阅读说明：本技术 一种含有lpxM基因的重组质粒及其制备方法和重组大肠杆菌 (Recombinant plasmid containing lpxM gene, preparation method thereof and recombinant escherichia coli ) 是由 郭美锦 张宁 洪琦 韦炎龙 于 2020-06-04 设计创作，主要内容包括：本发明公开了一种含有lpxM基因的重组质粒pCC1L及其制备方法,所述所述重组质粒pCC1L为在质粒pCC1FOS的基础上连接有lpxM基因。本发明还公开了一种重组大肠杆菌,转化所述重组质粒pCC1L得到的重组大肠杆菌。本发明的重组质粒上连接有lpxM基因,重组质粒可转化至大肠杆菌中,促进大肠杆菌的生长；本发明中的重组大肠杆菌,能够实现大肠杆菌的高生长和番茄红素的高生产。(The invention discloses a recombinant plasmid pCC1L containing lpxM gene and a preparation method thereof, wherein the recombinant plasmid pCC1L is connected with the lpxM gene on the basis of plasmid pCC1 FOS. The invention also discloses a recombinant Escherichia coli obtained by transforming the recombinant plasmid pCC 1L. The recombinant plasmid is connected with an lpxM gene, and can be transformed into escherichia coli to promote the growth of the escherichia coli; the recombinant escherichia coli can realize high growth of the escherichia coli and high production of lycopene.)

1. A recombinant plasmid pCC1L containing lpxM gene, wherein the recombinant plasmid pCC1L is a plasmid pCC1FOS to which lpxM gene is linked.

2. A method for preparing the recombinant plasmid pCC1L according to claim 1, comprising the steps of:

(1) the plasmid pET3b-lpxM was constructed as follows:

amplification of a target gene: using a target fragment containing an lpxM gene as a template and lpxM3 and lpxM5 as primers, and carrying out PCR amplification to obtain a 1.2kb fragment;

LPXM5：AAGAAGGAGATATACATatggaaacgaaaaaaaataatagc；

LPXM3：TTAGCAGCCGGATCCttatttgatgggataaagatcttt；

amplification of plasmid pET3 b: PCR amplification is carried out to obtain a 4.6kb fragment by taking the plasmid pET3b as a template and pET5 and pET3 as primers;

pET5：GGATCCGGCTGCTAACAAA；

pET3：ATGTATATCTCCTTCTTAAAGTTA；

③ in vitro recombination: connecting a target fragment containing the lpxM gene to the plasmid pET3b to obtain a plasmid pET3 b-lpxM;

fourthly, transformation and identification:

transforming the recombined plasmid pET3b-lpxM into a competent DMT-coated AmpR plate; carrying out PCR identification by taking T7/T7T as primers; identifying the fragment containing 1.2kb as positive clone, and extracting plasmid pET3 b-lpxM;

T7：TAATACGACTCACTATAGGG；

T7t：TGCTAGTTATTGCTCAGCGG；

(2) the construction procedure of recombinant plasmid pCC1L is as follows:

PCR is carried out to 1.2kb by taking the plasmid pET3b-lpxM as a template and BI5 and BI3 as primers, an LPXMCC fragment is recovered by electrophoretic gel, and the LPXMCC fragment is recombined with pCC1FOS in vitro to obtain a plasmid pCC 1L;

BI5：GTTTTCCCAGTCACGACAAGGAGATGGCGCCCAA；

BI3：CCATGATTACGCCAAGCCGGATATAGTTCCTCCTTT。

3. a recombinant escherichia coli comprising the recombinant plasmid pCC1L of claim 1 or 2, produced by transforming the recombinant plasmid pCC1L into escherichia coli DH 416.

4. The recombinant E.coli of claim 3, wherein said recombinant E.coli is screened with 25 μ g/mL chloramphenicol (Cm).

5. The recombinant Escherichia coli of claim 3, wherein the fermentation method of the recombinant Escherichia coli comprises the following steps:

1) culturing:

culturing the recombinant escherichia coli transformed with the recombinant plasmid pCC1L, which comprises the following steps: picking a single colony to a large test tube containing 5mL of liquid LB culture medium, and incubating for 10-12h at 37 ℃ and 220 rpm; eliminating the helper plasmid at 42 ℃;

2) fermentation:

picking single colony to a large test tube containing 5mL of liquid LB culture medium, and culturing overnight at 30 ℃ and 220 rpm; the next day, the cells were transferred to a self-induction medium and cultured to OD₆₀₀The culture was carried out at 30 ℃ and 220rpm for 24 hours at a temperature of 0.05.

6. The recombinant Escherichia coli of claim 5, wherein said LB medium comprises: peptone, yeast powder and NaCl.

7. The recombinant Escherichia coli of claim 6, wherein said LB medium comprises: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl.

