Recombinant bacterium and application thereof in production of tanshinone precursor 11, 20-dihydroxy ferruginol

Abstract

The invention discloses a recombinant bacterium and an application of the recombinant bacterium in production of a tanshinone precursor 11, 20-dihydroxy ferruginol. The recombinant bacterium contains or expresses diterpenoid synthase KSL1 and TPS1, and galactokinase, difunctional UDP glucose 4-isomerase / aldose 1-isomerase and hexose-1-phosphate uridine transferase are knocked out or inhibited. According to the invention, de novo synthesis of 11, 20-dihydroxy ferruginol is realized through a plurality of strategies, and the yield of 11, 20-dihydroxy ferruginol is obviously improved. The recombinant strain provided by the invention can be used for efficiently producing 11, 20-dihydroxy ferruginol and other tanshinone precursors, and has an important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant bacterium and its application in the production of tanshinone precursor 11,20-dihydroxyferric sulfate. Background Technology

[0002] Tanshinones are a class of secondary metabolites with significant research and medicinal value, including tanshinone IIA, tanshinone IIB, and cryptotanshinone, which possess antibacterial, anti-inflammatory, antitumor, and cardiovascular disease prevention effects. Currently, tanshinone compounds are mainly derived from plant extraction or chemical synthesis. However, due to the low content of plant secondary metabolites and the existence of multiple stereoconformations, extraction, purification, and chemical synthesis are significantly limited. Biosynthesis in a microbial heterologous host holds great potential to overcome these production obstacles. The production of tanshinone compounds using Saccharomyces cerevisiae as a host has attracted widespread attention. However, the long heterologous pathway involving multiple CYP450s in yeast remains a significant challenge, requiring multiple optimizations, including increasing precursor yield, cofactor engineering, endoplasmic reticulum membrane modification, and targeting metabolic regulation to improve the efficiency of rate-limiting enzymes, in order to increase yield.

[0003] 11,20-Dihydroxyferruginol is an important intermediate in the biosynthesis of tanshinone. Therefore, constructing engineered strains that produce high levels of 11,20-dihydroxyferruginol has significant application value. Summary of the Invention

[0004] The purpose of this invention is to provide a method for increasing the yield of 11,20-dihydroxyferric alcohol or a recombinant strain for producing 11,20-dihydroxyferric alcohol.

[0005] This invention first protects a recombinant strain that can be a yeast strain that has had or inhibited galactokinase GAL1, bifunctional UDP glucose 4-isomerase / aldose 1-isomerase GAL7 and / or hexose-1-phosphate uridine transferase GAL10 in vivo, and contains or expresses diterpene synthases KSL1 and / or TPS1.

[0006] In the aforementioned recombinant bacteria, KSL1 may be the tSmKSL1 gene (genbank number NC_080389.1) derived from Salvia miltiorrhiza Bunge. TPS1 may be the CfTPS1 gene (genbank number KF444506.1) derived from Coleus forskohlii.

[0007] The recombinant bacteria mentioned above integrated more than one copy (e.g., one, two, or three copies) of KSL1 and / or TPS1.

[0008] In the above-mentioned recombinant bacteria, the presence or expression of diterpenoid synthase KSL1 and / or TPS1 can be achieved by introducing the coding nucleic acid of diterpenoid synthase KSL1 and / or the coding nucleic acid of TPS1 into the starting bacteria.

[0009] In the aforementioned recombinant bacteria, the introduction of the coding nucleic acid for diterpenoid synthase KSL1 and / or the coding nucleic acid for TPS1 into the starting bacteria can be achieved by introducing an expression cassette containing the coding nucleic acids for diterpenoid synthase KSL1 and TPS1 into the starting bacteria. In an embodiment of the present invention, the introduction of the coding nucleic acid for diterpenoid synthase KSL1 and / or the coding nucleic acid for TPS1 into the starting bacteria can be achieved by introducing the repair fragment int4-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int4-ds into the starting bacteria.

[0010] In the above recombinant bacteria, knocking out or inhibiting galactokinase (GAL1), bifunctional UDP glucose 4-isomerase / aldose 1-isomerase (GAL7) and / or hexose-1-phosphate uridine transferase (GAL10) can be achieved by introducing the expression cassette knockout ΔGAL1 / 10 / 7 (nucleotide sequence from 5' to 3' as shown in SEQ ID NO:15) into the starting bacteria or any of the above recombinant bacteria.

[0011] The embodiments of the present invention found that recombinant bacteria integrating 3 copies of KSL1 and 3 copies of TPS1 and knocking out galactokinase (GAL1), bifunctional UDP glucose 4-isomerase / aldose 1-isomerase (GAL7) and hexose-1-phosphate uridine transferase (GAL10) produced a high level of tanshinone diene, with a content of 31.88 mg / L.

[0012] The recombinant bacteria described above may also contain or express ferric sulfate synthase CYP76AH1, ferric sulfate C11 hydroxylase CYP76AH3, 11-hydroxyferric sulfate C20 hydroxylase CYP76AK1, P450 reductase CPR and / or geraniol geraniol pyrophosphate synthase BTS1.

[0013] In the recombinant bacteria described above, the in vivo contents may also contain or express ferrous alcohol synthase CYP76AH1, ferrous alcohol C11 hydroxylase CYP76AH3, 11-hydroxyferrous alcohol C20 hydroxylase CYP76AK1, P450 reductase CPR, and / or geraniol geraniol pyrophosphate synthase BTS1. This can be achieved by introducing the encoding nucleic acid of ferrous alcohol synthase CYP76AH1, ferrous alcohol C11 hydroxylase CYP76AH3, 11-hydroxyferrous alcohol C20 hydroxylase CYP76AK1, P450 reductase CPR, and / or geraniol geraniol pyrophosphate synthase BTS1 into the starting bacteria or any of the recombinant bacteria described above.

[0014] In the above-mentioned recombinant bacteria, the step of introducing the coding nucleic acid of ferrous alcohol synthase CYP76AH1 into the starting bacteria or any of the above-mentioned recombinant bacteria may be to introduce an expression cassette containing the coding nucleic acid of ferrous alcohol synthase CYP76AH1 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0015] In the above-mentioned recombinant bacteria, the step of introducing the nucleic acid encoding ferrous alcohol C11 hydroxylase CYP76AH3 into the starting bacteria or any of the above-mentioned recombinant bacteria may be to introduce an expression cassette containing the nucleic acid encoding ferrous alcohol C11 hydroxylase CYP76AH3 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0016] In the above-mentioned recombinant bacteria, the step of introducing the nucleic acid encoding 11-hydroxyferric sulfate C20 hydroxylase CYP76AK1 into the starting bacteria or any of the above-mentioned recombinant bacteria can be to introduce an expression cassette containing the nucleic acid encoding 11-hydroxyferric sulfate C20 hydroxylase CYP76AK1 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0017] In the above-mentioned recombinant bacteria, the step of introducing the nucleic acid encoding P450 reductase CPR into the starting bacteria or any of the above-mentioned recombinant bacteria may be to introduce an expression cassette containing the nucleic acid encoding P450 reductase CPR into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0018] In the above-mentioned recombinant bacteria, the step of introducing the nucleic acid encoding geraniol geraniol pyrophosphate synthase BTS1 into the starting bacteria or any of the above-mentioned recombinant bacteria can be to introduce an expression cassette containing the nucleic acid encoding geraniol geraniol pyrophosphate synthase BTS1 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0019] The expression cassette may include a promoter, a nucleic acid encoding component, and a terminator.

[0020] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the originating bacteria or any of the above-described recombinant bacteria in the form of a plasmid and / or integrated into the chromosome.

[0021] The ferrous alcohol synthase CYP76AH1 can specifically be SmCYP76AH1 derived from Salvia miltiorrhiza Bunge (genbank number JX422213.1).

[0022] The ferrous ol C11 hydroxylase CYP76AH3 can specifically be SmCYP76AH3 derived from Salvia miltiorrhiza Bunge (genbank number KR140168.1).

[0023] The 11-hydroxyferric oxalool C20 hydroxylase CYP76AK1 can specifically be SmCYP76AK1 (genbank number KR140169.1) derived from Salvia miltiorrhiza Bunge.

[0024] P450 reductase CPR can be SmCPR1 (genbank number FR693803.1) from Salvia miltiorrhiza Bunge or AtCPR1 (genbank number NM_001203894.1) from Arabidopsis thaliana.

[0025] Geraniol geraniol pyrophosphate synthase BTS1 can specifically be BTS1 derived from Saccharomyces cerevisiae (genbank number NP_015256.1).

[0026] In the aforementioned recombinant bacteria, the promoter may be promoter GAL2p, promoter GAL7p, promoter TDH3p, or promoter PGK1p. The terminator may be terminator CYC1t, terminator IDP1t, terminator ADH1t, terminator FBAt, terminator PDC1t, or terminator ENO2t.

[0027] The recombinant bacteria may also contain or express acetyl-CoA thiolytic enzyme ERG10, hydroxymethylpentadiene-CoA reductase ERG13, hydroxymethylglutaryl-CoA reductase HMG1 or its truncated form tHMG1 and / or mevalonate kinase ERG12.

[0028] In the above-mentioned recombinant bacteria, the in vivo contents may also contain or express acetyl-CoA thiolysis enzyme ERG10, hydroxymethylpentadiene-CoA reductase ERG13, hydroxymethylglutaryl-CoA reductase HMG1 or its truncated form tHMG1 and / or mevalonate kinase ERG12. This can be achieved by introducing the coding nucleic acid of acetyl-CoA thiolysis enzyme ERG10, the coding nucleic acid of hydroxymethylpentadiene-CoA reductase ERG13, the coding nucleic acid of hydroxymethylglutaryl-CoA reductase HMG1 or its truncated form tHMG1 and / or the coding nucleic acid of mevalonate kinase ERG12 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0029] In the above-mentioned recombinant bacteria, the step of introducing the nucleic acid encoding acetyl-CoA thiolytic enzyme ERG10 into the starting bacteria or any of the above-mentioned recombinant bacteria can be to introduce an expression cassette containing the nucleic acid encoding acetyl-CoA thiolytic enzyme ERG10 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0030] In the above-mentioned recombinant bacteria, the step of introducing the nucleic acid encoding hydroxymethylpentadiene-coenzyme A reductase ERG13 into the starting bacteria or any of the above-mentioned recombinant bacteria can be the introduction of an expression cassette containing the nucleic acid encoding hydroxymethylpentadiene-coenzyme A reductase ERG13 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0031] In the above-mentioned recombinant bacteria, the step of introducing the nucleic acid encoding hydroxymethylglutaryl-CoA reductase HMG1 or its truncated form tHMG1 into the starting bacteria or any of the above-mentioned recombinant bacteria may be to introduce an expression cassette containing the nucleic acid encoding hydroxymethylglutaryl-CoA reductase HMG1 or its truncated form tHMG1 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0032] In the above-mentioned recombinant bacteria, the step of introducing the nucleic acid encoding mevalonate kinase ERG12 into the starting bacteria or any of the above-mentioned recombinant bacteria may be to introduce an expression cassette containing the nucleic acid encoding mevalonate kinase ERG12 into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0033] The expression cassette may include a promoter, a nucleic acid encoding component, and a terminator.

[0034] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the originating bacteria or any of the above-described recombinant bacteria in the form of a plasmid and / or integrated into the chromosome.

[0035] The specific type of ERG10 is ERG10 derived from Saccharomyces cerevisiae (genbank number NP_015297.1).

[0036] The specific type of hydroxymethylpentadiene coenzyme A reductase ERG13 can be ERG13 derived from Saccharomyces cerevisiae (genbank number NP_013580.1).

[0037] The truncated form of tHMG1 can specifically be tHMG1 derived from Saccharomyces cerevisiae (genbank number NP_013636.1).

[0038] The mevalonate kinase ERG12 can specifically be ERG12 derived from Saccharomyces cerevisiae (genbank number NP_013935.1).

[0039] In the aforementioned recombinant bacteria, the promoter may be promoter TDH3p-2, promoter PGK1p-2, promoter FBAp, promoter TPI1p, or promoter ENO2p. The terminator may be terminator CYC1t, terminator TEF1t, terminator ADH2t, terminator ENO2t, or terminator CCW12t.

