Gene combination, recombinant plasmid combination and strain for synthesizing luteoloside and method for heterologous biosynthesis of luteoloside

Abstract

The invention belongs to the field of gene engineering, and relates to a gene combination for synthesizing luteoloside, a recombinant plasmid combination, a strain and a method for heterologous biosynthesis of luteoloside. The gene combination comprises the following genes: a flavone hydroxylase gene TbFH; a flavone synthase II gene TbFNS-1 or TbFNS-2, and a flavone synthase II gene TbFNS-1 or TbFNS-2; the UDP glucosyltransferase gene is TbUGT-1, TbU75L1, U8089 or U16269, and the UDP glucosyltransferase gene is The invention provides a potential method for replacing traditional plant extraction or chemical synthesis, and lays a foundation for further transforming efficient biosynthesis through metabolic engineering.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a gene combination, recombinant plasmid combination, strain, and method for heterologous biosynthesis of luteolin. Specifically, it relates to the luteolin synthesis-related flavonoid hydroxylase gene (FH), flavonoid synthase II gene (FNSII), and UDP glucosyltransferase gene (UGT) from water chestnut, a recombinant expression vector containing said gene fragments, a genetically engineered Saccharomyces cerevisiae, and a method for heterologous biosynthesis of luteolin. Background Technology

[0002] Cynaroside is a flavonoid compound with significant pharmacological activity, exhibiting antioxidant, antitumor, and anti-inflammatory bioactivities. Traditional methods of obtaining it rely on plant extraction or chemical synthesis, leading to resource waste and environmental pollution. Microbial heterologous synthesis is a green and efficient production pathway, but the discovery of key enzyme genes and pathway optimization remain bottlenecks.

[0003] Water chestnut (Trapa bispinosa Roxb.) is a medicinal and edible plant rich in flavonoids, and the potential luteolin-synthesizing genes in its transcriptome have not yet been explored and utilized. This study, through transcriptome analysis of water chestnut, for the first time identified and validated key genes for luteolin synthesis, and constructed an efficient heterologous synthesis system. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for synthesizing luteolin glycoside using gene combinations, recombinant plasmid combinations, strains, and heterologous biosynthesis.

[0005] A first aspect of the present invention provides a gene combination for synthesizing luteolin, comprising the following gene fragments:

[0006]

[0007] The amino acid sequence is as shown in SEQ ID NO: 2; MGRFLIVEVAVAALLFFVTRLVVRSLLLRLQNRKLPPGPRGWPIIGALPLLGTMPHVALAKLAKKYGSVMYLKMGTCDMVVASTPDAARAFLKTLDINFSNRPPNAGATHLAYNAQDMVFAHYGPRWKLLRKLSNLHMLGGKALEDWAGVREDEVGHMLRAMCETSDRGEDVVVPEMLTYAMANMIGQVILSRRVFATKGSESNEFKDMVVELMTSSGYFNIGDFIPSIAWMDLQGIEKGMKRLHVKFDKLITEMIEQHSATAYRRKGKPDFLDQVMANSEMSDGERLNMTNVKALLLNLFTAGTDTSSSIIEWALAEMMKNPKIFRRAHEEMDRVIGRGRRLKESDIPNLPYLQAISKETMRKHPSTPLNLPRVSSEPCEVNGYYIPRGTRLSVNIWAIGRDPDVWESPLEFKPERFLEDERLSKIDPRGNSFELIPFGAGRRICAGTRMGIVLVEYVLGSLIHSFEWGLPPGTDDLDMNESFGLALQKKVPLTAVLTRRLSPTAYSS。

[0008]

[0009]

[0010] The amino acid sequence is as shown in SEQ ID NO: 6; MTMHNVLESVSVHAVLLALGLSVAATLLLRALKRHRKIPLPPSPPSLPVIGHFHLLSPLLHHSLSSLSSKYGPLIYLKLGSLPCVLASTPELAREFLKVNEHKFTSRKHSAAIDYLTYNNSSFAFAPYDAYWKFIKRVIQTELIGPRTLGQFLPVRTKELCYFLGTIHDMCQSGNSKINLTEELLKLTNNTISQMILSMRCSGTNSEADNVGSVIREVSEIFGEFNLSDFVWLCKHVDFQGFRKRFKDLQRRYDSILENIIETREGSRKAKRKELGPAAGSGPVDNQEIKDFLDMMLDIYEDPNSDMKVTRNNVKALILDLFTAATDTSATSLEWSLAELINNPRVLEKARQEIESVVGTTRLVQESDIPSLPYINAIIKETFRLHPPIPIIPRKSTEEAIINGYRIPAGSLLFVNMWAIARDPKVWDRPMEYEPERFLGKSGKESIEIKGHNYELLPFGTGRRGCPGISLAMLQVPVALSTIIQCFDWKPCGPHGQIVQSVDMSERPGLAVPRANDLICRLTPRLEVPALIK*。

[0011]

[0012] The amino acid sequence is as shown in SEQ ID NO: 8, MAMDVGSGSKLEIFFFPFLAPGHMIPVADIAKLFAARGAKSTLLTTTYYEQMFHRSIQRAQKLGSDLRILTMELPLQEVGLPEHCQNLDSISSIEMRMKYLMAVRKLEEQFERLVEEHRPDCVVSDMFLPWTVDVLAKFGIPRIVFHGTSHFAMGASECVRLYEPHTKVSADSEPFLIPNFPGEISMTGAKLPEYIREVTHMTEFNRQMMESETRSYGVIMNSFYELEPAYADYYGDALGRRLWSVGPVFLCTKDFEEKLQRWGKSASAGTGDGDDDGDGCLKWLDSKKPNSVVYICFGSMTKFTPLQLREIALGLEASGQQFVWVVKKEATIVEGGKEDWLPQGYEERMRDKGLIIRGWAPQVMILDHDAVGGFVTHCGWNSTLEGIAAGVPMVTWPVGAEQFYNEKLVTEVLRIGVQVGVQRWVKMTGDWVECGKVEAAVRRVMVGEEAEGMRVRAAQLSEAAKKAVEEGGSSHSDLTAL。

[0013]

