kg of eleocharis tuberosa peel.

[0003] Although eriodictyol can be synthesized from tyrosine or naringenin through microbial fermentation or biocatalysis, the conversion rate and yield are not ideal, and the highest fermentation level is 75% conversion rate and 3.3 g / L (Gao, S., et al. ACS Synth. Biol. 2020, 9, 3288-3297). This is mainly because the flavone 3β-hydroxylase and its coenzyme have poor performance.

[0004] Therefore, there is still a lack of strategies for large-scale production of eriodictyol, making the price of eriodictyol high. In summary, there is an urgent need in the art to provide a method for large-scale production of eriodictyol. SUMMARY

[0005] In order to obtain flavone 3β-hydroxylase and its coenzyme with good performance, the present application obtains flavone 3β-hydroxylase mutants and coenzyme mutants thereof with more than 10 times performance improvement through artificial intelligence assisted directed evolution.

[0006] The present application uses the Saccharomyces cerevisiae lysate converted by the flavone 3β-hydroxylase mutant and its coenzyme mutant to catalyze the production of 30 g / L of eriodictyol with a conversion rate of 95%, which is much higher than the highest value (75% conversion rate and 3.3 g / L) reported at present. This yield has met the production requirements of eriodictyol, and has great significance for promoting the large-scale industrial production of eriodictyol.

[0007] In a first aspect, the present application provides a flavone 3β-hydroxylase mutant HaF3'H-M5, characterized in that the amino acid sequence of the flavone 3β-hydroxylase mutant is shown as SEQ ID NO: 1.

[0008] In another preferred embodiment, the coding gene (HaF3'H-M5) of the flavone 3β-hydroxylase mutant has a nucleotide sequence shown as SEQ ID NO: 2.

[0009] In a second aspect of the present application, a flavone 3β-hydroxylase coenzyme mutant HaCPR-M3 is provided, wherein the amino acid sequence of the flavone 3β-hydroxylase coenzyme mutant is shown as SEQ ID NO: 3.

[0010] In another preferred embodiment, the nucleotide sequence of the coding gene (HaCPR-M3) of the flavone 3β-hydroxylase coenzyme mutant is shown as SEQ ID NO: 4.

[0011] In a third aspect of the present application, a recombinant strain is provided, wherein the recombinant strain expresses the flavone 3β-hydroxylase mutant HaF3'H-M5 of the first aspect of the present application, and / or the coenzyme mutant HaCPR-M3 of the second aspect of the present application.

[0012] In another preferred embodiment, the recombinant strain expresses the flavone 3β-hydroxylase mutant HaF3'H-M5 of the first aspect of the present application, and the coenzyme mutant HaCPR-M3 of the second aspect of the present application.

[0013] In a fourth aspect of the present application, a method for producing eriodictyol is provided, wherein the reaction is carried out in the presence of the lysate of the recombinant strain of the third aspect of the present application, with naringenin as the substrate, to obtain the eriodictyol.

[0014] In another preferred embodiment, the reaction comprises the following steps:

[0015] In the presence of the lysate of the recombinant strain, naringenin is 3'-hydroxylated in the presence of air, to obtain the eriodictyol.

[0016] In another preferred embodiment, the method further comprises carrying out the reaction as described above in the presence of glucose, phosphate, NADP+ and GDH.

[0017] In another preferred embodiment, the method comprises the following steps: in the presence of glucose, phosphate, NADP+ and GDH, a solution containing naringenin is added dropwise into a basic system in the presence of air, to obtain the eriodictyol.

