CN106434506B

CN106434506B - Construction method and application of recombinant bacterium for producing lycopene

Info

Publication number: CN106434506B
Application number: CN201610864886.3A
Authority: CN
Inventors: 张学礼; 李清艳; 唐金磊; 毕昌昊
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2016-09-29
Filing date: 2016-09-29
Publication date: 2019-12-31
Anticipated expiration: 2036-09-29
Also published as: CN106434506A

Abstract

The invention discloses a construction method and application of a recombinant bacterium for producing lycopene. The invention provides a recombinant bacterium which is any one of the following 1) to 3): 1) in order to improve the expression and/or activity of (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate synthetase gene ispG and (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate reductase gene ispH in the escherichia coli for producing lycopene, recombinant bacteria are obtained; 2) regulating and controlling the crt operon expression and/or activity of the recombinant bacteria shown in 1) to obtain recombinant bacteria; 3) regulating and controlling glpD gene expression and/or activity of the recombinant strain shown in 2) to obtain the recombinant strain. Experiments prove that by coordinately expressing the ispG and ispH genes in the recombinant escherichia coli with certain terpenoid synthesis capacity, the terpene compounds synthesis capacity of the recombinant escherichia coli, including beta-carotene, lycopene and other terpene compounds, can be effectively improved.

Description

Construction method and application of recombinant bacterium for producing lycopene

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a recombinant bacterium for producing lycopene.

Background

Terpenoids (terpenoids) are a class of compounds that occur in nature and are composed of isoprene as a building block. Many terpenoids have good pharmacological activity and are the main effective components of Chinese medicinal materials and natural plant medicines. Some terpenoids have been developed as effective drugs for wide clinical use.

Lycopene is a typical terpenoid; is a main component constituting tomato red pigment, and is an excellent antioxidant. Lycopene can prevent the damage of metabolite 'free radical' to human body tissue and organ, and can be used as natural health food or medicine. The health food containing lycopene can prevent senile vision deterioration, aging and cardiovascular diseases, and has certain effect in inhibiting digestive tract cancer, cervical cancer, breast cancer, skin cancer, bladder cancer, etc. Lycopene is nontoxic and harmless, and can be added into food such as ice cream, fruit juice, hard candy, bread, biscuit, cake, etc. like beta-carotene to improve its nutritive value. It has health promoting effect superior to vitamin E and beta-carotene.

Since terpenoids have wide application prospects and huge market demands, the method for efficiently producing terpenoids is always a research hotspot. The current methods for producing terpenoids mainly comprise three methods: chemical synthesis, plant extraction and microbial fermentation. The chemical synthesis method has the advantages of complex process flow, high energy consumption and large pollution; on the other hand, the content of terpenoids in plants is usually very low, and the plant extraction method causes serious damage to wild plant resources; in contrast, the microbial fermentation method is not limited by raw materials, and the production process is green and clean, so that the method has great advantages. As the genome sequence of Escherichia coli (Escherichia coli) is completely published, the genetic background and the metabolic pathway are very clear, and the method has the advantages of simple culture medium requirement, rapid growth and the like, and Escherichia coli is selected as a starting strain for modification and production of terpenoid in many researches. FIG. 1 shows the synthetic route of E.coli to produce terpene compounds after introduction of terpene synthesis genes.

Isopentenyl pyrophosphate (IPP) and Dimethylallyl pyrophosphate (DMAPP) are precursor compounds of all terpenoids, two synthetic pathways are currently known (Lee et al, 2002). One is the mevalonate Pathway (MVA Pathway), which is mainly present in the cytosol or endoplasmic reticulum of fungi and plants. The other is the MEP pathway, which is mainly present in bacteria, green algae and plant plastids. The starting materials for this pathway are glyceraldehyde-3-phosphate and pyruvate, which are catalyzed by a series of enzymes to form a mixture of IPP and DMAPP at a ratio of about 5: 1. IPP is isomerized into DMAPP by isovaleryl diphosphate isomerate (Idi). IPP and DMAPP are basic C5 units of terpenoids, and various terpenoids can be synthesized on the basis of the unit.

The colon bacillus has MEP path, can synthesize precursor IPP and DMAPP of the terpenoid, and can produce the terpenoid by introducing the synthetic gene of the terpenoid into the colon bacillus. However, since E.coli has a poor ability to synthesize IPP and DMAPP, the yield of terpenoids is generally low. Improving the metabolic flow of MEP pathway in escherichia coli and improving the capability of synthesizing IPP and DMAPP becomes the key for improving the yield of terpene compounds synthesized by escherichia coli. Numerous research groups have done a great deal of work in finding rate-limiting steps in the MEP pathway and increasing the expression intensity of key genes in the MEP pathway (Yoonet al, 2007; Ajikumar et al, 2010; Alper et al, 2005 a; Alper et al, 2005 b; Choi et al, 2010; Jin and Stephanopoulos, 2007; Yuan et al, 2006). Research shows that the expression strength of 1-deoxy-xylulose-5-phosphate synthase gene (dxs), 4-cytidine diphosphate-2-C-methyl-D erythritol synthase gene (ispD), 4-cytidine diphosphate-2-C-methyl-D erythritol kinase gene (ispF), 2-C-methyl-D-erythritol-2, 4-cyclic pyrophosphate synthase gene (ispE) and isovaleryl pyrophosphate isomerase gene (idi) in MEP pathway can improve the capability of recombinant escherichia coli to produce beta-carotene. However, increasing the expression intensity of 1-deoxy-xylulose 5-phosphate reductoisomerase gene (dxr), (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate synthase gene (ispG), (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate reductase gene (ispH) in MEP pathway, on the contrary, decreased the ability of recombinant escherichia coli to produce β -carotene (Yuan et al, 2006).

In order to identify the rate-limiting step of MEP pathway, Yuan et al use the homologous recombination method to replace the promoter of each gene of MEP pathway on chromosome with T5 promoter, and found that the yield of beta-carotene is increased by 100%, 40%, 20% and 40% after regulating dxs, idi, ispB and ispDF genes. After the 4 genes are combined and regulated by the T5 promoter, the yield of the beta-carotene is improved by 6.3 times, and reaches 6mg/g dry weight cells (Yuan et al, 2006). After Suh regulates key genes dxs, idi and ispDF of MEP pathway with strong promoter T5, the yield of beta-carotene was increased 4.5 times compared to control (Suh et al, 2012). In addition to the already identified rate limiting step in the MEP pathway, it is possible that other proteins in the MEP pathway are not found to be rate limiting because of low solubility at high expression (Zhou et al, 2012). The Zhao 2013 research finds that when the supply of the precursor is insufficient, the downstream beta-carotene synthesis gene is difficult to complete the high-intensity promoter regulation experiment, and when the supply of the precursor is sufficient, the high-intensity promoter regulation of the downstream gene is easy to realize, so that the strain condition is changed, and the speed-limiting step of producing the beta-carotene by escherichia coli is changed (Zhao et al, 2013).

Disclosure of Invention

An object of the present invention is to provide a recombinant bacterium.

The recombinant bacterium provided by the invention is any one of the following 1) -3):

1) in order to improve the expression and/or activity of (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate synthase gene ispG and (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate reductase gene ispH in the escherichia coli containing the crt operon and producing lycopene, recombinant bacteria are obtained;

2) regulating and controlling the crt operon expression and/or activity of the recombinant bacteria shown in 1) to obtain recombinant bacteria;

3) regulating and controlling glpD gene expression and/or activity of the recombinant strain shown in 2) to obtain the recombinant strain.

