A dual dynamic control system to improve the production of phenolic compounds synthesized by Saccharomyces cerevisiae

Abstract

The present invention discloses a set of dual dynamic regulation systems for improving the yield of phenolic compounds synthesized by Saccharomyces cerevisiae, which is composed of a glucose concentration-responsive gene circuit and an iron ion concentration-responsive gene circuit; the glucose concentration-responsive gene circuit is constructed based on the endogenous GAL regulation system of Saccharomyces cerevisiae after knocking out the GAL80 protein; the iron ion concentration-responsive dynamic regulation system is constructed based on the endogenous iron ion-repressible promoter Pfet3 and protein AFT1 / 2 of Saccharomyces cerevisiae; and the global regulation of de novo synthesis of phenolic compounds in Saccharomyces cerevisiae is realized. The recombinant Saccharomyces cerevisiae produced by the dual dynamic regulation system in coordination with ascorbic acid has a 72% higher de novo synthesis yield of tyrosol than the strain regulated by the single regulation-glucose concentration-responsive gene circuit, and the yield is as high as 750 mg / L. The development of this dual dynamic regulation system provides a new idea for enhancing the yield of de novo synthesis of phenolic compounds in Saccharomyces cerevisiae.

Description

Technical Field

[0001] The invention relates to a dual dynamic regulation system for improving the yield of phenolic compounds synthesized by brewer's yeast, and belongs to the technical field of microbial genetic engineering. Background Art

[0002] The 21st century is an era of challenges. Global issues such as resource shortages, environmental pollution, climate change and food crises are becoming more prominent. It is important to solve the above problems by developing emerging biotechnologies to replace traditional industrial technologies. The emergence of metabolic engineering has greatly promoted the development of emerging biotechnologies. At present, many high-value compounds in various industries have been reported to be synthesized by microorganisms. Saccharomyces cerevisiae is a commonly used chassis cell that is considered to be food-safe. To date, many synthetic pathways for target products have been successfully constructed in Saccharomyces cerevisiae, and the yield of target products has been improved through a variety of metabolic regulation methods. Phenolic compounds are a class of compounds with rich biological activities and many benefits to human health, such as antioxidant activity, anti-inflammatory and anti-cancer. Many typical phenolic compounds have been shown to be synthesized de novo in Saccharomyces cerevisiae through metabolic engineering. The metabolic regulation methods currently used are divided into static regulation and dynamic regulation. The dynamic regulation method can reasonably allocate carbon sources to achieve the maximum utilization of carbon atoms. The commonly used dynamic regulation method in Saccharomyces cerevisiae is the endogenous GAL regulation system with galactose as the sensor. In order to reduce costs, a glucose concentration-sensing GAL regulation system based on the knockout of the GAL80 repressor protein was developed. However, this dynamic regulation system is mainly responsible for regulating cell growth and product synthesis pathways, lacks regulation of product competition pathways, and has circuit leakage problems, which affects the yield of the target product and cannot maximize the yield. At the same time, hydroxyl groups are easily oxidized during polyphenol synthesis.

[0003] In Saccharomyces cerevisiae, the transcription factors AFT1 / 2 and Yap5 regulate iron metabolism in response to low and high iron levels, respectively. In addition to the production and assembly of iron cofactors, mitochondrial iron-sulfur (Fe / S) cluster biogenesis has emerged as a central player in iron sensing. Mitochondrial signals derived from Fe / S synthesis are exported and converted into Fe / S clusters, which directly interact with AFT1 / 2 and Yap5 proteins to regulate their transcriptional functions. Among them, the iron transport activator Aft1 / 2 can bind to iron-sulfur (2Fe-2S) clusters or directly bind to Fe when iron concentration levels are high. 2+ Binding results in dimerization, which results in inactivation of promoters of genes regulated by Pfet3. In contrast, iron-responsive promoters Pfet3 are activated in the absence of iron or in the presence of low iron concentrations. Summary of the invention

[0004] Technical issues:

[0005] The de novo synthesis of phenolic compounds by brewer's yeast has the advantages of being environmentally friendly and low-cost, but the static regulation method rigidly allocates carbon sources in the metabolic pathway. Although the commonly used glucose response circuit alleviates the rigid allocation of carbon sources, there is still the problem of circuit leakage.