[0014] The amino acid sequence is as shown in SEQ ID NO: 10, MASDPAHFLIVSLPAQGHINPSLQLAKLLVRQGALVTFATSVSVASRMLRRPDGGVGEGGVCFAPISDGYDQGFINDANSVQENMSELRRRGSESLRDLITSSAARGCPFSCIIYTLTLPWAAEVARELGVPSTLLWIQPAIVLDIYHYYFSGYKDSIVEVFSSASSIDLPGLPMKLDSRDLPSFLLPMDTYAFVLPTFQELLEVQEKEANQRVLVNTFDALEPEALKAIGKYRMTGIGPLVPSAFLGGRDPWDTSFGGDLFQDVSTKDYMQWLDSQAEGSVIYLSFGTIAVISKRQTEEVARGLLATGRPFLWVIRSKENKLHQKPQQQQEEEGGEADEHVNISCMEELEKQGMIVPWCSQLEVLSHRSVGCFLTHCGWNSTLESVVCGVPVVAFPLWSDQGTNAKLLEEAWGTGVRVVKKRAAAEEEDPGPEVVEASEIRRCVEAVMAERREEVKGNAMRLKEMAMEAVREGGSSERNLRAFIEDVNGSKHPS*。

[0015]

[0016] The amino acid sequence is as shown in SEQ ID NO: 12, MEWEILVVPFFGQGHLLPSIELCSHLASRNFSVNLVISSNLSSIVPTSLRSHPLVIITELPSSPAPPPPPPPSLPSLPPQQQNDPLQSHRSHHAKMAQGLKAVLSAHADGRSSRRIACAVIDVMMGWSGEIFQQFGVPTVGFFTSGACSAAMEFALWKADLEGLKPGEARILHGLPEAMALTEADLKRRPHGPPHLRNGGGGGLPFPSPPGGAGPKFMGPPQPGHQPPWTDEVAGSLALIFNTCDDLERPFIEYVADRVGKPVWGVGPLLPEKYWKSAGSLLHDGEIRTNRNSTVTEEEVVQWLDSKPPGSVLYVSFGSEVGPTMEEYPQLAEALAESSSPFIWVIQRGSGRSGPPRTFLGGRPDSDAGEDEGYFPHGLSDKVGERGLIIRGWAPQLLILSHPSTGGFLSHCGWNSTVEAVGRGVPFLTWPIRGDQYYNSKLIVAHLGAGYVVSDDMSRPVKKDQIMKGIERLMGDEGVRELAAQLRARFEIGFPASSVAGLDALGEFIRQRSA*。

[0017]

[0018] Amino acid sequence such as SEQ ID NO:14 shown as MPSSVVFVVTGSGQGHVHPCMELCRHLGSRDYHTTLVVPSSLSSAIPSSFSSHPFVSIAQITVPPGPPMPPSGPVAHQQAHQDLLAHLSAHSCDPSLPPALCAIVDFQLGWT KECFRKFQIPVISFFTFGACAAAMELGAWRAQASDLGPGESRPIVGLPEEMAVTYWDLKRKPMGPPRGSGAVSGGKHPGGPPRPGDMPPWVPQVEGPVGMMFNTCDDLERPFLEYMEAQM GMPAWGVGPLLPEPYWRSSDSLVRDGAVRQHTRHSNYSEEEVIQWLDRKPQRSVLYVAFGSEVGPAVEEYPELVAALEESSHPFIWAVQSKPGQPRYFPEGLDARVGDRGLVIDGWAPQL LILSHPSTGGFLSHCGWNSTAEAVGLGVPLLAWPIRGDQHYNAKLVVSHLRVGHRVADDLSENVKKGDILRGIEKLMGDEEAHERAARLKARFAGGFPASSAAALDAFQGYVISQKKG*.

[0019] In this invention, the flavonoid hydroxylase gene TbFH, flavonoid synthase II gene TbFNS-1, flavonoid synthase II gene TbFNS-2, UDP glucosyltransferase gene TbUGT-1, UDP glucosyltransferase gene TbU75L1, UDP glucosyltransferase gene U8089, and UDP glucosyltransferase gene U16269 are derived from water chestnut-related genes and obtained through gene base optimization based on the codon preference of Saccharomyces cerevisiae.

[0020] A second aspect of the present invention provides a recombinant plasmid combination for synthesizing luteolin, containing gene fragments from the gene combination; the recombinant plasmid is an Escherichia coli expression vector and / or a Saccharomyces cerevisiae expression vector; preferably, the Escherichia coli expression vector uses pET-28a as the original vector, and the Saccharomyces cerevisiae expression vector uses pESC-URA and pESC-LEU as the original vectors.

[0021] This invention provides a gene combination for synthesizing luteolin, comprising: a flavonoid hydroxylase gene TbFH, the nucleotide sequence of which is shown in SEQ ID NO: 1; a flavonoid synthase II gene TbFNS-1, the nucleotide sequence of which is shown in SEQ ID NO: 3; a flavonoid synthase II gene TbFNS-2, the nucleotide sequence of which is shown in SEQ ID NO: 5; a UDP glucosyltransferase gene TbUGT-1, the nucleotide sequence of which is shown in SEQ ID NO: 7; a UDP glucosyltransferase gene TbU75L1, the nucleotide sequence of which is shown in SEQ ID NO: 9; a UDP glucosyltransferase gene U8089, the nucleotide sequence of which is shown in SEQ ID NO: 11; and a UDP glucosyltransferase gene U16269, the nucleotide sequence of which is shown in SEQ ID NO: 13.

[0022] All of the above genes have been codon optimized in Saccharomyces cerevisiae. Among them, the genes derived from water chestnut (TbF3H, TbFNS-1, TbU75L1) have had their catalytic activity enhanced through directed evolution (such as the TbFNSL394I mutant).

[0023] A fourth aspect of the present invention provides a method for heterologous biosynthesis of luteolin, comprising: fermenting the above-mentioned strain to synthesize luteolin. Preferably, the fermentation conditions include: an initial pH of 5.8-6.2, an initial OD600 value of 0.8-1.2, a fermentation glucose concentration of 5.5-6.5%, an expansion culture medium volume of 150-500 mL, and a fermentation time of 55-65 h.

