CN105176905B - Synthesize Recombinant organism strain and the construction method of dammarendiol - Google Patents
Synthesize Recombinant organism strain and the construction method of dammarendiol Download PDFInfo
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Abstract
Recombinant organism strain and construction method the invention discloses synthesis dammarendiol, 1 gene of cytochromes NADPH reductases and dammarendiol synthase gene in the arabidopsis that the Recombinant organism strain for synthesizing dammarendiol contains the squalene synthetase gene for truncating 26 amino acid residues of C-terminal, 2,3 oxidosqualene synthase genes, truncation 45 amino acid residues of C-terminal.The present invention successfully constructs the Recombinant organism strain of synthesis dammarendiol.Compared with other host strains, the approach regulation and control of Escherichia coli synthesis dammarendiol are simple, are easy to carry out metabolic engineering, lay a good foundation for the Escherichia coli fermentation production of dammarane saponins.
Description
Technical field
The invention belongs to biological technical field, a kind of specific colibacillus engineering for synthesizing dammarendiol and structure side
Method.
Background technology
Ginseng, in disease treatment and in healthcare, always is research and using hot spot as a kind of rare traditional Chinese medicine.
Ginsenoside is a kind of triterpene compound, is the effective active composition in ginseng, can be divided into according to the difference of triterpenoid sapogenin
Dammarane saponins, oleanane glycoside.Wherein dammarane type ginsenoside such as Rb1, Rg3, Rh2, CK and sapogenin are former
Panoxadiol, has protection angiocarpy, antifatigue, anti-aging, anticancer, liver protection and the pharmacological action of strengthen immunity.Ginseng soap
The traditional mode of production main raw material(s) of glycosides is ginseng.Up to the present, China wild ginseng resource is endangered, ginseng planting
Easily influenced by pest and disease damage, Ginseng Products face the problem of high agriculture is residual, seriously affect the quality of China's ginseng class Related product.
In recent years, with the gradual parsing of ginsenoside route of synthesis, squalene synthetase, 2,3- oxidosqualenes synzyme,
A series of ginsenoside route of synthesis key genes such as dammarendiol synzyme, protopanoxadiol synzyme, glycosyl transferase
Successful excavation, from the beginning produce protopanoxadiol using microorganism, the report of Rh2, CK and Rg3 gradually appear.But these are reported
Road, as chassis cell, is engineered since the ergosterol synthesis pathway of yeast entogenous regulates and controls complexity using yeast
Bring difficulty.
Escherichia coli are the pattern microorganisms of microbe research and application, and fermentation operation and genetic manipulation are ripe.At present, greatly
Enterobacteria is successfully used for the biosynthesis of the prodrugs such as carotenoid, taxol, Arteannuic acid, but without biosynthesis
The report of dammarendiol.
Dammarendiol is the key precursor compound of dammarane type ginsenoside.
The content of the invention
The purpose of the present invention is overcome deficiency of the prior art, there is provided a kind of Escherichia coli base for synthesizing dammarendiol
Because of engineered strain.
Second object of the present invention is to provide a kind of structure for the Recombinant organism strain for synthesizing dammarendiol
Construction method.
Third object of the present invention is to provide the Recombinant organism strain of second of synthesis dammarendiol.
Fourth object of the present invention is to provide the Recombinant organism strain of second of synthesis dammarendiol
Construction method.
The 5th purpose of the present invention is to provide the Recombinant organism strain of three kinds of synthesis dammarendiols.
The 6th purpose of the present invention is to provide the structure of the Recombinant organism strain of three kinds of synthesis dammarendiols
Construction method.
The 7th purpose of the present invention is to provide the Recombinant organism strain of four kinds of synthesis dammarendiols.
The 8th purpose of the present invention is to provide the structure of the Recombinant organism strain of four kinds of synthesis dammarendiols
Construction method.
Technical scheme is summarized as follows：
It is a kind of synthesize dammarendiol Recombinant organism strain (bacterial strain 1), it is described it contain truncate C-terminal 26
The squalene synthetase gene of amino acid residue, 2,3- oxidosqualenes synthase gene, the plan south for truncating 45 amino acid residues of C-terminal
1 gene of cytochromes-NADPH- reductases and dammarendiol synthase gene in mustard.
The construction method of the Recombinant organism strain of dammarendiol is synthesized, is included the following steps：
(1) the squalene synthetase gene insertion Escherichia coli (Escherichia of 26 amino acid residues of C-terminal will be truncated
Coli) XbaI the and BamHI restriction enzyme sites of expression plasmid pET28a (+) obtain plasmid 1；By 2,3- oxidosqualene synthase genes
BamHI the and SacI restriction enzyme sites of plasmid 1 are inserted into, obtain plasmid 2；By in the arabidopsis for truncating 45 amino acid residues of C-terminal
1 gene of cytochromes-NADPH- reductases is inserted into HindIII the and XhoI restriction enzyme sites of plasmid 2, obtains plasmid 3；Agate will be reached
Enediol synthase gene is inserted into SacI the and HindIII sites of plasmid 3, obtains plasmid 5；
(2) plasmid 5 is converted into Escherichia coli, obtains the Recombinant organism strain of synthesis dammarendiol,
It is named as bacterial strain 1；
The sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with shown in SEQ ID NO.1；It is described
The sequence of 2,3- oxidosqualene synthase genes is with shown in SEQ ID NO.2；The plan south for truncating 45 amino acid residues of C-terminal
1 gene order of cytochromes-NADPH- reductases is with shown in SEQ ID NO.3 in mustard；The dammarendiol synthase gene
Sequence is with shown in SEQ ID NO.5.