[0006] Technical solution:

[0007] The present invention develops a dual dynamic regulation system for improving the synthesis of phenolic compounds by brewer's yeast. The iron ion responsive dynamic regulation system regulates the competitive path to compensate for the circuit leakage problem of the glucose concentration-sensing GAL regulation system. At the same time, the responsive element iron ions can complex the hydroxyl groups of phenolic compounds to achieve product pulling and hydroxyl protection. Finally, by adding ascorbic acid to further protect the hydroxyl groups, a high yield of the typical phenolic compound - tyrosol in brewer's yeast is achieved.

[0008] The present invention provides a dual dynamic control system for increasing the yield of phenolic compounds synthesized by brewer's yeast, namely, a primary glucose concentration-responsive dynamic control system and a secondary iron ion concentration-responsive dynamic control system. The dual dynamic control system constructed by the present invention dynamically controls the metabolic pathway of de novo synthesis of a typical phenolic compound-tyrosol in brewer's yeast, globally controls the cell growth pathway, product synthesis pathway, product competition pathway and realizes product pull. The development of this dual dynamic control system provides a new idea for increasing the yield of de novo synthesis of phenolic compounds in brewer's yeast.

[0009] Among them, the primary glucose concentration-responsive dynamic regulatory system is based on the glucose concentration-sensitive GAL regulatory system after the GAL80 repressor protein in the Saccharomyces cerevisiae genome is knocked out. Figure 2 a and 2c, which are responsible for regulating cell growth and product synthesis. In the synthesis of the phenolic compound tyrosol, it is responsible for regulating the synthesis pathway of the typical phenolic compound tyrosol and the cell growth of Saccharomyces cerevisiae. The GAL promoter is used to drive the expression of 3-deoxy-D-arabinoheptulose-7-phosphate synthase, chorismate mutase, tyrosine decarboxylase and phosphoketolase in the tyrosol synthesis pathway. The GAL promoter includes GAL2, GAL7, GAL1 and GAL10. The mechanism is as follows Figure 3 shown.

[0010] Among them, the secondary iron ion concentration-responsive dynamic regulation system is constructed based on the endogenous iron ion-repressible promoter Pfet3 and repressor protein AFT1 / 2 of Saccharomyces cerevisiae. Figure 2 b and 2c, which are responsible for regulating the product competition pathway and product complexation pull. In the synthesis of the phenolic compound tyrosol, the competition pathway for tyrosol synthesis and tyrosol complexation pull are responsible. The promoter Pfet3 is used to drive the expression of pyruvate decarboxylase isozymes, prephenate dehydratase, and indole glycerol phosphate synthase in the competition pathway, and iron ions are added to complex tyrosol. The mechanism is as follows Figure 3shown.

[0011] The first object of the present invention is to provide an iron ion concentration-responsive dynamic regulation system, comprising a promoter Pfet3 and a target gene coding sequence.

[0012] In one embodiment, the promoter Pfet3 is connected to the target gene coding sequence, and the promoter Pfet3 regulates the expression of the target gene in Saccharomyces cerevisiae.

[0013] The promoter Pfet3 is regulated by the iron ion concentration. The higher the iron ion concentration, the more restricted the expression of the repressor protein AFT1 / 2, thereby inducing the promoter Pfet3 to initiate the expression of downstream genes. Under the regulation of the promoter Pfet3, the expression of the target gene is weakened.

[0014] In one embodiment, the sequence of the promoter Pfet3 is shown as SEQ ID NO.9.

[0015] In one embodiment, the target gene includes the pyruvate decarboxylase isozyme gene pdc1, the prephenate dehydratase gene pha2 and / or the indoleglycerol phosphate synthase gene trp3.

[0016] The second object of the present invention is to provide a carrier, which comprises the iron ion concentration-responsive dynamic control system.

[0017] In one embodiment, the vector comprises a Saccharomyces cerevisiae expression vector.

[0018] The third object of the present invention is to provide a recombinant cell, which comprises the iron ion concentration-responsive dynamic regulatory system or the vector.

[0019] In one embodiment, the recombinant cell is hosted by Escherichia coli, Bacillus or yeast.