In the recombinant bacterium, the improvement of the expression and/or activity of (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate synthetase gene ispG and (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate reductase gene ispH in the lycopene-producing Escherichia coli is realized by the following (1) or (2):

(1) inserting a regulatory element mRSL-4 which is shown by a sequence 23 for starting the expression of the ispG gene in the lycopene-producing escherichia coli into the front of the ispG gene, i.e. ispG, and inserting an mRSL-14 which is shown by a sequence 32 for starting the expression of the ispH gene into the front of the ispH gene in the lycopene-producing escherichia coli, i.e. ispH;

(2) inserting an mRSL regulatory element mRSL-1 which is shown by a sequence 21 for starting the expression of the ispG gene in the lycopene-producing escherichia coli into the front of the ispG gene, i.e. ispG, and inserting an mRSL-14 which is shown by a sequence 32 for starting the expression of the ispH gene into the front of the ispH gene in the lycopene-producing escherichia coli, i.e. ispH;

the expression and/or activity of the crt operon of the recombinant bacteria shown in the regulation 1) is realized by replacing a promoter of the crt operon in the recombinant bacteria shown in the regulation 1) with an inducible promoter;

the expression and/or activity of the glpD gene of the recombinant strain shown in 2) is controlled by replacing the promoter of the glpD gene in the recombinant strain shown in 2) with an artificial control element M1-46.

The insertion positions are all inserted in front of the regulated gene and are close to the initiation codon of the regulated gene.

In the recombinant bacteria, the insertion is carried out by adopting a mode of genome site-specific editing or homologous recombination;

or the genome site-directed editing is specifically ZFN editing, TALEN editing or CRISPR/Cas9 editing;

or the homologous recombination is lambda-red homologous recombination or homologous recombination mediated by sacB gene mediated screening or homologous recombination mediated by integrating plasmid.

In the recombinant bacteria, the inducible promoter is a DNA molecule containing an inducible promoter Trc promoter and lacI;

or the nucleotide sequence of the DNA molecule containing the inducible promoter Trc promoter and lacI is sequence 33;

or the nucleotide sequence of the artificial regulatory element M1-46 is sequence 35;

or the lycopene-producing escherichia coli is obtained by knocking out crtX and crtY genes in an escherichia coli CAR001 strain beta-carotene synthesis gene cluster, constructing a synthetic lycopene strain, replacing original regulatory elements of an alpha-ketoglutarate dehydrogenase gene sucAB, a succinic acid dehydrogenase gene sdhABCD and a transaldolase gene talB with bacteria obtained by an artificial regulatory element M1-46, regulating crt operon, dxs and idi genes by an RBS library respectively, and performing combined regulation; specifically LYC 010.

In the recombinant bacteria, the preservation number of the recombinant bacteria shown in 3) is CGMCC No. 12883.

LYC029 is deposited in China general microbiological culture Collection center (CGMCC, address: No. 3 of Beijing university Hokko-Yang district, Xilu-1. Beichen, institute of microbiology, Chinese academy of sciences, zip code 100101) at 2016.8/19. 12883, and is classified and named as Escherichia coli (Escherichia coli).

The invention also aims to provide a method for constructing the recombinant bacterium, which comprises the following steps:

A-C：

a is the following 1) or 2):

1) inserting a regulatory element mRSL-4 which is shown by a sequence 23 for starting ispG gene expression in front of an ispG gene in the lycopene-producing escherichia coli, inserting an mRSL-14 which is shown by a sequence 32 for starting ispH gene expression in front of an ispH gene in the lycopene-producing escherichia coli, and obtaining a recombinant bacterium;

2) inserting an mRSL regulatory element mRSL-1 which is shown by a sequence 21 for starting the expression of the ispG gene in the lycopene-producing escherichia coli into the front of the ispG gene, i.e. ispG, and inserting an mRSL-14 which is shown by a sequence 32 for starting the expression of the ispH gene in the lycopene-producing escherichia coli into the front of the ispH gene, i.e. ispH, so as to obtain a recombinant bacterium;

B. replacing a promoter of the crt operon in the recombinant strain obtained in the step A) with an inducible promoter to obtain a recombinant strain;

C. replacing the promoter of the glpD gene in the recombinant strain obtained in the step B) with an artificial regulatory element M1-46 to obtain the recombinant strain.

In the method, the insertion is carried out by adopting a mode of genome site-specific editing or homologous recombination;

In the above method, the inducible promoter is a DNA molecule comprising an inducible promoter Trc promoter and lacI;

or the lycopene-producing Escherichia coli is LYC 010.

The application of the recombinant bacterium in the production of lycopene is also within the protection scope of the invention.

It is a third object of the present invention to provide a method for producing lycopene.

The method provided by the invention comprises the following steps: fermenting the recombinant bacteria to obtain the lycopene.

Experiments prove that in recombinant escherichia coli with certain terpenoid synthesis capacity, an MEP pathway intermediate HMBPP is accumulated in cells, and cytotoxicity can be caused; the coordination expression of the ispG and ispH genes can effectively improve the synthetic capacity of terpene compounds of the recombinant escherichia coli, including beta-carotene, lycopene and other terpene compounds.

Drawings

FIG. 1 shows the synthetic route of E.coli to produce terpene compounds after introduction of terpene synthesis genes.

FIG. 2 is a diagram of the regulation and determination of cell mass, relative yield of beta-carotene and the transcription level of the corresponding gene by library construction;

a is the cell amount and the relative yield of beta-carotene of the selected strain after dxr library construction; b is relative expression quantity of dxr of the selected strain after dxr is subjected to library construction; c is the cell mass and the relative yield of beta-carotene of the selected strain after the ispD is built; d is the relative expression quantity of the selected strain ispD after the ispD is used for establishing a library; e is the cell amount and the relative yield of beta-carotene of the selected strain after the ispE is used for building a library; f is the relative expression quantity of the selected strain ispE after the ispE is built into a library. G is the cell amount and the relative yield of beta-carotene of the selected strain after the library is built by ispG; h is the relative expression quantity of the selected strain ispG after the library of the ispG is built; i is cell amount and relative yield of beta-carotene of the selected strain after the construction of the ISpH library; j is the relative expression level of the selected strain with the ISpH after the ISpH is established.

FIG. 3 shows the variation in cell growth and β -carotene production of strains regulated by the combination of ispG and ispH genes. A is OD₆₀₀(ii) a B is the relative yield of beta-carotene.

FIG. 4 shows the variation of lycopene production by a combination of ispG and ispH genes in LYC010 regulated strain.

FIG. 5 shows the variation of lycopene production by strains regulated by the combination of ispG and ispH genes in LYC023 and LYC 029.

FIG. 6 is a diagram of a fermentation process.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Escherichia coli ATCC 8739, disclosed in The publication "Gunsalus IC, Hand DB. (1941). The use of bacteria in The chemical determination of total vitamin C.J. Biol chem.141: 853-;

recombinant strain M1-93 is disclosed in the literature "Lu, J., J.Tang, et al (2012.). Combinatorial modification of galP and glkgene expression for improved alternative glucose utilization. appl Microbiol Biotechnol93(6):2455 @", publicly available from the institute for Biotechnology in the Tianjin industry.

The sequence of the M1-93 artificial regulatory element is shown in a sequence 1.

Lycopene was purchased from Sigma under product catalog number L9879.

2-C-Methyl-D-erythroritol 4-phosphate (MEP) was purchased from Echelon Biosciences under catalog number I-M051.

1-Hydroxy-2-methyl-2-buten-4-yl 4-diphosphate (HMBPP) was purchased from Echelon Biosciences under catalog number I-M055.

1-Deoxy-D-xylulose 5-phosphate (DXP) was purchased from Echelon Biosciences under catalog number I-M050.

4-diphosphocytidol-2-C-methyl-D-erythroritol (CDP-ME) was purchased from Echelon Biosciences under catalog number I-M052.

2-C-Methyl-D-erythrothritol 2,4-cyclophosphate (MECPP) was purchased from Echelon Biosciences under catalog number I-M054.