It is a kind of synthesize dammarendiol Recombinant organism strain (bacterial strain 3), it is described it contain truncate C-terminal 26
The squalene synthetase gene of amino acid residue, 2,3- oxidosqualenes synthase gene, the plan south for truncating 45 amino acid residues of C-terminal
1 gene of cytochromes-NADPH- reductases in mustard, dammarendiol synthase gene, farnesyl pyrophosphate synthase gene and
Isopentenylpyrophosphate isomerase gene.
The construction method of the Recombinant organism strain of dammarendiol is synthesized, is included the following steps：
(1) the squalene synthetase gene insertion colibacillus expression plasmid pET28a of 26 amino acid residues of C-terminal will be truncated
XbaI the and BamHI restriction enzyme sites of (+) obtain plasmid 1；2,3- oxidosqualene synthase genes are inserted into the BamHI of plasmid 1
With SacI restriction enzyme sites, plasmid 2 is obtained；Cytochromes-NADPH- in the arabidopsis for truncating 45 amino acid residues of C-terminal is reduced
1 gene of enzyme is inserted into HindIII the and XhoI restriction enzyme sites of plasmid 2, obtains plasmid 3；Dammarendiol synthase gene is inserted
Enter SacI the and HindIII sites to plasmid 3, obtain plasmid 5；
(2) plasmid 5 is converted into Escherichia coli, obtains the Recombinant organism strain 1 of synthesis dammarendiol；
The sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with shown in SEQ ID NO.1；It is described
The sequence of 2,3- oxidosqualene synthase genes is with shown in SEQ ID NO.2；The plan south for truncating 45 amino acid residues of C-terminal
1 gene order of cytochromes-NADPH- reductases is with shown in SEQ ID NO.3 in mustard；The dammarendiol synthase gene
Sequence is with shown in SEQ ID NO.5.
(3) farnesyl pyrophosphate synthase gene is inserted into BamhI the and SacI restriction enzyme sites of plasmid pCDFDuet1,
Obtain plasmid 7；Isopentenylpyrophosphate isomerase gene is inserted into SacI the and HindIII restriction enzyme sites of plasmid 7, obtains matter
Grain 8；Plasmid 8 is transformed into the Recombinant organism strain 1 of synthesis dammarendiol, obtains synthesis dammarendiol
Recombinant organism strain, is named as bacterial strain 3；
The sequence of the farnesyl pyrophosphate synthase gene is with shown in SEQ ID NO.6；The isopentenylpyrophosphate is different
The sequence of structure enzyme gene is with shown in SEQ ID NO.7.
A kind of Recombinant organism strain (2) for synthesizing dammarendiol, it is residual that it contains truncation 26 amino acid of C-terminal
The squalene synthetase gene of base, 2,3- oxidosqualenes synthase gene, truncate it is thin in the saccharomyces cerevisiaes of 33 amino acid residues of C-terminal
Born of the same parents' pigment-NADPH- reductase genes and dammarendiol synthase gene.
The construction method of the Recombinant organism strain of dammarendiol is synthesized, is included the following steps：
(1) the squalene synthetase gene insertion colibacillus expression plasmid pET28a of 26 amino acid residues of C-terminal will be truncated
XbaI the and BamHI restriction enzyme sites of (+) obtain plasmid 1；2,3- oxidosqualene synthase genes are inserted into the BamHI of plasmid 1
With SacI restriction enzyme sites, plasmid 2 is obtained；By cytochromes-NADPH- in the saccharomyces cerevisiae for truncating 33 amino acid residues of C-terminal also
Nitroreductase gene is inserted into HindIII the and XhoI restriction enzyme sites of plasmid 2, obtains plasmid 4；Dammarendiol synthase gene is inserted
Enter SacI the and HindIII sites to plasmid 4, obtain plasmid 6；
(2) plasmid 6 is converted into Escherichia coli, obtains the Recombinant organism strain of synthesis dammarendiol,
It is named as bacterial strain 2；
The sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with shown in SEQ ID NO.1；It is described
The sequence of 2,3- oxidosqualene synthase genes is with shown in SEQ ID NO.2；The wine brewing for truncating 33 amino acid residues of C-terminal
Cytochromes-NADPH- reductase genes sequence is with shown in SEQ ID NO.4 in yeast；The dammarendiol synthase gene
Sequence is with shown in SEQ ID NO.5.
It is a kind of synthesize dammarendiol Recombinant organism strain (4), it is described it contain truncate 26 amino of C-terminal
Squalene synthetase gene, 2,3- oxidosqualenes synthase gene, the saccharomyces cerevisiae for truncating 33 amino acid residues of C-terminal of sour residue
Middle cytochromes-NADPH- reductase genes, dammarendiol synthase gene, farnesyl pyrophosphate synthase gene and isoamyl
Alkene pyrophosphoric acid isomerase gene.