[0020] In one embodiment, the recombinant cell is hosted by Saccharomyces cerevisiae.

[0021] The fourth object of the present invention is to provide a recombinant Saccharomyces cerevisiae, which uses the iron ion concentration responsive dynamic regulation system to regulate the expression of target genes, wherein the target genes include the expression of pyruvate decarboxylase isozyme gene pdc1, prephenate dehydratase gene pha2 and / or indoleglycerol phosphate synthase gene trp3; overexpression of 3-deoxy-D-arabinoheptulose acid-7-phosphate synthase mutant gene aro4 K229L , chorismate mutase mutant gene aro7 G141S , tyrosine decarboxylase gene pcaas opt , phosphoketolase gene Bbxfpk optand the mutant gene Trya M52I / A354V , and simultaneously knocked out the GAL80 gene, HO gene and LPP1 gene in the Saccharomyces cerevisiae genome.

[0022] In one embodiment, the use of the iron ion concentration-responsive dynamic regulatory system to regulate the expression of the target gene is to replace the promoter in front of the target gene in the Saccharomyces cerevisiae genome with the promoter Pfet3.

[0023] In one embodiment, the recombinant Saccharomyces cerevisiae is aro4 K229L 、aro7 G141S 、pcaas opt ,Bbxfpk opt 、Trya M52I / A354V It was integrated into the GAL80, GAL80, HO, HO, and LPP1 sites on the genome of the starting strain BY4741 in sequence.

[0024] In one embodiment, the aro4 K229L ,aro7 G141S , pcaas op ,Bbxfpk op and Trya M52I / A354V The amino acid sequences of the genes are shown in SEQ ID NO.1 to SEQ ID NO.5.

[0025] In one embodiment, the amino acid sequences of the pdc1, pha2 and trp3 genes are shown in SEQ ID NO.6 to SEQ ID NO.8.

[0026] The fifth object of the present invention is to provide a method for increasing tyrosol production, the method comprising utilizing the iron ion concentration-responsive dynamic regulation system to regulate genes related to product competition pathways, the genes comprising the pyruvate decarboxylase isozyme gene pdc1, the prephenate dehydratase gene pha2 and / or the indoleglycerol phosphate synthase gene trp3.

[0027] In one embodiment, the amino acid sequences of the pdc1, pha2 and trp3 genes are shown in SEQ ID NO.6 to SEQ ID NO.8.

[0028] The sixth object of the present invention is to provide a method for synthesizing tyrosol, which comprises inoculating the recombinant cerevisiae yeast into a fermentation system for fermentation preparation, and adding iron ions in the middle and late logarithmic phase of the strain growth to continue the fermentation.

[0029] In one embodiment, the recombinant yeast fermentation system is yeast extract (10 g / L), trypsin (20 g / L), sucrose (10 g / L), and glycerol (10 g / L).

[0030] In one embodiment, the fermentation system contains 0-1 g / L ascorbic acid.

[0031] In one embodiment, the recombinant yeast fermentation time is 96 hours.

[0032] In one embodiment, the iron ions are added in the form of 500-2500 μM ferric chloride.

[0033] Beneficial effects:

[0034] By constructing and testing the basic framework of the iron ion concentration-inhibitory gene circuit, the optimal inducing element for the system response was successfully screened out as trivalent iron ions. When the added trivalent iron ion concentration was 2500μM, the fluorescence value of the green fluorescent protein regulated by the iron ion concentration-inhibitory gene circuit decreased by 46% per OD, which fully proved that the promoter Pfet3 was regulated by the iron ion concentration. Through the dual dynamic regulation system and the coordinated regulation of ascorbic acid on the starting brewer's yeast strain YLYJ-006 that produced tyrosol, the optimal engineering strain YLYJ6-C was successfully constructed. The de novo synthesis yield of tyrosol was as high as 750mg / L, which was 72% higher than that of the starting strain YLYJ-006. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 To construct the basic framework and measure the performance of the dynamic control system responsive to iron ion concentration. Figure 1 a is an iron ion concentration inhibitory gene circuit, Figure 1 b is the effect of iron ion addition on cell growth, Figure 1 c shows the effect of iron ion addition on the expression of fluorescent protein;

[0036] Figure 2 The mechanism and ideal blueprint of the dual dynamic regulatory system for global regulation of the metabolic pathway of phenolic compound synthesis in Saccharomyces cerevisiae. Figure 2 a is a first-level glucose concentration-responsive dynamic control system, Figure 2 b is a secondary iron ion concentration responsive dynamic control system, Figure 2 c is a dual dynamic control system;

[0037] Figure 3 It is the regulation of the dual dynamic regulatory system in the metabolic pathway of de novo tyrosol synthesis in Saccharomyces cerevisiae.

