CN106434506A - Building method for producing lycopene recombinant bacteria and application - Google Patents

Abstract

The invention discloses a construction method and application of a recombinant bacterium producing lycopene. The invention provides a recombinant bacterium, which is any one of the following 1)-3): 1) for increasing the concentration of (E)-4-hydroxyl-3-methyl-2-butenyl-pyrogen in Escherichia coli producing lycopene The expression and/or activity of phosphate synthase gene ispG and (E)-4-hydroxyl-3-methyl-2-butenyl-pyrophosphate reductase gene ispH obtain recombinant bacteria; 2) is shown in regulation 1) The expression and/or activity of the crt operon of the recombinant bacterium to obtain the recombinant bacterium; 3) the expression and/or activity of the glpD gene of the recombinant bacterium shown in 2) to regulate the obtained recombinant bacterium. Experiments of the present invention prove that in recombinant Escherichia coli with a certain ability to synthesize terpenoids, the coordinated expression of ispG and ispH genes can effectively improve the ability to synthesize terpenes in recombinant Escherichia coli, including β-carotene and lycopene , and other terpenoids.

Description

Construction method and application of a recombinant bacterium producing lycopene

technical field

The invention belongs to the field of biotechnology, and in particular relates to a construction method and application of a recombinant bacterium producing lycopene.

Background technique

Terpenoids are a class of compounds that exist in nature and are composed of isoprene as a structural unit. Many terpenoids have good pharmacological activity and are the main active ingredients of traditional Chinese medicine and natural herbal medicine. Some terpenoids have been developed into effective drugs widely used in clinical practice.

Lycopene is a typical terpenoid; it is the main component of tomato red pigment and is an excellent antioxidant. Lycopene can prevent the damage caused by metabolite "free radicals" to human tissues and organs, and can be used as natural health food or medicine. Health food containing lycopene can prevent senile vision degradation, anti-aging and prevent cardiovascular disease. Lycopene also has a certain inhibitory effect on digestive tract cancer, cervical cancer, breast cancer, skin cancer, and bladder cancer. Lycopene is non-toxic and harmless. Like β-carotene, it can be added to ice cream, sherbet, hard candy, bread, biscuits, cakes and other foods to improve its nutritional value. Its health care function is better than vitamin E and β-carotene.

Since terpenoids have broad application prospects and huge market demand, methods for efficiently producing terpenoids have always been a research hotspot. At present, there are three main production methods of terpenoids: chemical synthesis, plant extraction and microbial fermentation. The chemical synthesis method has complicated process, high energy consumption and heavy 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 does not Due to the limitation of raw materials, the production process is green and clean, which has great advantages. Since the genome sequence of Escherichia coli (Escherichia coli) has been fully published, the genetic background and metabolic pathways are very clear, and it has the advantages of simple medium requirements and rapid growth, many studies have selected Escherichia coli as the starting strain to transform and produce terpenoids. Fig. 1 is the synthesis pathway of Escherichia coli to produce terpene compounds after the introduction of terpene synthesis genes.

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the precursor compounds of all terpenoids, and there are currently two known synthetic routes (Lee et al., 2002). One is the mevalonic acid pathway (Mevalonic Acid Pathway, MVA pathway), which mainly exists in the cytosol or endoplasmic reticulum of fungi and plants. The other is the MEP pathway, which mainly exists in bacteria, green algae and plant plastids. The initiators of this pathway are glyceraldehyde-3-phosphate and pyruvate, which are catalyzed by a series of enzymes to form an approximately 5:1 mixture of IPP and DMAPP. IPP isomerized to DMAPP under the catalysis of isopentenyl diphosphate isomerase (Idi). IPP and DMAPP are the basic C5 units of terpenoids, on which various terpenoids can be synthesized.

Escherichia coli has the MEP pathway, which can synthesize IPP and DMAPP, the precursors of terpenoids, and the synthesis gene of terpenoids is introduced into Escherichia coli, and Escherichia coli can produce terpenoids. However, due to the poor ability of E. coli to synthesize IPP and DMAPP, the yield of terpenoids is usually very low. Improving the metabolic flow of the MEP pathway in E. coli and improving the ability to synthesize IPP and DMAPP has become the key to increasing the production of terpenes synthesized by E. coli. Many research groups have done a lot of work in finding the rate-limiting steps in the MEP pathway and increasing the expression intensity of key genes in the MEP pathway (Yonet al., 2007; Ajikumar et al., 2010; Alper et al., 2005a; Alper et al. al., 2005b; Choi et al., 2010; Jin and Stephanopoulos, 2007; Yuan et al., 2006). Studies have shown that the 1-deoxy-xylulose-5-phosphate synthase gene (dxs), 4-diphosphate cytidine-2-C-methyl-D erythritol synthase gene (ispD) and 4-diphosphocytidine-2-C-methyl-D erythritol kinase gene (ispF), 2-C-methyl-D-erythritol-2,4-cyclic pyrophosphate synthase gene (ispE ), the expression intensity of isovaleryl pyrophosphate isomerase gene (idi) can improve the ability of recombinant Escherichia coli to produce β-carotene. However, the 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) expression intensity reduced the ability of recombinant E. coli to produce β-carotene (Yuan et al ., 2006).

In order to identify the rate-limiting steps of the MEP pathway, Yuan et al. used the method of homologous recombination to replace the promoters of the genes of the MEP pathway on the chromosome with the T5 promoter, and found that after regulating the dxs, idi, ispB, and ispDF genes, β - Carotene production increased by 100%, 40%, 20% and 40% respectively. After combined regulation of these 4 genes with the T5 promoter, the production of β-carotene was increased by 6.3 times, reaching 6 mg/g dry weight cells (Yuan et al., 2006). After Suh used the strong promoter T5 to regulate the key genes dxs, idi and ispDF of the MEP pathway, the production of β-carotene increased by 4.5 times compared with the control (Suh et al., 2012). In addition to the identified rate-limiting steps in the MEP pathway, there may be other proteins in the MEP pathway that have not been found to be rate-limiting because of their high expression and low solubility (Zhou et al., 2012). Zhao 2013 found that when the supply of precursor substances is insufficient, the downstream β-carotene synthesis gene is difficult to complete the high-strength promoter regulation experiment, but when the precursor substance supply is sufficient, it is easy to achieve high-strength promoter regulation of downstream genes, so It is believed that the rate-limiting step of E. coli producing β-carotene will change as the strain conditions change (Zhao et al., 2013).

Contents of the invention

One object of the present invention is to provide recombinant bacteria.

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

1) In order to improve (E)-4-hydroxyl-3-methyl-2-butenyl-pyrophosphate synthase gene ispG and (E)-4-hydroxyl in Escherichia coli containing crt operon and producing lycopene -expression and/or activity of 3-methyl-2-butenyl-pyrophosphate reductase gene ispH to obtain recombinant bacteria;

2) The recombinant bacteria obtained for regulating the expression and/or activity of the crt operon of the recombinant bacteria shown in 1);

3) A recombinant bacterium obtained for regulating the glpD gene expression and/or activity of the recombinant bacterium shown in 2).

In the above-mentioned recombinant bacteria, the (E)-4-hydroxyl-3-methyl-2-butenyl-pyrophosphate synthase gene ispG and (E)-4-hydroxyl-3 -expression and/or activity of the methyl-2-butenyl-pyrophosphate reductase gene ispH is achieved by the following (1) or (2):

(1) The ispG gene in the lycopene-producing Escherichia coli is inserted into the regulatory element mRSL-4::ispG shown in the sequence 23 that initiates the expression of the ispG gene, and the ispH gene in the lycopene-producing Escherichia coli is inserted into mRSL-14::ispH shown in the sequence 32 to start the expression of the ispH gene is inserted before the gene;

(2) inserting the ispG gene in the lycopene-producing Escherichia coli into the mRSL regulatory element mRSL-1::ispG shown in the sequence 21 that starts the expression of the ispG gene, and inserting the ispG gene in the lycopene-producing Escherichia coli mRSL-14::ispH shown in the sequence 32 inserted before the ispH gene to start the expression of the ispH gene;

The expression and/or activity of the crt operon of the recombinant bacteria shown in the regulation 1) is to replace the promoter of the crt operon in the recombinant bacteria shown in 1) with an inducible promoter;

The regulation of the glpD gene expression and/or activity of the recombinant bacteria shown in 2) is to replace the promoter of the glpD gene in the recombinant bacteria shown in 2) with the artificial regulatory element M1-46.