Isoprenyl pyrophosphate ternary salt solution (IPP) was purchased from Sigma and has catalog number I0503-1 VL.

pXZ-CS plasmids are disclosed in the literature "Tan Z, Zhu X, Chen J, Li Q, Zhang X (2013) Activating phosphoenolpyruvate carboxyylase and phosphoenolpyruvate carboxykinase inhibition for promoting secretion production. appl Environ Microbiol79(16): 4838-4844", publicly available from the institute of biotechnology and technology in the Tianjin industry.

pKD46 plasmid is described in the literature "Datsenko, wanner. one-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products. Proc Natl Acad Sci USA.2000.97 (12): 6640 and 6645; "the public is available from the institute of biotechnology in the Tianjin industry.

The recombinant Escherichia coli LYC010 is obtained by knocking out crtX and crtY genes in a beta-carotene synthetic gene cluster of a recombinant Escherichia coli CAR001 strain to construct a synthetic lycopene strain, replacing original regulating elements of an alpha-ketoglutarate dehydrogenase gene sucAB, a succinic acid dehydrogenase gene sdhABCD and a transaldolase gene talB with bacteria obtained by an artificial regulating element M1-46, regulating crt operon, dxs and idi genes by an RBS library respectively, and performing combined regulation; the specific construction method is disclosed in the invention patent with the invention name of 'a recombinant bacterium for producing lycopene and application thereof' with the publication number of ZL 201410029600.0, and can be obtained by the public from the institute of biotechnology in Tianjin industry. Has been preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Lu No.1 of Beijing university Hokko sunward, North Cheng, and the microbiological research institute of Chinese academy of sciences, zip code 100101) in 2013, 23 months and 9 months, and the preservation number is CGMCC No. 8238.

The recombinant bacterium CAR005 is characterized in that a beta-carotene synthesis gene cluster and a trc regulatory element are introduced into escherichia coli, and then the trc regulatory element of the beta-carotene synthesis gene cluster is replaced by an artificial regulatory element M1-93; replacing the original regulatory element of the 1-deoxy-xylulose-5-phosphate synthase gene dxs with an artificial regulatory element M1-37; replacing the original regulatory element of the isovaleryl pyrophosphate isomerase gene idi with an artificial regulatory element M1-46; and replacing original regulatory elements of the alpha-ketoglutarate dehydrogenase gene sucAB, the succinic dehydrogenase gene sdhABCD and the transaldolase gene talB with artificial regulatory elements M1-46 to obtain the strain, wherein the specific construction method is disclosed in the invention patent with the publication number of CN 103087972A and the invention name of 'recombinant microorganism for producing terpenoid and construction method', and the strain can be obtained by the institute of biotechnology in Tianjin industry.

Coli CAR005 having pKD46 is a bacterium obtained by introducing plasmid pKD46 into e.coli CAR 005.

The preparation method of the salt-free LB comprises the following steps:

50% sucrose solution: weighing 500g of sucrose, dissolving with a small amount of ultrapure water, and sterilizing at 115 deg.C for 20 min.

10% salt-free sucrose LB medium: 5g of yeast extract and 10g of peptone are weighed in 800ml of water and sterilized for 20min at 115 ℃. After sterilization, 200ml of a 50% sucrose solution was added.

6% salt-free sucrose LB medium: 5g of yeast extract, 10g of peptone and 15g of agar powder are weighed, dissolved in 880ml of water, sterilized at 115 ℃ for 20 min. After sterilization 120ml of a 50% sucrose solution were added.

In the examples, the concentrations of chloramphenicol, ampicillin and kanamycin were 34. mu.g/L, 50. mu.g/L and 50. mu.g/L, respectively.

The preparation method of the fermentation medium comprises the following steps:

1.10% glycerol mother liquor: 100g of glycerol is weighed, deionized water is added to 1L, and sterilization is carried out for 20min at 121 ℃.

2. And (3) a microelement mother solution: . Composition of the trace element solution: 1L of the solution contains 10g of FeSO₄·7H₂O、5.25g ZnSO₄·7H₂O、3.0g CuSO₄·5H₂O、0.5g MnSO₄·4H₂O、0.23g。

3. Weighing fermentMother powder: 10g/l, K₂HPO₄：10.5g/l、(NH₄)₂HPO₄6g/l, 15g/l of glycerin, citric acid: 1.84g/l, 10ml of microelement mother liquor in 700ml of water, sterilizing for 20min at 121 ℃. Adding sterilized MgSO₄·7H₂O solution and 100ml of 10% glycerol mother liquor, and finally adding sterile water to 1L

MgSO4 & 7H2O solution MgSO₄·7H₂O: 5g of the extract is dissolved in 20ml of deionized water and sterilized for 20min at the temperature of 121 ℃.

The preparation method of the high-density fermentation medium comprises the following steps:

1. a bottom material culture medium: yeast powder: 20g/l KH₂PO₄：10.5g/l、(NH₄)₂HPO₄6g/l, 30g/l of glycerin, citric acid: 1.84g/l, MgSO₄·7H₂10g/L and 15ml/L of microelement mother liquor.

2.100 times of microelement mother liquor: 1L of the solution contains 10g of FeSO₄·7H₂O、5.25g ZnSO₄·7H₂O、3.0g CuSO₄·5H₂O、0.5g MnSO₄·4H₂O、0.23g。

3. A supplemented medium: glycerol: 700 g/l.

TABLE 1 primers used in the present invention

TABLE 2 construction of strains of the invention

Example 1 relationship between expression intensity of MEP pathway ispG and ispH genes and beta-carotene production

In order to study the relationship between the expression intensity of each gene of the MEP pathway and the yield of beta-carotene in CAR005(Zhao J, Li Q, Sun T, et al. engineering Central metabolism modules of Escherichia coli for enhancing beta-carone production. Metabolic growth in EERING 2013; 17:42-50.CAR001 and CAR005D are all from this article), an mRS library was established to regulate dxr, ispD, ispE, ispG and ispH genes, 15 strains were randomly selected to determine the yield of beta-carotene, and several representative strains were selected to determine the gene expression level by a real-time quantitative PCR method based on the yield of beta-carotene to study the relationship between the expression level and the yield of beta-carotene.

Construction of mRS library to regulate dxr, ispD, ispE, ispG and ispH genes in CAR005

In the embodiment, mRS (mRNA stable region) library fragments are amplified by PCR, and are inserted before the initiation codons of several genes to be regulated, and original regulating elements are reserved. The prokaryote has a region between-10 and-35 called mRNA stable region, the variation of the nucleotide type and number of the region can affect the transcription activity of the gene, and the establishment of a library in the region can obtain promoters with different activities. The library sequence is shown in sequence 2.

dxr-1, dxr-2, dxr-3 … … dxr-15 are respectively mRSL-1:: dxr-mRSL-15:: dxr shown in Table 2, which are inserted before dxr gene in CAR005 genome in Escherichia coli for promoting expression of dxr gene.

ispD-1, ispD-2, ispD-3 … … ispD-15 are respectively mRSL-1:: ispD-mRSL-15:: ispD shown in Table 2 for promoting expression of ispD gene by inserting ispD gene in CAR005 genome in Escherichia coli before ispD gene.

ispE-1, ispE-2 and ispE-3 … … ispE-15 are respectively mRSL-1: (ispE-mRSL-15: (ispE) shown in Table 2, which is used for starting the expression of ispE genes by respectively inserting the ispE genes in the CAR005 genome in Escherichia coli before the ispE genes.

ispG-1, ispG-2 and ispG-3 … … ispG-15 are mRSL-1: (ispG-mRSL-15:) ispG shown in Table 2 for respectively inserting the ispG gene in the CAR005 genome in Escherichia coli before the ispG gene for starting the expression of the ispG gene.

ISpH-1, ISpH-2, and ISpH-3 … … ISpH-15 are mRSL-1:: ispH-mRSL-15:: ispH shown in Table 2 for promoting expression of ISpH genes, respectively, inserted before the ISpG gene in CAR005 genome in E.coli.

All the obtained bacteria constitute a library strain.