[0038] Figure 4 The effect of the dual dynamic regulation system on the yield of tyrosol synthesized from scratch by Saccharomyces cerevisiae. Figure 4a is the effect of iron ion responsive system regulation on tyrosol production, Figure 4 b: The iron ion responsive system regulates the gene expression of the inhibitory competition pathway. DETAILED DESCRIPTION

[0039] Biochemical materials:

[0040] Saccharomyces Cerevisiae By4741 and Escherichia coli BL21 (DE3) (our laboratory); Escherichia coli Top10 competent cells (Novagen, USA); tyrosol standard (Solarbio, China); restriction endonucleases, T4 DNA ligase, PrimeSTARMax DNA polymerase, loading buffer, LE agarose and S4 nucleic acid electrophoresis dye (Takara); antibiotics, yeast whole genome extraction kit and SanPrep column plasmid DNA small-scale extraction kit (Shanghai Shenggong Biotechnology Co., Ltd.); DNA5000ladder and DNA10000ladder (Shanghai Bao Biotechnology Co., Ltd.); tryptone and yeast extract (Oxoid Company, UK).

[0041] Culture medium:

[0042] YPD medium: glucose (20 g / L), tryptone (20 g / L), yeast extract (10 g / L), sterilized at 115°C for 30 min. Agar powder (15 g / L) is added to the solid medium, and antibiotics are added to the corresponding antibiotic selective medium.

[0043] YPSG medium: sucrose (10g / L), glycerol (10g / L), tryptone (20g / L), yeast extract (10g / L), sterilized at 115℃ for 30min. Agar powder (15g / L) is added to solid medium; if antibiotics or inducers are added, it is the corresponding antibiotic / inducer selective medium, such as YPSG-ascorbic acid medium (ascorbic acid 1g / L).

[0044] LB medium: NaCl (10 g / L), tryptone (10 g / L), yeast extract (5 g / L), sterilized at 120°C for 20 min. Agar powder (15 g / L) is added to the solid medium, and antibiotics are added to the corresponding antibiotic selective medium.

[0045] Strains, plasmids and primers:

[0046] Table 1 The strains and their genotypes are as follows

[0047]

[0048]

[0049] Table 2 Plasmids and their genotypes are as follows

[0050]

[0051] Table 3 Primers are as follows

[0052]

[0053]

[0054]

[0055] Basic operations in molecular biology:

[0056] (1) E. coli plasmid extraction, genome extraction, PCR product purification, and gel recovery were performed according to the instructions of the corresponding kit unless otherwise specified.

[0057] (2) DNA fragments were amplified using PCR technology, and different reaction systems and procedures were used according to different experimental requirements. The reaction system is shown in Table 4, and the reaction procedure is shown in Table 5.

[0058] Table 4 PCR reaction system

[0059]

[0060] Table 5 PCR reaction program

[0061]

[0062] (3) Enzyme digestion and ligation: DNA double enzyme digestion and T4 ligase reaction system and reaction conditions, unless otherwise specified, were performed according to the Takara company website (https: / / www.takarabiomed.com.cn / ).

[0063] (4) Fluorescence quantitative PCR: follow the kit instructions.

[0064] (5) Heat shock method was used for transformation of competent E. coli: the transformation method was carried out according to the instruction manual.

[0065] (6) Electroporation method was used for transformation of Saccharomyces cerevisiae competent cells: the transformation method was according to the instruction manual.