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

In the above-mentioned recombinant bacteria, the insertion is performed by site-directed genome editing or homologous recombination;

Or the targeted editing of the genome is specifically ZFN editing, TALEN editing or CRISPR/Cas9 editing;

Or the homologous recombination is specifically λ-red homologous recombination or homologous recombination mediated by sacB gene selection or integration plasmid mediated homologous recombination.

In the above-mentioned 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 knocked out of the crtX and crtY genes in the β-carotene synthesis gene cluster of the E. The original regulatory elements of the succinate dehydrogenase gene sdhABCD and the transaldolase gene talB were replaced by the artificial regulatory element M1-46, and then the RBS library was used to regulate the crt operon, dxs and idi genes respectively and combine them Regulatory acquisition; specifically LYC010.

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

LYC029 was deposited on August 19, 2016 in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) It is CGMCC No.12883, and the classification is named Escherichia coli (Escherichiacoli).

Another object of the present invention is to provide a method for constructing the above-mentioned recombinant bacteria, comprising the steps of:

A-C:

A is the following 1) or 2):

1) Pre-insert the ispG gene in lycopene-producing Escherichia coli into the regulatory element mRSL-4::ispG shown in Sequence 23 that initiates the expression of ispG gene, and insert the ispH gene in the lycopene-producing Escherichia coli The mRSL-14::ispH shown in the sequence 32 that starts the expression of the ispH gene is obtained to obtain the recombinant bacteria;

2) inserting the ispG gene in the lycopene-producing Escherichia coli into the mRSL regulatory element mRSL-1::ispG shown in the sequence 21 that initiates the expression of the ispG gene, and inserting the ispH gene in the lycopene-producing Escherichia coli mRSL-14::ispH shown in the sequence 32 to start the expression of the ispH gene is inserted before the gene to obtain the recombinant bacteria;

B, replacing the promoter of the crt operon in the recombinant bacterium obtained in A) with an inducible promoter to obtain the recombinant bacterium;

C. Replace the promoter of the glpD gene in the recombinant bacteria obtained in B) with the artificial regulatory element M1-46 to obtain the recombinant bacteria.

In the above method, the insertion is performed by means of genome-directed editing or homologous recombination;

Or the targeted editing of the genome is specifically ZFN editing, TALEN editing or CRISPR/Cas9 editing;

Or the homologous recombination is specifically λ-red homologous recombination or homologous recombination mediated by sacB gene selection or integration plasmid mediated homologous recombination.

In the above method, 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 LYC010.

The application of the above-mentioned recombinant bacteria in the production of lycopene is also within the protection scope of the present invention.

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

The method provided by the invention includes the following steps: fermenting the above-mentioned recombinant bacteria to obtain lycopene.

Experiments of the present invention prove that in recombinant Escherichia coli with a certain ability to synthesize terpenoids, the intracellular accumulation of the MEP pathway intermediate HMBPP can cause cytotoxicity; the coordinated expression of ispG and ispH genes can effectively increase the terpene content of recombinant Escherichia coli. Ability to synthesize compounds including β-carotene and lycopene, as well as other terpenoids.

Description of drawings

Fig. 1 is the synthesis pathway of Escherichia coli to produce terpene compounds after the introduction of terpene synthesis genes.

Figure 2 is the control and measurement of cell mass, relative production of β-carotene and transcription level of corresponding genes for library construction;

A is the cell mass and relative production of β-carotene of the selected strains after the establishment of the dxr bank; B is the relative expression of dxr in the selected strains after the establishment of the dxr bank; C is the cell mass and β-carotene of the selected strains after the establishment of the ispD bank D is the relative expression of ispD of the selected strains after the ispD library is built; E is the cell mass and relative production of β-carotene of the selected strains after the ispE library is built; F is the relative expression of ispE of the selected strains after the ispE library is built quantity. G is the cell mass and relative yield of β-carotene of the selected strains after the ispG library is built; H is the relative expression of ispG of the selected strains after the ispG library is built; I is the cell mass and β-carotene of the selected strains after the ispH library is built J is the relative expression level of ispH strains selected after ispH library construction.

Fig. 3 shows that the combination of ispG and ispH genes regulates the changes in cell growth and β-carotene production of the strain. A is OD ₆₀₀ ; B is the relative yield of β-carotene.

Figure 4 shows the changes in lycopene production of strains regulated by the combination of ispG and ispH genes in LYC010.

Figure 5 shows the changes in lycopene production of strains regulated by the combination of ispG and ispH genes in LYC023 and LYC029.

Figure 6 is a fermentation process diagram.

detailed description

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Escherichia coli ATCC 8739 has been disclosed in the document "Gunsalus IC, Hand DB. (1941). The use of bacteria in the chemical determination of total vitamin C.J Biol Chem.141:853-858." The public can obtain it from Tianjin Industrial Biotechnology Research obtained;

The recombinant strain M1-93 was disclosed in the document "Lu, J., J.Tang, et al. (2012). Combinatorial modulation of galP and glkgene expression for improved alternative glucoseutilization. ApplMicrobiolBiotechnol93 (6): 2455-2462.", public Available from Tianjin Institute of Industrial Biotechnology.

See sequence 1 for the sequence of M1-93 artificial regulatory element.

Lycopene was purchased from Sigma, catalog number L9879.

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

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

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

4-Diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) was purchased from Echelon Biosciences, catalog number I-M052.

2-C-Methyl-D-erythritol 2,4-cyclophosphate (MEcPP) was purchased from Echelon Biosciences with catalog number I-M054.

Isopentenyl pyrophosphate triammonium salt solution (IPP) was purchased from Sigma with catalog number I0503-1VL.

The pXZ-CS plasmid was disclosed in the document "Tan Z, Zhu X, Chen J, Li Q, Zhang X(2013) Activating phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase combination for improving succinate production. Appl Environ Microbiol79(16):4838-4844." , publicly available from Tianjin Institute of Industrial Biotechnology.

The pKD46 plasmid has been disclosed in the document "Datsenko, wanner. One-step inactivation of chromosomalgenes in Escherichia coli K-12 using PCR products. Proc Natl Acad SciUSA. 2000.97 (12): 6640-6645; ", and the public can obtain it from Tianjin Industrial Biotechnology obtained by the Institute.

Recombinant Escherichia coli LYC010 is to knock out the crtX and crtY genes in the β-carotene synthesis gene cluster of the recombinant Escherichia coli CAR001 strain to construct a strain that synthesizes lycopene, and then the α-ketoglutarate dehydrogenase gene sucAB, succinate The original regulatory elements of the dehydrogenase gene sdhABCD and the transaldolase gene talB were replaced with the artificial regulatory element M1-46, and then the RBS library was used to regulate the crt operon, dxs and idi genes, and combined regulation was obtained; The specific construction method has been disclosed in the invention patent with the publication number ZL 201410029600.0 and the invention title "A Recombinant Bacteria for Lycopene Production and Its Application", and the public can obtain it from Tianjin Institute of Industrial Biotechnology. On September 23, 2013, it was deposited in the General Microorganism Center of China Committee for Culture Collection of Microbial Cultures (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101), deposit The number is CGMCC No.8238.