[0040] The recombinant bacteria mentioned above may also contain or express β-isopropylpropionic acid dehydrogenase LEU2, phosphoribosylanthraquinone isomerase TRP1 and / or α-aminoadipic acid reductase LYS2.

[0041] In the above-mentioned recombinant bacteria, the in vivo contents may also contain or express β-isopropylpropionic acid dehydrogenase LEU2, phosphoribosylanthraquinone isomerase TRP1 and / or α-aminoadipic acid reductase LYS2. This can be achieved by introducing the nucleic acid encoding β-isopropylpropionic acid dehydrogenase LEU2, the nucleic acid encoding TRP1 and / or the nucleic acid encoding α-aminoadipic acid reductase LYS2 into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0042] The nucleotide sequence encoding the nucleic acid of the β-isopropylpropionic acid dehydrogenase LEU2 is shown in SEQ ID NO:48.

[0043] The nucleotide sequence encoding the nucleic acid of the phosphoribosylanthraquinone isomerase TRP1 is shown in SEQ ID NO:49.

[0044] The specific nucleic acid encoding the α-aminoadipic acid reductase LYS2 can be the encoding gene of LYS2 derived from Saccharomyces cerevisiae (genbank number NP_009673.1).

[0045] In embodiments of the present invention, the introduction of the nucleic acid encoding β-isopropylpropionic acid dehydrogenase LEU2, the nucleic acid encoding phosphoribosylanthraquinone isomerase TRP1, and / or the nucleic acid encoding α-aminoadipic acid reductase LYS2 into the starting bacteria or any of the above-mentioned recombinant bacteria may be performed by introducing plasmid p426-URA3-DPP1-gRNA, repair fragment DPP1Δ-us-LEU2 box-TRP1box-ENO2p, and repair fragment ENO2p-LYS2-TEF1t-DPP1Δ-ds into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0046] In the above-mentioned recombinant bacteria, the recombinant bacteria also have the galactose transcription factor protein GAL80 knocked out or inhibited in vivo and contain or express ferroreductase FDX and / or ferroreductase FDR.

[0047] In the above-mentioned recombinant bacteria, the presence or expression of ferroreductase FDX and / or ferroreductase FDR can be achieved by introducing the nucleic acid encoding ferroreductase FDX and / or the nucleic acid encoding ferroreductase FDR into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0048] The process of introducing the FDX-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can be described as introducing an expression cassette containing the FDX-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria.

[0049] The process of introducing the FDR-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria can be described as introducing an expression cassette containing the FDR-encoding nucleic acid into the starting bacteria or any of the aforementioned recombinant bacteria.

[0050] The expression cassette may include a promoter, a nucleic acid encoding component, and a terminator.

[0051] In the aforementioned recombinant bacteria, FDX can specifically be the SmFDX gene (genbank number XM_057925988.1) derived from Salvia miltiorrhiza Bunge. FDR can specifically be the SmFDR gene (genbank number XM_057943104.1) derived from Salvia miltiorrhiza Bunge.

[0052] In embodiments of the present invention, the introduction of the nucleic acid encoding ferroreductin FDX and / or the nucleic acid encoding ferroreductin reductase FDR into the starting bacteria or any of the above-mentioned recombinant bacteria may be performed by introducing plasmid p426-URA3-XII-4-gRNA, repair fragment XII-4-us-CCW12t-SmFDX-TDH3p-FBAt and repair fragment TDH3p-FBAt-SmFDR-HXT7p-XII-4-ds into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0053] In the above recombinant bacteria, knocking out or inhibiting the galactose transcription factor protein GAL80 can be achieved by introducing a GAL80 repair fragment (nucleotide sequence as shown in SEQ ID NO:58) into the originating bacteria or any of the above recombinant bacteria.

[0054] The present invention found that the YT071 strain constructed according to the above method (see examples) produced 22.69 mg / L of 11,20-dihydroxyferric rust alcohol. The culture medium was Deft medium containing 60 mg / L uracil (URA). The production process did not require the addition of galactose as an inducer, which can significantly reduce fermentation costs.

[0055] The recombinant bacteria mentioned above also contain substances that increase NADPH expression, thereby increasing the electron donor NADPH in the CYP450s reaction.

[0056] The substance that increases NADPH expression can be an overexpression of the NADPH gene; preferably, the NADPH gene is ZWF1 or PFK1. S724D and / or PFK2 S718D PFK1 S724D The nucleotide sequence of the gene is shown in SEQ ID NO:62. PFK2 S718D The nucleotide sequence of the gene is shown in SEQ ID NO:65.

[0057] The overexpression of ZWF1 can specifically involve introducing the plasmid p426-URA3-ZWF1p-gRNA and the repair fragment ZWF1p-us-PGK1p-ZWF1p-ds into the originating bacteria or any of the recombinant bacteria described above, that is, integrating the repair fragment ZWF1p-us-PGK1p-ZWF1p-ds into the originating bacteria or any of the recombinant bacteria described above.

[0058] The overexpression of PFK1 S724D Specifically, this can involve combining the plasmid p426-URA3-PFK1-gRNA and the repair fragment PFK1-us-PFK1. S724D -PFK1 ds introduces the originating bacteria or any of the recombinant bacteria described above, i.e., the repair fragment PFK1-us-PFK1. S724D -PFK1 ds are integrated into the originating bacteria or any of the recombinant bacteria described above.

[0059] The overexpression of PFK2 S718D Specifically, the gene could be the plasmid p426-URA3-PFK2-gRNA and the repair fragment PFK2-us-PFK2. S718D -PFK2-ds introduces the originating bacteria or any of the recombinant bacteria described above, that is, the repair fragment PFK2-us-PFK2. S718D -PFK2-ds are integrated into the originating bacteria or any of the recombinant bacteria described above.

[0060] In the above-mentioned recombinant bacteria, the recombinant bacteria also have transcription factors that inhibit phospholipid biosynthesis knocked out or inhibited, transcription factors that activate phospholipid biosynthesis are contained in or expressed, and / or heme synthase is contained in or expressed; the expression of heme synthase is used to increase the intracellular heme level of the recombinant bacteria.

[0061] In the aforementioned recombinant bacteria, the transcription factor that inhibits phospholipid biosynthesis may be the OPI1 gene. The transcription factor that activates phospholipid biosynthesis may be the INO2 gene (genbank number NP_010408.1) derived from Saccharomyces cerevisiae.

[0062] In the aforementioned recombinant bacteria, the knockout or inhibition of transcription factors that suppress phospholipid biosynthesis can be achieved by inserting plasmids...

[0063] p426-URA3-OPI1-gRNA and the repair fragment knockout OPI1 (nucleotide sequence as shown in SEQ ID NO:67) are introduced into the starting bacteria or any of the recombinant bacteria described above, that is, the repair fragment knockout OPI1 is integrated into the starting bacteria or any of the recombinant bacteria described above.

[0064] In the above-mentioned recombinant bacteria, the transcription factor containing or expressing activation of phospholipid biosynthesis can be introduced into the originating bacteria or any of the above-mentioned recombinant bacteria by introducing plasmid p426-URA3-int16-gRNA and repair fragment int16-us-PGK1p-INO2-IDP1t-int16-ds, that is, integrating the repair fragment int16-us-PGK1p-INO2-IDP1t-int16-ds into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0065] In the above recombinant bacteria, the expression of heme synthase is achieved by overexpressing heme synthase-related genes (such as the HEM13 gene).

[0066] In the above-mentioned recombinant bacteria, the presence or expression of heme synthase can be achieved by transferring plasmid p426-URA3-int16-gRNA, repair fragment int16-us-PGK1p-INO2-IDP1t-HIS3t, and repair fragment IDP1t-HIS3t-HEM13-GPM1p-int16-ds into the originating bacteria or any of the above-mentioned recombinant bacteria, that is, integrating the repair fragment int16-us-PGK1p-INO2-IDP1t-HIS3t and repair fragment IDP1t-HIS3t-HEM13-GPM1p-int16-ds into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0067] This application also attempts to modify iron transport to increase the yield of the target product 11,20-dihydroxyferric sulfate. The main strategies include: 1) increasing iron ion uptake and transport; 2) knocking out iron-suppressing genes and downregulating CCC1 gene expression (genbank number NP_013321.1).

[0068] The recombinant bacteria may also contain or express an iron transporter protein; preferably, the iron transporter protein is at least one of FIT2, FET3 and FTR1.

[0069] In the above-mentioned recombinant bacteria, the presence or expression of the iron ion transporter protein is achieved by introducing the coding nucleic acid of the iron ion transporter protein into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0070] In the above-mentioned recombinant bacteria, the step of introducing the nucleic acid encoding the iron ion transporter protein into the starting bacteria or any of the above-mentioned recombinant bacteria may be to introduce an expression cassette containing the nucleic acid encoding the iron ion transporter protein into the starting bacteria or any of the above-mentioned recombinant bacteria.

[0071] The expression cassette may include a promoter, a nucleic acid encoding component, and a terminator.

[0072] In the above-mentioned recombinant bacteria, the expression cassette is introduced into the originating bacteria or any of the above-described recombinant bacteria in the form of a plasmid and / or integrated into the chromosome.

[0073] The FIT2 mentioned above can specifically be FIT2 derived from Saccharomyces cerevisiae, with the genbank number NP_015027.1.

[0074] Specifically, the FET3 may be derived from Saccharomyces cerevisiae and its genbank number is NP_013774.1.

[0075] The FTR1 mentioned can specifically be FTR1 derived from Saccharomyces cerevisiae, with the genbank number NP_011072.1.

[0076] In the above-mentioned recombinant bacteria, the presence or expression of iron ion transporter protein can be achieved by transferring plasmid p426-URA3-X-1-gRNA, repair fragment X-1-us-CCW12p-FIT2-TDH2t-TPIp, repair fragment TDH2t-TPIp-FET3-ENO2t-PRM9t, and repair fragment ENO2t-PRM9t-FTR1-GPM1p-X1-ds into the originating bacteria or any of the above-mentioned recombinant bacteria, that is, integrating repair fragment X-1-us-CCW12p-FIT2-TDH2t-TPIp, repair fragment TDH2t-TPIp-FET3-ENO2t-PRM9t, and repair fragment ENO2t-PRM9t-FTR1-GPM1p-X1-ds into the originating bacteria or any of the above-mentioned recombinant bacteria.

[0077] The recombinant bacteria also have iron-associated protein and / or vacuole iron transporter CCC1 knocked out or inhibited in vivo.

[0078] Preferably, the iron-related protein is at least one of BolA protein BOL2, iron transcription factor protein YAP5, cytoplasmic glutathione protein GRX3, and cytoplasmic glutathione protein GRX4.

[0079] The BOL2 mentioned can specifically be BOL2 derived from Saccharomyces cerevisiae, with the genbank number NP_011296.1.

[0080] The YAP5 mentioned can specifically be YAP5 derived from Saccharomyces cerevisiae, with the genbank number NP_012283.1.

[0081] The GRX3 may specifically be a GRX3 derived from Saccharomyces cerevisiae with the genbank number NP_010382.3.

[0082] The GRX4 may specifically be a GRX4 derived from Saccharomyces cerevisiae with the genbank number NP_011101.3.

[0083] The knockout or inhibition of BOL2 can be achieved by transferring the plasmid p426-URA3-BOL2-gRNA and the repair fragment knocking out BOL2 (SEQ ID NO:74) into the starting bacteria or any of the recombinant bacteria described above, i.e., the strain that knocks out BOL2.

[0084] The knockout or inhibition of vacuole iron transporter CCC1 can be achieved by transferring plasmid p426-URA3-CCC1p-gRNA and repair fragment CCC1p-us-CYB2p-CCC1p-ds into the originating bacterium or any of the recombinant bacteria described above, that is, integrating the repair fragment CCC1p-us-CYB2p-CCC1p-ds into the originating bacterium or any of the recombinant bacteria described above.

[0085] The knockout or inhibition of GRX3 can be achieved by transferring the plasmid p426-URA3-GRX3-gRNA and the repair fragment that knocks out GRX3 (nucleotide sequence as shown in SEQ ID NO:78) into the starting bacteria or any of the recombinant bacteria described above, i.e., the strain that knocks out GRX3.