The construction method of the Recombinant organism strain of dammarendiol is synthesized, is included the following steps：
(1) the squalene synthetase gene insertion colibacillus expression plasmid pET28a of 26 amino acid residues of C-terminal will be truncated
XbaI the and BamHI restriction enzyme sites of (+) obtain plasmid 1；2,3- oxidosqualene synthase genes are inserted into the BamHI of plasmid 1
With SacI restriction enzyme sites, plasmid 2 is obtained；By cytochromes-NADPH- in the saccharomyces cerevisiae for truncating 33 amino acid residues of C-terminal also
Nitroreductase gene is inserted into HindIII the and XhoI restriction enzyme sites of plasmid 2, obtains plasmid 4；Dammarendiol synthase gene is inserted
Enter SacI the and HindIII sites to plasmid 3, obtain plasmid 6；
(2) plasmid 6 is converted into Escherichia coli, obtains the Recombinant organism strain 2 of synthesis dammarendiol；
The sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with shown in SEQ ID NO.1；It is described
The sequence of 2,3- oxidosqualene synthase genes is with shown in SEQ ID NO.2；The wine brewing for truncating 33 amino acid residues of C-terminal
Cytochromes-NADPH- reductase genes sequence is with shown in SEQ ID NO.4 in yeast；The dammarendiol synthase gene
Sequence is with shown in SEQ ID NO.5.
(3) farnesyl pyrophosphate synthase gene is inserted into BamhI the and SacI restriction enzyme sites of plasmid pCDFDuet1,
Obtain plasmid 7；Isopentenylpyrophosphate isomerase gene is inserted into SacI the and HindIII restriction enzyme sites of plasmid 7, obtains matter
Grain 8；Plasmid 8 is transformed into the Recombinant organism strain 2 of synthesis dammarendiol, obtains synthesis dammarendiol
Recombinant organism strain, is named as bacterial strain 4；
The sequence of the farnesyl pyrophosphate synthase gene is with shown in SEQ ID NO.6；The isopentenylpyrophosphate is different
The sequence of structure enzyme gene is with shown in SEQ ID NO.7.
The present invention successfully constructs the Recombinant organism strain of synthesis dammarendiol.With other host strain phases
Than the approach regulation and control of Escherichia coli synthesis dammarendiol are simple, are easy to carry out metabolic engineering, are dammarane saponins
Escherichia coli fermentation production is laid a good foundation.
Brief description of the drawings
The genetic map for the plasmid that Fig. 1 present invention uses.
A:The squalene synthetase gene insertion colibacillus expression plasmid pET28a of 26 amino acid residues of C-terminal will be truncated
XbaI the and BamHI restriction enzyme sites of (+) obtain plasmid 1.
B:2,3- oxidosqualene synthase genes are inserted into BamHI the and SacI restriction enzyme sites of plasmid 1, obtain plasmid 2.
C:1 gene order of cytochromes-NADPH- reductases in the arabidopsis for truncating 45 amino acid residues of C-terminal is inserted into
To HindIII the and XhoI restriction enzyme sites of plasmid 2, plasmid 3 is obtained.
D:Cytochromes-NADPH- reductase gene sequences are inserted in the saccharomyces cerevisiae that 33 amino acid residues of C-terminal will be truncated
Enter HindIII the and XhoI restriction enzyme sites to plasmid 2, obtain plasmid 4.
E:Dammarendiol synthase gene is inserted into SacI the and HindIII sites of plasmid 3, obtains plasmid 5.
F:Dammarendiol synthase gene is inserted into SacI the and HindIII sites of plasmid 4, obtains plasmid 6.
G：Farnesyl pyrophosphate synthase gene is inserted into BamhI the and SacI restriction enzyme sites of plasmid pCDFDuet1, is obtained
To plasmid 7.
H：Isopentenylpyrophosphate isomerase gene is inserted into SacI the and HindIII restriction enzyme sites of plasmid 7, obtains matter
Grain 8.
Fig. 2 Escherichia coli intracellular metabolite concentrations HPLC analyzes collection of illustrative plates.
A:Import 5 extractive from fermentative HPLC of the control strain recombinant bacterium analyses of pET28a (+) empty plasmid.
B：1 extractive from fermentative HPLC of bacterial strain is analyzed.
C：2 extractive from fermentative HPLC of bacterial strain is analyzed.
D：Dammarendiol standard items HPLC is analyzed.
E：Dammarendiol standard quality spectrogram.
F：Bacterial strain 1 synthesizes dammarendiol mass spectrogram.
G：Bacterial strain 2 synthesizes dammarendiol mass spectrogram.
Fig. 3 build bacterial strain dammarendiol content analysis.
Embodiment
Below by specific embodiment, the present invention is further illustrated.
Experimental method used in example below is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
Explanation：
1. the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is referred to as：ScERG9.
2. 2,3- oxidosqualene synthase genes are referred to as：McSE.
3. truncate 1 gene of cytochromes-NADPH- reductases in the arabidopsis of 45 amino acid residues of C-terminal to be referred to as：
ATR1。
4. truncate cytochromes-NADPH- reductase genes in the saccharomyces cerevisiae of 33 amino acid residues of C-terminal to be referred to as：
ScCPR。
5. dammarendiol synthase gene is referred to as：DS.