[0024] The technical route of the present invention is as follows: Figure 1 As shown: Transcriptomic analysis of six different growth stages of water chestnut was performed using a high-throughput sequencing platform to screen for flavonoid hydroxylase genes (FH), flavonoid synthase II genes (FNSII), and UDP glucosyltransferase genes (UGT) that may be involved in luteolin synthesis. Flavonoid hydroxylase genes TbFH, flavonoid synthase II genes TbFNS-1, TbFNS-2, and UDP glucosyltransferase genes TbUGT-1, TbU75L1, U8089, and U16269 were cloned from water chestnut and recombinant expression plasmids were constructed. These plasmids were transformed into *Saccharomyces cerevisiae* to construct recombinant *Saccharomyces cerevisiae* for heterologous expression. Fermentation screening of the transformed strains revealed luteolin-synthesizing genes TbFH, TbFNS-1, TbFNS-2, TbUGT-1, TbU75L1, U8089, and U16269. Finally, by optimizing specific genes, modifying chassis strains, and optimizing fermentation conditions, the recombinant brewing yeast was optimized to increase its luteolin yield.

[0025] The heterologous expression described in this invention specifically refers to: using cDNA obtained from reverse transcription of total RNA from water chestnut as a template, PCR amplification of candidate TbFH, TbFNS, and TbUGT expression sequence fragments, and ligation of each TbFH, TbFNS, and TbUGT expression fragment with E. coli expression vector pET-28a and Saccharomyces cerevisiae expression vectors pESC-URA and pESC-LEU by enzyme digestion to obtain a series of recombinant expression vectors, which are then transformed into E. coli BL21 or Saccharomyces cerevisiae BY4741 and BY4742 for expression verification.

[0026] The gene optimization described in this invention specifically refers to: gene optimization is the base adaptation optimization of genes with enzyme activity based on the codon preference of Saccharomyces cerevisiae.

[0027] The pathway required for the de novo synthesis of luteolin in plants as described in this invention is as follows: Figure 2 As shown. In plants, starting with phenylalanine, cinnamic acid is formed via phenylalanine ammonia-lyase (PAL). Then, through the pathway of 4-coumaric acid-co-coenzyme ligase (4CL) and trans-cinnamic acid-4 monooxygenase (CYP73A), coumaric acid and cinnamoyl-CoA are converted into p-coumaryl-CoA. Next, naringenin chalcone is formed via chalcone synthase (CHS). Naringenin is produced via chalcone isomerase (CHI). Subsequently, luteolin is formed via flavonoid hydroxylase (FH, CYP75A) and flavonoid synthase I / II (FNSI / II) through the intermediates of sennaol and apigenin. Finally, luteolin is produced by glycosyltransferase, which catalyzes 7-OH glycosylation.

[0028] The fermentation condition optimization described in this invention refers to optimizing conditions such as the initial pH of the culture medium, the initial OD600, the fermentation glucose concentration, the fermentation time, and the fermentation system.

[0029] Compared with existing technologies, this invention extracts one flavonoid hydroxylase gene (TbFH), two flavonoid synthase II genes (TbFNS-1 and TbFNS-2), and four UDP-glucosyltransferase genes (TbUGT-1, TbU75L1, U8089, and U16269) from the water chestnut transcriptome. Using genetic engineering and biotechnology, a yeast strain BY4741G was constructed to achieve heterologous biosynthesis of luteolin. By combining the TbFH, TbFNS-1, TbFNS-2, TbUGT-1, TbU75L1, U8089, and U16269 enzyme genes in the BY4742 strain, and optimizing the shake-flask fermentation conditions, the luteolin content produced reached a certain level. This provides a potential method to replace traditional plant extraction or chemical synthesis, laying the foundation for further efficient biosynthesis through metabolic engineering.

[0030] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0031] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0032] Figure 1 This is a technical roadmap for the present invention.

[0033] Figure 2 This is a diagram of the biosynthetic pathway of luteolin as described in this invention.

[0034] Figure 3 The images show the electrophoresis results of the PCR amplification products of the genes described in this invention, including: ARNA nucleic acid quality detection image; TbFH gene PCR amplification image; TbFNS-1 gene PCR amplification image; TbFNS-2 gene PCR amplification image; TbUGT-1 gene PCR amplification image; TbU75L1 gene PCR amplification image; U8089 gene PCR amplification image; U16269 gene PCR amplification image; and DNA marker.

[0035] Figure 4 This is a schematic diagram of the overexpression vector construction process described in this invention.

[0036] Figure 5 This is a schematic diagram of the inhibition vector construction process described in this invention.

[0037] Figure 6 The results of SDS-PAGE of prokaryotic expression of the gene recombinant plasmid vector described in this invention are as follows (M. Marker; 1, 2. TbFH before and after induction, 1 before induction, 2 after induction; 3, 4. TbFNS-1 before and after induction, 3 before induction, 4 after induction; 5, 6. TbFNS-2 before and after induction, 5 before induction, 6 after induction; 7, 8. TbUGT1 before and after induction, 7 before induction, 8 after induction; 9, 10. TbU75L1 before and after induction, 9 before induction, 10 after induction; 11, 12. U8089 before and after induction, 11 before induction, 12 after induction; 13, 14. U16269 before and after induction, 13 after induction, 14 before induction).

[0038] Figure 7 This is an HPLC chromatogram of the fermentation product of the Saccharomyces cerevisiae strain used to synthesize luteolin in this invention.

[0039] Figure 8 PCR verification images of URA-TbF3'5'H, URA-TbFNS, and URA-TbF3'5'H-TbFNS colonies.

[0040] Figure 9 Double enzyme digestion was used to construct and validate the defective vector of Saccharomyces cerevisiae (A. pESC-URA-TbFH; B. pESC-URA-TbFNS; C. pESC-LEU-TbUGT1).

[0041] Figure 10 The graph shows the results of fermentation and calculations on the changes in the activity of TbFN in Saccharomyces cerevisiae.

[0042] Figure 11 The expression levels of candidate pathway genes in different tissues of water chestnut are: A. TbFH, B. TbFNS-1, C. TbFNS-2, D. TbUGT1, E. TbU75L1, F. U8089, G. U16269.

[0043] Figure 12 The results are from the fermentation of strain 3TIV in a 5L bioreactor. Detailed Implementation

[0044] Preferred embodiments of the present invention will now be described in more detail. While preferred embodiments of the present invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0045] Example 1

[0046] Cloning, expression, and enzyme activity identification of candidate genes for luteolin synthesis in water chestnut: flavonoid hydroxylase gene TbFH, flavonoid synthase II genes TbFNS-1 and TbFNS-2, and UDP glucosyltransferase genes TbUGT-1, TbU75L1, U8089, and U16269.