[0086] The knockout or inhibition of GRX4 can be achieved by transferring the plasmid p426-URA3-GRX4-gRNA and the repair fragment that knocks out GRX4 (nucleotide sequence as shown in SEQ ID NO:79) into the originating bacteria or any of the recombinant bacteria described above, i.e., the strain that knocks out GRX4.

[0087] The knockout or inhibition of YAP5 can be achieved by transferring the plasmid p426-URA3-YAP5-gRNA and the repair fragment knocking out YAP5 (nucleotide sequence as shown in SEQ ID NO:46) into the starting strain or any of the recombinant strains described above, i.e., the strain that knocks out YAP5.

[0088] The starting strain may be *Saccharomyces cerevisiae*. Specifically, the *Saccharomyces cerevisiae* may be the BYHZ16 yeast strain or the BYHZ16::Cas9 strain.

[0089] Any of the recombinant bacteria mentioned above is a species of Saccharomyces cerevisiae.

[0090] Any of the recombinant bacteria mentioned above may be Saccharomyces cerevisiae.

[0091] This invention also protects the application of any of the recombinant bacteria described above, which may be S1) or S2).

[0092] S1) produces 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tungstenol, 11-hydroxytungstenol and / or 11,20-dihydroxytungstenol.

[0093] S2) produces downstream compounds of 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tungstenol, 11-hydroxytungstenol and / or 11,20-dihydroxytungstenol.

[0094] In the above S2), preferably, the downstream compound includes at least one of tanshinone IIA, tanshinone, 16-hydroxytanshinone, tanshinone IIB, cryptotanshinone, tanshinone I, dihydrotanshinone II, hydroxytanshinone, methyl tanshinone, and oxalic acid.

[0095] This invention also protects a method for producing 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tungsten, 11-hydroxytungsten, 11,20-dihydroxytungsten and / or "downstream compounds of 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tungsten, 11-hydroxytungsten and / or 11,20-dihydroxytungsten", which may include the following steps: fermentation Cultivate any of the recombinant bacteria described above, collect the fermentation products, and obtain 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tsucrose, 11-hydroxytsucrose, 11,20-dihydroxytsucrose and / or downstream compounds of 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tsucrose, 11-hydroxytsucrose and / or 11,20-dihydroxytsucrose.

[0096] In the above method, the downstream compound may include at least one of tanshinone IIA, tanshinone, 16-hydroxytanshinone, tanshinone IIB, cryptotanshinone, tanshinone I, dihydrotanshinone II, hydroxytanshinone, methyl tanshinone, and oxalic acid.

[0097] The inventors of this invention constructed a recombinant strain for producing 11,20-dihydroxyferric rust alcohol through extensive experiments. They divided the synthetic pathway of 11,20-dihydroxyferric rust alcohol into multiple modules for research and achieved de novo synthesis of 11,20-dihydroxyferric rust alcohol through various strategies. The starting strain was either the BYHZ16 yeast strain or the BYHZ16::Cas9 strain. For example, GGPP generates the key intermediate tanshinone diene under the catalysis of a fusion protein constructed from the diterpenoid synthase tSmKSL1 from Salvia miltiorrhiza Bunge and the diterpenoid synthase CfTPS1 from Coleus forskohlii; tanshinone diene forms the key intermediate ferrugin under the catalysis of SmCYP76AH1 from Salvia miltiorrhiza; ferrugin is ultimately converted into various tanshinone precursors under the catalysis of SmCYP76AH3 and SmCYP76AK1 from Salvia miltiorrhiza; among them, SmCYP76AH3 uses ferrugin as a substrate to generate 11-hydroxyferrugin, tsucrose, and 11-hydroxytsucrose, and SmCYP76AK1 uses 11-hydroxyferrugin and 11-hydroxytsucrose as substrates to generate 11,20-dihydroxyferrugin and 11,20-dihydroxytsucrose, respectively. Experiments have shown that the engineered strain constructed in this invention can efficiently produce 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tungsten, 11-hydroxytungsten, and / or 11,20-dihydroxytungsten, especially 11,20-dihydroxyferric alcohol. This invention has significant application value. Attached Figure Description

[0098] Figure 1 The biosynthetic pathway of 11,20-dihydroxyferric sulfate is shown.

[0099] Figure 2 The results of the first step in the example are the detection results of the tanshinone diene content.

[0100] Figure 3 The results of the detection of 20-dihydroxyferric alcohol content are shown in step 11 of the example.

[0101] Figure 4 The results of the detection of 20-dihydroxyferric alcohol content in step 3, 11, of the example are shown.

[0102] Figure 5 The results of the detection of 20-dihydroxyferric alcohol content are shown in step 4, 11 of the example.

[0103] Figure 6 The results of step 5, 11, in the embodiment show the detection results of the 20-dihydroxyferric alcohol content. Detailed Implementation

[0104] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0105] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0106] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0107] The primer names and their nucleotide sequences involved in the following examples are shown in Table 1.

[0108] Table 1

[0109]

[0110]

[0111]

[0112] The strain names and their genotypes involved in the following examples are shown in Table 2.

[0113] Table 2

[0114]

[0115]

[0116] The BYHZ16 yeast strain is described in the following literature: Hu T, Zhou J, Tong Y, Su P, Li X, Liu Y, et al. Engineering chimeric diterpene synthases and isoprenoid biosynthetic pathways enable high-level production of miltiradiene in yeast. Metab Eng 2020; 60:87-96.

[0117] LC-MS / MS was performed using an Agilent HPLC-QTOF system with an ESI detector and an Agilent EclipsePlus C18 column (RRHD 1.8 μm, 2.1 × 100 mm). Mobile phase A was 0.1% formic acid solution (solvent: water). Mobile phase B was 0.1% formic acid (solvent: acetonitrile). The flow rate was set to 0.3 mL / min. Mass spectrometry conditions were set to negative ion mode. The mobile phase gradient was: 0–8 min, 35% B–70% B; 8–10.5 min, 70% B–85% B; 10.5–12 min, 98% B; 12.5–13 min, 98% B–35% B; 13–15 min, 35% B. The UPLC system was a Waters UPLC system with PDA detectors (254 nm and 280 nm). The column was a BEH 5 cm × 2.1 mm C18 column (particle size 1.7 μm, Waters, Germany). The mobile phase gradient was: 0–8 min, 35% B–70% B; 8–10.5 min, 70% B–85% B; 10.5–12 min, 98% B; 12.5–13 min, 98% B–35% B; 13–15 min, 35% B. The GC-MS system was a Thermo Fisher Scientific system, and the column was an ATR-5ms capillary column (30m × 0.25mm id, 0.25μm film thickness, Thermo Fisher Scientific). The conditions were as follows: 0-5min, ramping at 40℃ / min to 170℃; 5-8.5min, ramping at 20℃ / min to 240℃; 8.5-10.5min, ramping at 40℃ / min to 300℃; 10.5-11min, holding at 300℃ for 1min.

[0118] In the following examples, plasmid p426-URA3-gRNA is a product of the EUROSCARF collection. Plasmid p426-URA3-gRNA carries the SNR52 promoter and the gRNA backbone.

[0119] In the following examples, p<0.05 indicates a significant difference, ** indicates p<0.01 indicates a significant difference, *** indicates p<0.001 indicates a significant difference, and **** indicates p<0.0001 indicates a significant difference.

[0120] Example

[0121] The inventors of this invention constructed a recombinant strain for producing the tanshinone precursor 11,20-dihydroxyferric sulfate through extensive experiments. The synthetic pathway of 11,20-dihydroxyferric sulfate was divided into five aspects for study, and de novo synthesis of 11,20-dihydroxyferric sulfate was achieved through various strategies. The starting strain was BYHZ16::Cas9 (described in the following literature: HU T, ZHOU J, TONG Y, et al. Engineering chimeric diterpenesynthases and isoprenoid biosynthetic pathways enables high-level production of miltiradiene in yeast[J]. Metab Eng, 2020, 60:87-96.), which is a Saccharomyces cerevisiae strain that integrates the Cas9 gene into the BYHZ16 yeast strain under the TEF1 promoter (the method of integrating the Cas9 gene can be found in the following literature: Yang S, Cao X, Yu W, Li S, Zhou YJ. Efficient targeted mutation of genomic essential genes). In yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 2020; 104:3037-47.). The genotype of the yeast strain BYHZ16 is MATα,LEU2Δ0,LYS2Δ0,MET15,URA3Δ0,TRP1Δ::HIS3-PGK1p-BTS1 / ERG20-ADH1t-TDH3p-SaGGPS-TPI1t-TEF1p-tHMG1-CYC1t,ROX1Δ,ERG9::Δ218-175,YJL064wΔ,YPL062wΔ.

[0122] The biosynthetic pathway of 11,20-dihydroxyferric sulfate is shown in the figure. Figure 1In Saccharomyces cerevisiae, the precursor of 11,20-dihydroxyferric ester, geraniylgeraniyl pyrophosphate (GGPP), is synthesized via the mevalonate (MVA) pathway. The first key intermediate, acetyl-coenzyme A (acetyl-CoA), is introduced, and under the catalysis of acetoacetyl-CoA thiolase (ERG10) and hydroxymethylglutaryl-CoA reductase (ERG13), hydroxymethylglutaryl-CoA (HMG-CoA) is generated. Subsequently, HMG-CoA is reduced to the key intermediate mevalonate under the catalysis of hydroxymethylglutaryl-CoA reductase (HMGR; the isoenzyme in Saccharomyces cerevisiae is HMG1) or a truncated HMGR1, tHMG1. MVA is converted into isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) by mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), mevalonate pyrophosphate decarboxylase (ERG19), and isopentenyl diphosphate isomerase (IDI1). These two compounds are then converted into farnesyl diphosphate (FPP) by farnesyl pyrophosphate synthase (ERG20). FPP is then converted into GGPP by geranylgeranyl diphosphate synthase (GGPPS). GGPP generates the key intermediate tanshinone diene under the catalysis of a fusion protein constructed from the diterpenoid synthase tSmKSL1 derived from Salvia miltiorrhiza Bunge and the diterpenoid synthase CfTPS1 derived from Coleus forskohlii. Tanshinone diene then forms the key intermediate ferrugin under the catalysis of SmCYP76AH1 from Salvia miltiorrhiza. Ferrulin, in turn, is catalyzed by SmCYP76AH3 and SmCYP76AK1 from Salvia miltiorrhiza, ultimately generating various tanshinone precursors.SmCYP76AH3 uses ferric alcohol as a substrate to generate 11-hydroxyferric alcohol, thodophenol and 11-hydroxythodophenol, while SmCYP76AK1 uses 11-hydroxyferric alcohol and 11-hydroxythodophenol as substrates to generate 11,20-dihydroxyferric alcohol and 11,20-dihydroxythodophenol, respectively.

[0123] I. Obtaining strains YT001, YT002, YT003, and YT004 and their application in the production of tanshinone diene.

[0124] (I) Obtaining strain YT001

[0125] The tSmKSL1 gene (genbank number NC_080389.1) from Salvia miltiorrhiza Bunge and the CfTPS1 gene (genbank number KF444506.1) from Coleus forskohlii were codon-optimized according to the preferences of Saccharomyces cerevisiae, and then integrated into the int4 site of BYHZ16::Cas9 using the CRISPR-Cas9 genome editing method.

[0126] 1. Construction of plasmid p426-URA3-int4-gRNA

[0127] (1) Using plasmid p426-URA3-gRNA as a template, AarI enzyme was used to digest the linearized plasmid p426-URA3-gRNA.

[0128] (2) Artificially synthesize int4-N20-F and int4-N20-R, mix them in equal amounts, treat at 95℃ for 10 min, cool to room temperature, and obtain annealed short nucleic acid sequences, which include a 4bp sequence homologous to the SNR52 promoter, a 20bp gRNA sequence, and a 4bp sequence homologous to the gRNA backbone.

[0129] (3) The annealed short nucleic acid sequence and the linearized plasmid p426-URA3-gRNA were ligated using T4 ligase to obtain plasmid p426-URA3-int4-gRNA.