The recombinant strain CAR005 is to introduce the β-carotene synthesis gene cluster and the trc regulatory element into Escherichia coli, and then replace the trc regulatory element of the β-carotene synthesis gene cluster with the artificial regulatory element M1-93; The original regulatory element of ketose-5-phosphate synthase gene dxs was replaced by artificial regulatory element M1-37; the original regulatory element of isovaleryl pyrophosphate isomerase gene idi was replaced by artificial regulatory element M1-46; The original regulatory elements of α-ketoglutarate dehydrogenase gene sucAB, succinate dehydrogenase gene sdhABCD and transaldolase gene talB were all replaced with artificial regulatory element M1-46. The specific construction method is in the publication number It is disclosed in the invention patent of CN 103087972A and the invention title is "Recombinant Microorganism for Producing Terpenoids and Construction Method", and the public can obtain it from Tianjin Institute of Industrial Biotechnology.

Escherichia coli CAR005 carrying pKD46 is a bacterium obtained by introducing plasmid pKD46 into Escherichia coli CAR005.

The preparation method of salt-free LB is as follows:

50% sucrose solution: Weigh 500g of sucrose, dissolve it in a small amount of ultrapure water, and dilute to 1L, sterilize at 115°C for 20 minutes.

10% salt-free sucrose LB medium: Weigh 5g of yeast extract, 10g of peptone in 800ml of water, sterilize at 115°C for 20min. Add 200ml of 50% sucrose solution after sterilization.

6% salt-free sucrose LB medium: Weigh 5g yeast extract, 10g peptone, 15g agar powder, dissolve in 880ml water, sterilize at 115°C for 20min. Add 120ml of 50% sucrose solution after sterilization.

The concentrations of chloramphenicol, ampicillin, and kanamycin in the examples were 34 μg/L, 50 μg/L, and 50 μg/L, respectively.

The preparation method of fermentation medium is as follows:

1. 10% glycerin mother solution: Weigh 100g glycerol, add deionized water to 1L, sterilize at 121°C for 20min.

2. Trace element mother liquor:. The composition of trace element solution: 1L solution contains 10g FeSO ₄ ·7H ₂ O, 5.25g ZnSO ₄ ·7H ₂ O, 3.0g CuSO ₄ ·5H ₂ O, 0.5g MnSO ₄ ·4H ₂ O, 0.23g.

3. Weigh yeast powder: 10g/l, K ₂ HPO ₄ : 10.5g/l, (NH ₄ ) ₂ HPO ₄ : 6g/l, glycerin: 15g/l, citric acid: 1.84g/l, 10ml trace elements The mother liquor was sterilized in 700ml of water at 121°C for 20 minutes. After sterilization, add sterilized MgSO ₄ 7H ₂ O solution and 100ml 10% glycerin mother solution, and finally add sterile water to 1L

MgSO4·7H2O solution: MgSO ₄ ·7H ₂ O: 5g was dissolved in 20ml deionized water, sterilized at 121°C for 20min.

The preparation method of high-density fermentation medium is as follows:

1. Base medium: yeast powder: 20g/l, KH ₂ PO ₄ : 10.5g/l, (NH ₄ ) ₂ HPO ₄ : 6g/l, glycerin: 30g/l, citric acid: 1.84g/l, MgSO ₄ ·7H ₂ O: 10g/L, 15ml/L trace element mother solution.

2. 100 times trace element mother solution: 1L solution contains 10g FeSO ₄ ·7H ₂ O, 5.25g ZnSO ₄ ·7H ₂ O, 3.0g CuSO ₄ ·5H ₂ O, 0.5g MnSO ₄ ·4H ₂ O, 0.23g.

3. Feed medium: glycerol: 700g/l.

Primers used in the present invention in table 1

Table 2. Bacterial strains constructed in the present invention

Example 1, the relationship between the MEP pathway ispG and ispH gene expression intensity and β-carotene output

In order to study the MEP pathway in CAR005 (Zhao J, Li Q, Sun T, et al. Engineering central metabolic modules of Escherichia coli for improving beta-carotene production. Metabolicengineering 2013; 17:42-50. Both CAR001 and CAR005D are from this article) The relationship between the expression intensity of each gene and the production of β-carotene, the mRS library was established to regulate dxr, ispD, ispE, ispG and ispH genes, 15 strains were randomly selected, and the production of β-carotene was determined. According to the production of β-carotene, Then select several representative strains and use the real-time quantitative PCR method to measure the gene expression level, and study the relationship between the expression level and the yield of β-carotene.

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

In this implementation case, the mRS (mRNA stable region) library fragment was amplified by PCR and inserted before the start codons of several genes to be regulated, and the original regulatory elements were retained. In prokaryotes, the region between -10 and -35 is called the mRNA stable region. Changes in the type and number of nucleotides in this region will affect the level of gene transcription activity. A library can be established in this region to obtain promoters with different activities. See sequence 2 for the library sequence.

dxr-1, dxr-2, dxr-3... dxr-15 are the mRSL-1::dxr shown in Table 2 for respectively inserting the dxr gene in the CAR005 genome in Escherichia coli into the expression of the dxr gene respectively -mRSL-15::dxr.

ispD-1, ispD-2, ispD-3...ispD-15 are the mRSL-1::ispD shown in Table 2 that are respectively inserted before the ispD gene in the CAR005 genome in Escherichia coli to initiate the expression of the ispD gene - mRSL-15::ispD, .

ispE-1, ispE-2, ispE-3...ispE-15 are the mRSL-1::ispE shown in Table 2 for respectively inserting the ispE gene in the CAR005 genome in Escherichia coli into the expression of the ispE gene respectively -mRSL-15::ispE.

ispG-1, ispG-2, ispG-3...ispG-15 are the mRSL-1::ispG shown in Table 2, respectively inserted before the ispG gene in the CAR005 genome in Escherichia coli to initiate the expression of the ispG gene -mRSL-15::ispG.

ispH-1, ispH-2, ispH-3...ispH-15 are the mRSL-1::ispH shown in Table 2 respectively inserted before the ispG gene in the CAR005 genome in Escherichia coli to initiate the expression of the ispH gene -mRSL-15::ispH.

All obtained strains constitute the library strains.

2. Production of β-carotene from library strains

Each of the library strains obtained in the above-mentioned one was singled out and colonized in a test tube of 4 ml of LB medium, and cultured overnight at 30°C and 250 rpm; The bacterial solution in the medium was transferred to a 100ml Erlenmeyer flask containing 10ml of culture solution, cultivated at 30°C and 250rpm; after 24 hours of cultivation. Take 500 μL of bacterial solution and centrifuge at 13,000 rpm for 3 minutes to discard the supernatant, wash the bacterial cells with sterilized water, add 1 ml of acetone to resuspend the bacterial cells, extract at 55°C in the dark for 15 minutes, and centrifuge at 13,000 rpm for 10 minutes to collect the supernatant. Take CAR005 as the control.

The absorption value of β-carotene in the supernatant was measured by an ultraviolet spectrophotometer at 453 nm.

The relative yield of β-carotene = the absorption value of β-carotene in the supernatant multiplied by the turbidity of the cells in the supernatant (OD600nm)

The results of the relative production of dxr-1, dxr-2, dxr-3...dxr-15β-carotene are shown in Figure 2A. Figure 2A shows that after the dxr gene of CAR005 was regulated by the mRS library, the β-carotene of 15 strains randomly selected -Carotene production is 0.5 to 0.99 times that of CAR005, and the cell volume is 0.66 to 0.9 times that of CAR005. Select the bacterial strain dxr-4 (regulatory element mRSL-4::dxr sequence see sequence 3) and dxr-6 (regulatory element mRSL-6::dxr sequence see sequence 4) with lower β-carotene production, β-carotene Strains dxr-2 (regulatory element mRSL-2::dxr sequence see sequence 5) and dxr-5 (regulatory element mRSL-5::dxr sequence see sequence 6) with medium yield, and strains with higher β-carotene production For dxr-11 (regulatory element mRSL-11::dxr sequence, see SEQ ID NO: 7) and dxr-15 (regulatory element mRSL-15::dxr sequence, see SEQ ID NO: 8), the expression level of dxr gene was determined by real-time quantitative PCR.

