CN110982770B - Method for enhancing naringenin synthesis by dynamically regulating fatty acid metabolic pathway - Google Patents
Method for enhancing naringenin synthesis by dynamically regulating fatty acid metabolic pathway Download PDFInfo
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Abstract
The invention discloses a method for dynamically regulating fatty acid metabolic pathway to strengthen naringenin synthesis, and belongs to the technical field of metabolic engineering. The invention is based on naringenin response protein Fde, utilizes regulation and control means such as gene overexpression technology and antisense RNA inhibition technology, and combines a synthesis way of forming and expressing naringenin, thereby establishing a dynamic circulation amplification system capable of sensing intracellular naringenin content, strengthening naringenin synthesis, and improving the yield of naringenin to 3.2 times of the original yield, and reaching 95.1 mg/L. The invention realizes the balance between cell growth and product synthesis, and the method provides reference for the metabolic engineering modification of other high value-added compound production strains.
Description
Technical Field
The invention relates to a method for strengthening naringenin synthesis by dynamically regulating fatty acid metabolic pathway, belonging to the technical field of metabolic engineering.
Background
The flavonoid compounds are natural products widely existing in plant tissues, have various physiological functions, such as anticancer, anti-inflammatory, anti-aging, human body hormone level regulation, antioxidation, liver protection and the like, and are widely applied to foods and medicines. All flavonoids contain a core skeleton consisting of two benzene rings connected by three carbon atoms. Naringenin is a flavone skeleton substance with most derivatives, and can be subjected to subsequent chemical or enzymatic catalytic modification, and catalytic reactions such as hydroxylation, methylation, glycosylation and the like to generate the flavonoid compound with higher added value and multiple functions.
In the process of synthesizing flavonoid compounds such as naringenin and the like by using escherichia coli, the supply shortage of intracellular malonyl-CoA is an important reason for lower final yield. Intracellular malonyl-CoA flows predominantly into the fatty acid synthesis pathway. In addition, acetyl-CoA which is a precursor substance of malonyl-CoA enters the TCA cycle for the most part, and provides necessary energy for the growth of thalli. These all result in a lower intracellular content of malonyl-CoA. Excessive accumulation of intracellular malonyl-CoA by metabolic engineering means not only affects the growth of thalli, but also causes excessive accumulation of intracellular fatty acyl-CoA derivatives, thereby further affecting the acylation level of the whole proteome of cells. It has been reported that researchers accumulate malonyl-CoA mainly by inhibiting the intracellular fatty acid synthesis pathway. Leonard et al obtained 700mg/L and 113mg/L of flavone and anthocyanin, respectively, by adding cerulenin to inhibit fatty acid synthesis-related genes (fabB/fabF). There has also been great progress in the accumulation of malonyl-CoA by potentiating the malonyl-CoA synthesis pathway. Leonard et al and Zhu et al greatly enhanced the production of flavonoids by overexpressing acetyl-CoA carboxylase (ACC) from C.glutamicum.
However, the static regulation system needs to add an inducer at a proper time for regulating the synthesis of malonyl-CoA, and the induction time, the concentration of the inducer, the growth state of the thallus and the like have great influence on the final yield. Meanwhile, since malonyl-CoA is closely related to cell growth, excessive accumulation of malonyl-CoA not only severely affects cell growth, but also affects the level of proteome acylation of cells to further alter the intracellular metabolic environment. Therefore, the construction of stable, efficient and dynamically adjustable naringenin production platform strain for cell growth and product synthesis is very important.
Disclosure of Invention
In order to solve the problems, the invention provides a method for dynamically responding to naringenin content to regulate fatty acid synthesis pathway, which strengthens intracellular malonyl-CoA accumulation, thereby further strengthening naringenin synthesis.
The first purpose of the invention is to provide a method for regulating and controlling a fatty acid synthesis pathway by dynamically responding to naringenin content, which is to regulate and control the fatty acid synthesis pathway by utilizing a biosensor dynamically responding to the naringenin content, wherein the biosensor comprises a naringenin-responsive regulatory protein Fder and a promoter P regulated and controlled by the FderfdeAThe amino acid sequence of the regulatory protein Fder is shown in SEQ ID NO. 1.