[0066] (7) Strain culture and fermentation method: Pick a single colony from the agar plate and inoculate it into 5 mL of YPD / LB liquid medium. Yeast is placed in a constant temperature shaker at 250 rpm and 30°C for overnight culture. Escherichia coli is placed in a constant temperature shaker at 200 rpm and 37°C for overnight culture. For the expansion and fermentation of Saccharomyces cerevisiae, take 1 mL of bacterial liquid and transfer it to 20 mL of YPD / YPSG medium for expansion and culture. Add the corresponding inducer, ascorbic acid and antibiotics according to the strain requirements.

[0067] Example 1: Construction of the basic framework of the iron ion concentration responsive dynamic control system and performance testing

[0068] (1) Using Pfet3-F(FseI) / Pfet3-F(Not1) as primers, the Pfet3 gene fragment with restriction sites was amplified from the whole genome of wild-type yeast By4741 by PCR. The primers are shown in Table 3. Then, the Pfet3 gene fragment with restriction sites and the recombinant plasmid pCKm-GFP carrying green fluorescent protein were double-digested. After gel recovery of the double-digested gene fragment and vector fragment, the gene fragment and the linear vector were cloned to obtain the recombinant plasmid pCKm-Pfet3-GFP. The plasmid genotype is shown in Table 2. The recombinant plasmid was introduced into the wild-type yeast By4741 to obtain the yeast engineering strain YLYJ-A. The genotype of YLYJ-A is shown in Table 1.

[0069] (2) The strain YLYJ-A was fermented in YPD medium for 72 h. At 48 h of fermentation, 500 μM, 1000 μM, 2000 μM, and 2500 μM of ferrous sulfate heptahydrate or ferric chloride were added, respectively. After the fermentation, the OD value of the fermentation broth was measured using a UV-visible spectrophotometer (T-6, Feller, Nanjing). 600 Test results such as Figure 1 As shown in b, when the added iron ions reach a certain concentration (2500 μM), iron ions promote cell growth, which is higher than the OD of the control strain without iron ions. 600 An increase of 8%-9%.

[0070] (3) The strain YLYJ-A was fermented in YPD medium for 72 h. At 48 h of fermentation, 500 μM, 1000 μM, 2000 μM and 2500 μM of ferrous sulfate heptahydrate or ferric chloride were added. After the fermentation, the fluorescence intensity of the precipitate after washing the fermentation liquid was measured using a fluorescence spectrometer (F-7000, Hitachi, Japan). The results are shown in Figure 2. Figure 1As shown in Figure c, the fluorescence intensity (OD / AU) of green fluorescent protein showed a downward trend with the increase of iron ion concentration, and the type of iron ions had no particularly obvious effect. When the iron ion concentration added reached 2500 μM, the fluorescence intensity (OD / AU) of YLYJ-A dropped to 177 and 169, respectively.

[0071] Example 2: Effect of the dual dynamic control system on the yield of tyrosol synthesized from scratch by Saccharomyces cerevisiae

[0072] 1. Construction of strains

[0073] (1) Ectyra M53I / A354V -1(EcoRI) / Ectyra M53I / A354V -2 were used as primers to amplify tyra from the whole genome of wild-type Escherichia coli BL21 (DE3) using PCR technology. M53I / A354V The upper part of the gene, Ectyra M53I / A354V -3 / Ectyra M53I / A354V -4 were used as primers to amplify tyra from the whole genome of wild-type Escherichia coli BL21 (DE3) using PCR technology. M53I / A354V The lower part of the gene, Ectyra M53I / A354V -5 / Ectyra M53I / A354V -6 (NotI) as primers, overlap extension PCR was used to amplify the above partial gene and the lower partial gene to obtain the final fragment tyra with restriction site M53I / A354V The recombinant plasmid pA-Tyra was cloned into the plasmid pUMRI-A-△LPP1 to obtain the recombinant plasmid pA-Tyra M53I / A354V .

[0074] The recombinant plasmid pA-Tyra was transformed into M53I / A354V The YLJ-006 yeast strain was obtained by integrating into the LPP1 site of strain YLYJ-004 and knocking out the LPP1 gene. The primers are shown in Table 3. The genotype of YLYJ-006 is shown in Table 2.