The results of the relative production of β-carotene from ispD-1 to ispD-15 are shown in Figure 2C, indicating that after the ispD gene of CAR005 was regulated by the mRS library, the β-carotene production of 15 randomly selected strains was 0.99 to 0.99% of that of CAR005. 1.05 times. The bacterial strain ispD-1 (regulatory element mRSL-1::ispD sequence is shown in sequence 9) and ispD-15 (regulatory element mRSL-15::ispD sequence is shown in sequence 10) and β-carotene with lower β-carotene production Strains ispD-6 (regulatory element mRSL-6::ispD sequence, see SEQ ID NO: 11) and ispD-13 (regulatory element mRSL-13::ispD sequence: see SEQ ID NO: 12) and strains with higher β-carotene production ispD-7 (see SEQ ID NO: 13 for the regulatory element mRSL-7::ispD sequence) and ispD-9 (see SEQ ID NO: 14 for the regulatory element mRSL-9::ispD sequence) were used to measure the expression level of ispD gene by real-time quantitative PCR.

The results of the relative production of β-carotene from ispE-1 to ispE-15 are shown in Figure 2E, indicating that after the ispE gene of CAR005 was regulated by the mRS library, the β-carotene production of 15 randomly selected strains was 0.97 to 0.97% of that of CAR005. 1.09 times. The strain ispE-5 (see sequence 15 for the regulatory element mRSL-5::ispE sequence) and ispE-15 (see sequence 16 for the regulatory element mRSL-15::ispE sequence) and β-carotene were selected for the lower production of β-carotene Strains ispE-3 (see SEQ ID NO: 17 for the regulatory element mRSL-3::ispE sequence) and ispE-12 (see SEQ ID NO: 18 for the regulatory element mRSL-12::ispE sequence) with medium yields, and strains with higher β-carotene production ispE-6 (see SEQ ID NO: 19 for the regulatory element mRSL-6::ispE sequence) and ispE-8 (see SEQ ID NO: 20 for the regulatory element mRSL-8::ispE sequence) were used to measure the expression level of the ispE gene by real-time quantitative PCR.

The results of the relative production of β-carotene from ispG-1 to ispG-15 are shown in Figure 2G, indicating that after the ispG gene of CAR005 was regulated by the mRS library, the production of β-carotene from 15 randomly selected strains was 0.11 to 0.11 to that of CAR005. 0.79 times, and the cell volume was 0.47 to 0.91 times that of CAR005. Select the lower bacterial strain ispG-1 (regulatory element mRSL-1::ispG sequence see sequence 21) and ispG-13 (regulatory element mRSL-13::ispG sequence see sequence 22) with lower β-carotene production, β-carotene The strain ispG-4 (regulatory element mRSL-4::ispG sequence is shown in SEQ ID NO: 23) and ispG-5 (regulatory element mRSL-5::ispG sequence is shown in SEQ ID NO: 24) with lower yield, and the lower yield of β-carotene For strains ispG-11 (regulatory element mRSL-11::ispG sequence, see SEQ ID NO: 25) and ispG-14 (regulatory element mRSL-14::ispG sequence, see SEQ ID NO: 26), real-time quantitative PCR method was used to measure the expression level of ispG gene.

The results of the relative production of β-carotene from ispH-1 to ispH-15 are shown in Figure 2I. After the ispH gene of CAR005 was regulated by the mRS library, the β-carotene production of 15 randomly selected strains was 0.98 to 1.06 times that of CAR005. Randomly select ispH-1 (regulatory element mRSL-1::ispH sequence see sequence 27), ispH-2 (regulatory element mRSL-2::ispH sequence see sequence 28), ispH-3 (regulatory element mRSL-3::ispH sequence See SEQ ID NO: 29 for the sequence), ispH-4 (see SEQ ID NO: 30 for the regulatory element mRSL-4::ispH sequence), ispH-5 (see SEQ ID NO: 31 for the regulatory element mRSL-5::ispH sequence) and ispH-14 (see SEQ ID NO: 31 for the regulatory element mRSL- 14::ispH sequence see sequence 32) real-time quantitative PCR method was used to measure the expression level of ispH gene.

For each of the above bacteria, the promoters of their respective genes were replaced with the regulatory elements in brackets.

3. Relationship between β-carotene production and corresponding gene expression

Extract the total RNA of strains dxr-4, dxr-6, dxr-2, dxr-5, dxr-11 and dxr-15, and reverse transcribe to obtain the first strand of cDNA; using the first strand cDNA as a template, dxr-610- f/dxr-802-r was used as a primer, and PCR was carried out in a real-time quantitative PCR instrument (Bio-Rad CFX Connect RealTime System) with iQSYBR Green RT-PCR kit (Bio-Rad Laboratories, Hercules, CA), and at the same time, in CAR005 The cDNA amount of dxr was used as a control, and the expression amount of dxr in the selected strains was determined. In order to ensure the consistency of the samples, 16S RNA was used as the internal reference gene, and the primers used for amplification were 16S-797-f/16S-963-r. The sequences of the primers used are shown in Table 1.

Real-time quantitative PCR amplification program:

1) 95℃10min, 1cycle

2) 95℃15s-60℃30s-72℃30s, 40cycles

3) 60°C-95°C, 0.2°C/s

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

Dilute the cDNA of CAR005 according to a certain concentration, measure the relative expression of dxr and 16S genes, draw a standard curve, calculate the relative expression of dxr and 16S genes in each sample according to the standard curve, and then do three replicates for each sample.

dxr sample = relative amount of dxr/relative amount of 16S

dxr relative expression intensity = dxr sample/dxrCAR005

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

The result is shown in Figure 2.

Figure 2B shows that the expression level of dxr in the 6 strains selected from the dxr gene mRS library of CAR005 is 0.68 to 42.86 times that of dxr in CAR005. The expression level of dxr gene in the strain dxr-4 with the lowest β-carotene production was the highest, which was 42.86 times that of dxr in CAR005; while the expression level of dxr gene in dxr-15 with the highest β-carotene production was the lowest, which was CAR005 0.68 times the expression level of dxr. The expression level of Dxr is inversely proportional to the production of β-carotene in the corresponding strain, that is, the higher the expression level of dxr in the strain, the lower the production of β-carotene.

Figure 2D shows that the expression level of ispD in the 6 strains selected by the ispD gene mRS library of CAR005 is 7 to 116 times that of ispD in CAR005. Compared with the starting strain CAR005, the β-carotene production and cell mass of the strain selected from the ispD gene mRS library have little change, but the ispD expression level is significantly different. Therefore, it is considered that there is no obvious correlation between the ispD expression level and the β-carotene production. .

Figure 2F shows that the expression level of ispE in the 6 strains selected from the ispE gene mRS library of CAR005 was 6 to 44.5 times that of ispE in CAR005. Compared with the original strain CAR005, the β-carotene production and cell mass of the selected strains of the ispE gene mRS library have little change, but the ispE expression level is significantly different. Therefore, it is considered that there is no obvious correlation between the ispE expression level and the β-carotene production. .

Figure 2H shows that the expression level of ispG in the 9 strains selected by the ispG gene mRS library of CAR005 is 5.09 to 75.83 times that of ispG in CAR005. The expression level of ispG in the strain ispG-1 with the lowest β-carotene production is the highest, which is 75.83 times that of ispG in CAR005; while the expression level of ispG gene in ispG-11 with the highest β-carotene production is the lowest, which is CAR005 5.09 times the expression level of ispG. The expression level of ispG is inversely proportional to the production of β-carotene in the corresponding strain, that is, the higher the expression level of ispG in the strain, the lower the production of β-carotene.

