CN109402154B - Method for improving isoorientin yield of recombinant bacteria by utilizing cooperative regulation and control strategy - Google Patents
Method for improving isoorientin yield of recombinant bacteria by utilizing cooperative regulation and control strategy Download PDFInfo
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Abstract
A method for improving the yield of isoorientin produced by recombinant bacteria by utilizing a cooperative regulation strategy is characterized in that the yield of isoorientin produced by the recombinant bacteria is greatly improved by introducing genes to regulate the supply of UDP-glucose, optimizing an induction transformation strategy and controlling the accumulation of acetic acid on the basis of the recombinant bacteria carrying a gene Gt6CGT of the carbon glycoside glycosyltransferase. By utilizing the strategy of the invention, the yield of the recombinant bacterium BL21-CGT-III isoorientin reaches 1371mg/L, the molar conversion rate of the substrate reaches 97.4 percent, which is 40 times of the highest yield at present, and the process for preparing the isoorientin is simple.
Description
Technical Field
The invention belongs to the fields of genetic engineering technology and microorganisms, and particularly relates to a method for improving the yield of isoorientin produced by recombinant bacteria by utilizing a cooperative regulation strategy.
Background
The C-glycoside flavone is a flavonoid compound with glycosyl directly connected to a flavone matrix by a C-C bond, and is widely present in extracts of wheat, corn, rice, mung bean, blueberry, lemon, black tea and bamboo leaf. Compared with oxygen glycoside flavone, carbon glycoside flavone has the characteristics of stable structure, capability of penetrating into focus parts and the like. Isoorientin (isoorientin), also known as luteolin 6-C-glucoside, is a luteolin glucoside type C-glycoside flavone compound, and is one of the main components of flavonoids in bamboo leaf extract. Researches in recent years find that isoorientin can remove free radicals, resist cell viability loss caused by oxidative damage, and has prevention and treatment effects on various diseases, such as improvement of relevant indications of diabetes, inhibition of inflammatory diseases, weakening of hepatic fibrosis development, induction of liver cancer cell apoptosis, virus resistance and other various effects, so that the isoorientin has a good application prospect.
Currently, isoorientin is mainly extracted from bamboo leaves, for example, the invention patent (201410416858.6) provides a method for preparing orientin and isoorientin, raw materials are firstly pretreated, and then the orientin and the isoorientin are prepared by using a preparation chromatograph. However, the content of isoorientin in the bamboo leaves is low, and simultaneously, the flavonoid compounds are complex in components, so that the extraction cost is higher. The key problem to be solved at present is to find a low-cost alternative method for preparing isoorientin.
The development of synthetic biology provides an efficient method for the preparation of natural products. At present, the recombinant bacterium can catalyze luteolin to generate isoorientin by cloning and expressing a carbon glycoside glycosyltransferase (Gt6CGT) gene derived from Gentiana triflora, but the highest yield is only 34mg/L (Applied Microbiology and Biotechnology (2018)102: 1251-1267). This results in too high production and extraction costs to be able to be industrialized. How to improve the yield of isoorientin produced by the recombinant strain becomes a key problem to be solved urgently. The yield of isoorientin generated by transforming luteolin with the recombinant strain is closely related to the expression of a gene of the carbon glycoside glycosyltransferase (Gt6CGT), the growth of recombination, the accumulation of acetic acid and the supply of coenzyme (UDP-glucose). The defects in either aspect result in a yield that cannot be greatly increased. Therefore, the method improves the yield of isoorientin produced by the recombinant strain by utilizing a synergistic regulation and control strategy.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides a method for improving the yield of isoorientin produced by recombinant bacteria by utilizing a cooperative regulation and control strategy, which is based on the recombinant bacteria carrying a gene Gt6CGT of carbon glycoside glycosyltransferase and greatly improves the yield of isoorientin produced by the recombinant bacteria by optimizing an induction transformation strategy, controlling acetic acid accumulation and regulating UDP-glucose supply.
The technical scheme is as follows: a method for improving the yield of isoorientin produced by recombinant bacteria by utilizing a cooperative regulation strategy is characterized in that the yield of isoorientin produced by the recombinant bacteria is greatly improved by introducing genes to regulate the supply of UDP-glucose, optimizing an induction transformation strategy and controlling the accumulation of acetic acid on the basis of the recombinant bacteria carrying a gene Gt6CGT of the carbon glycoside glycosyltransferase.