In one embodiment of the invention, the nucleotide sequence of the gene encoding the regulatory protein Fder is shown in SEQ ID NO. 2.
In one embodiment of the invention, the promoter PfdeAIncluding PfdeA(180-1)、PfdeA(223-1)、PfdeA(250-1)、PfdeA(288-1)、PfdeA(223-50+RBS)、PfdeA(223-83+RBS)、PfdeA(223 + RBS 135) -fdeA(223 + 180 RBS). The P isfdeAThe nucleotide sequence of (288-1) is shown in SEQ ID NO. 3.
With PfdeA(288-1) the number of 1 st nucleotide at the 3 'end and 288 th nucleotide at the 5' end, PfdeA(180-1)、PfdeA(223-1)、PfdeA(250-1)、PfdeA(223-50+RBS)、PfdeA(223-83+RBS)、PfdeA(223-135+RBS)、PfdeAThe (223-180+ RBS) promoter is located in the 180-1, 223-1, 250-1, 223-50, 223-80, 223-135 and 223-180 regions (5 '-3'), respectively, wherein PfdeA(223-50+RBS)、PfdeA(223-83+RBS)、PfdeA(223 + RBS 135) -fdeA(223-180+ RBS), wherein an RBS sequence is added at the 3' end, and the nucleotide sequence of the RBS is AAGGAG.
In one embodiment of the invention, said modulating the fatty acid synthesis pathway comprises the use of a promoter PfdeAAnd (3) regulating and controlling a target gene or antisense RNA thereof, thereby dynamically inhibiting the content of the intracellular active ACP.
In one embodiment of the invention, said modulating the fatty acid synthesis pathway comprises the use of a promoter PfdeAInhibits acpS and acpT gene expression, and overexpresses the acpH gene.
In one embodiment of the invention, the nucleotide sequence of acpH is shown in SEQ ID NO.4, the nucleotide sequence of antisense RNA of acpS is shown in SEQ ID NO.5, and the nucleotide sequence of antisense RNA of acpT is shown in SEQ ID NO. 6.
The antisense RNA contains a PT hairpin palindrome carrying a complementary sequence of a target gene and an rrnB terminator structure.
The second purpose of the invention is to provide a biosensor for dynamically responding the naringenin content, which comprises a fatty acid-responsive regulatory protein Fder and a promoter P regulated by the FderfdeAThe amino acid sequence of the regulatory protein Fder is shown in SEQ ID NO. 1.
The third purpose of the invention is to provide a carrier with dynamic response to the naringenin content, which contains the biosensor.
The fourth purpose of the invention is to provide an engineering strain for dynamically responding to the naringenin content, which takes the vector as an expression vector.
In one embodiment of the invention, the engineered strain is a host of escherichia coli.
In one embodiment of the invention, the engineered strain inhibits acpS and acpT gene expression and overexpresses the acpH gene.
The fifth purpose of the present invention is to provide the application of the above biosensor in the food or pharmaceutical field.
The invention has the advantages that:
the invention establishes a method for dynamically responding the intracellular naringenin content to regulate and control fatty acid synthesis, and the method strengthens the accumulation of intracellular malonyl-CoA by inhibiting the anabolism pathway of fatty acid and further promotes the synthesis of naringenin, thereby forming a dynamic circulation amplification system without adding an inducer. The method is a dynamic regulation and control method for producing flavonoids such as naringenin and the like by microbial fermentation, which is established for the first time, and provides an efficient metabolism regulation and control system for the microbial production of the flavonoids. Other target compounds with similar metabolic pathways may be synthesized using this method as well. Compared with the traditional static method, the method has the greatest advantages that no inducer is added, and the dynamic balance among cell growth, malonyl-CoA accumulation and naringenin synthesis can be realized. The balance of a microbial global metabolic network is realized, and the synthesis of a target compound is enhanced. By using the method, the yield of naringenin can be improved to 3.2 times of the original yield, and reaches 95.1 mg/L. In addition, the method also provides a certain reference significance for the dynamic synthesis of other high value-added compounds.