[0130] 2. Preparation of the repaired fragment int4-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int4-ds

[0131] The repair fragment int4-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int4-ds, from 5' to 3', consists of the upstream homologous arm int4-us (nucleotide sequence as shown in SEQ ID NO:1), the promoter GAL10p (nucleotide sequence as shown in SEQ ID NO:2), the gene fragment tSmKSL-GGGS-CfTPS1 (nucleotide sequence as shown in SEQ ID NO:3), the terminator CYC1t (nucleotide sequence as shown in SEQ ID NO:4), and the downstream homologous arm int4-ds (nucleotide sequence as shown in SEQ ID NO:5). The specific preparation process is as follows:

[0132] (1) Prepare the overlap system. The overlap system is either overlap system 1 or overlap system 2.

[0133] Overlap system 1 is 25 μL, consisting of 5 μL of 5×Prime STARbuffer, 2 μL of dNTPs (2.5 mM each), upstream homologous arm int4-us, downstream homologous arm int4-ds, expression cassette GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t (nucleotide sequence as shown in SEQ ID NO:6), 0.5 μL of Polymerase, and ddH2O.

[0134] The overlap system 2 consisted of 25 μL of PrimeStar STAR Max premix (2×) 12.5 μL, upstream homologous arm int4-us, downstream homologous arm int4-ds, expression cassette GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t (nucleotide sequence as shown in SEQ ID NO: 6), and ddH2O.

[0135] In the overlap system, the concentration of the upstream homologous arm int4-us was 0.03 pmol; the molar ratio of the upstream homologous arm int4-us, the downstream homologous arm int4-ds, and the expression cassette GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t was 1:3:1.

[0136] (2) Take the overlap system and react it to obtain the product.

[0137] The reaction conditions were: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 2 min, for 15 cycles; and a final extension at 72°C for 10 min.

[0138] (3) Take 2 μl of the product obtained in step (2) and use it as a template. Use primers composed of int4-up-F and int4-dw-R to perform PCR amplification to obtain the repair fragment int4-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int4-ds.

[0139] The reaction conditions were: denaturation at 98°C for 2 min; denaturation at 98°C for 10 sec, annealing at 55°C for 15 sec, extension at 72°C for 4 min, for 35 cycles; and a final extension at 72°C for 10 min.

[0140] 3. Obtaining strain YT001

[0141] (1) The plasmid p426-URA3-int4-gRNA obtained in step 1 and the repair fragment int4-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int4-ds obtained in step 2 were added to BYHZ16::Cas9 yeast competent cells in accordance with Table 3. After mixing, the cells were incubated at 30℃ for 30 min. Then, the cells were heat-shocked at 42℃ for 15 min, centrifuged at room temperature and 3800 rpm for 3-4 min, the cells were collected and resuspended in 100 μL of sterile water to obtain the resuspension.

[0142] Table 3

[0143]

[0144] Note: Salmon sperm single-stranded DNA needs to be denatured in a boiling water bath for 5 minutes, and then placed on ice for 2 minutes.

[0145] (2) Place the resuspended liquid droplet in the center of the SD-Ura plate, spread it evenly with a spreader until all the bacterial solution is completely absorbed, and incubate it upside down in a 30℃ incubator for 2-3 days.

[0146] SD-Ura plates: Add distilled water to 8g Ura minus medium (product of Pankino, catalog number YGM003A-3) and 20g agar powder, then bring the volume to 1L with distilled water and sterilize at 121℃ for 15min; finally, add glucose filtered through a 0.22μm filter membrane to a final concentration of 2%, pour into sterile plates and cool.

[0147] (3) Single clones were picked from the SD-Ura plate and colony PCR was performed. Specifically, the picked single clones were placed in 20 mmol / L NaOH solution and lysed at 99℃ for 20 min, then centrifuged for 2 min and the supernatant was collected. The supernatant was used as a template and PCR amplification was performed using specific primers to obtain the PCR amplification product.

[0148] The reaction conditions were: 98℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 55℃ annealing for 15 sec, 68℃ extension for 30-60 sec, 30 cycles; and a final extension at 68℃ for 10 min.

[0149] (4) Perform agarose gel electrophoresis on the PCR amplification products respectively, and then make the following judgment: if the PCR amplification product obtained by a certain single clone meets the expected DNA fragment size, then the single clone is a positive clone.

[0150] (5) Randomly select 4 positive clones and streak them on 5-FOA plates. Incubate them in a constant temperature incubator at 30℃ for 2-3 days. The resulting colonies are the strains that have lost the gRNA plasmid. The obtained strains are named YT001 yeast strain (hereinafter referred to as YT001).

[0151] 5-FOA plates: Add distilled water to 8g SC medium and 20g agar powder, then bring the volume to 1L with distilled water and sterilize at 121℃ for 20min; finally, when the temperature of the medium drops to about 60℃, add 5-FOA stock solution (obtained by dissolving 5-fluoroorotic acid (5-FOA) powder in dimethyl sulfoxide (DMSO)) in a sterile laminar flow hood to make the final concentration of 5-FOA 0.1%, pour into sterile plates and cool.

[0152] Compared to BYHZ16::Cas9, the YT001 yeast strain integrated 1 copy

[0153] GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t.

[0154] (II) Obtaining strain YT002

[0155] The tSmKSL1 gene (genbank number NC_080389.1) from Salvia miltiorrhiza Bunge and the CfTPS1 gene (genbank number KF444506.1) from Coleus forskohlii Plectranthus barbatus were integrated into the int14 site of YT001 using the CRISPR-Cas9 genome editing method.

[0156] 1. Construction of plasmid p426-URA3-int14-gRNA

[0157] Following the method in step (1), replace int4-N20-F with int14-N20-F and int4-N20-R with int14-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-int14-gRNA.

[0158] 2. The plasmid p426-URA3-int14-gRNA and the repair fragment int14 were added...

[0159] us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int14-ds was transduced into YT001 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30°C for 2-3 days. The resulting clones were the strains that had lost the gRNA plasmid. The repair fragment int14 us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int14-ds was integrated into the int14 site of YT001 cells. The resulting strain was named YT002 yeast strain (hereinafter referred to as YT002).

[0160] The repair fragment int14 us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int14-ds consists of, from 5' to 3', the upstream homologous arm int14-us (nucleotide sequence as shown in SEQ ID NO:7), the promoter GAL10p (nucleotide sequence as shown in SEQ ID NO:2), the gene fragment tSmKSL-GGGS-CfTPS1 (nucleotide sequence as shown in SEQ ID NO:3), the terminator CYC1t (nucleotide sequence as shown in SEQ ID NO:4), and the downstream homologous arm int14-ds (nucleotide sequence as shown in SEQ ID NO:8).

[0161] Compared to BYHZ16::Cas9, the YT002 yeast strain integrated 2 copies

[0162] GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t.

[0163] (III) Obtaining strain YT003

[0164] The galactose-consuming genes GAL1 / 10 / 7 (i.e., galactose-consuming genes GAL1, GAL10, and GAL7) in strain YT002 were knocked out using a one-step CRISPR-Cas9 genome editing method. GAL1 encodes galactokinase (genbank ID: NP_009576.1), GAL10 encodes a bifunctional UDP glucose 4-isomerase / aldose 1-isomerase (genbank ID: NP_009575.1), and GAL7 encodes hexose-1-phosphate uridine transferase (genbank ID: NP_009574.1).

[0165] 1. Construction of plasmid p426-URA3-GAL1 / 10 / 7-gRNA

[0166] Following the method in step (I), replace int4-N20-F with GAL1 / 10 / 7-N20-F and int4-N20-R with GAL1 / 10 / 7-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-GAL1 / 10 / 7-gRNA.

[0167] 2. Following the method in step (I), replace plasmid p426-URA3-int4-gRNA with plasmid p426-URA3-GAL1 / 10 / 7-gRNA to repair the fragment.

[0168] The expression cassette knockout ΔGAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int4-ds was replaced with expression cassette knockout ΔGAL1 / 10 / 7 (nucleotide sequence from 5' to 3' as shown in SEQ ID NO:15), and the BYHZ16::Cas9 yeast competent cells were replaced with YT002 yeast competent cells. All other steps remained unchanged, resulting in a strain with expression cassette knockout ΔGAL1 / 10 / 7 integrated into the GAL1 / 10 / 7 site of YT002. The resulting strain was named YT003 yeast strain (hereinafter referred to as YT003).

[0169] Compared to BYHZ16::Cas9, the YT003 yeast strain integrated 2 copies.

[0170] GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t, while simultaneously knocking out the galactose-consuming genes GAL1 / 10 / 7.

[0171] (iv) Obtaining strain YT004

[0172] The tSmKSL1 gene (genbank number NC_080389.1) from Salvia miltiorrhiza Bunge and the CfTPS1 gene (genbank number KF444506.1) from Coleus forskohlii Plectranthus barbatus were integrated into the int17 site of YT003 using the CRISPR-Cas9 genome editing method.

[0173] 1. Construction of plasmid p426-URA3-int17-gRNA

[0174] Following the method in step (I), replace int4-N20-F with int17-N20-F and int4-N20-R with int17-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-int17-gRNA.

[0175] 2. Plasmid p426-URA3-int17-gRNA and repair fragment

[0176] Transfecting YT003 yeast competent cells with int17-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int17-ds yielded positive clones. Randomly selected positive clones were streaked onto 5-FOA plates and incubated at 30°C for 2-3 days. The resulting clones were the strains that had lost the gRNA plasmid and were about to repair the fragment.

[0177] The int17-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int17-ds was integrated into the int17 site of YT003, and the resulting strain was named YT004 yeast strain (hereinafter referred to as YT004).

[0178] The repair fragment int17-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-int17-ds consists of, from 5' to 3', the upstream homologous arm int17-us (nucleotide sequence as shown in SEQ ID NO:9), the promoter GAL10p (nucleotide sequence as shown in SEQ ID NO:2), the gene fragment tSmKSL-GGGS-CfTPS1 (nucleotide sequence as shown in SEQ ID NO:3), the terminator CYC1t (nucleotide sequence as shown in SEQ ID NO:4), and the downstream homologous arm int17-ds (nucleotide sequence as shown in SEQ ID NO:10).

[0179] Compared to BYHZ16::Cas9, the YT004 yeast strain integrated 3 copies

[0180] GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t, while simultaneously knocking out the galactose-consuming genes GAL1 / 10 / 7.

[0181] (V) Application of YT001, YT002, YT003 and YT004 in the production of tanshinone diene

[0182] Three single clones from YT001, YT002, YT003, and YT004 were selected for parallel experiments. The experiments performed on each single clone are as follows:

[0183] 1. Add 1 ml of SC medium (solute and concentration: 8 g SC powder and 20 g / L glucose, solvent: water) to a 14 mL shaker tube, then inoculate with a single colony and incubate at 30℃ and 220 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a shaker flask (100 mL) containing 20 ml of YPD medium, then add galactose (as an inducer) to obtain the induction system; incubate the induction system at 30℃ and 200 rpm for 120 h to obtain the fermentation broth.

[0184] SC powder is a product of Pankino Corporation, with product catalog number YGM003A-1.

[0185] When the monoclonal strain is YT001 or YT002, the concentration of galactose in the induction system is 20 g / L.

[0186] When the monoclonal strain is YT003 or YT004, the concentration of galactose in the induction system is 10 g / L.

[0187] 2. Determine the OD of the fermentation broth. 600nm And take the average of 3 parallel experiments.

[0188] 3. Take 500 μL of the fermentation broth obtained in step 1, add 500 μL of chromatographically pure n-hexane, and vortex thoroughly for 5 min; then centrifuge at 13000 rpm for 12 min and collect the supernatant; finally, dilute the collected supernatant with 1 volume of n-hexane and perform GC-MS detection (the purpose is to detect the metabolic components of the strain).

[0189] Test results are shown Figure 2 The results showed that YT004, which integrates 3 copies of GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t and knocks out the galactose-consuming gene GAL1 / 10 / 7, produced the highest level of tanshinone diene, with a content of 31.88 mg / L.

[0190] II. Obtaining strains YT009, YT010, YT011, YT012, and YT016 and their application in the production of 11,20-dihydroxyferric rust alcohol.

[0191] A strain producing 11,20-dihydroxyferric rustol was constructed by selecting SmCYP76AH1, SmCYP76AH3, SmCYP76AK1 and SmCPR1 from Salvia miltiorrhiza Bunge and AtCPR1 from Arabidopsis thaliana.