The specific method is that Cellobiose phosphoribosylase gene from Saccharophagus degradans 2-40 and UTP-glucose-1-phosphate uridylyltransferase gene from Bifidobacterium bifidum are introduced into recombinant bacteria carrying carbon-glycoside glycosyltransferase Gt6CGT gene, the nucleic acid sequences of the genes are respectively shown as SEQ ID NO. 2 and SEQ ID NO. 3; based on a TB-Gly culture medium, the recombinant bacteria are induced to express the carbon glycoside glycosyltransferase Gt6CGT for 6h at the temperature of 20 ℃ by 0.1mM IPTG, and then a substrate luteolin is added for conversion.
The culture medium comprises 12g/L peptone, 24g/L yeast powder, 2.32g/L potassium dihydrogen phosphate, 12.54g/L dipotassium hydrogen phosphate and 10g/L glycerol.
The recombinant plasmid containing the gene sequence is pACYCDuet-Cep-UgpA.
The recombinant bacterium containing the recombinant plasmid is BL 21-CGT-III.
A method for improving the yield of isoorientin produced by recombinant bacteria by utilizing a cooperative regulation strategy comprises the steps of culturing the recombinant bacteria BL21-CGT-III in LB culture medium containing 50 mu g/mL streptomycin and 35 mu g/mL chloramphenicol overnight, transferring the recombinant bacteria BL21-CGT-III to TB-Gly culture medium containing 50 mu g/mL streptomycin and 35 mu g/mL chloramphenicol, carrying out shake culture at 37 ℃ and 200rpm until OD600When the concentration is 0.8, adding IPTG inducer with the final concentration of 0.1mM, and carrying out induced culture at 20 ℃ for 6 h; subsequently, 900mg/L of luteolin was added, and after 12 hours, 20g/L of cellobiose was added, followed by transformation and culture for 116 hours.
The method for improving the yield of isoorientin produced by the recombinant bacteria by utilizing the cooperative regulation strategy comprises the steps of centrifuging a conversion culture at 12000g for 20 minutes, taking a supernatant, collecting a sample, directly feeding the obtained supernatant into a column, wherein the filler of the column is AB-8 macroporous resin, the diameter of the column is 5cm, the length of the column is 30cm, balancing the column with deionized water, washing the column with 15 times of 3wt.% ethanol, eluting with 30 wt.% ethanol, and vacuum-drying the obtained sample to obtain an isoorientin refined product.
Has the advantages that: 1. the invention can reduce the influence of the substrate on the expression and the recombination growth of the carbon glycoside glycosyltransferase (Gt6CGT) to the minimum by adopting different induction transformation strategies. 2. According to the invention, the components of the culture medium are adjusted, so that the recombinant bacteria can well inhibit the accumulation of acetic acid in the transformation process, and the yield of isoorientin is greatly improved. 3. According to the invention, the yield of isoorientin is 826mg/L which is 8 times of that before optimization by inducing a transformation strategy and controlling acetic acid accumulation. 4. The invention provides a novel synthetic route for producing UDP-glucose by using cellobiose, which can slowly and continuously provide UDP-glucose by using cellobiose and can inhibit the accumulation of acetic acid. 5. By utilizing the strategy of the invention, the yield of the recombinant bacterium BL21-CGT-III isoorientin reaches 1371mg/L, the molar conversion rate of the substrate reaches 97.4 percent, and is 40 times of the highest yield at present. 6. The process for preparing isoorientin is simple.
Drawings
FIG. 1 is a graph showing the effect of different induced transformation strategies on the production of isoorientin by recombinant bacteria; (A) LB culture medium, (B) TB-Gly culture medium;
FIG. 2 is a graph showing the influence of different culture media on the accumulation of acetic acid in recombinant bacteria and the yield of isoorientin; (A) TB-Mal culture medium, (B) TB-Del culture medium, (C) TB-Gly culture medium, (D) TB-Glc culture medium;
FIG. 3 is a graph showing the influence of the introduction of a novel UDP-glucose synthesis pathway on the yield of isoorientin;
FIG. 4 is a time curve diagram of isoorientin production by recombinant bacteria BL 21-CGT-III.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
For further understanding of the present invention, the present invention will be described in detail with reference to examples, wherein, unless otherwise specified, the various reagents referred to in the examples are commercially available; unless otherwise specified, specific procedures described in the examples are described in the third edition of molecular cloning, laboratory Manual.
Example 1
1. Construction of recombinant bacterium BL21-CGT-I
Carbon glycoside glycosyltransferase Gt6CGT gene (AB985754.1) from Gentiana triflra is optimized according to an escherichia coli K12 dominant codon table (http:// www.kazusa.or.jp/codon /), codons with high amino acid synonymous codon usage frequency are selected as dominant codons, optimized carbon glycoside glycosyltransferase gene sequence information is finally obtained, the optimized carbon glycoside glycosyltransferase gene sequence information is synthesized by Shanghai Czeri bioengineering GmbH, and Nco I and BamH I enzyme digestion sites are respectively added at the N end and the C end of the gene to obtain the optimized Gt6CGT gene, wherein the gene sequence is shown as SEQ ID NO: 1.