Drawings
FIG. 1: promoter PfdeAStructurally optimized plasmid maps.
FIG. 2: promoters P of different lengths and different regionsfdeAThe expression intensity.
FIG. 3: naringenin response amplification system working principle. A: an amplification system schematic diagram of naringenin response, wherein a constitutive promoter expresses a naringenin synthesis pathway gene; b: module B metabolic pathway and working schematic; c: the expression of the acpH, asacpS and asacpT genes during fermentation is related to the change of naringenin content.
FIG. 4: the antisense RNA and acpH gene strains expressing the acpS, acpT, acpP and fabD genes in different combinations varied in intracellular acetyl-CoA, malonyl-CoA and CoA content. + represents overexpression, -represents that the expressed antisense RNA inhibits the gene, and 0 represents no change.
FIG. 5: dynamically regulating and controlling the growth trend chart of the strain on the coumaric acid, the naringenin and the thalli. A: a control strain containing only the naringenin synthesis pathway (block a); b: the combined fermentation result of an amplification module pACM-Fder-acpH-asacpT-asacpS and a module A is constructed by taking a low-copy plasmid pACM4 as a vector; c: an amplification module pCOM-Fder-acpH-asacpT-asacpS and a module A combined fermentation result are constructed by taking the medium copy plasmid pCOM4 as a vector; d: and (3) combining fermentation results of an amplification module pRSM-Fder-acpH-asapT-asapS and a module A which are constructed by taking the high-copy plasmid pRSM3 as a vector.
Detailed Description
The material and the method are as follows:
naringenin synthesis pathway genes were synthesized by Nanjing Kirsi and codon optimized, restriction enzymes and DNA polymerases were purchased from Saimeishi and Takara, respectively, MOPS was purchased from Biotechnology engineering (Shanghai) GmbH. Black fluorescent 96 latent well plates. A multifunctional microplate reader (BioTek) was used to detect the fluorescence intensity of the samples. Coli BL21(DE3) was used for protein expression and naringenin synthesis, and E.coli JM109 was used for molecular cloning.
A CoA quantifying method comprises the following steps: to quantify the intracellular contents of CoA, acetyl-CoA and malonyl-CoA, detection was carried out at 40 ℃ using Shim-pack VP-ODS (250 L.times.2.0) UPLC column and Shimadzu-Shimadzu LCMS-IT-TOF. The mobile phase was 15mM ammonium formate (A) dissolved in water and 10mM ammonium acetate (B) dissolved in methanol. 0.2 mL/min-1The flow rate gradient elution conditions are that, 0-5 min: 10-25% of B, 5-10 min: 25-100% B, 10-11 min: 100-10% B, 11-13 min: hold 10% B. Quantification of CoA, acetyl-CoA and malonyl-CoA was performed as integration of the area of the Extracted Ion Current (EIC) peak. EICs of CoA, acetyl-CoA and malonyl-CoA are [ M-H ] respectively]-＝766.5299m/z，[M+H]810.5813M/z and [ M-H +]-＝852.5764m/z。
RT-PCR method: total RNA of each strain was extracted separately using RNA extraction Kit RNAprep pure Cell/Bacteria Kit DP430 according to the Kit instructions. Total RNA was then reverse transcribed into cDNA using Takara Kit PrimeScript RT reagent Kit with gDNA Eraser RR047A, and genomic DNA was removed. The reverse transcribed cDNA was directly subjected to RT-qPCR machine experiment (Roche, LightCycler 480 II). The internal reference gene is 16S rRNA.
All of the bacterial cultures described in the figure and the following examples were cultured in LB medium at 37 ℃ and 220 rpm. Naringenin is fermented by MOPS culture medium under 30 deg.C and 220 rpm.
The sequences referred to in this application are shown in table 1.
Table 1 sequences to which the present application relates
Description of antisense RNA sequence: the bold sequence is the PT hairpin structure, the italic sequence is the rrnB terminator sequence, and the capital letters are the complementary region sequences of antisense RNA and mRNA.