[0047] 1.1 Source of water chestnut samples

[0048] In this study, fresh "Elian No. 5" water chestnuts with mud still attached were used, sourced from the water chestnut production base of Qiliang Company in Jiangxia District, Wuhan City. Based on their growth status, six different growth stages were selected, numbered S1, S2, S3, S4, S5, and S6 respectively. Sampling was conducted from July 25, 2021 to August 29, 2021, with sampling every 6 days. Samples were cut into small granules, flash-frozen in liquid nitrogen, and stored at -80℃.

[0049] 1.2 Cloning of candidate genes for the synthesis of luteolin in water chestnut

[0050] Based on the water chestnut transcriptome sequencing data previously obtained in our laboratory, this invention screened candidate enzyme genes related to luteolin biosynthesis, including: flavonoid hydroxylase gene TbFH; flavonoid synthase II genes TbFNS-1 and TbFNS-2; and UDP glucosyltransferase genes TbUGT-1, TbU75L1, U8089, and U16269. Primers were designed based on the gene sequences, and the sequences of the above-mentioned genes were obtained by polymerase chain reaction (PCR).

[0051] Using PCR technology, with cDNA obtained from reverse transcription of total RNA from water chestnuts as a template, primers were designed based on the sequence information of candidate enzyme genes obtained from water chestnut transcriptome sequencing, and restriction enzyme sites were added to both ends of the primers. The specific primer sequences are shown in Table 1.

[0052] Table 1: Specific primer sequences used for amplifying the TbFH, TbFNS-1, TbFNS-2, TbUGT-1, TbU75L1, U8089, and U16269 genes of water chestnut.

[0053]

[0054]

[0055] The PCR reaction system is shown in Table 2, with a total volume of 100 μL. The reaction conditions were set as follows: 98℃ for 30 s; 98℃ for 10 s, 55℃ for 5 s, extension at 72℃, for 28 cycles, followed by a final 72℃ for 10 min. A portion of the reaction solution was used for electrophoresis, and the remaining amplification reaction solution was stored at -20℃ with the gel images saved. The electrophoresis conditions were: 120 V for 25 min.

[0056] Table 2: PCR reaction system

[0057] Reagent Name Dosage Forward Primer (F) 10 pmol Reverse Primer(R) 10 pmol PrimeSTAR HS (Premix) 25μL Template 150ng <![CDATA[RNase dH2O]]> Up to 100 μL

[0058] Using reverse-transcribed cDNA as a template, bands of approximately 1500 bp were amplified. For example... Figure 3 As shown, the TbFH fragment size is 1527 bp, the TbFNS-1 fragment size is 1605 bp, the TbFNS-2 fragment size is 1599 bp, the TbUGT1 fragment size is 1446 bp, the TbU75L1 fragment size is 1485 bp, the U8089 fragment size is 1542 bp, and the U16269 fragment size is 1410 bp. The colony PCR results show that all gene sizes are correct, and the positive rate of transformed *E. coli* is good. Strains were screened, amplified, and plasmids were extracted.

[0059] 1.3 Expression level analysis of luteolin pathway genes in different parts of water chestnut

[0060] Four parts of water chestnut were selected, with EIF-5α as the housekeeping gene and the stem tissue group as the control group. The mRNA expression levels of genes along each pathway in different water chestnut tissues were calculated using the Relative mRNA expression = 2(-ΔΔCq). The results are as follows: Figure 11 As shown, TbFH was expressed at the highest level in the shell tissue and at the lowest level in the leaf tissue of water chestnut; TbFNS-1 and TbFNS-2 were expressed at the highest level in the root tissue and at the lowest level in the leaf tissue; TbUGT1 was expressed at the highest level in the root tissue and at the lowest level in the stem tissue; TbU75L1 was expressed at the highest level in the shell tissue and at the lowest level in the leaf tissue; U8089 was expressed at the highest level in the shell tissue and at the lowest level in the stem tissue; and U16269 was expressed at the highest level in the root tissue and at the lowest level in the stem tissue. The gene expression analysis results showed that the assembled fragments of each gene clustered well across different tissues, and the expression of different genes was selective across tissues. This suggests that there may be some differences in the accumulation of different flavonoid products among different tissues.

[0061] 1.4 Construction of prokaryotic and eukaryotic expression vectors for candidate genes for the synthesis of luteolin from water chestnut.

[0062] [1] The PCR products obtained in 1.2 were purified and recovered using the OMEGA PCR purification kit.

[0063] [2] Restriction endonucleases (selected according to the candidate gene settings) were used to digest the plasmid and the target gene fragment. The digestion reaction system is shown in Table 3:

[0064] Table 3 Enzyme digestion reaction system

[0065] Components Dosage Restriction endonucleases (2 types) 1μL DNA 5μg 0.1% BSA 10μL 10×H Buffer 10μL <![CDATA[RNase dH2O]]> Up to 100 μL

[0066] [3] Add the above components to centrifuge tubes, vortex to mix, and react in a water bath at 37°C for 4-6 hours. Take 10 μL of each enzyme digestion product for gel electrophoresis.

[0067] [4] The plasmid and the target gene vector fragment that were successfully excised by enzyme ligation were mixed with reagents in a reaction system of 12 μL and ligated overnight (16℃) to obtain a series of E. coli recombinant plasmids pET-28a-TbFH, pET-28a-TbFNS-1, pET-28a-TbFNS-2, pET-28a-TbUGT-1, pET-28a-TbU75L1, pET-28a-U8089, pET-28a-U16269 and a series of yeast recombinant plasmids pESC-URA-TbFH-TbFNS-1, pESC-URA-TbFH-TbFNS-2, pESC-LEU-TbUGT-1, pESC-LEU-TbU75L1, pESC-LEU-U8089, pESC-LEU-U16269 (s represents different genes). The enzyme components are shown in Table 4:

[0068] Table 4: Enzyme-linked reaction system

[0069] Components Dosage T4 DNA Ligase 1μL Target gene fragment: vector fragment Target gene: vector fragment = 6:1 (molar ratio) 10×T4 DNA Ligase Buffer 1μL <![CDATA[RNase dH2O]]> Up to 12μL

[0070] The ligation mixture and empty plasmid vector were transformed into *E. coli* and *Saccharomyces cerevisiae*, respectively. *E. coli* was transformed using the heat shock method, and *Saccharomyces cerevisiae* was transformed using the lithium acetate method. Single colonies were picked for colony PCR, and 5 μL of the reaction solution was subjected to agarose gel electrophoresis and imaging. Positive colonies were expanded, plasmids were extracted, and sent to a sequencing company for sequencing and validation.