Carrying out double enzyme digestion on the optimized Gt6CGT gene by using Nco I and BamH I, simultaneously carrying out double enzyme digestion on a recombinant plasmid pCDFDuet-1 by using Nco I and BamH I, respectively cutting and recovering glue, concentrating, connecting overnight at 16 ℃, transforming a connecting product into escherichia coli JM109 competent cells, screening positive clones, and carrying out sequence analysis; selecting the clone with correct sequence to extract plasmid, and obtaining recombinant plasmid pCDFDuet-Gt6 CGT.
The recombinant plasmid pCDFDuet-Gt6CGT is transformed into an escherichia coli BL21(DE3) host bacterium (Novagen) to obtain a recombinant bacterium BL 21-CGT-I.
2. Transforming luteolin into isoorientin by adopting different induced transformation strategies
The flavonoids compounds generally have antibacterial effect and can inhibit the growth of Escherichia coli. Therefore, the addition time of luteolin has important influence on the growth of recombinant bacteria and the expression of the carbonic glycoside glycosyltransferase Gt6 CGT. The recombinant strain BL21-CGT-I is taken as a research object, and the influence of the addition time of the luteolin on the yield of the isoorientin is researched through different induction transformation strategies.
Culturing the recombinant strain BL21-CGT-I in LB culture medium containing chloramphenicol (35. mu.g/mL) overnight, transferring to 50mL LB culture medium (LB culture medium: tryptone 10g/L, yeast powder 5g/L, sodium chloride 5g/L) or TB-Gly culture medium (peptone 12g/L, yeast powder 24g/L, potassium dihydrogen phosphate 2.32g/L, dipotassium hydrogen phosphate 12.54g/L, glycerol 10 g/L), culturing at 37 deg.C and 200rpm until OD is reached600When the concentration is 0.8, adding an isopropyl beta-D-thiogalactopyranoside (IPTG) inducer with the final concentration of 0.1mM, and carrying out induction culture at 20 ℃ for 0h, 3h, 6h and 9 h; adding 900mg/L luteolin, continuously transforming and culturing for 24h and 48h, and sampling to determine isoorientin yield. The research shows that the effect is best after the induction culture at 20 ℃ for 6h, and the yield of the isoorientin in an LB culture medium after 48h of transformation reaches 168mg/L which is 3 times of that in the induction culture at 20 ℃ for 0h, as shown in figure 1. The yield of isoorientin after 48 hours of conversion in a TB-Gly culture medium is 545mg/LInduced at 20 ℃ for 5.3 times of 0h (FIG. 1).
3. Influence of recombinant bacterium acetic acid accumulation on isoorientin yield
Acetic acid is a product of unbalanced metabolism of the recombinant bacteria and can influence the expression of the recombinant enzyme and the growth of the recombinant bacteria, so that the control of the accumulation of the acetic acid is very important for the recombinant bacteria to produce the isoorientin. The invention mainly researches the influence of acetic acid accumulation on the yield of isoorientin through different culture media, thereby obtaining the optimal conditions. The culture media used in the invention are respectively: TB-Gly medium: 12g/L peptone, 24g/L yeast powder, 2.32g/L potassium dihydrogen phosphate, 12.54g/L dipotassium hydrogen phosphate and 10g/L glycerol; TB-Glc medium: 12g/L peptone, 24g/L yeast powder, 2.32g/L potassium dihydrogen phosphate, 12.54g/L dipotassium hydrogen phosphate and 10g/L glucose; TB-Dex Medium: 12g/L of peptone, 24g/L of yeast powder, 2.32g/L of monopotassium phosphate, 12.54g/L of dipotassium phosphate and 10g/L of dextrin; TB-Mal medium: 12g/L of peptone, 24g/L of yeast powder, 2.32g/L of monopotassium phosphate, 12.54g/L of dipotassium phosphate and 10g/L of maltodextrin.