II, yield of library strain beta-carotene

Respectively picking single colonies of each obtained library strain in a test tube of 4ml LB culture medium, and culturing at 30 ℃ and 250rpm overnight; then transferring the bacterial liquid in the test tube into a 100ml triangular flask containing 10ml of culture solution according to the inoculation amount of 1% (volume percentage content), namely 100 mul of bacterial liquid, and culturing at 30 ℃ and 250 rpm; after 24h of incubation. Centrifuging 500 μ L of the bacterial solution at 13000rpm for 3min, discarding the supernatant, washing the thallus with sterilized water, adding 1ml of acetone to resuspend the thallus, extracting at 55 ℃ in the dark for 15min, centrifuging at 13000rpm for 10min, and collecting the supernatant. CAR005 was used as a control.

And measuring the absorption value of beta-carotene in the supernatant by using an ultraviolet spectrophotometer at 453 nm.

Relative yield of β -carotene uptake in supernatant multiplied by/supernatant cell turbidity (OD600nm)

The results of the relative yields of dxr-1, dxr-2, dxr-3 … … dxr-15 beta-carotene are shown in FIG. 2A, and FIG. 2A shows that the yields of beta-carotene of randomly selected 15 strains were 0.5 to 0.99 times that of CAR005 and the cell amount was 0.66 to 0.9 times that of CAR005, after the dxr gene of CAR005 was regulated by an mRS library. The dxr-4 (regulatory element mRSL-4:: dxr sequence is shown in sequence 3) and dxr-6 (regulatory element mRSL-6:: dxr sequence is shown in sequence 4) strains with low beta-carotene yield, dxr-2 (regulatory element mRSL-2:: dxr sequence is shown in sequence 5) and dxr-5 (regulatory element mRSL-5:: dxr sequence is shown in sequence 6) strains with medium beta-carotene yield, and dxr-11 (regulatory element mRSL-11:: dxr sequence is shown in sequence 7) and dxr-15 (regulatory element mRSL-15:: dxr sequence is shown in sequence 8) strains with high beta-carotene yield were selected to determine the expression level of dxr gene by a real-time quantitative PCR method.

The results of the relative yields of ispD-1 to ispD-15 β -carotene are shown in fig. 2C, which shows that the ispD gene of CAR005, regulated by the mRS library, yielded 0.99 to 1.05 times of the β -carotene of randomly selected 15 strains of CAR 005. The expression level of ispD gene was determined by a real-time quantitative PCR method using a selection of strains ispD-1 (regulatory element mRSL-1:: ispD sequence shown in SEQ ID NO: 9) and ispD-15 (regulatory element mRSL-15:: ispD sequence shown in SEQ ID NO: 10) with low β -carotene production, strains ispD-6 (regulatory element mRSL-6:: ispD sequence shown in SEQ ID NO: 11) and ispD-13 (regulatory element mRSL-13:: ispD sequence shown in SEQ ID NO: 12) with medium β -carotene production, and strains ispD-7 (regulatory element mRSL-7:: ispD sequence shown in SEQ ID NO: 12) and ispD-9 (regulatory element mRSL-9:: ispD sequence shown in SEQ ID NO: 14) with high β -carotene production.

The results of the relative yields of ispE-1 to ispE-15 β -carotene are shown in fig. 2E, which shows that the ispE gene of CAR005, regulated by the mRS library, yielded 0.97 to 1.09 times more β -carotene than CAR005 from randomly selected 15 strains. The amount of expression of the ispE gene was determined by a real-time quantitative PCR method using ispE-5 (regulatory element mRSL-5:: ispE sequence shown in SEQ ID NO: 15) and ispE-15 (regulatory element mRSL-15:: ispE sequence shown in SEQ ID NO: 16) strains with low β -carotene production, ispE-3 (regulatory element mRSL-3:: ispE sequence shown in SEQ ID NO: 17) and ispE-12 (regulatory element mRSL-12:: ispE sequence shown in SEQ ID NO: 18) strains with medium β -carotene production, and ispE-6 (regulatory element mRSL-6:: ispE sequence shown in SEQ ID NO: 19) and ispE-8 (regulatory element mRSL-8:: ispE sequence shown in SEQ ID NO: 20) strains with high β -carotene production.

The results of the relative yields of ispG-1 to ispG-15 β -carotene are shown in fig. 2G, which shows that after the ispG gene of CAR005 was regulated by the mRS library, the β -carotene yields of randomly selected 15 strains were 0.11 to 0.79 times that of CAR005, and the cell amounts were 0.47 to 0.91 times that of CAR 005. Strains ispG-1 (regulatory element mRSL-1:: ispG sequence see sequence 21) and ispG-13 (regulatory element mRSL-13:: ispG sequence see sequence 22) with low beta-carotene yield, strains ispG-4 (regulatory element mRSL-4:: ispG sequence see sequence 23) and ispG-5 (regulatory element mRSL-5:: ispG sequence see sequence 24) with low beta-carotene yield, and strains ispG-11 (regulatory element mRSL-11:: ispG sequence see sequence 25) and ispG-14 (regulatory element mRSL-14:: ispG sequence see sequence 26) with low beta-carotene yield are selected, and the expression level of the ispG genes is determined by a real-time quantitative PCR method.

Results of relative production of ispH-1 to ispH-15 β -carotene as shown in figure 2I, the β -carotene production of randomly selected 15 strains was 0.98 to 1.06 times that of CAR005 after the ispH gene of CAR005 was regulated by the mRS library. The expression level of the ispH gene was determined by a real-time quantitative PCR method by randomly selecting isppH-1 (see sequence 27 for regulatory element mRSL-1:: ispH sequence), isppH-2 (see sequence 28 for regulatory element mRSL-2:: ispH sequence), isppH-3 (see sequence 29 for regulatory element mRSL-3:: ispH sequence), isppH-4 (see sequence 30 for regulatory element mRSL-4:: ispH sequence), isppH-5 (see sequence 31 for regulatory element mRSL-5:: ispH sequence) and isppH-14 (see sequence 32 for regulatory element mRSL-14:: ispH sequence).

The above-mentioned bacteria have their gene promoters replaced with regulatory elements inside brackets.

Relation between yield of tri-beta-carotene and expression quantity of corresponding gene

Extracting total RNA of strains dxr-4, dxr-6, dxr-2, dxr-5, dxr-11 and dxr-15, and performing reverse transcription to obtain a first chain of cDNA; the first strand cDNA is used as a template, dxr-610-f/dxr-802-r is used as a primer, the iQSYBR Green RT-PCR kit (Bio-Rad Laboratories, Hercules, CA) is used for carrying out PCR in a real-time quantitative PCR instrument (Bio-Rad CFX Connect real time System), and meanwhile, the dxr expression quantity in the selected strain is determined by taking the dxr cDNA quantity in CAR005 as a control. In order to ensure the sample consistency, 16S RNA is used as an internal reference gene, the primer used for amplification is 16S-797-f/16S-963-r, and the sequence of the primer is shown in Table 1.

Real-time quantitative PCR amplification procedure:

1)95℃10min,1cycle

2)95℃15s-60℃30s-72℃30s,40cycles

3)60℃-95℃，0.2℃/s

ABI Prism 7000SDS software (Applied Biosystems) was used for data analysis.

Diluting cDNA of CAR005 according to a certain concentration, determining relative expression amounts of dxr and 16S genes, drawing a standard curve, calculating the relative expression amounts of the dxr and 16S genes in each sample according to the standard curve, and calculating three replicates for each sample.

dxr sample ═ dxr relative amount/16S relative amount

Relative dxr expression intensity dxr sample/dxrCAR 005

The relative expression of the corresponding genes of the selected strains of the library was determined by the same method, and the primers used are shown in Table 1.

The results are shown in FIG. 2.