6. farnesyl pyrophosphate synthase gene is referred to as：ispA.
7. isopentenylpyrophosphate isomerase gene is referred to as：idi.
1 dammarendiol of embodiment synthesizes the structure of expression vector
(1) acquisition of related gene
With saccharomyces cerevisiae W303-1a (U.S., ATCC) genome for template, with sequence SEQ ID NO.8 and SEQ ID
NO.9 is primer, and amplification obtains truncating the squalene synthetase gene ScERG9 (SEQ ID NO.1) of 26 amino acid residues of C-terminal.
According to the amino acid sequence of 2,3- oxidosqualenes synzyme in Merhylococcus capsulatus, carry out for the close of Escherichia coli
Numeral optimizes, and synthesizes 2,3- oxidosqualenes synthase gene McSE, (SEQ ID by Jin Sirui bio tech ltd
NO.2)。
According to the amino acid sequence of cytochromes-NADPH- reductases 1 in the arabidopsis for truncating 45 amino acid residues of C-terminal
Row, carry out the codon optimization for Escherichia coli, and are synthesized by Jin Wei intelligence bio tech ltd and truncate 45 ammonia of C-terminal
1 Gene A TR1 of cytochromes-NADPH- reductases (SEQ ID NO.3) in the arabidopsis of base acid residue.
Using saccharomyces cerevisiae W303-1a genomes as template, using sequence SEQ ID NO.18 and SEQ ID NO.19 as primer,
Amplification obtains cytochromes-NADPH- reductase gene ScCPR (SEQ in the saccharomyces cerevisiae of truncation 33 amino acid residues of C-terminal
ID NO.4)。
According to the amino acid sequence of dammarendiol synzyme in ginseng, the codon optimization for Escherichia coli is carried out,
And dammarendiol synthase gene DS (SEQ ID NO.5) is synthesized by Jin Wei intelligence bio tech ltd.
With e. coli bl21 (DE3) (U.S., ATCC) genome for template, with sequence SEQ ID NO.22 and SEQ ID
NO.23 is primer, and amplification obtains Escherichia coli farnesyl pyrophosphate synthase gene ispA (SEQ ID NO.6).
Using e. coli bl21 (DE3) genome as template, using sequence SEQ ID NO.26 and SEQ ID NO.27 to draw
Thing, amplification obtain Escherichia coli isopentenylpyrophosphate isomerase gene idi (SEQ ID NO.7).
(2) structure of dammarendiol synthesis expression vector
In order
XbaI the and BamHI restriction enzyme sites of ScERG9 insertion colibacillus expression plasmid pET28a (+) are obtained into plasmid 1.
Concretely comprise the following steps：With saccharomyces cerevisiae W303-1a (U.S., ATCC) genome for template, with sequence SEQ ID
NO.10 and SEQ ID NO.11 are primer, and amplification obtains ScERG9.
The genetic fragment is subjected to digestion with plasmid pET28a (+) with XbaI and BamHI restriction endonucleases, is connected, obtains plasmid 1
(Figure 1A).
2,3- oxidosqualene synthase genes are inserted into BamHI the and SacI restriction enzyme sites of plasmid 1, obtain plasmid 2.
Concretely comprise the following steps：With 2,3- oxidosqualenes synthetase gene sequence (SEQ ID NO.2) for template, with sequence SEQ
ID NO.12 and SEQ ID NO.13 are primer amplification McSE genes.By the genetic fragment and BamHI the and SacI inscribes of plasmid 1
Enzyme carries out digestion, connection, obtains plasmid 2 (Figure 1B).
1 gene order of cytochromes-NADPH- reductases is inserted into the arabidopsis that 45 amino acid residues of C-terminal will be truncated
HindIII the and XhoI restriction enzyme sites of plasmid 2, obtain plasmid 3.
Concretely comprise the following steps：With cytochromes-NADPH- reductase 1ATR1 gene orders (SEQ ID NO.3) in arabidopsis
For template, using sequence SEQ ID NO.16 and SEQ ID NO.17 as primer amplification ATR1 genes.By the genetic fragment and plasmid 2
Digestion, connection are carried out with HindIII and XhoI restriction endonucleases, obtains plasmid 3 (Fig. 1 C).
Cytochromes-NADPH- reductase genes sequence in the saccharomyces cerevisiae for truncating 33 amino acid residues of C-terminal is inserted into
To HindIII the and XhoI restriction enzyme sites of plasmid 2, plasmid 4 is obtained.
Concretely comprise the following steps：With cytochromes-NADPH- reductase genes sequence in saccharomyces cerevisiae (SEQ ID NO.4) for mould
Plate, using sequence SEQ ID NO.20 and SEQ ID NO.21 as primer amplification ScCPR genes.The genetic fragment and plasmid 2 are used
HindIII and XhoI restriction endonucleases carry out digestion, connection, obtain plasmid 4 (Fig. 1 D).
Dammarendiol synthase gene is inserted into SacI the and HindIII sites of plasmid 3, obtains plasmid 5.
Concretely comprise the following steps：With dammarendiol synthetase gene sequence (SEQ ID NO.5) for template, with sequence SEQ ID
NO.14 and SEQ ID NO.15 are primer amplification DS genes.By the genetic fragment and SacI the and HindIII restriction endonucleases of plasmid 3
Digestion, connection are carried out, obtains plasmid 5 (Fig. 1 E).