The recombinant strain BL21-CGT-I is cultured in LB culture medium containing chloramphenicol (35. mu.g/mL) overnight, and is respectively transferred to 50mL of TB-Gly culture medium added with 35. mu.g/mL chloramphenicol, TB-Glc culture medium, TB-Dex culture medium and TB-Mal culture medium at 37 ℃ and under the condition of shaking at 200rpm until OD is achieved600At 0.8, 0.1mM IPTG inducer is added to the solution, and the solution is subjected to induction culture at 20 ℃ for 6h, followed by addition of 900mg/L of luteolin. Sampling at different times to determine the amount of isoorientin and acetic acid. According to the research, as shown in FIG. 2, the highest output is TB-Gly medium, the isoorientin output reaches 826mg/L at 80h, and the isoorientin output in TB-Glc medium is only 113 mg/L. The yields of TB-Dex medium and TB-Mal medium were 358mg/L and 376mg/L, respectively. The highest amount of acetic acid accumulated is TB-Glc culture medium, and the amount of acetic acid accumulated in the TB-Dex culture medium and the TB-Mal culture medium reaches 1655mg/L after the TB-Glc culture medium is cultured for 32 hours, which is far higher than the amount of acetic acid in the TB-Dex culture medium and the TB-Mal culture medium. As can be seen from FIG. 2, the recombinant bacteria rapidly utilized glucose in TB-Glc medium to produce a large amount of acetic acid, while carbon source consumption rates in TB-Dex medium and TB-Mal medium were much lower than that in TB-Glc medium, and thus only a small amount of acetic acid was produced.Therefore, the production of isoorientin can be improved only by controlling the consumption rate of the carbon source to inhibit the production of acetic acid. On the other hand, too low carbon source consumption rate is not beneficial to the production of isoorientin by the recombinant bacteria. The glycerol consumption rate in the TB-Gly medium was higher than the consumption rates of dextrin and maltodextrin, and the isoorientin production in the TB-Gly medium was more than 2 times higher than that in the TB-Dex medium and the TB-Mal medium, although the acetic acid content in the TB-Gly medium was higher than that in the TB-Dex medium and the TB-Mal medium. Therefore, the TB-Gly culture medium provided by the invention is most beneficial to the recombinant bacteria to produce isoorientin.
Influence of the concentration of glycerol in the TB-Gly medium on the yield of isoorientin produced by recombinant bacteria
Changing the concentration of glycerol in a TB-Gly culture medium, and determining the isoorientin yield and acetic acid accumulation of the recombinant bacteria. The other components in the TB-Gly medium are kept unchanged, and the glycerol concentrations are respectively 5g/L, 10g/L, 20g/L, 30g/L and 40 g/L. The recombinant bacteria are respectively transformed in TB-Gly culture media with different glycerol concentrations for 80h according to the optimized culture conditions, and the amounts of isoorientin and acetic acid are measured by sampling. Researches find that the glycerol concentration of 10g/L is most beneficial to the recombinant bacteria to produce isoorientin.
5. Influence of introduction of novel UDP-glucose synthesis pathway on yield of isoorientin
5.1 construction of recombinant plasmid pACYCDuet-Cep-UgpA
Cellobiose phosphorylase gene (cep, ABD80580.1) derived from Saccharophagus degradans 2-40 is optimized according to an Escherichia coli K12 dominant codon table (http:// www.kazusa.or.jp/codon /), codons with high usage frequency of synonymous codons of amino acid are selected as dominant codons, finally optimized sequence information of the Cellobiose phosphorylase gene is obtained, the optimized sequence information is synthesized by Shanghai Jieli bioengineering GmbH, and Nco I and EcoR I enzyme cutting sites are respectively added at the N end and the C end of the gene to obtain the optimized cep gene, and the gene sequence is shown as SEQ ID NO: 2.
Carrying out double enzyme digestion on the optimized cep gene by using Nco I and EcoR I, simultaneously carrying out double enzyme digestion on the recombinant plasmid pACYCDuet-1 by using Nco I and EcoR I, respectively cutting and recovering glue, connecting overnight at 16 ℃ after concentrating, transforming a connecting product into escherichia coli JM109 competent cells, screening positive clones, and carrying out sequence analysis; selecting the clone with the correct sequence to extract the plasmid, and obtaining the recombinant plasmid pACYCDuet-Cep.
The UTP-glucose-1-phosphate uridyltransferase gene (ugpA, YP _003971086.1) from Bifidobacterium bifidum is optimized according to an escherichia coli K12 dominant codon table (http:// www.kazusa.or.jp/codon /), codons with high usage frequency of amino acid synonymous codons are selected as dominant codons, optimized gene sequence information is finally obtained and synthesized by Shanghai Jie Riyuri bioengineering limited company, Nde I enzyme cutting sites and Kpn I enzyme cutting sites are respectively added at the N end and the C end of the gene, and the optimized kupA gene is obtained, wherein the gene sequence is shown in SEQ ID NO: 3.