[0192] (I) Obtaining strains YT009, YT010, YT011, YT012 and YT016

[0193] 1. Construction of plasmids p426-URA3-X-4-gRNA, p426-URA3-YORWΔ17-gRNA, p426-URA3-YORWΔ22-gRNA, p426-URA3-AIF1-gRNA, p426-URA3-X-2-gRNA, p426-URA3-X-3-gRNA, and p426-URA3-YPRCδ15c-gRNA.

[0194] Following the method in step (I), replace int4-N20-F with X-4-N20-F and int4-N20-R with X-4-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-X-4-gRNA.

[0195] Following the method in step (I), replace int4-N20-F with YORWΔ17-N20-F and int4-N20-R with YORWΔ17-N20-R, keeping all other steps unchanged, to obtain plasmid p426-URA3-YORWΔ17-gRNA.

[0196] Following the method in step (I), replace int4-N20-F with YORWΔ22-N20-F and int4-N20-R with YORWΔ22-N20-R, keeping all other steps unchanged, to obtain plasmid p426-URA3-YORWΔ22-gRNA.

[0197] Following the method in step (I), replace int4-N20-F with AIF1-N20-F and int4-N20-R with AIF1-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-AIF1-gRNA.

[0198] Following the method in step (I), replace int4-N20-F with X-2-N20-F and int4-N20-R with X-2-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-X-2-gRNA.

[0199] Following the method in step (I), replace int4-N20-F with X-3-N20-F and int4-N20-R with X-3-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-X-3-gRNA.

[0200] Following the method in step (I), replace int4-N20-F with YPRCδ15c-N20-F and int4-N20-R with YPRCδ15c-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-YPRCδ15c-gRNA.

[0201] 2. Construction of the Expression Box

[0202] An expression cassette is constructed by connecting the components using the overlap method. The expression cassette consists of a promoter, the nucleotide sequence of the gene, and a terminator. The genes were SmCYP76AH1 (genbank number JX422213.1), SmCYP76AH3 (genbank number KR140168.1), SmCYP76AK1 (genbank number KR140169.1), and SmCPR1 (genbank number FR693803.1) from Salvia miltiorrhiza Bunge, AtCPR1 (genbank number NM_001203894.1) from Arabidopsis thaliana, BTS1 (genbank number NP_015256.1) from Saccharomyces cerevisiae, and CfTPS1 (genbank number KF444506.1) from Coleus forskohlii. The promoters are GAL2p (nucleotide sequence as shown in SEQ ID NO:11), GAL7p (nucleotide sequence as shown in SEQ ID NO:12), TDH3p (nucleotide sequence as shown in SEQ ID NO:13), or PGK1p (nucleotide sequence as shown in SEQ ID NO:14). The terminators are CYC1t (nucleotide sequence as shown in SEQ ID NO:4), IDP1t (nucleotide sequence as shown in SEQ ID NO:16), ADH1t (nucleotide sequence as shown in SEQ ID NO:17), FBAt (nucleotide sequence as shown in SEQ ID NO:18), PDC1t (nucleotide sequence as shown in SEQ ID NO:19), or ENO2t (nucleotide sequence as shown in SEQ ID NO:24).

[0203] Specifically, the constructed expression boxes are as follows: PGK1p-SmCPR1-CYC1t expression box, GAL2p-CYP76AH1-ENO2t expression box, GAL2p-CYP76AH3-FBAt expression box, GAL7p-CYP76AK1-PDC1t expression box, TDH3p-AtCPR1-ADH1t expression box, GAL2p-CYP76AH3-FBAt expression box and GAL7p-CYP76AK1-PDC1t expression box.

[0204] 3. Obtaining strain YT009

[0205] (1) Preparation of repair fragments

[0206] (1-1) The upstream homologous arm YORWΔ17-us (nucleotide sequence as shown in SEQ ID NO:20), the PGK1p-SmCPR1-CYC1t expression cassette, and the downstream homologous arm YORWΔ17-ds (nucleotide sequence as shown in SEQ ID NO:21) were ligated using the overlap method to obtain the repair fragment YORWΔ17-us-PGK1p-SmCPR1-CYC1t-YORWΔ17-ds. The repair fragment YORWΔ17-us-PGK1p-SmCPR1-CYC1t-YORWΔ17-ds consists of the upstream homologous arm YORWΔ17-us, the PGK1p-SmCPR1-CYC1t expression cassette, and the downstream homologous arm YORWΔ17-ds, sequentially from 5' to 3'.

[0207] (1-2) The upstream homologous arm YORWΔ22-us (nucleotide sequence shown in SEQ ID NO:22), the GAL2p-CYP76AH1-ENO2t expression cassette, and the downstream homologous arm YORWΔ22-ds (nucleotide sequence shown in SEQ ID NO:23) were ligated using the overlap method to obtain the repair fragment YORWΔ22-us-GAL2p-CYP76AH1-ENO2t-YORWΔ22-ds. The repair fragment YORWΔ22-us-GAL2p-CYP76AH1-ENO2t-YORWΔ22-ds consists of the upstream homologous arm YORWΔ22-us, the GAL2p-CYP76AH1-ENO2t expression cassette, and the downstream homologous arm YORWΔ22-ds, sequentially from 5' to 3'.

[0208] (1-3) The upstream homologous arm X-4-us (nucleotide sequence as shown in SEQ ID NO:25), the GAL2p-CYP76AH3-FBAt expression cassette, the GAL7p-CYP76AK1-PDC1t expression cassette, and the downstream homologous arm X-4-ds (nucleotide sequence as shown in SEQ ID NO:26) were ligated using the overlap method to obtain the repair fragment.

[0209] The repaired fragment X-4-us-FBAt-CYP76AH3-GAL2p-GAL7p-CYP76AK1-PDC1t-X-4-ds consists of the upstream homologous arm X-4-us, the GAL2p-CYP76AH3-FBAt expression cassette, the GAL7p-CYP76AK1-PDC1t-X-4-ds expression cassette, and the downstream homologous arm X-4-ds, from 5' to 3'.

[0210] (2) Plasmid p426-URA3-YORWΔ17-gRNA and repair fragment

[0211] YORWΔ17-us-PGK1p-SmCPR1-CYC1t-YORWΔ17-ds was transformed into YT004 yeast competent cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The repair fragment YORWΔ17-us-PGK1p-SmCPR1-CYC1t-YORWΔ17-ds was integrated into the YORWΔ17 site of YT004 to obtain intermediate strain 1.

[0212] (3) Plasmid p426-URA3-YORWΔ22-gRNA and repair fragment

[0213] YORWΔ22-us-GAL2p-CYP76AH1-ENO2t-YORWΔ22-ds was transformed into competent cells of intermediate strain 1 to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The repair fragment YORWΔ22-us-GAL2p-CYP76AH1-ENO2t-YORWΔ22-ds was integrated into the YORWΔ22 site of intermediate strain 1 to obtain intermediate strain 2.

[0214] (4) Plasmid p426-URA3-X-4-gRNA and repair fragment

[0215] X-4-us-FBAt-CYP76AH3-GAL2p-GAL7p-CYP76AK1-PDC1t-X-4-ds were transfected into competent cells of intermediate strain 2 to obtain positive clones. Randomly selected positive clones were streaked onto 5-FOA plates and incubated at 30°C for 2-3 days. The resulting clones were from strains that had lost the gRNA plasmid and were about to repair the fragment.

[0216] X-4-us-FBAt-CYP76AH3-GAL2p-GAL7p-CYP76AK1-PDC1t-X-4-ds was integrated into the X-4 site of intermediate strain 2, and the resulting strain was named YT009 yeast strain (hereinafter referred to as YT009).

[0217] 4. Obtaining strain YT010

[0218] (1) The upstream homologous arm X-2-us (nucleotide sequence as shown in SEQ ID NO:27), the TDH3p-AtCPR1-ADH1t expression cassette, and the downstream homologous arm X-2-ds (nucleotide sequence as shown in SEQ ID NO:28) were ligated using the overlap method to obtain the repair fragment X-2-us-ADH1t-AtCPR1-TDH3-X-2-ds.

[0219] The X-2-us-ADH1t-AtCPR1-TDH3-X-2-ds expression cassette, from 5' to 3', consists of the upstream homologous arm X-2-us, the TDH3p-AtCPR1-ADH1t expression cassette, and the downstream homologous arm X-2-ds, respectively.

[0220] (2) The plasmid p426-URA3-X-2-gRNA and the repair fragment X-2-us-ADH1t-AtCPR1-TDH3-X-2-ds were transformed into competent cells of YT009 yeast to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30℃ for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid and were about to have their repair fragments transferred.

[0221] X-2-us-ADH1t-AtCPR1-TDH3-X-2-ds was integrated into the X-2 site of YT009, and the resulting strain was named YT010 yeast strain (hereinafter referred to as YT010).

[0222] 5. Obtaining strain YT011

[0223] (1) Using the overlap method, the upstream homologous arm YPRCδ15c-us (nucleotide sequence as shown in SEQ ID NO:29), GAL2p-CYP76AH3-FBAt expression cassette, GAL7p-CYP76AK1-PDC1t expression cassette and the downstream homologous arm were combined.

[0224] The YPRCδ15c-ds nucleotide sequence (as shown in SEQ ID NO:30) was ligated to obtain the repair fragment YPRCδ15c-us-FBAt-CYP76AH3-GAL2p-GAL7p-CYP76AK1-PDC1t-YPRCδ15c-ds. The repair fragment YPRCδ15c-us-FBAt-CYP76AH3-GAL2p-GAL7p-CYP76AK1-PDC1t-YPRCδ15c-ds consists, from 5' to 3', the upstream homologous arm YPRCδ15c-us, the GAL2p-CYP76AH3-FBAt expression cassette, the GAL7p-CYP76AK1-PDC1t expression cassette, and the downstream homologous arm YPRCδ15c-ds.

[0225] (2) Plasmid p426-URA3-YPRCδ15c-gRNA and repair fragment

[0226] YPRCδ15c-us-FBAt-CYP76AH3-GAL2p-GAL7p-CYP76AK1-PDC1t-YPRCδ15c-ds were transfected into competent YT010 yeast cells to obtain positive clones. Randomly selected positive clones were streaked onto 5-FOA plates and incubated at 30°C for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid and were about to repair the fragment.

[0227] YPRCδ15c-us-FBAt-CYP76AH3-GAL2p-GAL7p-CYP76AK1-PDC1t-YPRCδ15c-ds was integrated into the YPRCδ15c site of YT010, and the resulting strain was named YT011 yeast strain (hereinafter referred to as YT011).

[0228] 6. Obtaining strain YT012

[0229] (1) The upstream homologous arm AIF1Δ-us (nucleotide sequence as shown in SEQ ID NO:31), the expression cassette GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t (nucleotide sequence as shown in SEQ ID NO:6), and the downstream homologous arm AIF1Δ-ds (nucleotide sequence as shown in SEQ ID NO:32) were ligated using the overlap method to obtain the repair fragment AIF1Δ-us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-AIF1Δ-ds.

[0230] The -us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-AIF1Δ-ds structure, from 5' to 3', consists of the upstream homologous arm AIF1Δ-us, the expression cassette GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t, and the downstream homologous arm AIF1Δ-ds.

[0231] (2) Plasmid p426-URA3-AIF1-gRNA and repair fragment AIF1Δ

[0232] Transfecting YT011 yeast competent cells with the GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-AIF1Δ-ds solution yielded positive clones. Randomly selected positive clones were streaked onto 5-FOA plates and incubated at 30°C for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid and were about to repair the AIF1Δ fragment.

[0233] -us-GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t-AIF1Δ-ds was integrated into the AIF1 site of YT011, resulting in yeast strain YT012 (hereinafter referred to as YT012) which integrates 1 copy of GAL10p-tSmKSL-GGGS-CfTPS1-CYC1t.

[0234] 7. Obtaining strain YT016

[0235] (1) The upstream homologous arm X-3-us (nucleotide sequence as shown in SEQ ID NO:33), the expression cassette GAL7p-BTS1-GGGS-CfTPS1-IDP1t (nucleotide sequence as shown in SEQ ID NO:35), and the downstream homologous arm X-3-ds (nucleotide sequence as shown in SEQ ID NO:34) were ligated using the overlap method to obtain the repair fragment.