Dammarendiol synthase gene is inserted into SacI the and HindIII sites of plasmid 4, obtains plasmid 6.
Concretely comprise the following steps：With dammarendiol synthetase gene sequence (SEQ ID NO.5) for template, with sequence SEQ ID
NO.14 and SEQ ID NO.15 are primer amplification DS genes.By the genetic fragment and SacI the and HindIII restriction endonucleases of plasmid 4
Digestion, connection are carried out, obtains plasmid 6 (Fig. 1 F).
(3) structure of farnesyl pyrophosphate synthase gene and isopentenylpyrophosphate isomerase gene over-express vector
1) by the BamHI and SacI of farnesyl pyrophosphate synthase gene (SEQ ID NO.6) insertion pCDFDuet1 plasmids
Restriction enzyme site, obtains plasmid 7.
Concretely comprise the following steps：With farnesyl pyrophosphate synthase gene (SEQ ID NO.6) for template, with sequence SEQ ID
NO.24 and SEQ ID NO.25 are primer amplification ispA genes.By the genetic fragment and plasmid pCDFDuet1 with BamHI and
SacI restriction endonucleases carry out digestion, connection, obtain plasmid 7 (Fig. 1 G).
By SacI the and HindIII digestions position of isopentenylpyrophosphate isomerase gene (SEQ ID NO.7) insertion plasmid 7
Point, obtains plasmid 8.Concretely comprise the following steps：With isopentenylpyrophosphate isomerase gene (SEQ ID NO.6) for template, with sequence SEQ
ID NO.28 and SEQ ID NO.29 are primer amplification idi genes.By the genetic fragment and plasmid 7 with SacI and HindIII
Enzyme cutting carries out digestion, connection, obtains plasmid 8 (Fig. 1 H).
Above-mentioned PCR amplification condition：95 DEG C, 10min；95 DEG C, 30S；55 DEG C, 30S, 30 circulations；72 DEG C, 1.5min；72
DEG C, 10min.
The structure of 2 dammarendiol E. coli production strain of embodiment
Plasmid 5 is converted into Escherichia coli (Escherichia coli) BL21 (DE3), obtains synthesis dammarendiol
Recombinant organism strain, be named as bacterial strain 1.
The Escherichia coli method for transformation of plasmid 5 is：Plasmid 5 is added into 50ul e. coli bl21s (DE3) competent cell
In, ice bath 30min, 42 DEG C of thermal shock 90s conversions.1ml LB culture mediums are added, 37 DEG C of rejuvenation 1h, collect thalline and be applied to kanamycins
In resistance screening LB tablets, 37 DEG C of overnight incubations.Bacterium colony is chosen after being incubated overnight and carries out bacterium colony PCR verifications, selects positive transformants
Son, carries out sequence verification, correct positive transformant is sequenced, is named as bacterial strain 1.
Embodiment 3 synthesizes the structure of the Recombinant organism strain of dammarendiol
Plasmid 6 is converted into Escherichia coli (Escherichia coli) BL21 (DE3), obtains synthesis dammarendiol
Recombinant organism strain, be named as bacterial strain 2.
The Escherichia coli method for transformation of plasmid 6 is the same as embodiment 2.
In order to carry out the control of recombinant bacterial strain, easy to the comparative analysis of metabolite, constructed according to above-mentioned method for transformation
Escherichia coli containing pET28a (+) empty plasmid, are named as bacterial strain 5.
Embodiment 4 synthesizes the structure of the Recombinant organism strain of dammarendiol
Plasmid 7 is converted into bacterial strain 1, obtains bacterial strain 3；
The conversion of plasmid 7 is into the method for bacterial strain 1：Plasmid 7 is added in the competent cell of 50ul bacterial strains 1, ice bath
30min, 42 DEG C of thermal shock 90s conversions.1ml LB culture mediums are added, 37 DEG C of rejuvenation 1h, collect thalline and be applied to kanamycins and strepto-
In plain resistance screening LB tablets, 37 DEG C of overnight incubations.Bacterium colony is chosen after being incubated overnight and carries out bacterium colony PCR verifications, selects positive turn
Beggar, carries out sequence verification, correct positive transformant is sequenced, is named as bacterial strain 3.
Embodiment 5 synthesizes the structure of the Recombinant organism strain of dammarendiol
Plasmid 8 is converted into bacterial strain 2, obtains bacterial strain 4；
The conversion of plasmid 8 is into the method for bacterial strain 2：Plasmid 8 is added in the competent cell of 50ul bacterial strains 2, ice bath
30min, 42 DEG C of thermal shock 90s conversions.1ml LB culture mediums are added, 37 DEG C of rejuvenation 1h, collect thalline and be applied to kanamycins and strepto-
In plain resistance screening LB tablets, 37 DEG C of overnight incubations.Bacterium colony is chosen after being incubated overnight and carries out bacterium colony PCR verifications, selects positive turn
Beggar, carries out sequence verification, correct positive transformant is sequenced, is named as bacterial strain 4.