Carrying out double enzyme digestion on the optimized upgA gene by Nde I and Kpn I, simultaneously carrying out double enzyme digestion on the recombinant plasmid pACYCDuet-Cep by Nde I and Kpn I, respectively cutting and recovering glue, concentrating, connecting overnight at 16 ℃, transforming a connecting product into escherichia coli JM109 competent cells, screening positive clones, and carrying out sequence analysis; selecting the clone with the correct sequence to extract the plasmid, and obtaining the recombinant plasmid pACYCDuet-Cep-UgpA.
5.2 obtaining of recombinant bacterium BL21-CGT-III
The recombinant plasmid pCDFDuet-Gt6CGT and the recombinant plasmid pACYCDuet-Cep-UgpA were co-transformed into Escherichia coli BL21(DE3) host bacteria (Novagen), and cultured overnight at 37 ℃ on an LB plate containing streptomycin (50. mu.g/mL) and chloramphenicol (35. mu.g/mL) to obtain recombinant bacteria BL 21-CGT-III.
5.3 study on production of isoorientin by recombinant bacterium BL21-CGT-III
The recombinant strain BL21-CGT-III was cultured overnight in LB medium containing streptomycin (50. mu.g/mL) and chloramphenicol (35. mu.g/mL), transferred to 50mL TB-Gly medium supplemented with streptomycin (50. mu.g/mL) and chloramphenicol (35. mu.g/mL), cultured at 37 ℃ with shaking at 200rpm to OD600When the concentration is 0.8, adding IPTG inducer with the final concentration of 0.1mM, and carrying out induced culture at 20 ℃ for 6 h; adding luteolin 900mg/L and cellobiose (0g/L, 1g/L, 5g/L, 10g/L, 20g/L, 40g/L) at different concentrations, continuing transformation culture, sampling and testingAnd (3) determining the yield of isoorientin. According to the research, as shown in FIG. 3, when 20g/L of cellobiose is added, the yield of isoorientin produced by the recombinant strain is the highest, and the yield reaches 1176mg/L within 104h of transformation culture.
The recombinant strain BL21-CGT-III was cultured overnight in LB medium containing streptomycin (50. mu.g/mL) and chloramphenicol (35. mu.g/mL), transferred to 50mL TB-Gly medium supplemented with streptomycin (50. mu.g/mL) and chloramphenicol (35. mu.g/mL), cultured at 37 ℃ with shaking at 200rpm to OD600When the concentration is 0.8, adding IPTG inducer with the final concentration of 0.1mM, and carrying out induced culture at 20 ℃ for 6 h; followed by 900mg/L luteolin and at different times 20g/L cellobiose (0 h, 12h, 24h, 36h, 48h, 60h after substrate addition). The research shows that when the cellobiose is added after 12 hours of luteolin addition, the isoorientin yield of the recombinant strain is the highest, and the isoorientin yield reaches 1206mg/L after 104 hours of transformation culture, as shown in Table 1.
TABLE 1 Effect of cellobiose addition timing on isoorientin production by recombinant bacteria
6. Time curve of isoorientin produced by recombinant bacterium BL21-CGT-III
The recombinant strain BL21-CGT-III was cultured overnight in LB medium containing streptomycin (50. mu.g/mL) and chloramphenicol (35. mu.g/mL), transferred to 50mL TB-Gly medium supplemented with streptomycin (50. mu.g/mL) and chloramphenicol (35. mu.g/mL), cultured at 37 ℃ with shaking at 200rpm to OD600When the concentration is 0.8, adding an isopropyl beta-D-thiogalactopyranoside (IPTG) inducer with the final concentration of 0.1mM, and carrying out induction culture at 20 ℃ for 6 h; followed by addition of 900mg/L luteolin and after 12h addition of 20g/L cellobiose and sampling at different time intervals to determine the amount of isoorientin and substrate. As shown in FIG. 4, the yield reaches 1371mg/L in 116h of transformation culture, and the molar conversion rate of the substrate is 97.4%, which is 40 times higher than the highest yield reported at present.
7. Separating and purifying isoorientin
12000g of fermentation liquor obtained by conversion is centrifuged for 20 minutes, supernatant is taken, the obtained supernatant is directly fed into a column, the filler of the chromatographic column is AB-8 macroporous resin (the diameter is 5cm, the length of the column is 30cm), the chromatographic column is balanced by deionized water, after the sample feeding is finished, the chromatographic column is washed by 3 percent ethanol with 15 times of volume, then the ethanol with 30 percent is used for elution, the obtained sample is dried in vacuum to obtain the isoorientin refined product with the purity of 95 percent, and the yield of the isoorientin is 81.5 percent (Table 2).