Figure 2J shows that the expression level of ispH in the 6 strains selected by the ispH gene mRS library of CAR005 is 4 to 36.4 times that of ispH in CAR005. Compared with the starting strain CAR005, the β-carotene production and cell mass of the strain selected from the ispH gene mRS library have little change, but the ispH expression level is significantly different. Therefore, it is considered that there is no obvious correlation between the ispH expression level and the β-carotene production. .

4. Recombinant bacteria obtained by combined regulation of ispG and ispH

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

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

Recombinant Escherichia coli G1H3 is the mRSL regulatory element mRSL-1::ispG (SEQ ID NO: 21) that pre-inserts the ispG gene in the genome of the starting bacteria CAR005 to start the expression of the ispG gene, and inserts the front of the ispH gene in the genome of the starting bacteria CAR005 to start the ispH gene Expressed mRSL-3::ispH (SEQ ID NO: 29);

Recombinant Escherichia coli G1H4 is the mRSL regulatory element mRSL-1::ispG (SEQ ID NO: 21) that pre-inserts the ispG gene in the genome of the starting bacteria CAR005 to start the expression of the ispG gene, and inserts the front of the ispH gene in the genome of the starting bacteria CAR005 to start the ispH gene Expressed mRSL-4::ispH (SEQ ID NO: 30);

Recombinant Escherichia coli G1H14 is the mRSL regulatory element mRSL-1::ispG (SEQ ID NO: 21) that pre-inserts the ispG gene in the genome of the starting bacteria CAR005 to start the expression of the ispG gene, and inserts the front of the ispH gene in the genome of the starting bacteria CAR005 to start the ispH gene Expressed mRSL-14::ispH (SEQ ID NO: 32).

Recombinant Escherichia coli G4H3 is the mRSL regulatory element mRSL-4::ispG (sequence 23) that inserts the ispG gene in the genome of the starting bacteria CAR005 to start the expression of the ispG gene, and inserts the ispH gene in the genome of the starting bacteria CAR005 to start the ispH gene Expressed mRSL-3::ispH (SEQ ID NO: 29).

Recombinant Escherichia coli G4H4 is the mRSL-4::ispG (sequence 23) that pre-inserts the ispG gene in the genome of the starting bacterium CAR005 to start the expression of the ispG gene, and inserts the ispH gene in the genome of the starting bacterium CAR005 to start the ispH gene Expressed mRSL-4::ispH (SEQ ID NO: 30).

Recombinant Escherichia coli G4H14 is the mRSL regulatory element mRSL-4::ispG (sequence 23) that inserts the ispG gene in the genome of the starting bacteria CAR005 to start the expression of the ispG gene, and inserts the ispH gene in the genome of the starting bacteria CAR005 to start the ispH gene Expressed mRSL-14::ispH (SEQ ID NO: 32).

Recombinant Escherichia coli G11H3 is the mRSL regulatory element mRSL-11::ispG (sequence 25) that inserts the ispG gene in the genome of the starting bacteria CAR005 to start the expression of the ispG gene, and inserts the ispH gene in the genome of the starting bacteria CAR005 to start the ispH gene Expressed mRSL-3::ispH (SEQ ID NO: 29).

Recombinant Escherichia coli G11H4 is the mRSL regulatory element mRSL-11::ispG (sequence 25) that inserts the ispG gene in the genome of the starting bacteria CAR005 to start the expression of the ispG gene, and inserts the ispH gene in the genome of the starting bacteria CAR005 to start the ispH gene Expressed mRSL-4::ispH (SEQ ID NO: 30).

Recombinant Escherichia coli G11H14 is the mRSL regulatory element mRSL-11::ispG (sequence 25) that pre-inserts the ispG gene in the genome of the starting bacteria CAR005 to start the expression of the ispG gene, and inserts the front of the ispH gene in the genome of the starting bacteria CAR005 to start the ispH gene Expressed mRSL-14::ispH (SEQ ID NO: 32).

The above-mentioned recombinant Escherichia coli all use the two-step homologous recombination method to insert artificial regulatory elements. Taking the construction method of G1H3, G1H4, and G1H14 as an example, the details are as follows:

(1) Using the pXZ-CS plasmid as a template, ispH-cat-up and ispH-cat–down as primers, a DNA fragment I of about 3000 bp was obtained by PCR; after DpnI treatment, the DNA fragment I was electrotransferred into Escherichia coli with pKD46 ispG-1. Take 200 μl of the transformed bacteria solution and spread it on the LB plate containing chloramphenicol and ampicillin, after culturing overnight at 30°C, screen it on the LB plate containing chloramphenicol and ampicillin and the LB plate containing kanamycin respectively to obtain the It does not grow on the Namycin LB plate, and the clone that grows on the Chloramphenicol-Ampicillin LB plate was verified by colony PCR using primers cat-up and ispH-395-down. The result was that the recombinant bacteria were correct, and the recombinant bacteria were pre-ispH genes. Escherichia coli ispG-1 of cat-sacB, which can be used for the second step of homologous recombination.

(2) Using the genomic DNA of ispH-3, ispH-4 and ispH-14 as templates respectively, the amplification primers are ispH-440-up/ispH-395-down, and the DNA fragment II of about 940bp is obtained by PCR amplification; this The fragment includes corresponding regulatory elements and about 400 bp upstream and downstream homology arms, and this fragment is electrotransformed into the strain containing pKD46 obtained in step (1). Transfer the transformed bacteria solution into 50ml of salt-free LB+10% surcose medium, culture at 37°C and 250rpm for 24 hours with shaking, then streak on a salt-free LB+6%surcose plate, and culture overnight at 41°C to remove the pKD46 plasmid. Screen on LB plates containing chloramphenicol and LB plates without antibiotics respectively, and obtain clones that do not grow on LB plates containing chloramphenicol, and perform PCR verification using primers P-up/ispH-395-down verify. The screening and verification were correct, and they were sent for sequencing, and the strains with correct sequencing were named G1H3, G1H4, and G1H14. The primers used in the case are shown in Table 1, and the constructed strains are shown in Table 2.

(3) Using the methods described in (1) and (2) above, use the ispH artificial regulatory elements of ispH-3, ispH-4 and ispH-14 to regulate the ispH gene in ispG-4 and ispG-11 respectively, and obtain the recombinant large intestine Bacteria G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14. The primers used are shown in Table 1, and the constructed strains are shown in Table 2.

2. Determination of β-carotene in recombinant Escherichia coli G1H3, G1H4, G1H14, G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14

Recombinant Escherichia coli G1H3, G1H4, G1H14, G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14 were fermented and cultured according to the method in Example 1, and the yield of β-carotene was measured. Yield. The result is shown in Figure 3.

The results showed that the production of B-carotene in G1H3, G1H4, and G1H14 was 0.16, 0.47, and 0.55 times that of CAR005, respectively. The production of -carotene increases with the increase of ispH gene expression; the production of B-carotene in G4H3, G4H4, and G4H14 is 0.57, 1.70, and 1.76 times that of CAR005, respectively (the expression of ispG in G4H3, G4H4, and G4H14 is 10.84 times that of CAR005 times), the production of B-carotene in G11H3, G11H4 and G11H14 was 0.93, 1.66 and 1.72 times that of CAR005, respectively (the expression of ispG in G11H3, G11H4 and G11H14 was 5.09 times that of CAR005); when the expression of ispG was low, β-carotene The production of carotene increases with the expression of ispH, and when the expression of ispH increases to a certain extent, the production of β-carotene no longer increases. And the trend of cell growth and β-carotene production of each strain was the same.

The strain G4H14 with the highest β-carotene production was named CAR015.

CAR015 was deposited on August 19, 2016 in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) It is CGMCC No.12884, and its classification name is Escherichia coli.

Example 2. The combination of ispG and ispH regulates the production of lycopene

In the above example 1, it can be seen that in the experiment of increasing the production of β-carotene by the combination regulation of ispG and ispH, the combination with the highest production of β-carotene is G4H14, that is, the corresponding promoters in mRSL-4::ispG and mRSL-14::ispH Combination, can effectively increase the expression of ispG and ispH, whether it has the same effect on all terpenoids.