FIG. 2B shows that the dxr expression level in 6 strains selected from the dxr gene mRS library of CAR005 is 0.68 to 42.86 times the dxr expression level in CAR 005. The dxr expression level of the strain dxr-4 with the lowest beta-carotene yield is the highest, and the expression level is 42.86 times of that of CAR 005; and the expression level of dxr gene in dxr-15 with highest beta-carotene yield is the lowest and is 0.68 times of the expression level of dxr in CAR 005. The expression level of Dxr is inversely proportional to the beta-carotene yield of the corresponding strain, i.e., the higher the expression level of Dxr in the strain, the lower the beta-carotene yield.

FIG. 2D shows that the expression level of ispD in 6 strains selected from the ispD gene mRS library of CAR005 is 7 to 116 times the expression level of ispD in CAR 005. Compared with the original strain CAR005, the yield and the cell quantity of beta-carotene of the strain selected by the ispD gene mRS library do not change greatly, and the expression level of ispD is obviously different, so that the expression level of ispD and the yield of beta-carotene have no obvious correlation.

FIG. 2F shows that the amount of expression of ispE in 6 strains selected from the ispE gene mRS library of CAR005 is 6 to 44.5 times the amount of expression of ispE in CAR 005. Compared with the original strain CAR005, the yield and the cell quantity of beta-carotene of the strain selected by the ispE gene mRS library are not greatly changed, and the expression level of the ispE is obviously different, so that the expression level of the ispE and the yield of the beta-carotene have no obvious correlation.

FIG. 2H shows that the expression level of ispG in the 9 strains selected from the ISpG gene mRS library of CAR005 is 5.09 to 75.83 times higher than the expression level of ispG in CAR 005. The expression level of ispG in the strain ispG-1 with the lowest yield of beta-carotene is the highest, and the expression level is 75.83 times of that of ispG in CAR 005; the expression level of the ispG gene in the ispG-11 with the highest beta-carotene yield is the lowest and is 5.09 times of that in CAR 005. The expression level of ispG is inversely proportional to the yield of beta-carotene of the corresponding strain, i.e., the higher the expression level of ispG in the strain, the lower the yield of beta-carotene.

FIG. 2J shows that the expression level of ISpH in 6 strains selected from the ISpH gene mRS library of CAR005 is 4 to 36.4 times the expression level of ISpH in CAR 005. Compared with the original strain CAR005, the yield and the cell quantity of beta-carotene of the strain selected by the ispH gene mRS library do not change greatly, and the expression level of the ispH gene is obviously different, so that the expression level of the ispH gene and the yield of the beta-carotene have no obvious correlation.

Recombinant bacterium obtained by combined regulation and control of IV, ispG and ispH

The artificial regulatory elements for ispG-1, ispG-4 and ispG-11 in the ispG library regulation, and the artificial regulatory elements for isppH-3, isppH-4 and isppH-14 in the ispH library were combined and regulated in CAR005, respectively.

1. Construction of recombinant Escherichia coli G1H3, G1H4, G1H14, G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14

The recombinant Escherichia coli G1H3 is characterized in that an mRSL regulatory element mRSL-1 for starting the expression of the ispG gene is inserted in front of the ispG gene in the starting bacteria CAR005 genome, i.e., ispG (sequence 21), and an mRSL-3 for starting the expression of the ispH gene is inserted in front of the ispH gene in the starting bacteria CAR005 genome, i.e., ispH (sequence 29);

the recombinant Escherichia coli G1H4 is characterized in that an mRSL regulatory element mRSL-1 for starting the expression of the ispG gene is inserted in front of the ispG gene in the starting bacteria CAR005 genome, i.e., ispG (sequence 21), and an mRSL-4 for starting the expression of the ispH gene is inserted in front of the ispH gene in the starting bacteria CAR005 genome, i.e., ispH (sequence 30);

the recombinant Escherichia coli G1H14 is formed by inserting an mRSL regulatory element mRSL-1 for starting the expression of the ispG gene in front of the ispG gene in the starting bacteria CAR005 genome, i.e., ispG (sequence 21), and inserting an mRSL-14 for starting the expression of the ispH gene in front of the ispH gene in the starting bacteria CAR005 genome, i.e., ispH (sequence 32).

The recombinant Escherichia coli G4H3 is formed by inserting an mRSL regulatory element mRSL-4:: ispG (sequence 23) for starting the expression of the ispG gene in front of the ispG gene in the CAR005 genome of the starting bacterium, and inserting an mRSL-3:: ispH (sequence 29) for starting the expression of the ispH gene in front of the ispH gene in the CAR005 genome of the starting bacterium.

The recombinant Escherichia coli G4H4 is formed by inserting an mRSL regulatory element mRSL-4:: ispG (sequence 23) for starting the expression of the ispG gene in front of the ispG gene in the CAR005 genome of the starting bacterium, and inserting an mRSL-4:: ispH (sequence 30) for starting the expression of the ispH gene in front of the ispH gene in the CAR005 genome of the starting bacterium.

The recombinant Escherichia coli G4H14 is formed by inserting an mRSL regulatory element mRSL-4:: ispG (sequence 23) for starting the expression of the ispG gene in front of the ispG gene in the CAR005 genome of the starting bacterium, and inserting an mRSL-14:: ispH (sequence 32) for starting the expression of the ispH gene in front of the ispH gene in the CAR005 genome of the starting bacterium.

The recombinant Escherichia coli G11H3 is formed by inserting an mRSL regulatory element mRSL-11:: ispG (sequence 25) for starting the expression of the ispG gene in front of the ispG gene in the CAR005 genome of the starting bacterium, and inserting an mRSL-3:: ispH (sequence 29) for starting the expression of the ispH gene in front of the ispH gene in the CAR005 genome of the starting bacterium.

The recombinant Escherichia coli G11H4 is formed by inserting an mRSL regulatory element mRSL-11:: ispG (sequence 25) for starting the expression of the ispG gene in front of the ispG gene in the CAR005 genome of the starting bacterium, and inserting an mRSL-4:: ispH (sequence 30) for starting the expression of the ispH gene in front of the ispH gene in the CAR005 genome of the starting bacterium.

The recombinant Escherichia coli G11H14 is formed by inserting an mRSL regulatory element mRSL-11 for starting the expression of the ispG gene in front of the ispG gene in the starting bacteria CAR005 genome, i.e., ispG (sequence 25), and inserting an mRSL-14 for starting the expression of the ispH gene in front of the ispH gene in the starting bacteria CAR005 genome, i.e., ispH (sequence 32).

The recombinant escherichia coli is inserted with an artificial regulatory element by a two-step homologous recombination method, and the construction method of G1H3, G1H4 and G1H14 is taken as an example, and the method comprises the following steps:

firstly, taking pXZ-CS plasmid as a template and ispH-cat-up and ispH-cat-down as primers, and carrying out PCR to obtain a DNA fragment I with about 3000 bp; after the DpnI treatment, the DNA fragment I was electroporated into E.coli ispG-1 carrying pKD 46. 200 mul of transformed bacterial liquid is taken to be coated on an LB plate containing chloramphenicol and ampicillin, after overnight culture at 30 ℃, clones which do not grow on the LB plate containing chloramphenicol and on the LB plate containing kanamycin are obtained and are respectively screened, colony PCR verification is carried out by using primers cat-up and ispH-395-down, and as a result, a recombinant bacterium is correct, the recombinant bacterium is escherichia coli ispG-1 with cat-sacB in front of ispH gene, and the bacterium can be used for second homologous recombination.

(II) respectively taking genome DNA of ispH-3, ispH-4 and ispH-14 as templates, carrying out PCR amplification to obtain a DNA fragment II of about 940bp, wherein the amplification primer is ispH-440-up/ispH-395-down; this fragment, which includes the corresponding regulatory elements and homology arms of about 400bp upstream and downstream, was electrically transferred to the pKD 46-containing strain obtained in step (one). The transformed bacterial liquid is transferred into 50ml of salt-free LB + 10% sucrose culture medium, and after shaking culture at 37 ℃ and 250rpm for 24h, streaking is carried out on a salt-free LB + 6% sucrose plate, overnight culture is carried out at 41 ℃, and the pKD46 plasmid is removed. Clones that did not grow on the chloramphenicol-containing LB plates were obtained by screening on chloramphenicol-containing LB plates and antibiotic-free LB plates, respectively, and verified by PCR using the primers P-up/ispH-395-down. The strains which were screened and verified to be correct, sent to be sequenced and named as G1H3, G1H4 and G1H 14. The primers used in the cases are shown in Table 1, and the strains constructed are shown in Table 2.