[0018] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described in the following (e.g., the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. DETAILED DESCRIPTION

[0019] The present application first obtains flavone 3beta-hydroxylase and coenzyme for catalyzing efficient synthesis of eriodictyol from naringenin, and then obtains flavone 3beta-hydroxylase mutant and coenzyme mutant with improved performance through artificial intelligence assisted directed evolution technology, so as to realize high-yield synthesis of eriodictyol. Specifically as follows:

[0020] Search for flavone 3beta-hydroxylase and coenzyme on Uniprot website to obtain flavone 3beta-hydroxylase and coenzyme sequence from sunflower (Helianthus Annuus) (Uniprot ID: A0A251SE34 and A0A251TTX6). After codon optimization of R26-V505 of Uniprot ID: A0A251SE34 and G98-W707 of Uniprot ID: A0A251TTX6, they are inserted into EcoRI and SacI and BamHI and XhoI of pESC-His respectively to obtain plasmid pESC-His_HaF3'H_HaCPR. Then, HaF3'H and HaCPR in pESC-His_HaF3'H_HaCPR are subjected to 5 rounds and 3 rounds of artificial intelligence assisted directed evolution to obtain HaF3'H-M5 and HaCPR-M3 respectively. The final plasmid pESC-His_HaF3'H-M5_HaCPR-M3 catalyzes the production of eriodictyol in the lysate of transformed Saccharomyces cerevisiae, which is 10 times that of the original plasmid.

[0021] The amino acid sequence of the flavone 3beta-hydroxylase mutant HaF3'H-M5 obtained by the present application is shown in SEQ ID NO: 1:

[0022] MRHSKRLPPGPAPWPIVGNLPHLSTLPHHSLTALAKKYGPLMHLRFGFVDVVVVTSASVAAQIIKVHDVNFASRPPNSGAKEMAVNYHDLVWAPYGPRWRMLRKICVLNLFSAKALANFRHVRQEEVAKLTRALVGAGQSTVRLGQLINVCTTNALARVLLGRRVFDDSDPKANEFKEMVIELLRLGAEFNIGDFIPALDWLDLQGITKKMKKLHDRFDSFINVIFEEHKSANRGEPHHVDLLTTLKNDVDGEGSKLSDIEIKALYLNLFTAGTDPTNSTVEWAIAELIRHPQILKQAQKELDIVVGQNRLVTELDLGQLTIIQAITKETFRLCPPSAIVLPRIASESCEVDKYYIPKGSMLLFNLWAIARDPKLWTDPLEFKPTRFLPGGEKSNVDIRGNDFEVMPFGAARRMCVGMSLGLRMTQLLIATLVQSFDWELANGLEPEKLNMDESYGITLEKAEPLMVHPKPRLAPHVYQCV*

[0023] The nucleotide sequence of its encoding gene (HaF3'H-M5) is shown as SEQ ID NO: 2:

[0024]

[0025] The amino acid sequence of the flavone 3β-hydroxylase coenzyme mutant HaCPR-M3 obtained by the application is shown as SEQ ID NO: 3:

[0026] MGKKKVTVFFGTQTGTAEGFAKALVEEAKARYEKAVFKVIDLDDYAADDEEYEEKLKKESLAFFFLATYGDGEPTDNAARFYKWFTEGEEKGEWLTKLQYGVFGLGNRQYEYFNKIAKVVDDGLTEQGAKRLVPVGLGDDDQCIEDDFTAWKELVWPELDQLLRDEDDTTVATPYTAAVSEYRVVFHDKPDMFAEDQSMTNGHAVHDAQHPCRSNVAVKKELHTPESDRSCTHLEFDISNTGLTYETGDFVGVYCENLSEVVDEAERLVGLPPDTYFSVHTDNEDGTPLSGASLPPPFPPCTLRKALACYADVLSSPKKSALLALAAHATDPSEADRLKFLASPAGKDEYSQWIVASHRSLLEVMEAFPSAKPPLGVFFASVAPRLQPRYNTISSSPKMAPNRIHVTCALVYEKTPAGRVHKGVCSTWMKNAVPMTENQDCSWAPVYVRTSNFRLPSDPTVPVIMIGPGTGLAPFRGFLQERLALKEAGTELGSSVLFFGCRNRKVDFIYENEFNNFVESGALSELIVAFSREGPTKEYVQHKMSEKASDIWNLLSEGAYLYVCGNAKGMAKDVHRTLHTIVQEQGSLDSSKAELYVKDLQMSGRYLRDVW*

[0027] The nucleotide sequence of the encoding gene (HaCPR-M3) is shown as SEQ ID NO: 4:

[0028]

[0029] In the present application, in a preferred embodiment, the amino acid sequence shown in SEQ ID NO: 1 and 3 can have substitution, deletion, and / or addition of one or several amino acid residues, provided that the function is not affected, and such functionally equivalent derivative proteins also fall within the scope of the present application.