The structure of blank control bacterial strain
In order to carry out the control of recombinant bacterial strain, easy to the comparative analysis of metabolite, constructed according to above-mentioned method for transformation
E. coli recombinant stain 5 containing pET28a (+) empty plasmid.
PET28a (+) conversion is into the method for bacterial strain 1：PET28a (+) is added into 50ul e. coli bl21s (DE3)
In competent cell, ice bath 30min, 42 DEG C of thermal shock 90s conversions.1ml LB culture mediums are added, 37 DEG C of rejuvenation 1h, collect thalline and apply
Screened in kalamycin resistance in LB tablets, 37 DEG C of overnight incubations.Bacterium colony is chosen after being incubated overnight and carries out bacterium colony PCR verifications, is chosen
Positive transformant is selected, is named as bacterial strain 5.
Fermentation process and the metabolin extraction of 6 recombinant bacterial strain of embodiment and measure
(1) fermentation process of recombinant bacterium is extracted with metabolin
The go bail for recombinant bacterial strain 1 deposited, bacterial strain 2 and bacterial strain 5 is transferred in liquid LB test tubes of the 4mL containing kanamycins, 37 DEG C,
After 200rpm is incubated overnight, OD600 is measured, it is 0.05 that bacteria concentration is diluted to OD600, and 30mL is inoculated in by 3% inoculum concentration
In Shake flask medium (containing kanamycins), 37 DEG C, 200rpm cultivate to OD600 be 0.6-0.8 when, the IPTG for adding 0.2mM is lured
The expression of gene is led, after cultivating 24h, takes 20mL zymotic fluids to centrifuge at twice respectively, removes supernatant, 2mL acetone is added, is used on ice
Ultrasonic cell disintegration instrument crushes thalline, extracts product.
The go bail for recombinant bacterial strain 3 deposited, bacterial strain 4 is transferred in 4mL containing in the liquid LB test tubes of kanamycins and streptomysin, and 37
DEG C, after 200rpm is incubated overnight, OD600 is measured, it is 0.05 that bacteria concentration is diluted to OD600, is inoculated in by 3% inoculum concentration
In 30mL Shake flask mediums (containing kanamycins and streptomysin), 37 DEG C, 200rpm cultivate to OD600 be 0.6-0.8 when, addition
The expression of the IPTG induced genes of 0.2mM, after cultivating 24h, takes 20mL zymotic fluids to centrifuge at twice respectively, removes supernatant, adds 2mL
Acetone, crushes thalline with ultrasonic cell disintegration instrument on ice, extracts product.
(2) the HPLC measure and GC-MS measure of metabolite
HPLC is measured
Liquid phase chromatogram condition：20 μ L of sample size, chromatographic column for Dalian according to the C18 columns of Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S., specification is 5 μ, 250mm*
4.6mm, mobile phase are methanol：Acetonitrile=4:6, flow velocity：1mL/min, ultraviolet detection wavelength are 203nm, and column temperature is 30 DEG C.Standard
Curve quantitative analysis.
GC-MS is measured
Sampling volume 1uL, solvent delay 12min, chromatographic column：HP-5ms(30m×0.25×0.5um)；Chromatographic condition：80
DEG C, 1min；20 DEG C of min-1 to 300 DEG C of insulation 18min；MS scans ion range:100-500Da.
(3) measurement result
A：Dammarendiol (Fig. 2-A) is not detected by E. coli recombinant stain 5 containing pET28a (+) empty plasmid.
B：Measured in bacterial strain 1, bacterial strain 2 dammarendiol (Fig. 2-B, Fig. 2-C, Fig. 2-D, Fig. 2-E, Fig. 2-F, Fig. 2-
G)。
C：The content of dammarendiol is respectively in recombinant bacterial strain 1, recombinant bacterial strain 2, recombinant bacterial strain 3 and recombinant bacterial strain 4：
1.64mg/L, 0.97mg/L, 8.63mg/L and 6.47mg/L (Fig. 3).
Claims (8)
1. it is a kind of synthesize dammarendiol Recombinant organism strain, it is characterized in that it is described it contain truncate C-terminal 26
The squalene synthetase gene of amino acid residue, 2,3- oxidosqualenes synthase gene, the plan south for truncating 45 amino acid residues of C-terminal
1 gene of cytochromes-NADPH- reductases and dammarendiol synthase gene in mustard；Described truncation 26 amino acid of C-terminal are residual
The sequence of the squalene synthetase gene of base is with shown in SEQ ID NO.1；The sequence of the 2,3- oxidosqualenes synthase gene with
Shown in SEQ ID NO.2；1 gene of cytochromes-NADPH- reductases in the arabidopsis for truncating 45 amino acid residues of C-terminal
Sequence is with shown in SEQ ID NO.3；The dammarendiol synthetase gene sequence is with shown in SEQ ID NO.5.