Example 1PfdeAThe promoter has simple structure
Recognition of the promoter P by the naringenin response protein FderfdeAThe minimum structure of (A) is not clear, and a minimum length of P must be used in order to construct an efficient regulatory systemfdeAA promoter. Thus, using a promoter truncation strategy, from PfdeAThe promoters were truncated at both ends to different extents (FIG. 1). The pCDM4 is used as a starting plasmid, and is connected with EGFP after SpeI/SalI double enzyme digestion to construct the pCDM-EGFP plasmid.
With PfdeA(288-1) the promoter is used as a template, PfdeA (M) -F/PfdeA (N) -RBS-R or PfdeA (M) -F/PfdeA (1) -R (the number of the first base at the downstream of the PfdeA promoter is 1, M is the upstream position of the PfdeA promoter amplified by the upstream primer, N is the downstream position of the PfdeA promoter amplified by the downstream primer) is used as a primer pair, and different regions of the PfdeA promoter are amplified. The PfdeA promoter region obtained by amplification is as follows: 180-1, 223-1, 250-1, 288-1, 223-50+ RBS, 223-83+ RBS, 223-.
Respectively combining P of different areasfdeAThe promoter is connected to the EGFP upstream of the pCDM-EGFP plasmid which is subjected to KpnI/SpeI double digestion.Finally, 8 plasmids were obtained, namely pCDM-PfdeA (180-1) -EGFP, pCDM-PfdeA (223-1) -EGFP, pCDM-PfdeA (250-1) -EGFP, pCDM-PfdeA (288-1) -EGFP, pCDM-PfdeA (223-50+ RBS) -EGFP, pCDM-PfdeA (223-83+ RBS) -EGFP, pCDM-PfdeA (223 + 135+ RBS) -EGFP and pCDM-PfdeA (223 + 180+ RBS) -EGFP (FIG. 1).
The pRSF-Duet-1 plasmid is used as a base, and an FdeR regulatory protein gene is connected with an XbaI/KpnI site, so that a pRSFR vector is constructed. Simultaneous transformation of pRSFR and pCDM-P in E.colifdeAEGFP plasmid, induced by adding naringenin at different concentrations, and the intensity of green fluorescent protein was measured. The results show that: pfdeAThe promoter length is in the range of 223-135bp, the expressed fluorescent protein has higher intensity (figure 2), reaching 62789 fluorescent intensity/OD600The promoter length is 4.3 times of that of an unclipped promoter, and the promoter length is shortest, so that the promoter can be used for subsequent metabolic regulation. The promoter is named as PfdeA(223-135)。
Example 2 fatty acid synthetic pathway optimization
Based on the embodiment 1, the P with a simplified structure is utilizedfdeAThe promoter was constructed as a regulatory system as shown in FIGS. 3A and 3B. The vector pACM4 is used as a framework, the Escherichia coli is used as a host, and P is extracted based on the principle of ePathbrickfdeA(223-135)The promoters are respectively cloned to the antisense RNAs upstream of the acpS, acpT, acpP and fabD genes to regulate the expression of the genes, and P is used for regulating the expression of the genesfdeA(223-135)The promoter over-expresses the acpH gene and expresses the genes in combination to obtain recombinant plasmids of different combinations (fig. 4), which are respectively: pACM-Fder-acpH, pACM-Fder-acpH-asacpT-asacpS-asacpP, pACM-Fder-acpH-asacpT-asacpS-asfabD and pACM-Fder-acpH-asacpT-asacpS-asacpP-asfabD.
The above recombinant plasmid was transformed into E.coli BL21(DE 3). After the single clones were cultured overnight at 37 ℃ and 220rpm in LB medium, respectively, the single clones were inoculated at 1% inoculum size the next day into MOPS medium and cultured at 30 ℃ and 220rpm for 2 hours. And detecting the contents of intracellular CoA, acetyl-CoA and malonyl-CoA. The results show that: by combining antisense RNA of over-expressed acpS and acpT and the acpH gene, the contents of intracellular CoA, acetyl-CoA and malonyl-CoA are highest and are respectively 0.442nmol/mg DW, 0.241nmol/mg DW and 0.967nmol/mg DW, which are respectively improved by 1.56, 4.21 and 7.45 times compared with the original strain of escherichia coli BL21(DE 3).