TABLE 2 purification Process of Isoorientin
Sequence listing
<110> Nanjing university of forestry
<120> method for improving yield of isoorientin produced by recombinant bacteria by utilizing synergistic regulation and control strategy
<160> 3
<170> SIPOSequenceListing 1.0
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<213> Gentiana triflora (Gentiana triflora)
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ggcgtggtta atccgatgct gcgcttaggc aaagcctttg cctctaaagg cctgttagtg 120
accctgagcg ccccggaaat tgttggcacc gaaattcgta aagccaataa cctgaatgat 180
gatcagccga ttaaagtggg tagcggtatg attcgctttg agttctttga tgatggttgg 240
gaatcagtta atggctctaa accgtttgat gtttgggtgt atattaatca cctcgatcag 300
accggtcgtc agaaactgcc gattatgctg aaaaaacatg aagaaaccgg taccccggtg 360
tcttgtctga ttctgaatcc gttagtgccg tgggttgcag atgttgcaga tagcttacag 420
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ggcatgccgc tgttaaaata tgatgaagtt ccggattatc tgcatccgcg caccccgtat 600
ccgttcttcg gtaccaatat tctgggtcag tttaaaaatc tgtctaaaaa tttttgtatt 660
ctgatggata ccttttatga actggaacat gaaattattg ataatatgtg taaactgtgt 720
ccgattaaac cgattggccc gctgtttaaa attccgaaag atccgtctag taatggcatt 780
accggcaatt ttatgaaagt ggatgattgt aaagaatggt tagatagtcg tccgacctct 840
accgtggtgt atgtgagcgt tggtagcgtt gtgtatctga aacaggaaca ggtgaccgaa 900
atggcctatg gcattctgaa tagcgaagtg agctttctgt gggtgctgcg cccgccgagc 960
aaacgcattg gtaccgaacc gcatgtgctg ccggaagaat tttgggaaaa agcaggcgat 1020
cgcggcaaag ttgttcagtg gagtccgcag gaacaggtgc tggcccatcc ggccaccgtg 1080
ggctttctga cccattgtgg ctggaatagt acccaggaag ccattagctc aggcgtgccg 1140
gttattacct ttccgcagtt tggcgatcag gtgaccaatg ccaaatttct ggttgaagag 1200
tttaaagtgg gcgttcgctt aggtcgcggc gaactggaaa atcgtattat tacccgcgat 1260
gaagttgaac gcgccttacg cgaaattacc agcggcccga aagcagaaga agttaaagaa 1320
aatgcactga agtggaagaa gaaagcagaa gaaaccgttg ccaaaggcgg ctatagcgaa 1380
cgtaatctgg tgggctttat tgaagaagtt gcacgcaaaa ccggcaccaa ataa 1434
<210> 2
<211> 2436
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgaaatttg gtcattttga tgataaagca cgcgaatatg ttattaccga tccgaaaacc 60
ccgtatccgt ggattaatta tctgggtaat gaagattttt ttagcttagt gagcaataca 120
ggcggcggct atagctttta taaagatgcc aaatttcgtc gtctgacccg ctatcgctat 180
aataatgttc cggtggataa tggcggcaaa tatttttata ttaatgatag cggcgatgtg 240
tggagtccgg gttggaagcc cgttaaagca gaactggatg catattcttg tgcacatggt 300
ctgagctata cccgcattac aggagaacgc aatggcattc aggccgaagt gctgagcttt 360
attccgttag gtacgtgggc cgaaattcag aaagtttcac tgaaaaatac cagcggtgcc 420
accaaaaaat ttaaactgtt tagctttgca gaatggtgtc tgtggaatgc agaagatgat 480
atgaccaatt ttcagcgcaa ttttagtacc ggcgaagtgg aagtggaaga tagcgtgatt 540
tatcataaaa ccgaatttaa agaacgtcgt aatcattatg ccttttatag cgttaatgcc 600
ccgattcagg gctttgatac cgatcgcgat aaatggaaag gtctgtataa tgattttgat 660
aaaccggatg cagtgtttga aggcgaaccg cgcaattcag aagcacatgg ttggagtccg 720
attgcctcac attatttaga agttgaactg gcgcctggcg aatctaagga tctgattttt 780
gtgctgggct atattgaagt tgccccggaa aataaatggg aatctaaagg cgttattaat 840
aaatctccgg ccaaagaact gattgcacgc tttgatagcg tggaaaaagt ggatgcagaa 900
ctgaccaaac tggccgatta ttgggccaat ctgctgtcaa cctatagtgt tgaatcaggc 960
gatgaaaaac tggatcgcat ggttaatatt tggaatcagt atcagtgtat ggtgaccttt 1020
aatatgtctc gtagcgccag cttttttgaa agcggcattg gtcgcggtat gggctttcgc 1080
gatagtaatc aggatctgat tggctttgtt catcaggttc cggaacgtgc acgtgaacgc 1140
attattgata ttgcctcaac ccagtttgaa gatggtagcg cctatcatca gtatcagccg 1200
ctgaccaaac gcggtaataa tgccattggc ggcaatttta atgatgatcc gctgtggtta 1260