2. the construction method of the Recombinant organism strain of claim 1 synthesis dammarendiol, it is characterized in that including such as
Lower step：
(1) it will truncate the squalene synthetase gene insertion colibacillus expression plasmid pET28a's (+) of 26 amino acid residues of C-terminal
XbaI and BamHI restriction enzyme sites obtain plasmid 1；2,3- oxidosqualene synthase genes are inserted into the BamHI and SacI of plasmid 1
Restriction enzyme site, obtains plasmid 2；By 1 base of cytochromes-NADPH- reductases in the arabidopsis for truncating 45 amino acid residues of C-terminal
Because being inserted into HindIII the and XhoI restriction enzyme sites of plasmid 2, plasmid 3 is obtained；Dammarendiol synthase gene is inserted into matter
SacI the and HindIII sites of grain 3, obtain plasmid 5；
(2) plasmid 5 is converted into Escherichia coli, obtains the Recombinant organism strain of synthesis dammarendiol, name
For bacterial strain 1；
The sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with shown in SEQ ID NO.1；The 2,3-
The sequence of oxidosqualene synthase gene is with shown in SEQ ID NO.2；In the arabidopsis for truncating 45 amino acid residues of C-terminal
1 gene order of cytochromes-NADPH- reductases is with shown in SEQ ID NO.3；The dammarendiol synthetase gene sequence
With shown in SEQ ID NO.5.
3. it is a kind of synthesize dammarendiol Recombinant organism strain, it is characterized in that it is described it contain truncate C-terminal 26
The squalene synthetase gene of amino acid residue, 2,3- oxidosqualenes synthase gene, the plan south for truncating 45 amino acid residues of C-terminal
1 gene of cytochromes-NADPH- reductases in mustard, dammarendiol synthase gene, farnesyl pyrophosphate synthase gene and
Isopentenylpyrophosphate isomerase gene；The sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with SEQ
Shown in ID NO.1；The sequence of the 2,3- oxidosqualenes synthase gene is with shown in SEQ ID NO.2；The truncation C-terminal 45
1 gene order of cytochromes-NADPH- reductases is with shown in SEQ ID NO.3 in the arabidopsis of amino acid residue；It is described to reach agate
Enediol synthetase gene sequence is with shown in SEQ ID NO.5；The sequence of the farnesyl pyrophosphate synthase gene is with SEQ
Shown in ID NO.6；The sequence of the isopentenylpyrophosphate isomerase gene is with shown in SEQ ID NO.7.
4. the construction method of the Recombinant organism strain of claim 3 synthesis dammarendiol, it is characterized in that including such as
Lower step：
(1) it will truncate the squalene synthetase gene insertion colibacillus expression plasmid pET28a's (+) of 26 amino acid residues of C-terminal
XbaI and BamHI restriction enzyme sites obtain plasmid 1；2,3- oxidosqualene synthase genes are inserted into the BamHI and SacI of plasmid 1
Restriction enzyme site, obtains plasmid 2；By 1 base of cytochromes-NADPH- reductases in the arabidopsis for truncating 45 amino acid residues of C-terminal
Because being inserted into HindIII the and XhoI restriction enzyme sites of plasmid 2, plasmid 3 is obtained；Dammarendiol synthase gene is inserted into matter
SacI the and HindIII sites of grain 3, obtain plasmid 5；
(2) plasmid 5 is converted into Escherichia coli, obtains the Recombinant organism strain 1 of synthesis dammarendiol；
The sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with shown in SEQ ID NO.1；The 2,3-
The sequence of oxidosqualene synthase gene is with shown in SEQ ID NO.2；In the arabidopsis for truncating 45 amino acid residues of C-terminal
1 gene order of cytochromes-NADPH- reductases is with shown in SEQ ID NO.3；The dammarendiol synthetase gene sequence
With shown in SEQ ID NO.5；
(3) farnesyl pyrophosphate synthase gene is inserted into BamhI the and SacI restriction enzyme sites of plasmid pCDFDuet1, is obtained
Plasmid 7；Isopentenylpyrophosphate isomerase gene is inserted into SacI the and HindIII restriction enzyme sites of plasmid 7, obtains plasmid 8；
Plasmid 8 is transformed into the Recombinant organism strain 1 of synthesis dammarendiol, obtains the large intestine of synthesis dammarendiol
Oxydans genetic engineering bacterial strain, is named as bacterial strain 3；
The sequence of the farnesyl pyrophosphate synthase gene is with shown in SEQ ID NO.6；The isopentenylpyrophosphate isomerase
The sequence of gene is with shown in SEQ ID NO.7.
5. it is a kind of synthesize dammarendiol Recombinant organism strain, it is characterized in that it is described it contain truncate C-terminal 26
The squalene synthetase gene of amino acid residue, 2,3- oxidosqualenes synthase gene, the wine brewing for truncating 33 amino acid residues of C-terminal
Cytochromes-NADPH- reductase genes and dammarendiol synthase gene in yeast, described truncation 26 amino acid of C-terminal are residual
The sequence of the squalene synthetase gene of base is with shown in SEQ ID NO.1；The sequence of the 2,3- oxidosqualenes synthase gene with
Shown in SEQ ID NO.2；Cytochromes-NADPH- reductase bases in the saccharomyces cerevisiae for truncating 33 amino acid residues of C-terminal
Because sequence is with shown in SEQ ID NO.4；The dammarendiol synthetase gene sequence is with shown in SEQ ID NO.5.