Embodiment 3 dynamic regulation and control system construction and functional verification
Based on our previous studies (Zhou SH et al, 2019, Biotechnol Bioeng,116:1392-1404, DOI: 10.1002/bit.26941), an optimized naringenin synthesis pathway module A (pCDM-P) was obtainedssrA-UTRrpsT-CHS-PUTRglpDCHI and pETM-PUTRtrxA-TAL-PUTRtalB-4CL), combined with the optimization result module B of example 2 (pACM-FdeR-acpH-asacpT-asacpS), plasmids of module a and module B (fig. 3A, 3B) were combined to construct a dynamic cyclic amplification system of naringenin response, i.e., pACM-FdeR-acpH-asacpT-asacpS were transformed into pCDM-P alonessrA-UTRrpsT-CHS-PUTRglpDCHI and pETM-PUTRtrxA-TAL-PUTRtalBSingle colonies were obtained from 9G3 strain of-4 CL.
In order to verify the amplification effect of naringenin on the system, the single colony is selected and cultured in an LB culture medium overnight; the next day, the cells were inoculated into 25mL of MOPS medium at an inoculum size of 1%, and cultured at 220rpm at 30 ℃ for 48 hours. Samples were taken at different times and naringenin production, antisense RNA to acpS and acpT, and the expression level of the acpH gene were determined simultaneously. Analyzing the change relation between the gene expression level and the naringenin yield. And (3) displaying a detection result: as the fermentation proceeded, naringenin production gradually increased, while the antisense RNAs of acpS and acpT and mRNA expression levels of the acpH gene were linearly related to naringenin production (fig. 3C). Therefore, the constructed dynamic regulation and control system has a sensitive response effect.
Example 4 dynamic regulatory intensity optimization
The amplification intensity of the dynamic amplification system constructed in example 3 directly affected the inhibition intensity of the fatty acid synthesis pathway (fig. 3A). Too strong inhibition of fatty acid synthesis will affect cell growth, while too weak inhibition will not achieve the effect of enhancing malonyl-CoA accumulation. Thus, to modulate the dynamic regulatory response intensity of fig. 3A, three different copy number tables of plasmids (pRSM3 (high copy), pCOM4 (medium copy), and pACM4 (low copy)) were usedExpressing the module B gene. The modules B obtained by construction are named as: pRSM-Fder-acpH-asacpT-asacpS, pCOM-Fder-acpH-asacpT-asacpS and pACM-Fder-acpH-asacpT-asacpS were transferred to a medium containing only pCDM-PssrA-UTRrpsT-CHS-PUTRglpDCHI and pETM-PUTRtrxA-TAL-PUTRtalBSingle colonies were obtained from 9G3 strain of-4 CL.
Shaking flask fermentation (picking the single colony to LB medium overnight culture; inoculating to 25mL MOPS medium with 1% inoculum size the next day, culturing at 30 deg.C and 220 rpm) was performed, sampling at different times to detect naringenin production (FIG. 5). And (3) displaying a detection result: the accumulation of coumaric acid by the dynamically regulated strain is obviously reduced (> 55%). Meanwhile, the research finds that the growth of the original strain and the growth of the dynamically regulated strain have no significant difference. Of the three modules, the final yields of pACM-Fder-acpH-asacpT-asacpS and pRSM-Fder-acpH-asacpT-asacpS were essentially the same, so that it is considered to be more advantageous that the pACM-Fder-acpH-asacpT-asacpS module with low copy number can save more cell growth resources. Compared with a control strain, after the pACM-Fder-acpH-asacpT-asacpS module is utilized, the yield of naringenin is increased to 3.2 times of the original yield, and reaches 95.1 mg/L.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for enhancing naringenin synthesis by dynamically regulating fatty acid metabolic pathway
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Arg Ala Glu Val Leu Lys Asp Ala Val His Asp Val Leu Arg Arg Ile
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Pro Arg Val Leu Ala Arg Ala His Ala Glu Gly Lys His Ile Arg Phe