[0030] In the present application, in a preferred embodiment, the nucleotide sequence shown in SEQ ID NO: 2 can have substitution, deletion, and / or addition of one or several nucleotides, provided that the function is not affected, and such functionally equivalent nucleotide sequences also fall within the scope of the present application.

[0031] In addition, the recombinant bacteria containing the coding gene also fall within the scope of the present application.

[0032] Abbreviations

[0033] YPD: yeast extract peptone dextrose medium

[0034] YPG: yeast extract peptone galactose medium

[0035] SD / -His: yeast minimal medium lacking histidine

[0036] FMN: riboflavin-5-phosphate

[0037] FAD: flavin adenine dinucleotide

[0038] NADPH: nicotinamide adenine dinucleotide phosphate

[0039] NADP+: oxidized form of reduced coenzyme II (NADPH)

[0040] GDH: glucose dehydrogenase

[0041] Tris: tris(hydroxymethyl)aminomethane

[0042] DMSO: dimethyl sulfoxide

[0043] HPLC: high performance liquid chromatography

[0044] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are usually carried out according to the conventional conditions, or according to the conditions suggested by the manufacturers. Unless otherwise specified, the percentages and parts are calculated by weight. Other materials, reagents, etc. used, if not otherwise specified, are reagents and materials obtained from commercial channels.

[0045] Example 1

[0046] Artificial intelligence assisted directed evolution of HaF3'H and HaCPR

[0047] The binding structure of HaF3'H with naringenin, hematin and the binding structure of HaCPR with FMN, FAD, NADPH were obtained using software such as AlphaFold2 and RosettaDock.

[0048] Using artificial intelligence algorithm deep neural network DNN to design 100 (HaF3'H) or 50 (HaCPR) single point mutations within 10 angstroms of the ligand, construct and screen the activity of these mutants.

[0049] Using artificial intelligence algorithm to comprehensively evaluate the relative activity of these designed mutants, these data are used to further train the artificial intelligence algorithm. Then, the trained artificial intelligence algorithm predicts 1000 mutants with activity improvement (containing 1-3 site mutations), constructs and screens the activity of these mutants. The best mutant is defined as "M1", and all data are used to further train the artificial intelligence algorithm.

[0050] The same prediction of mutants and screening is carried out on the basis of M1, and "M2", "M3", etc. are obtained in turn. HaF3'H undergoes 5 rounds of artificial intelligence assisted directed evolution to obtain "HaF3'H-M5", and HaCPR undergoes 5 rounds of artificial intelligence assisted directed evolution to obtain "HaCPR-M3". The pESC-His_HaF3'H_HaCPR and its mutant plasmid is transformed into INVSc1 Saccharomyces cerevisiae competent cells (Unidobiology), and cultured in 5mL SD / -His medium (Unidobiology) at 30 degrees Celsius, 250rpm for 24 hours, then diluted to OD600=0.3 in 5mL YPD medium to OD600=2-3, and then replaced with 5mL YPG induction medium (Unidobiology) for further culture for 36 hours. The bacterial cells are collected, 0.75mL lysis buffer solution is added, and ultrasonic is used to obtain cell lysate. The selection is 100μL reaction, the conditions are 110mM naringenin, 220mM glucose, 0.1mM NADP+, 1μM GDH, 500mM Tris (pH=8), 20% DMSO (v / v), 2.0mL centrifuge tube, 1000rpm, 37 degrees Celsius, reaction for 1 hour. After the reaction is completed, 300μL acetonitrile is used to quench the reaction, the supernatant is taken after centrifugation and detected by HPLC. The transformation rate of pESC-His_HaF3'H_HaCPR is 9.5%, and the transformation rate of pESC-His_HaF3'H-M5_HaCPR-M3 is 95%.