[0236] X-3-us-GAL7p-BTS1-GGGS-CfTPS1-IDP1t-X-3-ds. (Repair fragment)

[0237] The X-3-us-GAL7p-BTS1-GGGS-CfTPS1-IDP1t-X-3-ds structure consists of the upstream homologous arm X-3-us, the expression cassette GAL7p-BTS1-GGGS-CfTPS1-IDP1t, and the downstream homologous arm X-3-ds, sequentially from 5' to 3'.

[0238] (2) Plasmid p426-URA3-X-3-gRNA and repair fragment

[0239] X-3-us-GAL7p-BTS1-GGGS-CfTPS1-IDP1t-X-3-ds was transformed into competent cells of YT012 yeast to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The repair fragment X-3-us-GAL7p-BTS1-GGGS-CfTPS1-IDP1t-X-3-ds was integrated into the X-3 site of YT012 to obtain the YT016 yeast strain with integrated GAL7p-BTS1-GGGS-CfTPS1 fusion protein, abbreviated as YT016.

[0240] (II) Application of YT009, YT010, YT011, YT012 and YT016 in the production of 11,20-dihydroxyferric alcohol

[0241] Four single clones from YT009, YT010, YT011, YT012, and YT016 were selected for parallel experiments. The experiments performed on each single clone are as follows:

[0242] 1. Add 1 ml of SC medium (solutes and concentrations of 8 g / L SC powder and 20 g / L glucose, solvent: water) to a 14 mL shaker tube, then inoculate a single colony and incubate at 30℃ and 220 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a 100 mL shaker flask containing 20 ml of YPD medium (solutes and concentrations of 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose, solvent: water), then add galactose (as an inducer) to obtain the induction system. The concentration of galactose in the induction system is 10 g / L. Incubate the induction system at 30℃ and 200 rpm for 120 h to obtain the fermentation broth.

[0243] 2. Determine the OD of the fermentation broth. 600nm And take the average of 3 parallel experiments.

[0244] 3. Take 500 μL of the fermentation broth obtained in step 1, add 500 μL of chromatographically pure n-hexane, and vortex thoroughly for 5 min; then centrifuge at 13000 rpm for 12 min and collect the supernatant; finally, dilute the collected supernatant with 1 volume of n-hexane and perform GC-MS detection (the purpose is to detect the metabolic components of the strain).

[0245] 4. Take 500 μL of the fermentation broth obtained in step 1, add 500 μL of chromatographically pure ethyl acetate, and vortex thoroughly for 5 min; then centrifuge at 13000 rpm for 5 min, collect 300 μL of the ethyl acetate layer, evaporate to dryness, add 300 μL of chromatographically pure methanol, and perform UPLC detection or LC-MS / MS.

[0246] Test results are shown Figure 3 The results showed that strain YT016 produced the highest content of 11,20-dihydroxyferric sulfate.

[0247] III. Enhancing 11,20-dihydroxyferric sulfate production through GGPP pathway modification, defective gene replacement, and FDR-FDX overexpression.

[0248] (I) Obtaining strains YT034, YT060, and YT071

[0249] 1. Plasmid p426-URA3-XI-3-gRNA, plasmid p426-URA3-XII-4-gRNA and plasmid

[0250] Construction of p426-URA3-XII-5-gRNA

[0251] Following the method in step (I), replace int4-N20-F with XI-3-N20-F and int4-N20-R with XI-3-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-XI-3-gRNA.

[0252] Following the method in step (I), replace int4-N20-F with XII-4-N20-F and int4-N20-R with XII-4-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-XII-4-gRNA.

[0253] Following the method in step (I), replace int4-N20-F with XII-5-N20-F and int4-N20-R with XII-5-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-XII-5-gRNA.

[0254] Following the method in step (I), replace int4-N20-F with DPP1-N20-F and int4-N20-R with DPP1-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-DPP1-gRNA.

[0255] Following the method in step (I), replace int4-N20-F with GAL80-N20-F and int4-N20-R with GAL80-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-GAL80-gRNA.

[0256] 2. Construction of the Expression Box

[0257] An expression cassette was constructed by connecting the components using an overlap method. The expression cassette consists of a promoter, the nucleotide sequence of the gene, and a terminator. The genes associated with GGPP metabolic flux are ERG10 (genbank ID NP_015297.1), ERG13 (genbank ID NP_013580.1), tHMG1 (genbank ID NP_013636.1), and ERG12 (genbank ID NP_013935.1) from *Saccharomyces cerevisiae*. The promoters are TDH3p-2 (nucleotide sequence as shown in SEQ ID NO:45), PGK1p-2 (nucleotide sequence as shown in SEQ ID NO:44), FBAp (nucleotide sequence as shown in SEQ ID NO:36), TPI1p (nucleotide sequence as shown in SEQ ID NO:37), or ENO2p (nucleotide sequence as shown in SEQ ID NO:47). The terminator is CYC1t (nucleotide sequence as shown in SEQ ID NO:4), TEF1t (nucleotide sequence as shown in SEQ ID NO:39), ADH2t (nucleotide sequence as shown in SEQ ID NO:40), ENO2t (nucleotide sequence as shown in SEQ ID NO:24), or CCW12t (nucleotide sequence as shown in SEQ ID NO:55).

[0258] 3. Obtaining strain YT034

[0259] (1) Preparation of repair fragments

[0260] (1-1) The upstream homologous arm XII-5-us (nucleotide sequence as shown in SEQ ID NO:42), promoter PGK1p, ERG10 gene, terminator TEF1t, promoter FBAp and ERG13 gene were ligated using the overlap method to obtain the repair fragment XII-5-us-PGK1p-ERG10-TEF1t-FBAp-ERG13, which was named fragment 1.

[0261] (1-2) The ERG13 gene, terminator ADH2t, promoter TDH3p, tHMG1 gene and terminator CYC1t were linked using the overlap method to obtain the repair fragment ERG13-ADH2t-TDH3p-tHMG1-CYC1t, which was named fragment 2.

[0262] (1-3) The terminator CYC1t, promoter TPI1p, ERG12 gene, terminator ENO2t and downstream homologous arm XII-5-ds (nucleotide sequence as shown in SEQ ID NO:43) were ligated using the overlap method to obtain the repair fragment CYC1t-TPI1p-ERG12-ENO2t-XII-5-ds, which was named fragment 3.

[0263] (2) Plasmid p426-URA3-XII-5-gRNA, fragment 1, fragment 2 and fragment 3 were transformed into competent cells of YT016 yeast to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, fragments 1, 2 and 3 were integrated into the XII-5 site of YT016 to obtain the yeast strain YT034, abbreviated as YT034.

[0264] 4. Obtaining strain YT060

[0265] (1) The upstream homologous arm DPP1Δ-us (nucleotide sequence as shown in SEQ ID NO:50), the gene expression box encoding LEU2 (nucleotide sequence as shown in SEQ ID NO:48), the gene expression box encoding TRP1 (nucleotide sequence as shown in SEQ ID NO:49) and the promoter ENO2p were linked using the overlap method to obtain the repair fragment DPP1Δ-us-LEU2 box-TRP1 box-ENO2p.

[0266] (2) The promoter ENO2p, the coding gene of LYS2 from Saccharomyces cerevisiae (genbank number NP_009673.1), the terminator TEF1t, and the downstream homologous arm DPP1Δ-ds (nucleotide sequence as shown in SEQ ID NO:51) were ligated using the overlap method to obtain the repair fragment ENO2p-LYS2-TEF1t-DPP1Δ-ds.

[0267] (3) Plasmid p426-URA3-DPP1-gRNA, repair fragment DPP1Δ-us-LEU2 box-TRP1 box-ENO2p, and repair fragment ENO2p-LYS2-TEF1t-DPP1Δ-ds were transformed into competent cells of YT034 yeast to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30℃ for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid, i.e., the repair fragments DPP1Δ-us-LEU2 box-TRP1 box-ENO2p and the repair fragment...

[0268] ENO2p-LYS2-TEF1t-DPP1Δ-ds was integrated into the DPP1 site of YT034 (i.e., LEU2, TRP1 and LYS2 were integrated into the DPP1 site of YT034), resulting in yeast strain YT060, abbreviated as YT060.

[0269] 5. Obtaining strain YT071

[0270] SmFDX and SmFDR were integrated into strain YT060, and GAL80 was knocked out to obtain strain YT071. Details are as follows:

[0271] (1) The upstream homologous arm XII-4-us (nucleotide sequence as shown in SEQ ID NO:53), the terminator CCW12t, the SmFDX gene from Salvia miltiorrhiza Bunge (genbank number XM_057925988.1), the promoter TDH3p, and the terminator FBAt were ligated using the overlap method to obtain the repair fragment.

[0272] XII-4-us-CCW12t-SmFDX-TDH3p-FBAt.

[0273] (2) The promoter TDH3p, terminator FBAt, SmFDR gene from Salvia miltiorrhiza Bunge (genbank number XM_057943104.1), promoter HXT7p (nucleotide sequence as shown in SEQ ID NO:56) and downstream homologous arm XII-4-ds (nucleotide sequence as shown in SEQ ID NO:54) were ligated using the overlap method to obtain the repair fragment TDH3p-FBAt-SmFDR-HXT7p-XII-4-ds.

[0274] (3) The plasmid p426-URA3-XII-4-gRNA, the repair fragment XII-4-us-CCW12t-SmFDX-TDH3p-FBAt and the repair fragment TDH3p-FBAt-SmFDR-HXT7p-XII-4-ds were transformed into competent cells of YT060 yeast to obtain positive clones. The positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The repair fragments XII-4-us-CCW12t-SmFDX-TDH3p-FBAt and TDH3p-FBAt-SmFDR-HXT7p-XII-4-ds were integrated into the XII-4 site of YT060 to obtain intermediate strain a.

[0275] (4) The plasmid p426-URA3-GAL80-gRNA and the GAL80 knockout repair fragment (nucleotide sequence as shown in SEQ ID NO: 58) were transformed into competent cells of intermediate strain a to obtain positive clones; positive clones were randomly selected and streaked on 5-FOA plates and cultured in a constant temperature incubator at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, which were the yeast strain YT071, abbreviated as YT071.

[0276] (II) Application of YT034, YT060 and YT071 in the production of 11,20-dihydroxyferric alcohol

[0277] Three single clones of YT034, YT060, and YT071 were selected for parallel experiments. The experiments performed on each single clone are as follows:

[0278] 1. Add 1 ml of SC medium to a 14 mL shake tube, and then inoculate single clones of YT034, YT071 and YT060 respectively. Incubate at 30℃ and 220 rpm for 18-24 h to obtain YT034 bacterial suspension, YT071 bacterial suspension and YT060 bacterial suspension respectively.

[0279] 2. After completing step 1, inoculate the YT034 bacterial culture into a shake flask (100mL) containing 20ml of YPD medium (galactose as an inducer) containing 10g / L galactose, and incubate at 30℃ and 200rpm for 120h to obtain the YT034 fermentation broth.

[0280] 3. After completing step 1, inoculate the YT060 bacterial culture into a shake flask (100mL) containing 20ml of Deft medium (galactose as an inducer) containing 10g / L galactose and 60mg / L uracil (URA), and incubate at 30℃ and 200rpm for 120h to obtain the YT060 fermentation broth.

[0281] The solutes and their concentrations in the Deft medium were 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 ml / L trace metal solutions, and 1 ml / L vitamin solutions, with water as the solvent.

[0282] 4. After completing step 1, inoculate the YT071 bacterial culture into a shake flask containing 20 ml of Deft medium (without galactose) containing 60 mg / L uracil (URA) (100 mL) and incubate at 30°C and 200 rpm for 96 h to obtain the YT071 fermentation broth.

[0283] 5. Determine the OD of the fermentation broth (YT034 fermentation broth, YT060 fermentation broth, or YT071 fermentation broth). 600nm And take the average of 3 parallel experiments.

[0284] 6. Take 500 μL of fermentation broth (YT034 fermentation broth, YT060 fermentation broth or YT071 fermentation broth), add 500 μL of chromatographically pure n-hexane, and vortex thoroughly for 5 min; then centrifuge at 13000 rpm for 12 min and collect the supernatant; finally, dilute the collected supernatant with 1 volume of n-hexane and perform GC-MS detection (the purpose is to detect the metabolic components of the strain).