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Ala Leu Met Pro Gln Val Gln Asp Pro Thr Arg Ser Leu Asp Arg Ala
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<213> Artificial sequence
<400> 6
gcggccgcag gaggaattaa ccatgcagtg gtggtggtgg tggtgcatag atgatctccg 60
gtagcgggga aagcgtgtgc gaaagcaatg cacgccccgc cagccagcgt tcgcgtcgtg 120
gaccttgcgg tgcttgctcg cgtaaacccg gtggcagtgg agctgcgctt aaggtcgaaa 180
ctttccccag aactatccga tacatatcag ggccaacgtt taatggaaaa tgaaagtgcg 240
tatcgtatca cttgtcgcct catcccggta accgactttt cggtctgccc ggccccagta 300
aaatcgccag tttgctaccg cctttgagca ccaccaccac caccactgca tggttaattc 360
ctccttagtt ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc 420
agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac 480
cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat 540
gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 600
ctttcgtttt atctgcctag g 621
<210> 7
<211> 502
<212> DNA
<213> Artificial sequence
<400> 7
gcggccgcag gaggaattaa ccatgcagtg gtggtggtgg tggtgcggtg tcaagagaat 60
ccgcgcccag gtcttcaacg aaagaagcat tgttggtaac ttcttcctgc ttaacgccca 120
gctgttcgcc gataattttc ttaacgcgtt cttcgatagt gctcatactc ttaaatttcc 180
tatcaaaact cgctttcgcg atggttgagc accaccacca ccaccactgc atggttaatt 240
cctccttagt tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg 300
cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga 360
ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca 420
tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg 480
cctttcgttt tatctgccta gg 502
<210> 8
<211> 572
<212> DNA
<213> Artificial sequence
<400> 8
gcggccgcag gaggaattaa ccatgcagtg gtggtggtgg tggtgcgaag cttcagcaaa 60
cgtttcttcg acaattggat agctcgccgc catatcagcc agcattccaa cggtttgaga 120
accctgtcca gggaacacaa atgcaaattg cgtcatgttt taatccttat cctagaaacg 180
aaccagcgcg gagccccagg tgaatccacc gccaaaggct tcaagcagaa ccaactgccc 240
cggcttaatg cgcccgtcgc gtacagcttc atccaggagc accaccacca ccaccactgc 300
atggttaatt cctccttagt tttggcggat gagagaagat tttcagcctg atacagatta 360
aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg 420
tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg 480
ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg 540
aaagactggg cctttcgttt tatctgccta gg 572
<210> 9
<211> 88
<212> DNA
<213> Artificial sequence
<400> 9
aatgtattga tgccgtccat atcatgaatc aaaacaatcc atttgatcaa tatcaagctc 60
actcttaagc ttcactcatc cgctgcat 88
<210> 10
<211> 51
<212> DNA
<213> Artificial sequence
<400> 10
ctcggaggag gccatggtac ctgatcaata tcaagctcac tcttaagctt c 51
<210> 11
<211> 48
<212> DNA
<213> Artificial sequence
<400> 11
ctcggaggag gccatggtac caatgtattg atgccgtcca tatcatga 48
<210> 12
<211> 42
<212> DNA
<213> Artificial sequence
<400> 12
ctcggaggag gccatggtac cctcggatag acgacggatg gg 42
<210> 13
<211> 41
<212> DNA
<213> Artificial sequence
<400> 13
ctcggaggag gccatggtac cggcgctggt ctccgttgtt g 41
<210> 14
<211> 47
<212> DNA
<213> Artificial sequence
<400> 14
ttctccctta cccatactag tgtctgtctc cttgtctcct accctgc 47
<210> 15
<211> 46
<212> DNA
<213> Artificial sequence
<400> 15
ttctccctta cccatactag tctccttcgc gttgtcatgg ccaggc 46
<210> 16
<211> 46
<212> DNA
<213> Artificial sequence
<400> 16
ttctccctta cccatactag tctccttatc cagtgcgagt gcgccg 46
<210> 17
<211> 52
<212> DNA
<213> Artificial sequence
<400> 17
ttctccctta cccatactag tctccttatg cagcggatga gtgaagctta ag 52
<210> 18
<211> 56
<212> DNA
<213> Artificial sequence
<400> 18
ttctccctta cccatactag tctccttaat ggattgtttt gattcatgat atggac 56
Claims (6)
1. A method for regulating fatty acid synthetic pathway by dynamically responding naringenin content utilizes a biosensor for dynamically responding naringenin content to regulate fatty acid synthetic pathway, wherein the biosensor comprises a regulatory protein Fder of naringenin response and a promoter P regulated by FderfdeAThe amino acid sequence of the regulatory protein Fder is shown as SEQ ID NO. 1; the regulation of fatty acid synthesis pathway is realized by using promoter PfdeAInhibits acpS and acpT gene expression, and overexpresses the acpH gene.