And thirdly, using the methods of the first step and the second step to respectively regulate the ISpH genes in the ispG-4 and the ispG-11 by using artificial regulation elements of the ISpH-3, the ISpH-4 and the ISpH-14 to obtain the recombinant escherichia coli G4H3, G4H4, G4H14, G11H3, G11H4 and G11H 14. The primers used are shown in Table 1, and the strains constructed are shown in Table 2.

2. Beta-carotene assay for recombinant E.coli G1H3, G1H4, G1H14, G4H3, G4H4, G4H14, G11H3, G11H4, and G11H14

Recombinant E.coli G1H3, G1H4, G1H14, G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14 were subjected to fermentation culture according to the method of example 1, and the beta-carotene production was measured while the relative beta-carotene production was calculated using CAR005 as a control. The results are shown in FIG. 3.

The results show that the yields of B-carotene in G1H3, G1H4 and G1H14 are respectively 0.16, 0.47 and 0.55 times of CAR005, namely when the expression level of ispG is high (the expression level of ispG in G1H3, G1H4 and G1H14 is 75.8 times of that of CAR 005), the yield of beta-carotene is improved along with the increase of the expression level of the ispH gene; the yields of B-carotene in G4H3, G4H4, and G4H14 were 0.57, 1.70, and 1.76 times higher than that of CAR005 (the yields of ispG in G4H3, G4H4, and G4H14 were 10.84 times higher than that of CAR 005), and the yields of B-carotene in G11H3, G11H4, and G11H14 were 0.93, 1.66, and 1.72 times higher than that of CAR005 (the yields of ispG 11H3, G11H4, and G11H14 were 5.09 times higher than that of CAR 005), respectively; at low ispG expression levels, beta-carotene production increased with increasing expression levels of isppH, and when expression levels of isppH increased to some extent, beta-carotene production did not increase. And the cell growth and beta-carotene production trends of each strain were the same.

The strain with the highest β -carotene production, G4H14, was designated CAR 015.

CAR015 was deposited in China general microbiological culture Collection center (CGMCC, accession No. 3, Ministry of microbiology, Japan institute of China academy of sciences, postal code 100101) at 2016, 8, 19, 8, 9, with a collection number of CGMCC No.12884, and a classification name of Escherichia coli (Escherichia coli).

Example 2, ispG and ispH combination Regulation of lycopene production

In the above example 1, it is shown that the combination of ispG and ispH for improving the yield of beta-carotene has the highest yield of beta-carotene G4H14, i.e., mRSL-4:: ispG and mRSL-14:: corresponding promoter combinations in ispH, can effectively improve the expression of ispG and ispH, and whether the effect is the same for all terpenoids.

In this example, lycopene was regulated using a combination of mRSL-4:: ispG and mRSL-14:: ispH.

Gene for regulating and controlling ISpH in LYC010

The recombinant Escherichia coli LYC013 is mRSL-14 for starting expression of the ispH gene inserted in front of the ispH gene in the genome of the starting bacterium LYC010 (sequence 32).

The replacement is carried out by regulating the ispH gene by a two-step method to construct LYC013 strain, which comprises the following steps.

Firstly, taking pXZ-CS plasmid as a template and ispH-cat-up and ispH-cat-down as primers, and carrying out PCR to obtain a DNA fragment I with about 3000 bp; after the DpnI treatment, the DNA fragment I was electroporated into E.coli LYC010 carrying pKD 46. 200 mul of transformed bacterial liquid is taken to be coated on an LB plate containing chloramphenicol and ampicillin, after overnight culture at 30 ℃, clones which do not grow on the LB plate containing chloramphenicol and on the LB plate containing kanamycin are obtained by screening respectively, colony PCR verification is carried out by using primers cat-up and isppH-395-down, and as a result, a recombinant bacterium is correct, the recombinant bacterium is escherichia coli LYC010 with cat-sacB in front of ispH gene, and the bacterium can be used for the second step of homologous recombination.

Secondly, using genome DNA of ispH-14 as a template, using an amplification primer of ispH-440-up/ispH-395-down, and carrying out PCR amplification to obtain a DNA fragment II of about 940 bp; this fragment, which includes the corresponding regulatory elements and homology arms of about 400bp upstream and downstream, was electrically transferred to the pKD 46-containing strain obtained in step (one). The transformed bacterial liquid is transferred into 50ml of salt-free LB + 10% sucrose culture medium, and after shaking culture at 37 ℃ and 250rpm for 24h, streaking is carried out on a salt-free LB + 6% sucrose plate, overnight culture is carried out at 41 ℃, and the pKD46 plasmid is removed. Clones that did not grow on the chloramphenicol-containing LB plates were obtained by screening on chloramphenicol-containing LB plates and antibiotic-free LB plates, respectively, and verified by PCR using the primers P-up/ispH-395-down. The strain is screened and verified to be correct, sent for sequencing, and named as LYC 013. The primers used in the cases are shown in Table 1, and the strains constructed are shown in Table 2.

Secondly, regulating and controlling ispG gene in LYC010 and LYC013

The LYC011 is characterized in that an mRSL regulatory element mRSL-1 for starting the expression of an ispG gene is inserted in front of the ispG gene in the genome of a starting bacterium LYC010 (sequence 21).

LYC012 is mRSL-4 which is an mRSL regulatory element used for starting the expression of ispG gene and is inserted in front of the ispG gene in the genome of the starting bacterium LYC010 (sequence 23).

LYC014 was prepared by pre-inserting the ispG gene in LYC010 genome of original bacterium into mRSL regulatory element mRSL-1 for starting the expression of ispG gene (sequence 21), and pre-inserting the ispH gene in LYC010 genome of original bacterium into mRSL-14 for starting the expression of ispH gene (sequence 32).

LYC015 is characterized in that an mRSL regulatory element mRSL-4 for starting the expression of the ispG gene is inserted in front of the ispG gene in the genome of the starting bacterium LYC010, i.e., ispG (sequence 23), and an mRSL-14 for starting the expression of the ispH gene is inserted in front of the ispH gene in the genome of the starting bacterium LYC010, i.e., ispH (sequence 32).

The strain uses the artificial control elements (sequence 21 and sequence 23) of ispG-1 and ispG-4 to control the ispG gene by two steps from LYC010 and LYC013 respectively, so as to construct LYC011, LYC012, LYC014 and LYC015 strains.

Firstly, PCR is carried out by taking pXZ-CS plasmid as a template and ispG-cat-up and ispG-cat-down as primers to obtain DNA fragment I of about 3000 bp; after the DpnI treatment, the DNA fragment I was transformed into E.coli LYC010 and LYC013, respectively, carrying pKD 46. And (2) coating 200 mu l of transformed bacterial liquid on an LB plate containing chloramphenicol and ampicillin, culturing overnight at 30 ℃, screening on the LB plate containing chloramphenicol ampicillin and the LB plate containing kanamycin respectively to obtain clones which do not grow on the LB plate containing chloramphenicol and grow on the LB plate containing chloramphenicol, and performing colony PCR verification by using primers cat-up and ispG-305-down to obtain correct recombinant bacteria, wherein the recombinant bacteria are escherichia coli LYC010 and LYC013 with cat-sacB in front of ispG gene and are used for the second homologous recombination.