8. The recombinant E.coli of claim 5, wherein said self-induction medium comprises: peptone, yeast powder, NaH₂PO₄·2H₂O、K₂HPO₄·3H₂O, NaCl, Tween 80, glycerol, MgSO₄Glucose, L-arabinose; wherein, the MgSO₄Preparing mother liquor with the glucose.

9. The recombinant E.coli of claim 8, wherein said self-induction medium comprises: 15g/L peptone, 12g/L yeast powder and 3g/L NaH₂PO₄·2H₂O, 7g/L K₂HPO₄·3H₂O, 2.5g/L NaCl, 5g/L Tween 80, 10g/L glycerol and 50g/L MgSO₄200g/L glucose, 200 g/L-arabinose; wherein, the MgSO₄Preparing mother liquor with the glucose.

Technical Field

The invention relates to the technical field of gene recombination, in particular to a recombinant plasmid containing lpxM gene, a preparation method thereof and recombinant escherichia coli obtained by using the recombinant plasmid.

Background

Tomato redHormone (Lycopenene, C)₄₀H₅₆) The red pigment is a deep red natural pigment, is a C40 terpenoid, has the relative molecular mass of 536.9, belongs to carotenoid, has the functions of improving immunity, resisting aging, protecting vision, preventing cardiovascular diseases and cancers and the like, is one of the well-marketed health care products at present, and is mostly present in blood plasma, liver, adrenal gland, lung, prostate and skin. Lycopene is a carotenoid with high economic value, and is widely applied in the industries of feed, food, health products, cosmetics, medicines and the like.

At present, lycopene is produced industrially mainly through three ways of plant extraction method, chemical synthesis method and microbial synthesis method. The plant extraction method is the most common method in China at present, and the method takes tomato fruits or residues as raw materials and utilizes organic reagents to extract and obtain the lycopene. However, the lycopene content in the fruit is typically less than 1% of the total carbon elements, and therefore the cost of the process is high. In addition, the use of plants as raw materials takes up a lot of land and is climate limited. Although the chemical synthesis method can reduce the production cost of the lycopene, the process is mature, but the method has a plurality of synthesized byproducts, and chemical reaction substances remained in the lycopene product have unclear structures and cannot be removed, so that the lycopene product prepared by the method is not suitable for long-term use as food, health care products and the like. The microbial synthesis method is to produce lycopene by using microorganisms, is not limited by the outside and has great development space. Meanwhile, the microorganism is used as a renewable raw material, and is also advantageous in the aspect of environmental resource protection. Coli, one of the most thoroughly studied species, has become a common host for producing lycopene by virtue of the advantages of clear genetic background, high growth speed and simple and mature genetic engineering technology.

Therefore, it is highly desirable to provide a recombinant escherichia coli capable of achieving high growth of escherichia coli and high production of lycopene to solve the above problems.

Disclosure of Invention

The invention aims to provide a recombinant plasmid pCC1L containing lpxM gene, a preparation method thereof and recombinant escherichia coli, which can promote the high growth of the escherichia coli on the basis of high lycopene production and further improve the lycopene yield.

In order to achieve the purpose, the invention adopts the following technical scheme.

The invention provides a recombinant plasmid pCC1L containing an lpxM gene, wherein the recombinant plasmid pCC1L is connected with the lpxM gene on the basis of a plasmid pCC1 FOS.

The invention also provides a recombinant plasmid pCC1L (Cm)^R) The preparation method comprises the following steps:

(1) the plasmid pET3b-lpxM was constructed as follows:

amplification of a target gene: using a target fragment containing an lpxM gene as a template and lpxM3 and lpxM5 as primers, and carrying out PCR amplification to obtain a 1.2kb fragment;

LPXM5：AAGAAGGAGATATACATatggaaacgaaaaaaaataatagc；

LPXM3：TTAGCAGCCGGATCCttatttgatgggataaagatcttt；

amplification of plasmid pET3 b: PCR amplification is carried out to obtain a 4.6kb fragment by taking the plasmid pET3b as a template and pET5 and pET3 as primers;

pET5：GGATCCGGCTGCTAACAAA；

pET3：ATGTATATCTCCTTCTTAAAGTTA；

③ in vitro recombination: connecting a target fragment containing the lpxM gene to the plasmid pET3b to obtain a plasmid pET3 b-lpxM;

fourthly, transformation and identification:

transforming the recombined plasmid pET3b-lpxM into a competent DMT-coated AmpR plate; carrying out PCR identification by taking T7/T7T as primers; identifying the fragment containing 1.2kb as positive clone, and extracting plasmid pET3 b-lpxM;

T7：TAATACGACTCACTATAGGG；

T7t：TGCTAGTTATTGCTCAGCGG；

(2) the construction procedure of recombinant plasmid pCC1L is as follows:

the plasmid pET3b-lpxM is used as a template, BI5 and BI3 are used as primers, PCR is about 1.2kb, an electrophoretic gel recovers LPXMCC fragments, and the LPXMCC fragments are recombined with pCC1FOS in vitro to obtain a plasmid pCC1L (Cm)^R)；

BI5：GTTTTCCCAGTCACGACAAGGAGATGGCGCCCAA；

BI3：CCATGATTACGCCAAGCCGGATATAGTTCCTCCTTT。

The invention also provides a recombinant Escherichia coli containing the recombinant plasmid pCC1L, which is prepared by mixing the recombinant plasmid pCC1L (Cm)^R) Transformed into Escherichia coli DH 416.