[0285] 7. Take 500 μL of fermentation broth (YT034 fermentation broth, YT060 fermentation broth or YT071 fermentation broth), add 500 μL of chromatographic grade ethyl acetate, and vortex thoroughly for 5 min; then centrifuge at 13000 rpm for 5 min, collect 300 μL of ethyl acetate layer, evaporate to dryness, add 300 μL of chromatographic methanol, and perform UPLC detection.

[0286] Test results are shown Figure 4The results showed that strain YT071 produced 22.69 mg / L of 11,20-dihydroxyferric oxalate in a Deft medium containing 60 mg / L uracil (URA). The production process did not require the addition of galactose as an inducer, which significantly reduced fermentation costs.

[0287] IV. Enhancing 11,20-dihydroxyferric sulfate production by increasing NADPH levels, expanding the endoplasmic reticulum membrane, and overexpressing heme synthesis genes.

[0288] To further increase the yield of 11,20-dihydroxyferric rust alcohol, the inventors of this application overexpressed the NADPH-producing genes (ZWF1, PFK1) in strain YT071. S724D and PFK2 S718D To increase the electron donor NADPH in the CYP450s reaction, the endoplasmic reticulum membrane expansion genes (INO2 and OPI1) are mediated by knocking out the gene OPI1 that negatively regulates phospholipid biosynthesis or overexpressing the transcription factor INO2 that positively regulates phospholipid biosynthesis. This is achieved by overexpressing the heme synthase-related gene (HEM13) to increase the intracellular heme level in Saccharomyces cerevisiae.

[0289] (I) Construction of strain YT080

[0290] 1. Construction of plasmids

[0291] Following the method in step (I), replace int4-N20-F with ZWF1p-N20-F and int4-N20-R with ZWF1p-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-ZWF1p-gRNA.

[0292] Following the method in step (I), replace int4-N20-F with PFK1-N20-F and int4-N20-R with PFK1-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-PFK1-gRNA.

[0293] Following the method in step (I), replace int4-N20-F with PFK2-N20-F and int4-N20-R with PFK2-N20-R, keeping all other steps unchanged, to obtain plasmid p426-URA3-PFK2-gRNA.

[0294] Following the method in step (I), replace int4-N20-F with OPI1-N20-F and int4-N20-R with OPI1-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-OPI1-gRNA.

[0295] Following the method in step (I), replace int4-N20-F with int16-N20-F and int4-N20-R with int16-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-int16-gRNA.

[0296] 2. Obtaining strain YT074

[0297] (1) The upstream homologous arm ZWF1p-us (nucleotide sequence as shown in SEQ ID NO:59), the promoter PGK1p, and the downstream homologous arm ZWF1p-ds (nucleotide sequence as shown in SEQ ID NO:60) were connected by the overlap method to obtain the repair fragment ZWF1p-us-PGK1p-ZWF1p-ds.

[0298] (2) The plasmid p426-URA3-ZWF1p-gRNA and the repair fragment ZWF1p-us-PGK1p-ZWF1p-ds were transformed into competent cells of YT071 yeast to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30℃ for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid and were about to have their repair fragments removed.

[0299] ZWF1p-us-PGK1p-ZWF1p-ds was integrated into the ZWF1p site of YT071 to obtain yeast strain YT074, abbreviated as YT074.

[0300] 3. Obtaining strain YT078

[0301] (1) Using the overlap method, the upstream homologous arm PFK1-us (nucleotide sequence as shown in SEQ ID NO:61) and PFK1 were combined. S724D The gene (nucleotide sequence shown in SEQ ID NO:62) and the downstream homologous arm PFK1 ds (nucleotide sequence shown in SEQ ID NO:63) are ligated to obtain the repair fragment PFK1-us-PFK1. S724D -PFK1 ds.

[0302] (2) Plasmid p426-URA3-PFK1-gRNA and repair fragment PFK1-us-PFK1 S724D -PFK1 ds were transfected into competent YT074 yeast cells to obtain positive clones; randomly selected positive clones were streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid and were about to repair the PFK1-us-PFK1 fragment. S724D-PFK1d was integrated into the PFK1 site of YT074 to obtain yeast strain YT078, abbreviated as YT078.

[0303] 4. Obtaining strain YT080

[0304] (1) Using the overlap method, the upstream homologous arm PFK2-us (nucleotide sequence as shown in SEQ ID NO:64) and PFK2 were combined. S718D The gene (nucleotide sequence shown in SEQ ID NO:65) and the downstream homologous arm PFK2-ds (nucleotide sequence shown in SEQ ID NO:66) are ligated to obtain the repair fragment PFK2-us-PFK2. S718D -PFK2-ds.

[0305] (2) Plasmid p426-URA3-PFK2-gRNA and repair fragment PFK2-us-PFK2 S718D Transfecting PFK2-ds cells into competent YT078 yeast cells yielded positive clones. Randomly selected positive clones were streaked onto 5-FOA plates and incubated at 30°C for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid and were about to repair the PFK2-us-PFK2 fragment. S718D -PFK2-ds was integrated into the PFK2 site of YT078 to obtain yeast strain YT080, abbreviated as YT080.

[0306] (II) Obtaining the YT086 and YT097 strains through endoplasmic reticulum membrane modification

[0307] 1. The plasmid p426-URA3-OPI1-gRNA and the repair fragment knocking out OPI1 (nucleotide sequence as shown in SEQ ID NO:67) were transformed into competent cells of YT080 yeast to obtain positive clones. The positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, that is, YT080 yeast strain YT086, which was obtained by knocking out the transcription factor OPI1 that negatively regulates phospholipid biosynthesis.

[0308] 2. Obtaining strain YT087

[0309] (1) The upstream homologous arm int16-us (nucleotide sequence as shown in SEQ ID NO:68), promoter PGK1p-2, INO2 gene from Saccharomyces cerevisiae (genbank number NP_010408.1), terminator IDP1t and downstream homologous arm int16-ds (nucleotide sequence as shown in SEQ ID NO:70) were ligated using the overlap method to obtain the repair fragment int16-us-PGK1p-INO2-IDP1t-int16-ds.

[0310] (2) The plasmid p426-URA3-int16-gRNA and the repair fragment int16-us-PGK1p-INO2-IDP1t-int16-ds were transformed into competent cells of YT080 yeast to obtain positive clones. The positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid. The repair fragment int16-us-PGK1p-INO2-IDP1t-int16-ds was integrated into the int16 site of YT080 to obtain the yeast strain YT087, abbreviated as YT087.

[0311] 3. Obtaining strain YT097

[0312] Following the method in step (I), replace int4-N20-F with int16-N20-F and int4-N20-R with int16-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-int16-gRNA.

[0313] Plasmid p426-URA3-int16-gRNA and repair fragment int16-us-PGK1p-INO2-IDP1t-int16-ds were transformed into competent YT086 yeast cells to obtain positive clones. Randomly selected positive clones were streaked onto 5-FOA plates and incubated at 30°C for 2-3 days. The resulting clones were from strains that had lost the gRNA plasmid and were about to have their repair fragments removed.

[0314] int16-us-PGK1p-INO2-IDP1t-int16-ds was integrated into the int16 site of YT086 to obtain yeast strain YT097, abbreviated as YT097.

[0315] (III) Obtaining strain YT107

[0316] (1) The upstream homologous arm int16-us (nucleotide sequence as shown in SEQ ID NO:68), promoter PGK1p, INO2 gene, terminator IDP1t and terminator HIS3t (nucleotide sequence as shown in SEQ ID NO:38) were ligated using the overlap method to obtain the repair fragment int16-us-PGK1p-INO2-IDP1t-HIS3t.

[0317] (2) The terminator IDP1t, terminator HIS3t, HEM13 gene from Saccharomyces cerevisiae (genbank number NP_010329.1), promoter GPM1p (nucleotide sequence as shown in SEQ ID NO:41) and downstream homologous arm int16-ds were ligated using the overlap method to obtain the repair fragment IDP1t-HIS3t-HEM13-GPM1p-int16-ds.

[0318] (3) The plasmid p426-URA3-int16-gRNA, the repair fragment int16-us-PGK1p-INO2-IDP1t-HIS3t, and the repair fragment IDP1t-HIS3t-HEM13-GPM1p-int16-ds were transformed into competent cells of YT086 yeast to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30℃ for 2-3 days. The clones that grew were from strains that had lost the gRNA plasmid, i.e., the repair fragments int16-us-PGK1p-INO2-IDP1t-HIS3t and the repair fragment IDP1t-HIS3t-HEM13-GPM1p-int16-ds.

[0319] IDP1t-HIS3t-HEM13-GPM1p-int16-ds was integrated into the int16 site of YT086 to obtain yeast strain YT107, abbreviated as YT107.

[0320] (iv) Application of YT080, YT086, YT097 and YT107 in the production of 11,20-dihydroxyferric alcohol

[0321] Three single clones from YT080, YT086, YT097, and YT107 were selected for parallel experiments. The experiments performed on each single clone are as follows:

[0322] 1. Add 1 ml of SC medium to a 14 mL shaker tube, then inoculate with a single clone of YT074, YT078, YT080, YT086, YT087, YT096, YT097, or YT107. Incubate at 30℃ and 200 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a 100 mL shaker flask containing 20 ml of Deft medium and incubate at 30℃ and 200 rpm for 96 h to obtain the fermentation broth. Measure the OD of the fermentation broth. 600nm And take the average of 3 parallel experiments.

[0323] 2. Take 500 μL of the fermentation broth obtained in step 1, add 500 μL of chromatographically pure n-hexane, and vortex thoroughly for 5 min; then centrifuge at 13000 rpm for 12 min and collect the supernatant; finally, dilute the collected supernatant with 1 volume of n-hexane and perform GC-MS detection (the purpose is to detect the metabolic components of the strain).

[0324] 3. Take 500 μL of the fermentation broth obtained in step 1, add 500 μL of chromatographically pure ethyl acetate, and vortex thoroughly for 5 min; then centrifuge at 13000 rpm for 5 min, collect 300 μL of the ethyl acetate layer, evaporate to dryness, add 300 μL of chromatographically pure methanol, centrifuge at 13000 rpm for 12 min, and perform UPLC detection.

[0325] Some test results can be found Figure 5 The results showed that the NADPH-modified YT080 strain produced the highest yield of 11,20-dihydroxyferric ferrol at 29.43 mg / L; after endoplasmic reticulum modification, the YT097 strain, which knocked out OPI1 and overexpressed INO2, produced the highest yield of 11,20-dihydroxyferric ferrol at 39.12 mg / L; overexpression of HEM13 increased the yield, with the YT107 strain producing the highest yield of 11,20-dihydroxyferric ferrol at 41.49 mg / L.

[0326] V. Increasing the yield of 11,20-dihydroxyferric alcohol by modifying iron-related transport genes.

[0327] In *Saccharomyces cerevisiae*, no modification of iron metabolism (improving intracellular iron ion levels) has been found to enhance the catalytic activity of CYP450s and thus improve catalytic efficiency. This application attempts to modify iron transport to increase the yield of the target product, 11,20-dihydroxyferric sulfate. The main strategies include: 1) increasing iron ion uptake and transport (overexpressing iron transport genes such as FIT2, FET3, and FTR1); 2) knocking out iron-inhibiting genes (such as BOL2, YAP5, GRX3, and GRX4) and downregulating CCC1 gene expression (genbank number NP_013321.1).

[0328] (I) Construction of strains that overexpress iron transport genes and knock out or downregulate the iron-suppressing CCC1 gene

[0329] 1. Construction of plasmids

[0330] Following the method in step (I), replace int4-N20-F with CCC1p-N20-F and int4-N20-R with CCC1p-N20-R, keeping all other steps unchanged, to obtain plasmid p426-URA3-CCC1p-gRNA.

[0331] Following the method in step (I), replace int4-N20-F with BOL2-N20-F and int4-N20-R with BOL2-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-BOL2-gRNA.

[0332] Following the method in step (I), replace int4-N20-F with YAP5-N20-F and int4-N20-R with YAP5-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-YAP5-gRNA.