In this example, the combination of mRSL-4::ispG and mRSL-14::ispH is used to regulate lycopene.

1. Regulation of ispH gene in LYC010

Recombinant Escherichia coli LYC013 is the mRSL-14::ispH (sequence 32) in which the ispH gene in the genome of the starting strain LYC010 is pre-inserted for initiating the expression of the ispH gene.

The above replacement regulates the ispH gene through a two-step method to construct the LYC013 strain, and the specific steps are as follows.

(1) Using the pXZ-CS plasmid as a template, ispH-cat-up and ispH-cat–down as primers, a DNA fragment I of about 3000 bp was obtained by PCR; after DpnI treatment, the DNA fragment I was electrotransferred into Escherichia coli with pKD46 LYC010. Take 200 μl of the transformed bacteria solution and spread it on the LB plate containing chloramphenicol and ampicillin, after culturing overnight at 30°C, screen it on the LB plate containing chloramphenicol and ampicillin and the LB plate containing kanamycin respectively to obtain the It does not grow on the Namycin LB plate, and the clone that grows on the Chloramphenicol-Ampicillin LB plate was verified by colony PCR using primers cat-up and ispH-395-down. The result was that the recombinant bacteria were correct, and the recombinant bacteria were pre-ispH genes. Escherichia coli LYC010 of cat-sacB, which can be used for the second step of homologous recombination.

(2) Using the genomic DNA of ispH-14 as a template, the amplification primers are ispH-440-up/ispH-395-down, and a DNA fragment II of about 940bp is obtained by PCR amplification; this fragment includes corresponding regulatory elements and upstream and downstream 400bp Left and right homology arms, electrotransform this fragment into the strain containing pKD46 obtained in step (1). Transfer the transformed bacteria solution into 50ml of salt-free LB+10% surcose medium, culture at 37°C and 250rpm for 24 hours with shaking, then streak on a salt-free LB+6%surcose plate, and culture overnight at 41°C to remove the pKD46 plasmid. Screen on LB plates containing chloramphenicol and LB plates without antibiotics respectively, and obtain clones that do not grow on LB plates containing chloramphenicol, and perform PCR verification using primers P-up/ispH-395-down verify. The screening verification was correct, and it was sent for sequencing, and the strain with correct sequencing was named LYC013. The primers used in the case are shown in Table 1, and the constructed strains are shown in Table 2.

2. Regulation of the ispG gene in LYC010 and LYC013

LYC011 is the mRSL-1::ispG regulatory element mRSL-1::ispG (SEQ ID NO: 21 ), which is inserted in front of the ispG gene in the genome of the starting strain LYC010 to initiate the expression of the ispG gene.

LYC012 is the mRSL regulatory element mRSL-4::ispG (SEQ ID NO: 23) that is inserted in front of the ispG gene in the genome of the starting strain LYC010 to initiate the expression of the ispG gene.

LYC014 is the mRSL regulatory element mRSL-1::ispG (sequence 21) that inserts the ispG gene in the genome of the starting bacteria LYC010 to start the expression of the ispG gene, and inserts the ispH gene in the genome of the starting bacteria LYC010 to start ispH Gene expression of mRSL-14::ispH (SEQ ID NO: 32).

LYC015 is the mRSL regulatory element mRSL-4::ispG (sequence 23) that inserts the ispG gene in the genome of the starting bacteria LYC010 to start the expression of the ispG gene, and inserts the ispH gene in the genome of the starting bacteria LYC010 to start ispH Gene expression of mRSL-14::ispH (SEQ ID NO: 32).

The artificial regulatory elements of ispG of ispG-1 and ispG-4 (sequence 21 and sequence 23, respectively) were used in each of the above strains to start from LYC010 and LYC013, respectively, to regulate the ispG gene in two steps, and construct LYC011, LYC012, LYC014, and LYC015 strains ,Specific steps are as follows.

(1) Using the pXZ-CS plasmid as a template, ispG-cat-up and ispG-cat-down as primers, PCR obtained a DNA fragment I of about 3000 bp; after DpnI treatment, the DNA fragment I was electrotransferred to the large intestine with pKD46 respectively Bacillus LYC010, LYC013. Take 200 μl of the transformed bacteria solution and spread it on the LB plate containing chloramphenicol and ampicillin, after culturing overnight at 30°C, screen it on the LB plate containing chloramphenicol and ampicillin and the LB plate containing kanamycin respectively to obtain the It does not grow on the Namycin LB plate, but the clone that grows on the Chloramphenicol Ampicillin LB plate was verified by colony PCR using primers cat-up and ispG-305-down. The result was that the recombinant bacteria were correct. Escherichia coli LYC010 and LYC013 of cat-sacB for the second step of homologous recombination.

(2) Using the genomic DNA of ispG-1 and ispG-4 as a template, the amplification primers are ispG-471-up/ispG-305-down, and PCR amplification obtains a DNA fragment II of about 770bp; this fragment includes the corresponding regulatory elements And the homology arm of about 400bp upstream and downstream, this fragment was electrotransformed into the strain containing pKD46 obtained in step (1). Transfer the transformed bacteria solution into 50ml of salt-free LB+10% surcose medium, culture at 37°C and 250rpm for 24 hours with shaking, then streak on a salt-free LB+6%surcose plate, and culture overnight at 41°C to remove the pKD46 plasmid. Screen on LB plates containing chloramphenicol and LB plates without antibiotics respectively, and obtain clones that do not grow on LB plates containing chloramphenicol, and perform PCR verification using primers P-up/ispG-305-down verify. The screening and verification were correct, and they were sent for sequencing. The strains with correct sequencing were named LYC011, LYC012, LYC014, and LYC015. The primers used in the case are shown in Table 1, and the constructed strains are shown in Table 2.

ispG-1 replaces the promoter of the ispG gene in the CAR005 genome of Escherichia coli with mRSL-1::ispG shown in Table 2 (see sequence 21 for the sequence of the regulatory element mRSL-1::ispG).

ispG-4 replaces the promoter of the ispG gene in the CAR005 genome of Escherichia coli with mRSL-4::ispG shown in Table 2 (see sequence 23 for the sequence of the regulatory element mRSL-4::ispG).

3. Determination of lycopene in recombinant Escherichia coli LYC010, LYC011, LYC012, LYC014, LYC015.

Recombinant Escherichia coli LYC011, LYC012, LYC014, LYC015 and LYC010 single colonies were placed in 4ml test tubes of LB medium, and cultivated overnight at 250rpm at 37°C for 24 hours; The seed solution was transferred to a small shake flask (100 ml) containing 10 ml of LB medium, and cultured at 37° C. and 250 rpm in the dark for 24 hours, then samples were taken to determine the lycopene production. Take LYC010 as the control.

The determination method is as follows:

Take 0.5ml of the cultured bacteria solution and centrifuge at 14000rpm for 3min, wash with sterile water, suspend the precipitate with 1ml of acetone, extract at 55°C for 15 minutes in the dark, then centrifuge the sample at 14000rpm for 10 minutes, the supernatant is red, while The strain changed from red to white after being extracted with acetone, and the supernatant containing lycopene was filtered through a 0.45 μm microporous membrane, and the content was determined by HPLC.

Detection conditions: VWD detector, Symmetry C18 chromatographic column (250mm×4.6mm, 5μm), mobile phase is methanol: acetonitrile: dichloromethane (21:21:8), flow rate 1.0mL/min, column temperature 30°C, detection The wavelength is 480nm, and the measurement time is 20min. Each sample to be tested has 3 parallel samples, and the experimental results are obtained from the average of the 3 parallel samples. Or use a Symmetry C18 chromatographic column (100mm×4.6mm, 5μm), the mobile phase is methanol: acetonitrile: dichloromethane (21:21:8), the flow rate is 1.2mL/min, and the measurement takes 10min.