6. the construction method of the Recombinant organism strain of claim 5 synthesis dammarendiol, it is characterized in that including such as
Lower step：
(1) it will truncate the squalene synthetase gene insertion colibacillus expression plasmid pET28a's (+) of 26 amino acid residues of C-terminal
XbaI and BamHI restriction enzyme sites obtain plasmid 1；2,3- oxidosqualene synthase genes are inserted into the BamHI and SacI of plasmid 1
Restriction enzyme site, obtains plasmid 2；By cytochromes-NADPH- reductase bases in the saccharomyces cerevisiae for truncating 33 amino acid residues of C-terminal
Because being inserted into HindIII the and XhoI restriction enzyme sites of plasmid 2, plasmid 4 is obtained；Dammarendiol synthase gene is inserted into matter
SacI the and HindIII sites of grain 4, obtain plasmid 6；
(2) plasmid 6 is converted into Escherichia coli, obtains the Recombinant organism strain of synthesis dammarendiol, name
For bacterial strain 2；
The sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with shown in SEQ ID NO.1；The 2,3-
The sequence of oxidosqualene synthase gene is with shown in SEQ ID NO.2；The saccharomyces cerevisiae for truncating 33 amino acid residues of C-terminal
Middle cytochromes-NADPH- reductase genes sequence is with shown in SEQ ID NO.4；The dammarendiol synthetase gene sequence
With shown in SEQ ID NO.5.
7. it is a kind of synthesize dammarendiol Recombinant organism strain, it is characterized in that it is described it contain truncate C-terminal 26
The squalene synthetase gene of amino acid residue, 2,3- oxidosqualenes synthase gene, the wine brewing for truncating 33 amino acid residues of C-terminal
Cytochromes-NADPH- reductase genes in yeast, dammarendiol synthase gene, farnesyl pyrophosphate synthase gene and
Isopentenylpyrophosphate isomerase gene, the sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with SEQ
Shown in ID NO.1；The sequence of the 2,3- oxidosqualenes synthase gene is with shown in SEQ ID NO.2；The truncation C-terminal 33
Cytochromes-NADPH- reductase genes sequence is with shown in SEQ ID NO.4 in the saccharomyces cerevisiae of amino acid residue；It is described to reach agate
Enediol synthetase gene sequence is with shown in SEQ ID NO.5, the sequence of the farnesyl pyrophosphate synthase gene is with SEQ
Shown in ID NO.6；The sequence of the isopentenylpyrophosphate isomerase gene is with shown in SEQ ID NO.7.
8. the construction method of the Recombinant organism strain of claim 7 synthesis dammarendiol, it is characterized in that including such as
Lower step：
(1) it will truncate the squalene synthetase gene insertion colibacillus expression plasmid pET28a's (+) of 26 amino acid residues of C-terminal
XbaI and BamHI restriction enzyme sites obtain plasmid 1；2,3- oxidosqualene synthase genes are inserted into the BamHI and SacI of plasmid 1
Restriction enzyme site, obtains plasmid 2；By cytochromes-NADPH- reductase bases in the saccharomyces cerevisiae for truncating 33 amino acid residues of C-terminal
Because being inserted into HindIII the and XhoI restriction enzyme sites of plasmid 2, plasmid 4 is obtained；Dammarendiol synthase gene is inserted into matter
SacI the and HindIII sites of grain 3, obtain plasmid 6；
(2) plasmid 6 is converted into Escherichia coli, obtains the Recombinant organism strain 2 of synthesis dammarendiol；
The sequence of the squalene synthetase gene for truncating 26 amino acid residues of C-terminal is with shown in SEQ ID NO.1；The 2,3-
The sequence of oxidosqualene synthase gene is with shown in SEQ ID NO.2；The saccharomyces cerevisiae for truncating 33 amino acid residues of C-terminal
Middle cytochromes-NADPH- reductase genes sequence is with shown in SEQ ID NO.4；The dammarendiol synthetase gene sequence
With shown in SEQ ID NO.5；
(3) farnesyl pyrophosphate synthase gene is inserted into BamhI the and SacI restriction enzyme sites of plasmid pCDFDuet1, is obtained
Plasmid 7；Isopentenylpyrophosphate isomerase gene is inserted into SacI the and HindIII restriction enzyme sites of plasmid 7, obtains plasmid 8；
Plasmid 8 is transformed into the Recombinant organism strain 2 of synthesis dammarendiol, obtains the large intestine of synthesis dammarendiol
Oxydans genetic engineering bacterial strain, is named as bacterial strain 4；
The sequence of the farnesyl pyrophosphate synthase gene is with shown in SEQ ID NO.6；The isopentenylpyrophosphate isomerase
The sequence of gene is with shown in SEQ ID NO.7.
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| CN107828709B (en) * | 2017-11-09 | 2021-06-25 | 天津大学 | Recombinant escherichia coli for heterologous synthesis of ambergris alcohol and construction method thereof |
| CN108949788B (en) * | 2018-07-10 | 2020-07-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Lycopene synthesis related gene and application thereof |
| CN111378681B (en) * | 2018-12-27 | 2023-01-17 | 中国医学科学院药物研究所 | Recombinant bacterium for producing dammarenediol-II glucoside and application thereof |
| CN110438145A (en) * | 2019-07-15 | 2019-11-12 | 天津大学 | The corynebacterium glutamicum of synthesis geraniol and construction method and application |
| CN112322675B (en) * | 2020-11-05 | 2022-07-05 | 苏州大学 | Preparation method of epoxy squalene and engineering bacteria |
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