[0071] like Figure 4 The image shown is a map of the *E. coli* expression plasmid of the gene described in this invention (taking TbFH as an example). Figure 5 The image shown is a map of the Saccharomyces cerevisiae expression plasmid of the gene described in this invention (taking TbFH as an example).

[0072] 1.5 Construction of Escherichia coli and Saccharomyces cerevisiae expression strains for candidate genes related to luteolin synthesis

[0073] The recombinant plasmids pET-28a-TbFH, pET-28a-TbFNS-1, pET-28a-TbFNS-2, pET-28a-TbUGT-1, pET-28a-TbU75L1, pET-28a-U8089, and pET-28a-U16269, which were verified correctly in section 1.3, were transformed into Rosetta Escherichia coli expression strains. After colony PCR verification, E. coli expression strains were obtained.

[0074] The recombinant plasmids pESC-URA-TbFH-TbFNS-1, pESC-URA-TbFH-TbFNS-2, pESC-LEU-TbUGT-1, pESC-LEU-TbU75L1, pESC-LEU-U8089, and pESC-LEU-U16269 successfully constructed in section 1.3 were transformed into competent cells of *Saccharomyces cerevisiae* BY4741 via lithium acetate conversion. After transformation, the bacterial culture was plated on SC plates (lacking the corresponding amino acids) and incubated in a biochemical incubator for 3-4 days (28℃). The *Saccharomyces cerevisiae* expression strain was obtained after colony PCR verification.

[0075] 1.6 SDS-PAGE determination of fermentation products

[0076] Choose Bioshap's SDS-PAGE kit to prepare protein gels:

[0077] [1] Pick a single colony (expression strain) of Escherichia coli that has been identified in the previous LB resistance plate, inoculate it into 5 mL of LB resistance liquid medium, and incubate overnight (37℃, 220 rpm).

[0078] [2] The seed culture was inoculated into LB liquid medium (containing 1% glucose) at an inoculation rate of 2% and cultured for 3-4 hours to reach OD. 600 It is 0.6.

[0079] [3] Take an appropriate amount of bacterial solution and store it in a 1.5 mL centrifuge tube. Calculate the volume of the remaining bacterial solution, add IPTG (0.5 mM), and continue culturing for 4 h.

[0080] [4] After the culture is completed, take 1 mL of bacterial solution, centrifuge, and retain the bacterial precipitate.

[0081] [5] Select PBS to resuspend the bacterial cells, wash the bacterial cells and centrifuge (repeat twice), add 200 μL PBS to resuspend the bacterial cells, and vortex to mix.

[0082] Pipette 20 μL of the bacterial cell mixture into a 1.5 mL centrifuge tube, add 1 / 4 volume of protein loading buffer, mix well, and boil for 10 min. After the solution cools, detect the expression of the fusion protein by SDS-PAGE electrophoresis. Prepare the protein gel according to Table 5. The results are as follows: Figure 6As shown in the figure, M: Marker; 2\4: TbFH protein expression map; 6\8\10\12: TbUGT-1 protein expression map. Compared with the blank control group, specific protein bands appeared in the experimental group (within the red box in the figure). The size of the recombinant TbFH enzyme gene was approximately 55 kDa (theoretical predicted value 55.6 kDa), and the size of the TbUGT-1 enzyme gene recombinant plasmid vector was approximately 85 kDa (theoretical predicted value 86.9 kDa). The actual values ​​were close to the predicted values. This indicates that the recombinant protein was successfully expressed in E. coli, and the protein size was roughly consistent with the predicted value.

[0083] Table 5: SDS-PAGE Protein Gel Formulation

[0084] Element 8% separating gel (mL) 5% buildup adhesive (mL) 10% APS 0.05 0.02 distilled water 1.7 1.4 1M Tris, pH 6.8 - 0.25 1M Tris, pH 8.8 1.9 - 30% Acr-Bis(29:1) 1.3 0.33 TEMED 0.003 0.002 10% SDS 0.05 0.02

[0085] 1.7 Identification of candidate genes for the synthesis of luteolin in water chestnut and functional identification using fermentation of yeast expression strains.

[0086] [1] Ferment the yeast strain carrying the gene vector constructed in 1.4 for about 3-4 days, take 10 mL of bacterial solution into a 15 mL centrifuge tube, and add twice the volume of ethyl acetate.

[0087] [2] After mixing by inverting the tubes, place them in an ultrasonic cleaner and ultrasonically break the strains for 60 minutes. Then remove them and let them stand overnight.

[0088] [3] Take the upper organic phase of the overnight sample into a clean centrifuge tube for later detection, and save the remaining samples.

[0089] [4] The sample needs to be derivatized. Take 1 mL of sample (fermentation sample of Saccharomyces cerevisiae) into a clean centrifuge tube (10 mL), set the nitrogen blowing flow rate, blow dry the fermentation sample for about 40 min.

[0090] [5] Add 100 μL of MSTFA silanizing reagent, derivatize at 75-80 °C for 30 min, and after cooling, add 400 μL of chromatographic grade acetonitrile. The sample preparation is complete.

[0091] [6] Select capillary column model HP-5MS and Agilent GC-MS detector. The carrier gas is helium (1.0 mL / min), the temperature is 250℃, the injection volume is 1 μL (splitless mode), the starting temperature is 80℃, the temperature is increased to 300℃ (20℃ / min), and held for 15 min.

[0092] [7] The ion source temperature was 230℃ and the electron energy was 70eV. The detection of the product for functional verification in yeast was performed in full scan mode with a scan range of 50 to 1000 m / z.

[0093] [8] Collect the bacterial culture, add an equal volume of ethyl acetate, sonicate for 1 h, extract the upper organic phase, repeat the process twice to collect the organic phase, and concentrate it to a volume of 0.5 mL using a nitrogen blower.