Secondly, using the genome DNA of ispG-1 and ispG-4 as a template, using an amplification primer ispG-471-up/ispG-305-down, and carrying out PCR amplification to obtain a DNA fragment II of about 770 bp; this fragment, which includes the corresponding regulatory elements and homology arms of about 400bp upstream and downstream, was electrically transferred to the pKD 46-containing strain obtained in step (one). The transformed bacterial liquid is transferred into 50ml of salt-free LB + 10% sucrose culture medium, and after shaking culture at 37 ℃ and 250rpm for 24h, streaking is carried out on a salt-free LB + 6% sucrose plate, overnight culture is carried out at 41 ℃, and the pKD46 plasmid is removed. Clones that did not grow on the chloramphenicol-containing LB plates were obtained by screening on chloramphenicol-containing LB plates and antibiotic-free LB plates, respectively, and verified by PCR using the primers P-up/ispG-305-down. The strains are selected and verified to be correct, sent for sequencing, and named as LYC011, LYC012, LYC014 and LYC 015. The primers used in the cases are shown in Table 1, and the strains constructed are shown in Table 2.

ispG-1 is the replacement of the promoter of the ispG gene in the CAR005 genome in E.coli with mRSL-1:: ispG shown in Table 2 (see sequence 21 for regulatory elements mRSL-1:: ispG sequence).

ispG-4 the promoter of the ispG gene in the CAR005 genome in E.coli was replaced with mRSL-4:: ispG as shown in Table 2 (regulatory elements mRSL-4:: ispG sequences see sequence 23).

Thirdly, lycopene determination of recombinant escherichia coli LYC010, LYC011, LYC012, LYC014 and LYC 015.

Picking single colonies of recombinant Escherichia coli LYC011, LYC012, LYC014, LYC015 and LYC010 in a test tube of 4ml LB culture medium, and culturing at 37 ℃ and 250rpm overnight for 24 hours; then, according to the inoculation amount of 1 percent of the volume percentage, the seed liquid cultured overnight is transferred into a small shaking flask (100ml) containing 10ml of LB culture medium, and after 24 hours of light-shielding culture at 37 ℃ and 250rpm, a sample is taken to determine the yield of the lycopene. LYC010 was used as control.

The measurement method is as follows:

centrifuging 0.5ml of the cultured bacterial solution at 14000rpm for 3min, washing with sterile water, suspending and precipitating with 1ml of acetone, extracting at 55 ℃ in the dark for 15min, centrifuging the sample at 14000rpm for 10min, wherein the supernatant is red, the strain is red-white after acetone extraction, filtering the supernatant containing lycopene with a 0.45 μm microporous filter membrane, and measuring the content by HPLC.

Detection conditions are as follows: VWD detector, Symmetry C18 chromatography column (250mm × 4.6mm, 5 μm), mobile phase methanol: acetonitrile: dichloromethane (21:21:8), flow rate of 1.0mL/min, column temperature of 30 deg.C, detection wavelength of 480nm, and measurement time of 20 min. Each sample to be tested has 3 parallel samples, and the experimental result is taken from the mean value of the 3 parallel samples. Alternatively, using a Symmetry C18 column (100mm × 4.6mm, 5 μm), the mobile phase was methanol: acetonitrile: dichloromethane (21:21:8) was added at a flow rate of 1.2mL/min, and the measurement was carried out for 10 min.

Lycopene standards were purchased from sigma corporation, usa (cat. No. l 9879). The peak time in the sample was the same as that of lycopene in the standard (11.3 min) by HPLC. Each sample to be tested has 3 parallel samples, and the experimental result is the average value of the three parallel samples.

The results are shown in fig. 4, the growth and lycopene production of the LYC011 and LYC012 strains with high expression of ispG gene are properly inhibited, the cell amount is 76% and 91% of the original LYC010 strain, and the lycopene production is 12% and 40% of LYC010 strain; LYC014 and LYC015 are obtained after controlling ISpH on the basis of LYC011 and LYC012 in a combined mode, cell growth is recovered, the lycopene yield is obviously improved, the lycopene yield is respectively 7.97 times and 4.43 times of that of an original strain (LYC010), the lycopene yield of the strain LYC015 with the highest yield is 44.38mg/L, the unit cell dry weight yield is 27.97mg/g DCW, and the unit cell yield is 1.82 times of that of LYC 010.

Example 3 construction of LYC023 Strain and determination of lycopene production

The promoter of the crt operon in LYC015 strain is a constitutive artificial regulatory element M1-93, and the constitutive promoter is replaced by an inducible promoter trc promoter, which is induced by IPTG. This example investigated the effect of constitutive and inducible promoters on lycopene production.

First, the crt operon promoter in LYC015 strain is replaced by an inducible promoter

LYC023 is a recombinant bacterium obtained by inserting an mRSL regulatory element mRSL-4 for starting the expression of the ispG gene in front of the ispG gene in the genome of the original bacterium LYC010 (sequence 23), and inserting mRSL-14 for starting the expression of the ispH gene in front of the ispH gene in the genome of the original bacterium LYC010 (sequence 32), and replacing the promoter of the crt operon in the genome of LYC010 with a DNA molecule containing an inducible promoter Trc promoter and lacI (sequence 33).

Starting from LYC015, the crt operon is regulated and controlled by a two-step method to construct LYC023 strain, and the specific steps are as follows.

Firstly, PCR is carried out by taking pXZ-CS plasmid as a template and ldhA-cat-up and crtE-cat-down as primers to obtain a DNA fragment I of about 3000 bp; after the DpnI treatment, the DNA fragment I was electroporated into E.coli LYC015 carrying pKD 46. 500 mul of transformed bacterial liquid is taken to be coated on an LB plate containing chloramphenicol and ampicillin, after overnight culture at 30 ℃, clones which do not grow on the LB plate containing chloramphenicol and grow on the LB plate containing chloramphenicol are obtained by screening on the LB plate containing chloramphenicol and the LB plate containing kanamycin respectively, colony PCR verification is carried out by using primers cat-up and crtE-340-down, and as a result, a recombinant bacterium is correct, and the recombinant bacterium is escherichia coli with cat-sacB in front of a crt operon and can be used for the second step of homologous recombination.

(II) taking the total DNA of a QL002 strain (ZHao J, Li Q, Sun T, et al. engineering Central metabolism modules of Escherichia coli for improving beta-carotenes production. Metabolic growth in 2013; 17:42-50.CAR001 and CAR005D are both from the article) as a template, carrying out PCR amplification to obtain a DNA fragment II of 2340bp or so; this fragment, which includes the Trc promoter and lacI sequence and the homology arms of about 400bp upstream and downstream (see sequence 33 for Trc promoter and lacI sequence), was electrically transferred to the pKD 46-containing strain obtained in step (I). The transformed bacterial liquid is transferred into 50ml of salt-free LB + 10% sucrose culture medium, and after shaking culture at 37 ℃ and 250rpm for 24h, streaking is carried out on a salt-free LB + 6% sucrose plate, overnight culture is carried out at 41 ℃, and the pKD46 plasmid is removed. Clones that did not grow on the chloramphenicol-containing LB plates were selected separately from LB plates containing chloramphenicol, and those that did not grow on the chloramphenicol-containing LB plates were verified by PCR using the primers ldhA-up/crtE-340-down. The strain is screened and verified to be correct, sent for sequencing, and named as LYC 023. The primers used in the cases are shown in Table 1, and the strains constructed are shown in Table 2.

Secondly, lycopene determination of recombinant escherichia coli LYC015 and LYC 023.

Recombinant Escherichia coli LYC015 and LYC023 are subjected to fermentation culture according to the method of example 2, the seed solution after overnight culture is transferred into a small shake flask (100ml) containing 10ml of fermentation medium, after the overnight culture is carried out for 4 hours at 37 ℃ and 250rpm in the dark, the expression of the crt operon is induced by 1mM IPTG, and after the 24 hours of culture, a certain amount of bacteria are taken to determine the yield of lycopene. The results are shown in Table 3.