[0075] (2) Using his3-F (BamHI) / his3-R (Fse1), leu2-F (BamHI) / leu2-R (Fse1) and ura2-F (BamHI) / ura2-R (Fse1) as primers, the his3, leu2 and ura3 fragments with restriction sites were amplified from the pESC-HIS, pESC-URA and pESC-LEU plasmids by PCR technology. The primers are shown in Table 3. Then, the his3, leu2 and ura3 gene fragments with restriction sites and the plasmid pCKm-Pfet3-GFP constructed in Example 1 were double-digested. After the double-digested gene fragments and vector fragments were recovered by gel, the gene fragments and the linear vector were cloned to obtain recombinant plasmids, and the recombinant plasmids pCKm-Pfet3-his3, pCKm-Pfet3-leu2 and pCKm-Pfet3-ura3 were obtained. The plasmid phenotypes are shown in Table 2.

[0076] The upstream homology arm of pdc1 was amplified from the whole genome of wild-type Saccharomyces cerevisiae By4741 using PCR technology using leu2-pdc1-Pfet3-1 / leu2-pdc1-Pfet3-2 as primers, the pdc1 gene was amplified from the whole genome of wild-type Saccharomyces cerevisiae By4741 using PCR technology using leu2-pdc1-Pfet3-3 / leu2-pdc1-Pfet3-4 as primers, and the pdc1 gene was amplified from the whole genome of wild-type Saccharomyces cerevisiae By4741 using PCR technology. Using dc1-Pfet3-5 / leu2-pdc1-Pfet3-6 as primers, the downstream homology arm of pdc1 was amplified from the whole genome of wild-type Saccharomyces cerevisiae By4741 by PCR technology, and then using leu2-pdc1-Pfet3-1 / leu2-pdc1-Pfet3-6 as primers, the above upstream homology arm, gene and downstream homology arm were amplified to obtain the final long gene fragment leu2-pdc1 ​​by overlap extension PCR technology. Similarly, the upstream homology arm of trp3 was amplified from the whole genome of wild-type Saccharomyces cerevisiae By4741 using primers his3-trp3-Pfet3-1 / his3-trp3-Pfet3-2 by PCR technology, the trp3 gene was amplified from the whole genome of wild-type Saccharomyces cerevisiae By4741 using primers his3-trp3-Pfet3-3his3-trp3-Pfet3-4 by PCR technology, and the trp3 gene was amplified from the whole genome of wild-type Saccharomyces cerevisiae By4741 using primers his3 -trp3-Pfet3-5 / his3-trp3-Pfet3-6 were used as primers to amplify the downstream homology arm of trp3 from the whole genome of wild-type Saccharomyces cerevisiae By4741 by PCR technology, and then the upstream homology arm, gene and downstream homology arm were amplified by overlap extension PCR technology to obtain the final fragment long gene fragment his3-trp3 using primers his3-trp3-1 / his3-trp3-Pfet3-6. The gene fragment of ura3-pha2 was amplified from the whole genome of wild-type Saccharomyces cerevisiae By4741 by PCR technology. The primers are shown in Table 3.

[0077] The long gene fragments leu2-pdc1, his3-trp3 and ura3-pha2 were respectively introduced into the parent strain YLYJ-006 to obtain the recombinant yeast engineering strains YLYJ6-A, YLYJ6-B and YLYJ6-C. The strain genotypes are shown in Table 1.

[0078] 2. Fermentation preparation of tyrosol

[0079] (1) The recombinant yeast strains YLYJ-006, YLYJ6-A, YLYJ6-B and YLYJ6-C constructed in step 1 were fermented in YPSG medium and YPSG-ascorbic acid medium for 96 hours, respectively. When the strains in the YPSG-ascorbic acid medium were fermented for 48 hours, 2500 μM ferric chloride was added to start the secondary dynamic regulation. The results are shown in Figure 4 As shown in a, after ascorbic acid and the activation of the secondary regulatory system, the tyrosol production of all strains was greatly improved. In terms of the iron ion responsive system regulation, YLYJ6-C had the best effect, with a final yield of up to 750 mg / L.

[0080] (2) The recombinant yeast strains YLYJ-006, YLYJ6-A, YLYJ6-B and YLYJ6-C were fermented in YPSG medium for 48 hours. One batch was added with 2500 μM ferric chloride to start the secondary dynamic regulation, and the other batch was a control without adding iron ions. The results are shown in Figure 4 As shown in b, the secondary regulatory dynamic system was activated by the addition of iron ions. The corresponding mRNA levels of pdc1, pha2 and trp3 showed a downward trend after the secondary regulatory dynamic system was activated, verifying that the secondary dynamic regulation had an inhibitory effect on the gene expression of the competing pathway.