Further, the recombinant E.coli was screened with 25. mu.g/mL chloramphenicol (Cm).

Further, the fermentation method of the recombinant escherichia coli comprises the following steps:

1) culturing:

culturing the recombinant escherichia coli transformed with the recombinant plasmid pCC1L, which comprises the following steps: picking a single colony to a large test tube containing 5mL of liquid LB culture medium, and incubating for 10-12h at 37 ℃ and 220 rpm; eliminating the helper plasmid at 42 ℃;

2) fermentation:

picking single colony to a large test tube containing 5mL of liquid LB culture medium, and culturing overnight at 30 ℃ and 220 rpm; the next day, the cells were transferred to a self-induction medium (FMGAT medium) and cultured to OD fermentation initiation₆₀₀The culture was carried out at 30 ℃ and 220rpm for 24 hours at a temperature of 0.05.

Further, the LB medium includes: peptone, yeast powder and NaCl.

Further, the LB medium includes: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl.

Further, the self-induction medium comprises: peptone, yeast powder, NaH₂PO₄·2H₂O、K₂HPO₄·3H₂O, NaCl, Tween 80, glycerol, MgSO₄Glucose, L-arabinose; wherein, the MgSO4 and the glucose are separately prepared into mother liquor.

Further, the self-induction medium (FMGAT medium) comprises: 15g/L peptone, 12g/L yeast powder and 3g/L NaH₂PO₄·2H₂O, 7g/L K₂HPO₄·3H₂O, 2.5g/L NaCl, 5g/L Tween 80, 10g/L glycerol and 50g/L MgSO₄200g/L glucose and 200 g/L-arabinose.

Further, in the FMGAT medium, the MgSO₄Preparing mother liquor with the glucose.

In the present invention, the plasmid pCC1FOS is transformed into the E.coli DH416 by a transformation method which is conventional in the art.

In the present invention, the target gene fragment can be obtained by a conventional method.

In the present invention, the plasmid pET3b and the plasmid pCC1FOS were used as commercially available products.

In a specific embodiment of the invention, the plasmid pET3b was purchased from Novagen; the plasmid pCC1FOS was purchased from Epicentre.

The DH416 strain of the present invention has a deletion of the lpxM site.

Lycopene and other terpenoids share common precursors: isopentenyl diphosphate (IPP) and Dimethylallyl Diphosphate (DMAPP). These two substances are obtained in vivo from the Mevalonate (MVA) pathway or the 2-C-methyl-D-erythritol-4-phosphate (methyl-D-erythrol 4-phosphate, MEP) pathway (fig. 9). The MEP pathway is mainly present in the cytoplast of gram-negative bacteria and green algae, and the precursors are Glyceraldehyde 3-phosphate (glycoaldehyde 3-phosphate) and Pyruvate (Pyruvate); the MVA pathway is mainly present in the cytoplasm of most eukaryotes, archaea and gram-positive cocci, the precursor being Acetyl-CoA (Acetyl-CoA). Therefore, by introducing a heterologous MVA approach into Escherichia coli, precursor accumulation is increased, and lycopene yield can be effectively improved.

The invention has the beneficial effects that:

the method successfully connects the lpxM gene to the plasmid to obtain the recombinant plasmid pCC1L, and successfully converts the recombinant plasmid into the escherichia coli to obtain the recombinant escherichia coli, thereby promoting the growth of thalli and further improving the yield of lycopene. The invention can realize the high growth of the escherichia coli and the high production of the lycopene.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a schematic diagram of the recombinant plasmid and transformation according to the present invention.

FIG. 2 is a diagram showing the sizes of the lpxM gene fragment and pET3b backbone in the present invention.

FIG. 3 shows the result of PCR product identification of the lpxM-pET3b recombinant system of the present invention.

FIG. 4 shows the result of PCR product identification of recombinant plasmid pCC1L in the present invention.

Fig. 5 is a graph of a standard lycopene graph according to the present invention.

FIG. 6 is a histogram showing the comparison of cell growth in test example 3 of the present invention.

FIG. 7 is a histogram comparing cell growth in test example 4 of the present invention.

FIG. 8 is a graph showing the comparison of the growth of cells and lycopene production after fermentation of DH411 and DH 416.

FIG. 9 is a schematic diagram of the metabolic pathway of lycopene in the present invention.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. 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.