[0094] [9] An Agilent ZORBAX EclipsePlus C18 Analytical 4.6 x 250 mm 5-Micron liquid chromatography column was used. The mobile phase composition was as follows: 0.5% formic acid in water (A), methanol (B), and 0.5% formic acid in methanol (C). Elution was performed at gradients of 0-82, 15-60, 25-60, 30-82, and 35-82 (time min - A%), with a total time of 35 min, a run time of 5 min, a column temperature of 30 °C, a flow rate of 0.8 mL / min, and an injection volume of 10 μL.

[0095] Candidate genes were expressed using the engineered Saccharomyces cerevisiae strain constructed in Example 1. Based on GC-MS results, the candidate genes with enzyme activity were pESC-LEU-TbUGT-1, pESC-URA-TbFH-TbFNS-1, and pESC-URA-TbFH-TbFNS-2. The experimental results are as follows: Figure 7 and Figure 8 The above plasmids were co-transformed into a single BY4741 strain, resulting in two combined strains: pESC-URA-TbFH-TbFNS-1 and pESC-LEU-TbUGT-1, and pESC-URA-TbFH-TbFNS-2 and pESC-LEU-TbUGT-1. This successfully achieved de novo synthesis of luteolin in the same *Saccharomyces cerevisiae* strain. Experimental results are as follows: Figure 7 As shown, Methanol: methanol blank control group; AHF: HPLC detection curves of pESC-URA-TbFH-TbFNS-1 and pESC-LEU-TbUGT-1 samples; BHF: HPLC detection curves of pESC-URA-TbFH-TbFNS-2 and pESC-LEU-TbUGT-1 samples; Betulinic: luteolin standard.

[0096] 1.8 Expression and Fermentation Activity Verification of Candidate Genes for Luteolin Synthesis in Saccharomyces cerevisiae (Saccharomyces cerevisiae)

[0097] Using the already constructed pET-28a vector as a template, the target gene fragment was amplified. The target gene was reconstructed into the pESC-URA and pESC-LEU vectors, creating pESC-URA-TbFH, pESC-URA-TbFNS-1, pESC-URA-TbFNS-2, pESC-URA-75A-FNS1, and pESC-LEUTbUGT1. These vectors were then transformed into the BY4741 Saccharomyces cerevisiae strain. Figure 8 and Figure 9 The results include PCR gel images of the relevant bacterial colonies and double enzyme digestion verification.

[0098] Positive strains were amplified in SD deficient medium until an OD value of approximately 1.6 was obtained, serving as the seed culture for fermentation. Fermentation was conducted in YPD medium with 1% raffinose and 2% galactose added as carbon sources and inducers, and ascorbic acid at a final concentration of 0.5 mmol as an antioxidant. Depending on the specific pathway genes loaded, different substrates were used for fermentation to verify the presence of the required activity for each pathway. Specifically, pESC-URA-TbFH strains were tested for the formation of senna and luteolin products using naringenin and apigenin, respectively; pESC-URA-TbFNS-1 and pESC-URA-TbFNS-2 strains were tested for the formation of apigenin and luteolin products using naringenin and senna, respectively; and pESC-URA-75A-FNS1 strain was tested for the formation of senna, apigenin, and luteolin products using naringenin as a substrate.

[0099] Example 2: Optimized gene cloning and vector construction for water chestnut

[0100] Constructing optimized gene expression vectors and dual-gene vectors in Saccharomyces cerevisiae

[0101] The *Saccharomyces cerevisiae* expression vectors pESC-URA and pESC-LEU were reconstructed, and the TbUGT-1, TbU75L1, U8089, and U16269 genes were ligated with TbFH, TbFNS-1, and TbFNS-2 to construct the following combinations of *Saccharomyces cerevisiae* plasmids: pESC-URA-TbFH-TbFNS-1-TbUGT-1, pESC-URA-TbFH-TbFNS-2-TbUGT-1, pESC-URA-TbFH-TbFNS-1-TbU75L1, pESC-URA-TbFH-TbFNS-2-TbU75L1, pESC-URA-TbFH-TbFNS-1-U8089, and pESC-URA-TbFH-TbFNS-2-U8089. , pESC-URA-TbFH-TbFNS-1-U16269, pESC-URA-TbFH-TbFNS-2-U16269 and pESC-LEU-TbUGT-1

[0102] The method and steps for constructing the brewing yeast carrier are the same as in Example 1, Section 1.4.

[0103] like Figure 3 and 4As shown, the constructed recombinant plasmid vector was subjected to colony PCR, positive strains were picked and cultured, and the plasmid was extracted and digested with two enzymes. The appearance of the correct band indicates that the dual-gene vector was successfully constructed.

[0104] Example 3: Directed Evolution of Flavonoid Synthesase II (TbFNSII) in the Luteolin Synthesis Pathway

[0105] 3.1 Directed Evolution of the Flavonoid Synthase II (TbFNSII) Gene

[0106] 3.1.1 Mutation hotspot analysis and alanine scan

[0107] The amino acid sequence of TbFNSII was analyzed using the online program Hotspot Wizard 3.0, identifying 15 mutation hotspots. These hotspots all had mutable scores greater than 7, indicating high variability. Alanine scans were performed on these sites to verify their impact on enzyme activity. The alanine scan results are shown below. Figure 10 As shown, the activities of four sites, I391, I393, L394 and R509, changed significantly, with the activity of site L394 showing the most significant change.

[0108] 3.1.2 Saturation Mutation and Activity Verification

[0109] Saturation mutations were performed at four sites: I391, I393, L394, and R509. A mutant library was constructed and validated through fermentation in *Saccharomyces cerevisiae*. Fermentation results showed that the TbFNSIIL394I mutant exhibited twice the catalytic activity of the wild type, significantly improving the conversion efficiency of naringenin to apigenin. Molecular docking results indicated that this activity enhancement might be due to the closer spatial distance between the substrate catalytic site and the heme iron at the enzyme's active site in the mutant.