attctgtcaa ccaccgatta tattaaagaa accggcgatt ttagtattct ggaagaacag 1320
gttccgtatg ataatgatgc aagcaaagcc acctctcatt ttgaacatct gaaacgtagc 1380
ttttatcata ccgttaataa tctgggtccg catggcctgc cgctgattgg tcgcgccgat 1440
tggaatgatt gtctgaatct gaattgtttt agcgaagatc cgaatgagtc ctttcagacc 1500
accggcaata aaaccgggcg taccgcagaa tcactgatga ttgcaggcct gtttgtgctg 1560
tatggcaatg aatttgttaa actgtgtcgc gaaattggtc aggatggtga agccgccgaa 1620
gcccaggccc atattgatca gatggtggaa gccgttaaaa aacatggttg ggatggcgaa 1680
tggtttctgc gcgcctatga ttattatggc aaaaaagtgg gctctaaaga aaatgaagaa 1740
ggtaaaattt ttattgaatc acagggcttt tgtggcatgg caggcattgg cttagaagat 1800
ggcttagttg aaaaatctat ggatagtgtt aaagaatggt tagattgtga ttatggcatt 1860
gtgttacagc agccggcctt taccaaatat tatattgaat atggcgaaat ttctacctat 1920
ccggcaggct ataaagaaaa tgcaggcatt ttttgtcata ataatccgtg gattatgatt 1980
accgaaaccc tgctgggtcg cggcgataaa gcctttgaat attatcgcaa aattgccccg 2040
gcctatctgg aagaaatttc tgacctgcat aaagttgaac cgtatgcata ttgtcagatg 2100
attgcaggta aagatgccta tctgccgggt gaaggtaaaa attcttggtt aaccggcacc 2160
gcctcttgga attttgcagc cattacccag tatattctgg gcgttaaacc ggattatagc 2220
ggcttagcca ttaatccgtg tattccctcg tcttgggatg gctttaaagt gacccgcaaa 2280
tatcgcggtg ccacctataa tattattgtg accaatccga cccatgtgag caaaggcgtt 2340
aaatcactga ccctgaatgg caatgccatt gatggctata ttgttccgcc gcagcaggca 2400
gggacagtgt gtaatgtgga agtgacctta ggctaa 2436
<210> 3
<211> 1467
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgtttgcgg aggatctgaa acgcaccgaa aaaatgaccg tggatgatgt gtttgaacag 60
agcgcccaga aaatgcgcga acagggtatg agcgaaattg caatctcaca gtttcgtcat 120
gcatatcatg tgtgggcaag tgaaaaagaa agtgcttgga ttcgcgaaga tgcagtggaa 180
ccactgcatg gcgttcgctc ttttcatgat gtgtataaga ccattgatca tgataaagcg 240
gttcatgcct ttgccaaaac cgcctttctg aaactgaatg gtggcctggg tacatctatg 300
ggcctccagt gtgccaaatc actgttacca gttcgtcgtc ataaagcacg tcagatgcgt 360
tttctggata ttatcttagg tcaggtgctg acagctcgta cacgcctgaa tgttccactg 420
ccggtgactt tcatgaactc ttttcgtaca agcgatgata ctatgaaagc cttacgtcat 480
cagcgcaaat tcaaacagac ggacatcccg ttagaaatca ttcagcatca ggaacctaaa 540
atcgatgctg ctacgggcgc cccagcctct tggccggcta atccagacct ggaatggtgt 600
cctccgggtc atggtgacct gtttagtacc ctgtgggaaa gcggcttact ggatacctta 660
ctggaacatg gctttgaata tctgtttatt agtaatagtg ataatctggg cgctcgtccg 720
agtcgcacat tagcacagta ttttgaagat acaggtgctc cttttatggt ggaagttgct 780
aatcgcacct atgcggatcg taaaggcggc catattgtgc gtgatacggc aacgggtcgc 840
ttaatcttac gcgaaatgtc tcaggtgcat ccggatgata aagatgcggc acaggatatt 900
gccaaacatc cgtattttaa taccaacaac atctgggttc gcatcgatgt gttaagggtt 960
atgctggcag aacatgatgg cgtgctgcca ttaccagtta ttatcaacaa caaaacggtt 1020
gatccgaccg atcctcagag tccagccgtt gtgcagctgg aaacggctat gggcgccgca 1080
atcggtctgt ttgaaggcgc catttgcgtg caggttgata gaatgcgttt tctgccggtt 1140
aaaacgacga atgatctgtt tatcatgcgt agcgatcgct ttcatctgac cgatagctat 1200
gagatggaag atggtaatta tatctttcca aatgtggact tagacccacg ctattacaag 1260
aatatcgaag attttaacga acgctttccg tataatgttc ctagcttagc agccgccaat 1320
tcagtgagca tcaaaggcga ttggaccttt ggtcgcgatg tgattatgtt tgccgatgcc 1380
cgcttagaag atcgcaacga gccgagctat gttcctaatg gcgaatatgt tggtcctatg 1440
ggtatcgaac cgggcgattg ggtttaa 1467
Claims (6)