[0333] Following the method in step (I), replace int4-N20-F with X-1-N20-F and int4-N20-R with X-1-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-X-1-gRNA.

[0334] Following the method in step (I), replace int4-N20-F with GRX3-N20-F and int4-N20-R with GRX3-N20-R, keeping all other steps unchanged, to obtain plasmid p426-URA3-GRX3-gRNA.

[0335] Following the method in step (I), replace int4-N20-F with GRX4-N20-F and int4-N20-R with GRX4-N20-R, keeping all other steps unchanged, to obtain plasmid p426-URA3-GRX4-gRNA.

[0336] Following the method in step (I), replace int4-N20-F with YAP5-N20-F and int4-N20-R with YAP5-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-YAP5-gRNA.

[0337] Following the method in step (I), replace int4-N20-F with BOL2-N20-F and int4-N20-R with BOL2-N20-R, while keeping all other steps unchanged, to obtain plasmid p426-URA3-BOL2-gRNA.

[0338] 2. Obtaining strain YT113

[0339] The genbank number for FTR1 (derived from *Saccharomyces cerevisiae*) is NP_011072.1; for FIT2, it is NP_015027.1; for FET3, it is NP_013774.1; for BOL2, it is NP_011296.1; for YAP5, it is NP_012283.1; for GRX3, it is NP_010382.3; and for GRX4, it is NP_011101.3.

[0340] (1) The upstream homologous arm X-1-us (nucleotide sequence as shown in SEQ ID NO:52), promoter CCW12p (nucleotide sequence as shown in SEQ ID NO:57), FIT2 gene (genbank number NP_015027.1), terminator TDH2t (nucleotide sequence as shown in SEQ ID NO:69) and promoter TPIp (nucleotide sequence as shown in SEQ ID NO:71) were ligated using the overlap method to obtain the repair fragment X-1-us-CCW12p-FIT2-TDH2t-TPIp.

[0341] (2) Using the overlap method, the terminator TDH2t, promoter TPIp, and FET3 gene (genbank number [missing information]) were linked together.

[0342] The NP_013774.1), terminator ENO2t, and terminator PRM9t (nucleotide sequence as shown in SEQ ID NO:72) were linked to obtain the repair fragment TDH2t-TPIp-FET3-ENO2t-PRM9t.

[0343] (3) Using the overlap method, the terminator ENO2t, terminator PRM9t, and FTR1 genes (genbank number:

[0344] The NP_011072.1), promoter GPM1p (nucleotide sequence as shown in SEQ ID NO:41), and downstream homologous arm X1-ds (nucleotide sequence as shown in SEQ ID NO:73) were ligated to obtain the repair fragment ENO2t-PRM9t-FTR1-GPM1p-X1-ds.

[0345] (4) Plasmid p426-URA3-X-1-gRNA, repair fragment X-1-us-CCW12p-FIT2-TDH2t-TPIp, repair fragment TDH2t-TPIp-FET3-ENO2t-PRM9t, and repair fragment ENO2t-PRM9t-FTR1-GPM1p-X1-ds were transformed into competent YT107 yeast cells to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30℃ for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid and were intended to be used for expression cassettes.

[0346] CCW12p-FIT2-TDH2t-TPIp-FET3-ENO2t-PRM9t-FTR1-GPM1p was integrated into the X-1 site to obtain strain YT113, abbreviated as YT113.

[0347] 3. Obtaining strain YT125

[0348] The plasmid p426-URA3-BOL2-gRNA and the BOL2 knockout fragment (SEQ ID NO:74) were transformed into competent YT113 yeast cells to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, i.e., strains with BOL2 knockout, and were named strain YT125, or YT125 for short.

[0349] 4. Obtaining strain YT133

[0350] (1) The upstream homologous arm CCC1p-us (nucleotide sequence as shown in SEQ ID NO:75), the promoter CYB2p (nucleotide sequence as shown in SEQ ID NO:76), and the downstream homologous arm CCC1p-ds (nucleotide sequence as shown in SEQ ID NO:77) were connected by the overlap method to obtain the repair fragment CCC1p-us-CYB2p-CCC1p-ds.

[0351] (2) The plasmid p426-URA3-CCC1p-gRNA and the repair fragment CCC1p-us-CYB2p-CCC1p-ds were transformed into competent YT125 yeast cells to obtain positive clones. Positive clones were randomly selected and streaked onto 5-FOA plates and cultured at 30℃ for 2-3 days. The resulting clones were strains that had lost the gRNA plasmid and were about to have their repair fragments removed.

[0352] CCC1p-us-CYB2p-CCC1p-ds was integrated into the CCC1p site to obtain strain YT133, abbreviated as YT133.

[0353] 5. Obtaining strain YT139

[0354] The plasmid p426-URA3-GRX3-gRNA and the repair fragment knocking out GRX3 (nucleotide sequence as shown in SEQ ID NO:78) were transformed into competent cells of YT133 yeast to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, i.e., YT133 strains with GRX3 knocked out, resulting in strain YT139, abbreviated as YT139.

[0355] 6. Obtaining strain YT141

[0356] The plasmid p426-URA3-GRX4-gRNA and the repair fragment knocking out GRX4 (nucleotide sequence shown in SEQ ID NO:79) were transformed into competent cells of YT133 yeast to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, i.e., YT133 strain with GRX4 knocked out, resulting in strain YT141, abbreviated as YT141.

[0357] 7. Obtaining strain YT142

[0358] The plasmid p426-URA3-YAP5-gRNA and the repair fragment knocked out YAP5 (nucleotide sequence shown in SEQ ID NO:46) were transformed into competent cells of YT133 yeast to obtain positive clones. Positive clones were randomly selected and streaked on 5-FOA plates and cultured at 30°C for 2-3 days. The clones that grew were strains that had lost the gRNA plasmid, i.e., YT133 strain with YAP5 knocked out, resulting in strain YT142, abbreviated as YT142.

[0359] (II) Application of YT113, YT125, YT133, YT139, YT141 and YT142 in the production of 11,20-dihydroxyferric alcohol

[0360] Three single clones from YT113, YT125, YT133, YT139, YT141, and YT142 were selected for parallel experiments. The experiments performed on each single clone are as follows:

[0361] 1. Add 1 ml of SC medium to a 14 mL shaker tube, then inoculate with a single clone of YT113, YT125, YT133, YT139, YT141, or YT142. Incubate at 30℃ and 200 rpm for 18-24 h to obtain the bacterial culture. Inoculate the bacterial culture into a 100 mL shaker flask containing 20 ml of Deft medium and incubate at 30℃ and 200 rpm for 96 h to obtain the fermentation broth. Measure the OD of the fermentation broth. 600nm And take the average of 3 parallel experiments.

[0362] 2. Take 500 μL of the fermentation broth obtained in step 1, add 500 μL of chromatographically pure n-hexane, and vortex thoroughly for 5 min; then centrifuge at 13000 rpm for 12 min and collect the supernatant; finally, dilute the collected supernatant with 1 volume of n-hexane and perform GC-MS detection (the purpose is to detect the metabolic components of the strain).

[0363] 3. Take 500 μL of the fermentation broth obtained in step 1, add 500 μL of chromatographically pure ethyl acetate, and vortex thoroughly for 5 min; then centrifuge at 13000 rpm for 5 min, collect 300 μL of the ethyl acetate layer, evaporate to dryness, add 300 μL of chromatographically pure methanol, centrifuge at 13000 rpm for 12 min, and perform UPLC detection.

[0364] Some test results can be found Figure 6 The results showed that the YT142 strain, which increased intracellular iron transport (by overexpressing iron transport genes FIT2, FET3, and FTR1) while simultaneously knocking out iron-inhibiting genes (BOL2 and YAP5), produced the highest iron yield of 67.69 mg / L.

[0365] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A recombinant strain of yeast that has in vivo knocked out or inhibited galactokinase GAL1, bifunctional UDP glucose 4-isomerase / aldose 1-isomerase GAL7 and / or hexose-1-phosphate uridine transferase GAL10 and contains or expresses diterpene synthase KSL1 and / or TPS1.

2. The recombinant bacteria according to claim 1, characterized in that: The recombinant bacteria also contain or express ferric sulfate synthase CYP76AH1, ferric sulfate C11 hydroxylase CYP76AH3, 11-hydroxyferric sulfate C20 hydroxylase CYP76AK1, P450 reductase CPR and / or geraniol geraniol pyrophosphate synthase BTS1.

3. The recombinant bacteria according to claim 1 or 2, characterized in that: The recombinant bacteria also contain or express acetyl-CoA thiolytic enzyme ERG10, hydroxymethylpentadiene-CoA reductase ERG13, hydroxymethylglutaryl-CoA reductase HMG1 or its truncated form tHMG1 and / or mevalonate kinase ERG12.

4. The recombinant bacteria according to any one of claims 1 to 3, characterized in that: The recombinant bacteria also contain or express β-isopropylpropionic acid dehydrogenase LEU2, phosphoribosylanthraquinone isomerase TRP1, and / or α-aminoadipic acid reductase LYS2.

5. The recombinant bacteria according to any one of claims 1 to 4, characterized in that: The recombinant bacteria also have the galactose transcription factor protein GAL80 knocked out or inhibited in vivo, and contain or express ferroreductin FDX and ferroreductin reductase FDR.

6. The recombinant bacteria according to any one of claims 1 to 4, characterized in that: The recombinant bacteria also contain substances that increase NADPH expression, thereby increasing the electron donor NADPH in the CYP450s reaction.

7. The recombinant bacteria according to claim 6, characterized in that: The substance that increases NADPH expression is an overexpression of the NADPH gene; preferably, the NADPH gene is ZWF1 or PFK1. S724D and / or PFK2 S718D .

8. The recombinant bacteria according to any one of claims 1 to 7, characterized in that: The recombinant bacteria also have transcription factors that inhibit phospholipid biosynthesis knocked out or inhibited, transcription factors that activate phospholipid biosynthesis are contained in or expressed, and / or heme synthase is contained in or expressed; the expression of heme synthase is used to increase the intracellular heme level of the recombinant bacteria.

9. The recombinant bacteria according to any one of claims 1 to 8, characterized in that: The recombinant bacteria also contain or express iron ion transport proteins. Preferably, the iron transporter is at least one of FIT2, FET3 and FTR1.

10. The recombinant bacteria according to any one of claims 1 to 9, characterized in that: The recombinant bacteria also have iron-associated protein and / or vacuole iron transporter CCC1 knocked out or inhibited in vivo. Preferably, the iron-related protein is at least one of BolA protein BOL2, iron transcription factor protein YAP5, cytoplasmic glutathione protein GRX3, and cytoplasmic glutathione protein GRX4.

11. The application of the recombinant bacteria according to any one of claims 1 to 10, wherein S1) or S2) is: S1) Produces 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tungsten, 11-hydroxytungsten and / or 11,20-dihydroxytungsten; S2) produces downstream compounds of 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tungstenol, 11-hydroxytungstenol and / or 11,20-dihydroxytungstenol. Preferably, the downstream compound includes at least one of tanshinone IIA, tanshinone, 16-hydroxytanshinone, tanshinone IIB, cryptotanshinone, tanshinone I, dihydrotanshinone II, hydroxytanshinone, methyl tanshinone, and oxalic acid.

12. A method for producing 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tsucrose, 11-hydroxytsucrose, 11,20-dihydroxytsucrose and / or downstream compounds of 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tsucrose, 11-hydroxytsucrose and / or 11,20-dihydroxytsucrose, comprising the following steps: fermentation culture The recombinant bacteria described in any one of 1 to 10 are used to collect the fermentation product and obtain 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tsucrose, 11-hydroxytsucrose, 11,20-dihydroxytsucrose and / or "downstream compounds of 11,20-dihydroxyferric alcohol, tanshinone diene, ferric alcohol, 11-hydroxyferric alcohol, tsucrose, 11-hydroxytsucrose and / or 11,20-dihydroxytsucrose"; Preferably, the downstream compound includes at least one of tanshinone IIA, tanshinone, 16-hydroxytanshinone, tanshinone IIB, cryptotanshinone, tanshinone I, dihydrotanshinone II, hydroxytanshinone, methyl tanshinone, and oxalic acid.

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