[0110] 3.2 Construction of recombinant plasmids and transformation of strains

[0111] 3.2.1 Construction of Recombinant Plasmids

[0112] The codon-optimized TbFH, TbFNSIIL394I, and glycosyltransferase genes WsGT / VlGT2 were integrated into the pESC-URA vector to construct a full-pathway gene vector; ScPGM2 and LjCPR were integrated into the pESC-LEU vector to construct an overexpression vector; and dCas9-KRAB and sgEXG-sgSPR were integrated into the pESC-TRP vector to construct a repression vector. The specific construction process is as follows:

[0113] The pESC-URA vector was double-digested with restriction endonucleases, and the TbFH, TbFNSIIL394I, and WsGT / VlGT2 gene fragments were ligated into the vector to construct pESC-URA-TbFH-TbFNSIIL394I-WsGT / VlGT2.

[0114] Using the same method, the ScPGM2 and LjCPR gene fragments were ligated into the pESC-LEU vector to construct pESC-LEU-ScPGM2-LjCPR.

[0115] The dCas9-KRAB and sgEXG-sgSPR gene fragments were ligated into the pESC-TRP vector to construct pESC-TRP-dCas9-KRAB-sgEXG-sgSPR.

[0116] 3.2.2 Transformation of strains

[0117] The recombinant plasmid constructed above was co-transformed into *Saccharomyces cerevisiae* strain BY4741, and the recombinant plasmid was introduced into the yeast cells via lithium acetate transformation. After transformation, the bacterial culture was plated on SD medium containing the appropriate antibiotic and incubated at 30°C for 3-4 days. Positive colonies were selected for scale-up culture for subsequent fermentation experiments.

[0118] Example 4: Optimization of fermentation conditions for brewer's yeast

[0119] 4.1 Fermentation condition optimization and product detection

[0120] 4.1.1 Optimization of fermentation conditions

[0121] To increase the yield of luteolin, the fermentation conditions of the transformed Saccharomyces cerevisiae strain were systematically optimized. The main parameters optimized included culture medium composition, fermentation temperature, fermentation time, and stirring speed. The specific optimization process is as follows:

[0122] 1. Optimization of culture medium composition

[0123] The composition of the culture medium has a significant impact on the yield of fermentation products. In this study, single-factor gradient experiments were conducted on the carbon source, nitrogen source, CaCl2, and FeSO4 components in the culture medium to determine the optimal culture medium formulation.

[0124] Carbon source optimization: The 3EIV-2sg strain was fermented for 3 days in YPD medium containing different concentrations of glucose (2%, 4%, 6%, 8%, 10%, 12%). HPLC analysis after fermentation showed that with increasing carbon source concentration, the content of apigenin first increased and then decreased, reaching a maximum of 8.94 mg / L at 8% carbon source; the content of sennaol first increased with increasing carbon source concentration, reaching a maximum of 246.45 mg / L at 6% carbon source, and then decreased to a minimum of 114.65 mg / L at 8% carbon source; the content of luteolin increased and then decreased, reaching a maximum of 29.7 mg / L at a carbon source concentration of 4%. Therefore, 4% glucose was ultimately selected as the optimized carbon source concentration for the culture medium.

[0125] Nitrogen source optimization: The 3EIV-2sg strain was fermented for 3 days in YPD medium containing different concentrations of tryptone (1%, 2%, 4%, 6%, 8%, 10%, 12%). HPLC analysis after fermentation showed that with increasing nitrogen source concentration, the content of apigenin slowly increased, the content of sennae continuously decreased, and the content of luteolin initially increased and then decreased, reaching a maximum of 29.7 mg / L at a nitrogen source concentration of 4%. Therefore, 4% tryptone was ultimately selected as the optimized nitrogen source concentration for the culture medium.

[0126] CaCl2 concentration optimization: The 3EIV-2sg strain was fermented for 3 days in YPD medium containing different concentrations of CaCl2 (0M, 0.005M, 0.01M, 0.05M, 0.1M, 0.2M). HPLC analysis after fermentation showed that the content of apigenin gradually decreased with increasing calcium salt concentration. The content of sennaol initially increased with increasing calcium salt concentration, reaching a maximum of 279.45 mg / L at 0.005M, and then decreased with further increases, reaching a minimum of 68.29 mg / L at 0.2M. The content of luteolin initially increased and then decreased, reaching a maximum of 6.01 mg / L at 0.01M. Therefore, 0.01M CaCl2 was ultimately selected as the optimized calcium salt concentration for the culture medium.

[0127] FeSO4 concentration optimization: The 3EIV-2sg strain was fermented for 3 days in YPD medium containing different concentrations of FeSO4 (0M, 0.005M, 0.01M, 0.05M, 0.1M, 0.5M). HPLC analysis after fermentation showed that the contents of apigenin, sennaol, and luteolin did not change significantly or showed no obvious trend with increasing iron salt concentration. The luteolin content was highest at 0M FeSO4. Therefore, FeSO4 salt was not added as a supplement to the medium in subsequent fermentations.

[0128] Based on the above optimization results, the final culture medium formula is determined to be: 2% yeast extract, 4% tryptone, 4% glucose, and 0.01M CaCl2.

[0129] 2. Fermentation temperature optimization

[0130] Fermentation temperature has a significant impact on yeast growth and metabolism. Fermentation experiments were conducted at different temperatures (25℃, 28℃, 30℃, and 32℃) under optimized culture conditions. The results showed that the highest yield of fermentation products was achieved at 28℃; therefore, 28℃ was selected as the optimal fermentation temperature.

[0131] 3. Fermentation time optimization

[0132] Fermentation time determines the degree of substrate transformation and product accumulation. Fermentation experiments were conducted under optimized culture medium and temperature conditions for different fermentation times (1 day, 2 days, 3 days, and 4 days). The results showed that the luteolin content reached its highest level of 23.1 mg / L after 4 days of fermentation, while the sennaol content was 147 mg / L. Therefore, 4 days was selected as the optimal fermentation time.

[0133] 4. Optimized stirring speed

[0134] Stirring speed affects the mixing of the fermentation broth and oxygen transfer. Fermentation experiments were conducted at different stirring speeds (180 rpm, 200 rpm, 230 rpm, and 250 rpm) under optimized culture medium, temperature, and fermentation time conditions. The results showed that the highest yield of fermentation products was achieved at 230 rpm; therefore, 230 rpm was selected as the optimal stirring speed.

[0135] The final fermentation conditions were determined as follows: YPD medium containing 2% yeast extract, 4% tryptone, 4% glucose, and 0.01M CaCl2; fermentation temperature of 28℃; fermentation time of 4 days; and stirring speed of 230 rpm.