2. The method according to claim 1, wherein the nucleotide sequence of the gene encoding the regulatory protein Fder is shown in SEQ ID No. 2.
3. The method of claim 1, wherein the promoter P isfdeAIncluding PfdeA(180-1)、PfdeA(223-1)、PfdeA(250-1)、PfdeA(288-1)、PfdeA(223-50+RBS)、PfdeA(223-83+RBS)、PfdeA(223 + RBS 135) -fdeA(223-180+ RBS); the P isfdeAThe nucleotide sequence of (288-1) is shown as SEQ ID NO.3, and the nucleotide sequence of RBS is AAGGAG.
4. The method according to claim 1, wherein the nucleotide sequence of acpH is shown as SEQ ID No.4, the nucleotide sequence of the antisense RNA of acpS is shown as SEQ ID No.5, and the nucleotide sequence of the antisense RNA of acpT is shown as SEQ ID No. 6.
5. An engineering strain for dynamically responding to naringenin content is characterized in that a vector for dynamically responding to naringenin content is taken as an expression vector; the expression vector contains a fatty acid response regulatory protein FdeR and a promoter P regulated by the FdeRfdeAThe amino acid sequence of the regulatory protein Fder is shown as SEQ ID NO. 1; the engineering strain takes escherichia coli as a host; the engineering strain inhibits the expression of acpS and acpT genes and overexpresses an acpH gene.
6. The use of the engineered strain of claim 5 in the food or pharmaceutical field.
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Citations (3)
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| CN1948338A (en) * | 2006-11-10 | 2007-04-18 | 中国科学院遗传与发育生物学研究所 | Transcription factor of regulating and controlling vegetable fatty acid metabolism and its coding gene and application |
| CN104387360A (en) * | 2014-11-21 | 2015-03-04 | 段煜 | Naringenin fatty acid ester and preparation method thereof as well as pharmaceutical composition with naringenin fatty acid ester as active component and application of pharmaceutical composition |
| CN106755163A (en) * | 2016-12-14 | 2017-05-31 | 吴银娣 | A kind of method for producing Flavonoid substances |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1948338A (en) * | 2006-11-10 | 2007-04-18 | 中国科学院遗传与发育生物学研究所 | Transcription factor of regulating and controlling vegetable fatty acid metabolism and its coding gene and application |
| CN104387360A (en) * | 2014-11-21 | 2015-03-04 | 段煜 | Naringenin fatty acid ester and preparation method thereof as well as pharmaceutical composition with naringenin fatty acid ester as active component and application of pharmaceutical composition |
| CN106755163A (en) * | 2016-12-14 | 2017-05-31 | 吴银娣 | A kind of method for producing Flavonoid substances |
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| Fine‐tuning the (2S)‐naringenin synthetic pathway using an an iterative high‐throughput balancing strategy;Jingwen Zhou et al;《Biotechnology and Bioengineering》;20190211;第116卷;全文 * |
| 基于静态与动态调控策略的柚皮素生物合成研究;周胜虎;《中国博士学位论文全文数据库 工程科技Ⅰ辑》;20200515(第05期);全文 * |
| 柚皮素合成关键基因表达水平对目标产物积累水平的量化影响;焦亭亭等;《生物工程学报》;20190319;第35卷(第7期);全文 * |
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