[0081] Although there is some background noise, so that the yield cannot reach the ideal state, the present invention provides a new idea for regulating the production of tyrosol. In order to further increase the yield of tyrosol, the regulation can be further refined by establishing a promoter library.

[0082] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An iron ion concentration responsive dynamic control system, characterized in that: The invention comprises a promoter Pfet3 and a target gene coding sequence as shown in SEQ ID NO.9; the promoter Pfet3 is connected to the target gene coding sequence, and the promoter Pfet3 regulates the expression of the target gene in Saccharomyces cerevisiae, The target gene is the indole glycerol phosphate synthase gene trp3, and the cerevisiae yeast is the overexpression 3-deoxy-D-arabinoheptulose acid-7-phosphate synthase mutant gene aro4 K229L , chorismate mutase mutant gene aro7 G141S , tyrosine decarboxylase gene pcaas opt , phosphoketolase gene Bbxfpk opt and the mutant gene Trya M53I / A354V , Saccharomyces cerevisiae BY4741, in which the GAL80 gene, HO gene and LPP1 gene were knocked out simultaneously, pcaas opt and Bbxfpk opt The nucleotide sequences of the genes are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively; The iron ion concentration is 2500 μM, and the system is also regulated by ascorbic acid at a concentration of 1 g / L.

2. A carrier, characterized in that The vector comprises the promoter Pfet3 of the iron ion concentration responsive dynamic regulation system according to claim 1 and a target gene.

3. A recombinant cell, characterized in that The recombinant cell comprises the vector according to claim 2.

4. A recombinant Saccharomyces cerevisiae, characterized in that: The recombinant Saccharomyces cerevisiae is Saccharomyces cerevisiae BY4741 containing the promoter Pfet3 of the iron ion concentration-responsive dynamic regulation system of claim 1 and the target gene; the Saccharomyces cerevisiae BY4741 overexpresses the mutant gene aro4 of 3-deoxy-D-arabinoheptulose-7-phosphate synthase K229L , chorismate mutase mutant gene aro7 G141S , tyrosine decarboxylase gene pcaas opt , phosphoketolase gene Bbxfpk opt and the mutant gene Trya M53I / A354V , and simultaneously knocked out the GAL80 gene, HO gene and LPP1 gene in the genome.

5. The recombinant Saccharomyces cerevisiae according to claim 4, characterized in that The target gene of the iron ion concentration-responsive dynamic regulation system is the indole glycerol phosphate synthase gene trp3.

6. The recombinant Saccharomyces cerevisiae according to claim 4, characterized in that The recombinant Saccharomyces cerevisiae is aro4 K229L 、aro7 G141S 、pcaas opt ,Bbxfpk opt 、Trya M53I / A354V It was integrated into the GAL80, GAL80, HO, HO, and LPP1 sites on the genome of the starting strain BY4741 in sequence.

7. The recombinant Saccharomyces cerevisiae according to claim 4 or 6, characterized in that The aro4 K229L ,aro7 G141S and Trya M53I / A354V The nucleotide sequences of the genes are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.5 respectively.

8. The recombinant Saccharomyces cerevisiae according to claim 5, characterized in that The nucleotide sequence of the trp3 gene is shown in SEQ ID NO.

8.

9. A method for increasing tyrosol production, characterized in that: The method is to use the iron ion concentration responsive dynamic control system of claim 1 to control the indole glycerol phosphate synthase gene trp3, and the control method is to replace the promoter in front of the trp3 gene in the Saccharomyces cerevisiae genome with the promoter Pfet3.

10. A method for synthesizing tyrosol, characterized in that: The method comprises inoculating the recombinant brewer's yeast of any one of claims 4 to 8 into a fermentation system for fermentation preparation, adding iron ions in the middle and late logarithmic phase of the strain growth to continue fermentation, the fermentation system contains 1 g / L ascorbic acid, and the iron ions are added in the form of 2500 μM ferric chloride.

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