The standard lycopene was purchased from Sigma, USA (Cat.No.L9879). Tested by HPLC, the peak time in the sample is the same as that of lycopene in the standard (the peak time is 11.3 min). Each sample to be tested has three parallel samples, and the experimental results take the average value of the three parallel samples.

The results are shown in Figure 4. The growth and lycopene production of LYC011 and LYC012 strains with high expression of ispG gene alone were properly inhibited, and the cell mass was 76% and 91% of that of the starting strain LYC010, respectively, and the lycopene production was respectively 12% and 40% of LYC010; on the basis of LYC011 and LYC012, LYC014 and LYC015 were obtained after combined regulation of ispH, cell growth was restored, and lycopene production was significantly increased, and the lycopene production was 7.97 times that of the starting strain (LYC010), respectively and 4.43 times, the lycopene yield of the strain LYC015 with the highest yield was 44.38mg/L, the yield per unit cell dry weight was 27.97mg/g DCW, and the yield per unit cell was 1.82 times that of LYC010.

Example 3, LYC023 strain construction and lycopene yield determination

LYC015 is the mRSL regulatory element mRSL-4::ispG (sequence 23) that inserts the ispG gene in the genome of the starting bacteria LYC010 to start the expression of the ispG gene, and inserts the ispH gene in the genome of the starting bacteria LYC010 to start ispH Gene expression of mRSL-14::ispH (SEQ ID NO: 32).

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

1. Replace the crt operon promoter in the LYC015 strain with an inducible promoter

LYC023 is the mRSL regulatory element mRSL-4::ispG (sequence 23) that inserts the ispG gene in the genome of the starting bacteria LYC010 to start the expression of the ispG gene, and inserts the ispH gene in the genome of the starting bacteria LYC010 to start the ispH Gene expressed mRSL-14::ispH (SEQ ID NO: 32), and the promoter of the crt operon in the LYC010 genome was replaced with a DNA molecule (SEQ ID NO: 33) containing an inducible promoter Trc promoter and lacI to obtain a recombinant bacterium.

Starting from LYC015, the crt operon was regulated in two steps to construct LYC023 strain, the specific steps are as follows.

(1) Using the pXZ-CS plasmid as a template, ldhA-cat-up and crtE-cat–down as primers, a DNA fragment I of about 3000 bp was obtained by PCR; after DpnI treatment, the DNA fragment I was electrotransferred into Escherichia coli with pKD46 LYC015. Take 500 μl of the transformed bacteria solution and spread it on the LB plate containing chloramphenicol and ampicillin, after culturing overnight at 30°C, screen on the LB plate containing chloramphenicol and ampicillin and the LB plate containing kanamycin respectively, and obtain the It does not grow on the Namycin LB plate, but the clone that grows on the Chloramphenicol Ampicillin LB plate is verified by PCR with primers cat-up and crtE-340-down, and the result is that the recombinant bacteria are correct, and the recombinant bacteria are the proband of the crt operon Escherichia coli with cat-sacB, which can be used for the second step of homologous recombination.

(2) Take QL002 strain (Zhao J, Li Q, Sun T, et al. Engineering central metabolic modules of Escherichia coli for improving beta-carotene production. Metabolicengineering 2013; 17:42-50. Both CAR001 and CAR005D are from this article) The total DNA is used as a template, and the amplification primers are ldhA-up/crtE-340-down, and PCR amplification obtains about 2340bp DNA fragment II; this fragment includes Trc promoter and lacI sequence and about 400bp upstream and downstream homologous arms ( See sequence 33 for the Trc promoter and lacI sequence), and electrotransfer this fragment into the strain containing pKD46 obtained in step (1). Transfer the transformed bacteria solution into 50ml of salt-free LB+10% surcose medium, culture at 37°C and 250rpm for 24 hours with shaking, then streak on a salt-free LB+6%surcose plate, and culture overnight at 41°C to remove the pKD46 plasmid. Screen on LB plates containing chloramphenicol and LB plates without antibiotics respectively, and obtain clones that do not grow on LB plates containing chloramphenicol, and perform PCR verification using primers ldhA-up/crtE-340-down verify. The screening verification was correct, and it was sent for sequencing, and the strain with correct sequencing was named LYC023. The primers used in the case are shown in Table 1, and the constructed strains are shown in Table 2.

2. Determination of lycopene in recombinant Escherichia coli LYC015 and LYC023.

Recombinant Escherichia coli LYC015 and LYC023 were fermented and cultured according to the method of Example 2, and the overnight cultured seed liquid was transferred to a small shake flask (100ml) containing 10ml of fermentation medium, and cultured at 37°C and 250rpm in the dark for 4h 1mM IPTG was used to induce the expression of the crt operon, and after 24 hours of culture, a certain amount of bacteria was taken to measure the lycopene production. The results are shown in Table 3.

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

As shown in Table 3, LYC023 grew slightly better than LYC015, OD600 was 1.06 times that of LYC015, and the yield and unit yield were slightly lower, respectively 76% and 72% of LYC015, but the cells were more stable.

Implementation case 4. Construction of LYC029 strain and determination of lycopene yield

1. The M1-46 artificial regulatory element regulates the glpD gene of LYC023

The recombinant bacterium LYC029 is the mRSL regulatory element mRSL-4::ispG (sequence 23) that will be used to start the mRSL regulatory element mRSL-4::ispG (sequence 23) in the ispG gene in the genome of the starting bacteria LYC010 genome, and the ispH gene in the genome of the starting bacteria LYC010 will be inserted before for mRSL-14::ispH (SEQ ID NO: 32) that initiates ispH gene expression, and the promoter of the crt operon in the LYC010 genome is replaced with a DNA molecule (SEQ ID NO: 33) containing an inducible promoter Trc promoter and lacI, and the LYC010 The promoter of the glpD gene in the genome is replaced with an artificial regulatory element M1-46 to obtain a recombinant bacterium.

See SEQ ID NO: 34 for the promoter sequence of the glpD gene, and see SEQ ID NO: 35 for the sequence of the replaced artificial regulatory element M1-46.

Using the method of two-step homologous recombination, artificial regulatory elements are inserted, as follows:

(1) Using the pXZ-CS plasmid as a template, glpD-cat-up and glpD-cat–down as primers, a DNA fragment I of about 3000 bp was obtained by PCR; after DpnI treatment, the DNA fragment I was electrotransferred into Escherichia coli with pKD46 LYC023. Take 200 μl of the transformed bacteria solution and spread it on the LB plate containing chloramphenicol and ampicillin, after culturing overnight at 30°C, screen it on the LB plate containing chloramphenicol and ampicillin and the LB plate containing kanamycin respectively to obtain the It does not grow on the Namycin LB plate but grows on the Chloramphenicol Ampicillin LB plate. Use primers cat-up and glpD-373-I-r to enter the colony PCR to verify that the recombinant bacteria are correct. The recombinant bacteria are preceded by the glpD gene. Escherichia coli LYC023 of cat-sacB, which can be used for the second step of homologous recombination.

(2) Using the genomic DNA of M1-46 as a template, the amplification primers are glpD-p-up/glpD-RBS-down, and the DNA fragment II of about 200bp is amplified by PCR; this fragment includes the corresponding regulatory elements and the origin of glpD The homology arm of about 50bp upstream and downstream of the initiation codon was electrotransformed into the strain containing pKD46 obtained in step (1). Transfer the transformed bacteria solution into 50ml of salt-free LB+10% surcose medium, culture at 37°C and 250rpm for 24 hours with shaking, then streak on a salt-free LB+6%surcose plate, and culture overnight at 41°C to remove the pKD46 plasmid. Screen on the LB plate containing chloramphenicol and the LB plate without antibiotics, and obtain the clones that do not grow on the LB plate containing chloramphenicol, and perform PCR verification, using primers P-up/glpD-373-I-r verify. The screening verification was correct, and it was sent for sequencing, and the strain with correct sequencing was named LYC029. The primers used in the case are shown in Table 1, and the constructed strains are shown in Table 2.