1. A method for improving the yield of isoorientin produced by recombinant bacteria by using a cooperative regulation strategy is characterized in that a gene sequence is shown as SEQ ID NO. 1Gentiana trifloraThe gene sequence of the Gt6CGT gene is shown in SEQ ID NO 2Saccharophagus degradans2-40 derived Cellobiose phosphorinase gene, and gene sequence shown as SEQ ID NO. 3Bifidobacterium bifidumThe UTP-glucose-1-phosphate uridylyl transferase gene from the source is co-transferred into escherichia coli to construct recombinant bacteria, and luteolin is used as a substrate to be converted into isoorientin.
2. The method for improving the yield of isoorientin produced by recombinant bacteria by utilizing the cooperative regulation and control strategy according to claim 1, characterized in that the method comprises the step ofSaccharophagus degradans2-40 derived Cellobiose phosphorinase gene andBifidobacterium bifidumintroducing a source UTP-glucose-1-phosphate uridyltransferase gene into a recombinant bacterium carrying a carbon glycoside glycosyltransferase Gt6CGT gene; on the basis of a TB-Gly culture medium, inducing the recombinant bacteria to express the carbon glycoside glycosyltransferase Gt6CGT for 6h at the temperature of 20 ℃ by using 0.1mM IPTG, and then adding a substrate luteolin for conversion; the culture medium consists of 12g/L peptone, 24g/L yeast powder, 2.32g/L potassium dihydrogen phosphate, 12.54g/L dipotassium hydrogen phosphate and 10g/L glycerol.
3. The method for improving the yield of isoorientin produced by recombinant bacteria by utilizing the cooperative control strategy according to claim 2, wherein the recombinant plasmid containing the Cellobiose phosphoriylase gene sequence and the UTP-glucose-1-phosphate uridyltransferase gene sequence is pACYCDuet-Cep-UgpA.
4. The method for improving the yield of isoorientin produced by recombinant bacteria by utilizing a synergistic regulation and control strategy according to claim 3, wherein the recombinant bacteria containing the recombinant plasmid according to claim 3 is BL 21-CGT-III.
5. The method for improving the yield of isoorientin produced by recombinant bacteria by utilizing a synergistic regulation strategy according to claim 4, wherein the recombinant bacteria BL21-CGT-III are cultured overnight in LB culture medium containing 50 ug/mL streptomycin and 35 ug/mL chloramphenicol, transferred into TB-Gly culture medium added with 50 ug/mL streptomycin and 35 ug/mL chloramphenicol, and subjected to shaking culture at 37 ℃ and 200rpm until OD is reached600When the concentration is 0.8, adding IPTG inducer with the final concentration of 0.1mM, and carrying out induced culture at 20 ℃ for 6 h; subsequently, 900mg/L of luteolin was added, and after 12 hours, 20g/L of cellobiose was added, followed by transformation and culture for 116 hours.
6. The method for improving the yield of isoorientin produced by recombinant bacteria by utilizing the synergistic regulation strategy according to claim 5, characterized in that the transformed culture is centrifuged at 12000g for 20 minutes, a supernatant is taken to collect a sample, the obtained supernatant is directly put on a column, the filler of the chromatographic column is AB-8 macroporous resin, the diameter of the chromatographic column is 5cm, the length of the column is 30cm, the chromatographic column is balanced with deionized water, after the sample is loaded, 3wt.% ethanol with 15 times volume is used for washing the chromatographic column, then 30 wt.% ethanol is used for elution, and the obtained sample is dried in vacuum to obtain an isoorientin refined product.
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