[0051] Example 2

[0052] Production of Saccharomyces cerevisiae engineering bacteria lysate

[0053] The above-mentioned transformed pESC-His_HaF3'H-M5_HaCPR-M3 Saccharomyces cerevisiae engineering bacteria were inoculated into 5 L fermentation broth (20 g / L peptone, 10 g / L yeast powder, 30 g / L glucose, 5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, pH 5.5) at a 10% (v / v) inoculation amount, and cultured at 30°C, 200-1000 rpm, 1 vvm air, 30% dissolved oxygen, and pH 5.5 controlled by 5% ammonia water and 6M hydrochloric acid for 10 h. Then, 2 vvm air was supplied and 0.5 L (50% glucose) of the batch feeding medium was added. After another 14 h of fermentation, the temperature was reduced to 25°C, and 2.5 L (40% galactose, 10% glucose, 3 g / L peptone, 3 g / L yeast powder) of the induction medium was added for further fermentation for 72 h. Centrifugation yielded 205 g / L of the engineering bacteria, and 1 L of lysis buffer was added. After lysis using a high-pressure homogenizer, the engineering bacteria lysis solution was obtained.

[0054] Example 3

[0055] Production of Eriodictyol

[0056] In a 1 L reaction kettle, 150 mM glucose, 20 mM phosphate, 0.1 mM NADP+, 1 μM GDH, and 500 mL of the above-mentioned engineering bacteria lysis solution were added, and 1 L / min of air was supplied while stirring at 500 rpm. The pH was controlled at 8.0 using 6M sodium hydroxide, and the temperature was controlled at 37°C. Then, 110 mL of 1M naringenin methanol solution was added dropwise, and the naringenin was dropped for 5 h. The total reaction time was 6 h. At this time, the conversion rate of naringenin in the solution was 95%.

[0057] After concentration under reduced pressure, 1 L of ethyl acetate was used for extraction three times, and the combined solution was concentrated under reduced pressure. 50 mL of methanol was added to dissolve the recrystallization, water was added to precipitate the crystals, and the crystals were filtered and dried to obtain 28.5 g of eriodictyol with a purity of 97.5% and a total yield of 90%.

[0058] All the documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various modifications and changes can be made to the present application by those skilled in the art upon reading the above description of the present application, and these equivalent forms should fall within the scope of the appended claims of the present application.

Claims

1. A flavonoid 3β-hydroxylase mutant HaF3'H-M5, characterized in that The amino acid sequence of the flavone 3β-hydroxylase mutant is shown in SEQ ID NO:

1.

2. A flavonoid 3β-hydroxylase coenzyme mutant HaCPR-M3, characterized in that: The amino acid sequence of the flavone 3β-hydroxylase coenzyme mutant is shown in SEQ ID NO:

3.

3. A recombinant strain expressing the flavonoid 3β-hydroxylase mutant according to claim 1 and / or the coenzyme mutant according to claim 2.

4. The recombinant strain according to claim 3, characterized in that The recombinant strain expresses the flavone 3β-hydroxylase mutant as claimed in claim 1 and the coenzyme mutant as claimed in claim 2.

5. A method for producing eriodictyol, characterized in that: In the presence of the lysate of the recombinant strain as claimed in claim 3, the reaction is carried out with naringenin as a substrate to obtain the eriodictyol.

6. The method according to claim 5, wherein The reaction comprises the steps of: In the presence of the recombinant strain lysate and in the presence of air, naringenin is 3'-hydroxylated to obtain the eriodictyol.

7. The method according to claim 6, wherein The method further comprises: carrying out the reaction as claimed in claim 6 in the presence of glucose, phosphate, NADP+ and GDH.

8. The method according to claim 5, wherein The method comprises the steps of: in the presence of glucose, phosphate, NADP+ and GDH, and in the presence of air, dropwise adding a solution containing naringenin into an alkaline system, thereby obtaining the eriodictyol.