2. Determination of lycopene in recombinant Escherichia coli LYC023 and LYC029.

Recombinant Escherichia coli LYC023 and LYC029 were fermented and cultured according to the method of Example 2, and the overnight cultured seed liquid was transferred to a small shaker flask (100ml) containing 10ml of fermentation medium, and cultured at 37°C and 250rpm in the dark for 4h 1mM IPTG was used to induce the expression of the crt operon, and after 24 hours of culture, a certain amount of bacteria was taken to measure the lycopene production. The results are shown in Table 3.

As shown in Table 3, after regulating glpD on the basis of LYC023, both cell growth and lycopene production were improved, and the growth and production were adjusted by 22% and 7%, respectively.

LYC029 was deposited on August 19, 2016 in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) It is CGMCC No.12883, and the classification is named as Escherichia coli (Escherichia coli).

Example 5, Lycopene High Density Fermentation of Recombinant Escherichia coli LYC023 and LYC029

Recombinant Escherichia coli LYC023 and LYC029 were subjected to high-density fermentation in a 7L fermenter (Labfors 4; Infors Biotechnology Co. Ltd.). Methods as below:

The process flow is shown in Figure 6, specifically as follows:

1. Take the lycopene strain from the -80°C refrigerator, streak it on the LB plate, and place it in a 37°C incubator for 15 hours.

2. Pick a single colony and inoculate it into a Erlenmeyer flask containing 120ml of LB medium, place it on a shaker at 37°C, and cultivate it at 250rpm until the OD ₆₀₀ is 3.0-4.0, and the obtained bacterial liquid is the seed liquid for high-density fermentation. .

3. Inoculate the prepared seed solution into a 5L fermenter, culture at 37°C, pH 7.0, dissolved oxygen at 20%, cascade dissolved oxygen with stirring and ventilation, and adjust the speed and temperature through the intelligent control system of the instrument Aeration maintained dissolved oxygen at 20%. After the carbon source in the initial medium is exhausted, the dissolved oxygen will suddenly rise. At this time, the feeding is started, and the feeding rate is adjusted by the DO-STAT method to maintain the dissolved oxygen in an appropriate range.

4. Add 0.1mM IPTG to induce when the cell OD grows to about 90. Cultivate 48 hours and ferment and finish.

According to the method in Example 2, the fermentation broth at different time points was taken to measure the cell lycopene production and OD ₆₀₀ .

The results are shown in Figure 5. After 48 hours of culture in LYC023, the OD ₆₀₀ of the cells was 344, the yield of lycopene was 3.14 g/l, and the unit yield was 28.3 mg/g (Figure 5A). LYC029 was cultured for 48 hours, the OD ₆₀₀ of the bacteria was 420, the yield of lycopene was 4.64g/l, which was 47.8% higher than that of LYC023, and the unit yield was 34.2mg/g, which was 20.8% higher than that of LYC023 (Figure 5B).

Claims (10)

1. recombinant bacterium, for following 1) -3) in any one：

1) be improve containing crt operator and produce lycopene Escherichia coli in (E) -4- hydroxy-3-methyl -2- cyclobutenyl - The expression of pyrophosphate synthetase gene ispG and (E) -4- hydroxy-3-methyl -2- cyclobutenyl-pyrophosphoric acid reductase gene ispH And/or activity, obtain recombinant bacterium；

2) be regulation and control 1) shown in recombinant bacterium crt operator expression and/or activity, the recombinant bacterium obtaining；

3) be regulation and control 2) shown in the glpD gene expression of recombinant bacterium and/or activity, the recombinant bacterium obtaining.

2. recombinant bacterium according to claim 1 it is characterised in that：Described raising produces (E) -4- in lycopene Escherichia coli Hydroxy-3-methyl -2- cyclobutenyl-pyrophosphate synthetase gene ispG and (E) -4- hydroxy-3-methyl -2- cyclobutenyl-pyrophosphoric acid The expression of reductase gene ispH and/or activity are to be realized by following (1) or (2)：

(1) insertion before the described ispG gene producing in lycopene Escherichia coli is started shown in the sequence 23 of ispG gene expression Controlling element mRSL-4::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started ispH base Because of the mRSL-14 shown in the sequence 32 of expression::ispH；

(2) insertion before the described ispG gene producing in lycopene Escherichia coli is started shown in the sequence 21 of ispG gene expression MRSL controlling element mRSL-1::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started The mRSL-14 shown in sequence 32 of ispH gene expression::ispH；

Described regulation and control 1) shown in the crt operator expression of recombinant bacterium and/or activity be by 1) shown in crt operator in recombinant bacterium Promoter replace with inducible promoter；

Described regulation and control 2) shown in the glpD gene expression of recombinant bacterium and/or activity be by 2) shown in glpD gene in recombinant bacterium Promoter is substituted for artificial regulatory element M1-46.

3. recombinant bacterium according to claim 1 and 2 it is characterised in that：

Described insertion is to carry out by the way of genome pinpoints editor or homologous recombination；

Or described genome fixed point editor is specially that ZFN edits, TALEN edits or CRISPR/Cas9 edits；

Or described homologous recombination is specially λ-red homologous recombination or the homologous recombination of sacB gene mediated screening or integrated plasmid is situated between The homologous recombination led.

4. according to described recombinant bacterium arbitrary in claim 1-3 it is characterised in that：

Described inducible promoter is the DNA molecular containing inducible promoter Trc promoter and lacI；

Or the described nucleotides sequence containing inducible promoter Trc promoter and the DNA molecular of lacI is classified as sequence 33；

Or the nucleotides sequence of described artificial regulatory element M1-46 is classified as sequence 35；

Or described product lycopene Escherichia coli be LYC010.

5. according to described recombinant bacterium arbitrary in claim 1-4 it is characterised in that：

3) preserving number of the recombinant bacterium shown in is CGMCC No.12883.

6. a kind of method building arbitrary described recombinant bacterium in claim 1-5, comprises the steps：

A-C：

A is following 1) or 2)：

1) before the ispG gene that will produce in lycopene Escherichia coli, insertion starts the tune shown in the sequence 23 of ispG gene expression Control element mRSL-4::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started ispH gene table The mRSL-14 shown in sequence 32 reaching::IspH, obtains recombinant bacterium；

2) insertion before the described ispG gene producing in lycopene Escherichia coli is started shown in the sequence 21 of ispG gene expression MRSL controlling element mRSL-1::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started The mRSL-14 shown in sequence 32 of ispH gene expression::IspH, obtains recombinant bacterium；

B, by A) promoter of crt operator in the recombinant bacterium that obtains replaces with inducible promoter, obtains recombinant bacterium；

C, by B) promoter of glpD gene in the recombinant bacterium that obtains is substituted for artificial regulatory element M1-46, obtains recombinant bacterium.

7. method according to claim 6 it is characterised in that：

Described insertion is to carry out by the way of genome pinpoints editor or homologous recombination；

Or described genome fixed point editor is specially that ZFN edits, TALEN edits or CRISPR/Cas9 edits；

Or described homologous recombination is specially λ-red homologous recombination or the homologous recombination of sacB gene mediated screening or integrated plasmid is situated between The homologous recombination led.

8. the method according to claim 6 or 7 it is characterised in that：Described inducible promoter is to start containing induction type Sub- Trc promoter and the DNA molecular of lacI；

Or the described nucleotides sequence containing inducible promoter Trc promoter and the DNA molecular of lacI is classified as sequence 33；

Or the nucleotides sequence of described artificial regulatory element M1-46 is classified as sequence 35；

Or described product lycopene Escherichia coli be LYC010.

9. application in producing lycopene for arbitrary described recombinant bacterium in claim 1-5.

10. a kind of method producing lycopene, comprises the steps：Arbitrary described recombinant bacterium in fermentation claim 1-5, Obtain lycopene.