Table 3 shows the effect of induced expression of the crt operon and glpD regulation on lycopene production

As shown in Table 3, LYC023 grows slightly better than LYC015, OD600 is 1.06 times of LYC015, yield and unit yield are slightly reduced, and are 76% and 72% of LYC015 respectively, but the thallus is more stable.

Example 4 construction of LYC029 Strain and determination of lycopene production

M1-46 artificial regulatory element for regulating glpD gene of LYC023

The recombinant LYC029 is a recombinant bacterium obtained by inserting an mRSL regulatory element mRSL-4 for starting the expression of the ispG gene in front of the ispG gene in the genome of the original bacterium LYC010, ispG (sequence 23), inserting an mRSL-14 for starting the expression of the ispH gene in front of the ispH gene in the genome of the original bacterium LYC010, ispH (sequence 32), replacing a promoter of a crt operon in the genome of LYC010 with a DNA molecule containing an inducible promoter Trc promoter and lacI (sequence 33), and replacing a promoter of a glpD gene in the genome of LYC010 with an artificial regulatory element M1-46.

The promoter sequence of the glpD gene is shown in sequence 34, and the replaced artificial regulatory element M1-46 sequence is shown in sequence 35.

The method of two-step homologous recombination is used for inserting artificial regulatory elements, and comprises the following steps:

firstly, taking pXZ-CS plasmid as a template and glpD-cat-up and glpD-cat-down as primers, and carrying out PCR to obtain a DNA fragment I of about 3000 bp; after the DpnI treatment, the DNA fragment I was electroporated into E.coli LYC023 harboring pKD 46. 200 mul of transformed bacterial liquid is taken to be coated on an LB plate containing chloramphenicol and ampicillin, after overnight culture at 30 ℃, clones which do not grow on the kanamycin LB plate and grow on the chloramphenicol ampicillin LB plate are obtained by screening on the LB plate containing chloramphenicol and the LB plate containing kanamycin respectively, colony PCR verification is carried out by using primers cat-up and glpD-373-I-r, and as a result, a recombinant bacterium is correct, and the recombinant bacterium is escherichia coli LYC023 with cat-sacB in front of the glpD gene and can be used for the second step of homologous recombination.

Secondly, using genome DNA of M1-46 as a template, using an amplification primer of glpD-p-up/glpD-RBS-down, and carrying out PCR amplification to obtain a DNA fragment II of about 200 bp; this fragment, which includes the corresponding regulatory elements and homology arms of about 50bp upstream and downstream of the start codon of glpD, was electrically transferred to the pKD 46-containing strain obtained in step (I). The transformed bacterial liquid is transferred into 50ml of salt-free LB + 10% sucrose culture medium, and after shaking culture at 37 ℃ and 250rpm for 24h, streaking is carried out on a salt-free LB + 6% sucrose plate, overnight culture is carried out at 41 ℃, and the pKD46 plasmid is removed. Clones that did not grow on the LB plate containing chloramphenicol were obtained by screening on LB plate containing chloramphenicol and LB plate containing no antibiotic, respectively, and verified by PCR using the primer P-up/glpD-373-I-r. The strain is screened and verified to be correct, sent to be sequenced, and named as LYC 029. The primers used in the cases are shown in Table 1, and the strains constructed are shown in Table 2.

And secondly, detecting lycopene by using recombinant escherichia coli LYC023 and LYC 029.

Recombinant Escherichia coli LYC023 and LYC029 were subjected to fermentation culture according to the method of example 2, and the overnight-cultured seed solution was transferred to a small shake flask (100ml) containing 10ml of fermentation medium, and after culturing at 37 ℃ for 4h in the dark at 250rpm, the crt operon expression was induced with 1mM IPTG, and after culturing for 24 hours, a certain amount of bacteria was taken to determine the lycopene production. The results are shown in Table 3.

As shown in Table 3, after regulation of glpD based on LYC023, cell growth and lycopene yield were both improved, with growth and yield being regulated by 22% and 7%, respectively.

LYC029 is deposited in China general microbiological culture Collection center (CGMCC, address: No. 3 of Beijing university institute of microbiology, postal code 100101) at 2016, 8.19.2016 under the name of CGMCC, with the collection number of CGMCC No.12883, and is classified and named as Escherichia coli.

Example 5 high Density fermentation of lycopene by recombinant E.coli LYC023 and LYC029

Recombinant E.coli LYC023 and LYC029 were subjected to high density fermentation in a 7L fermentor (Labfors 4; InfrBiotechnoligy Co. Ltd.). The method comprises the following steps:

the process flow is shown in fig. 6, and specifically comprises the following steps:

1. taking out lycopene strain from refrigerator at-80 deg.C, streaking on LB plate, and placing in incubator at 37 deg.C for 15 h.

2. A single colony was picked and inoculated into a triangular flask containing 120ml of LB medium, and cultured at 37 ℃ in a shaker at 250rpm to OD₆₀₀3.0-4.0, and the obtained bacterial liquid is the seed liquid of high-density fermentation. .

3. Inoculating the prepared seed solution into a 5L fermentation tank, culturing at 37 deg.C with pH of 7.0 and dissolved oxygen constant at 20%, cascading dissolved oxygen with stirring and ventilation, and regulating rotation speed and ventilation by intelligent control system to maintain dissolved oxygen at 20%. After the carbon source in the initial culture medium is exhausted, the dissolved oxygen is suddenly increased, at the moment, the feeding is started, and the feeding rate is adjusted by a DO-STAT method to maintain the dissolved oxygen in a proper range.

4. When the OD of the cells reached about 90, 0.1mM IPTG was added for induction. The fermentation was completed after 48 hours of cultivation.

The fermentation broths at different time points were used to determine the lycopene production and OD of the cells according to the method of example 2₆₀₀。

As shown in FIG. 5, LYC023 was cultured for 48 hours, and the microbial OD was obtained₆₀₀344, lycopene production was 3.14g/l, unit yield 28.3mg/g (FIG. 5A). Culturing LYC029 for 48 hr to obtain thallus OD₆₀₀420, the yield of lycopene is 4.64g/l, which is 47.8% higher than LYC023, and the unit yield is 34.2mg/g, which is 20% higher than LYC 023.8% (fig. 5B).

Claims

1. The recombinant bacterium is any one of the following 1) to 3):

3) regulating and controlling glpD gene expression and/or activity of the recombinant strain shown in 2) to obtain a recombinant strain;

the improvement of the expression and/or activity of (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate synthase gene ispG and (E) -4-hydroxy-3-methyl-2-butenyl-pyrophosphate reductase gene ispH in lycopene-producing Escherichia coli is realized by (1) or (2) as follows:

2. The recombinant bacterium according to claim 1, wherein:

the insertion is performed by means of genome site-directed editing or homologous recombination.

3. The recombinant bacterium according to claim 2, wherein:

the genome fixed-point editing is ZFN editing, TALEN editing or CRISPR/Cas9 editing;

the homologous recombination is lambda-red homologous recombination or homologous recombination mediated by sacB gene mediated screening or homologous recombination mediated by integrating plasmid.

4. The recombinant bacterium according to any one of claims 1 to 3, wherein:

the inducible promoter is a DNA molecule containing an inducible promoter Trc promoter and lacI.

5. The recombinant bacterium according to claim 4, wherein:

the nucleotide sequence of the DNA molecule containing the inducible promoter Trc promoter and lacI is sequence 33;

the nucleotide sequence of the artificial regulatory element M1-46 is sequence 35;

the lycopene-producing Escherichia coli is LYC010 CGMCC No. 8238.

6. The recombinant bacterium according to any one of claims 1 to 3, wherein:

3) the preservation number of the recombinant bacteria is CGMCC number 12883.

7. A method for constructing the recombinant bacterium according to any one of claims 1 to 6, comprising the steps of:

A-C：

a is the following 1) or 2):

8. Use of the recombinant bacterium of any one of claims 1-6 for the production of lycopene.

9. A method for producing lycopene comprising the steps of: fermenting the recombinant bacterium of any one of claims 1-6 to obtain lycopene.