[0136] 4.1.2 Product Detection

[0137] After fermentation, the fermentation broth was collected for product analysis. High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the fermentation products. The results showed that under optimized fermentation conditions, *Saccharomyces cerevisiae* could efficiently synthesize luteolin, achieving a high yield. Specific results are as follows: luteolin yield: 4.2 mg / L, luteolin-based yield: 34.8 mg / L, and sennaol yield: 147 mg / L.

[0138] Through the above optimization conditions, the efficient synthesis of luteolin was successfully achieved, laying the foundation for subsequent industrial production.

[0139] In a 5L bioreactor, YPD medium was used for scale-up cultivation with the addition of PTM1 metal ion solution and vitamin solution as excipients. High-concentration YPD medium and glucose solution were added daily for fermentation. Fermentation was carried out sequentially on strains 3EIV and 3TIV, respectively. Figure 12 As shown, the fermentation results indicated that the 3TIV sample produced higher levels of apigenin, sennaol, luteolin, and luteolinoglycoside compared to the 3EIV sample. Sennaol reached its highest level of 720 mg / L at 75 h, while apigenin and luteolin reached their highest levels of 19 mg / L and 34.8 mg / L, respectively, at 120 h. Luteolinoglycoside reached its highest level of 4.2 mg / L at 65 h.

[0140] This invention identifies one flavonoid hydroxylase gene (TbFH), two flavonoid synthase II genes (TbFNS-1 and TbFNS-2), and four UDP-glucosyltransferase genes (TbUGT-1, TbU75L1, U8089, and U16269) from the water chestnut transcriptome. Using genetic engineering and biotechnology, a yeast strain BY4741G was constructed to achieve heterologous biosynthesis of luteolin. By combining the TbFH, TbFNS-1, TbFNS-2, TbUGT-1, TbU75L1, U8089, and U16269 enzyme genes in the BY4742 strain, and optimizing the shake-flask fermentation conditions, a high level of luteolin content was achieved. This provides a potential method to replace traditional plant extraction or chemical synthesis, laying the foundation for further efficient biosynthesis through metabolic engineering.

[0141] The CYP450s and UGT enzyme genes obtained in this invention increase the diversity of luteolin-producing enzyme genes, laying the foundation for the subsequent microbial metabolic engineering production of luteolin. Since *Saccharomyces cerevisiae* cells grow rapidly and are easy to ferment, and there is a mature platform for genetic manipulation of *Saccharomyces cerevisiae*, it is expected that the fermentation yield of luteolin can be significantly increased in the future through metabolic engineering combined with fermentation engineering technology. Therefore, this invention is a luteolin biosynthesis and genetic engineering technology with promising industrial application prospects.

Claims

1. A gene combination for synthesizing luteolin, comprising the following gene fragments: The flavonoid hydroxylase gene TbFH has the nucleotide sequence shown in SEQ ID NO: 1; The nucleotide sequence of the flavonoid synthase II gene TbFNS-1 is shown in SEQ ID NO: 3; The nucleotide sequence of the flavonoid synthase II gene TbFNS-2 is shown in SEQ ID NO: 5; The nucleotide sequence of the UDP glucosyltransferase gene TbUGT-1 is shown in SEQ ID NO: 7; The nucleotide sequence of the UDP glucosyltransferase gene TbU75L1 is shown in SEQ ID NO: 9; The nucleotide sequence of the UDP glucosyltransferase gene U8089 is shown in SEQ ID NO: 11; The nucleotide sequence of the UDP glucose transferase gene U16269 is shown in SEQ ID NO:

13.

2. The gene combination for synthesizing luteolin glycoside according to claim 1, wherein, The flavonoid hydroxylase gene TbFH, flavonoid synthase II gene TbFNS-1, flavonoid synthase II gene TbFNS-2, UDP glucosyltransferase gene TbUGT-1, UDP glucosyltransferase gene TbU75L1, UDP glucosyltransferase gene U8089, and UDP glucosyltransferase gene U16269 were derived from water chestnut-related genes and optimized based on the codon preference of Saccharomyces cerevisiae.

3. A recombinant plasmid combination for synthesizing luteolin, containing gene fragments from the gene combination of claim 1 or 2; wherein the recombinant plasmid is an Escherichia coli expression vector and / or a Saccharomyces cerevisiae expression vector; preferably, the Escherichia coli expression vector uses pET-28a as the original vector, and the Saccharomyces cerevisiae expression vector uses pESC-URA and pESC-LEU as the original vectors.

4. The recombinant plasmid combination according to claim 3, wherein the recombinant plasmid combination is selected from the following combinations: (i) Combination of yeast recombinant plasmid pESC-URA-TbFH-TbFNSL394I-TbU75L1 and yeast recombinant plasmid pESC-LEU-LjCPR-ScPGM2; (ii) A combination of yeast recombinant plasmid pESC-TRP-dCas9-KRAB-sgEXG1-sgSPR1 as a repression vector.

5. A strain for synthesizing luteolin, comprising the recombinant plasmid combination as described in claim 3 or 4; wherein the strain is *Saccharomyces cerevisiae*, preferably strain BY4741.

6. The strain according to claim 5, wherein the genome of the strain integrates overexpression frames of the rate-limiting enzyme genes HMG1 and ERG20 of the Saccharomyces cerevisiae MVA pathway.

7. A method for heterologous biosynthesis of luteolin, comprising: The strain described in claim 5 or 6 is fermented to synthesize luteolin.

8. The method according to claim 7, wherein the fermentation culture conditions include: Culture medium: 2% yeast extract, 4% tryptone, 4% glucose, 0.01M CaCl2; Substrate: Naringenin (final concentration 0.5 g / L); Antioxidant: Ascorbic acid (final concentration 0.01mM); Fermentation time: 4 days; Temperature: 28℃, shaking speed: 220rpm.

9. The method according to claim 7 or 8, wherein the yield of luteolin reaches 4.2 mg / L and the yield of its precursor luteolin reaches 34.8 mg / L.

10. The method for heterologous biosynthesis of luteolin glycoside according to claim 9, wherein, The fermentation conditions include: an initial pH of 5.8-6.2, an initial OD600 value of 0.8-1.2, a fermentation glucose concentration of 5.5-6.5%, an expansion culture medium volume of 150-500 mL, and a fermentation time of 55-65 h.

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