CN110551748A - super-recombinant mycobacterium smegmatis producing nicotinic acid and construction method thereof - Google Patents

Abstract

the invention provides a construction method of super-recombinant mycobacterium smegmatis for producing nicotinic acid, which comprises the steps of 1) constructing a plasmid pMHS-NrtR _ms, 2) constructing a plasmid pHAGE-NrtR _ms, 3) preparing a recombinant TM4 bacteriophage, 4) constructing nrtR _ms gene deletion strain mycobacterium smegmatis mc ² 100, 5) amplifying a nucd gene and constructing a plasmid pMV361-NudC _ms, and 6) constructing nrtR gene knockout/nucd gene overexpression super-recombinant mycobacterium smegmatis.

Description

Super-recombinant mycobacterium smegmatis producing nicotinic acid and construction method thereof

Technical Field

the invention relates to the technical field of nicotinic acid production, and particularly relates to super recombinant mycobacterium smegmatis for producing nicotinic acid and a construction method thereof.

Background

Nicotinic acid, 3-picolinic acid, also known as vitamin B3, belongs to the B vitamin group, is a water-soluble vitamin, belongs to the B vitamin group, and is one of the 13 vitamins essential to the human body. Nicotinic acid is an important factor for resisting a hypecopathy and a hydrogen carrier in body tissues, and has the effects of maintaining the health of skin and nerves and promoting digestion. In the absence of it, pellagra may occur, manifested as dermatitis, glossitis, oropharynx, diarrhea, dysphoria, insomnia, and paresthesia. Nicotinamide, collectively known as vitamin PP, is used for combating pellagra and also as a vasodilator. As a medical intermediate, is used for producing isoniazid, nicotinamide, nicousamide, inositol nicotinate and the like. Niacin is also used in food products, meat additives and feed additives to prevent pellagra. Especially, the feed added with nicotinic acid can improve the disease resistance of livestock and poultry, accelerate the growth, improve the utilization rate of the feed, save a large amount of feed and reduce the feeding cost. Compared with the control group, the milk yield of the dairy cows fed with the feed added with the nicotinic acid in the United states can be improved by 15 to 20 percent. In addition, nicotinic acid can also be used as a deodorant for biochemical hormones, air and exhaust gas which form activated sludge. Nicotinic acid also has certain application in the dye industry, the photosensitive material industry, hair dyeing auxiliaries, detergents and the like.

The most commonly used method is to synthesize nicotinic acid by using 3-methylpyridine as a raw material and using an air oxidation method, the 3-methylpyridine, air and ammonia gas are proportionally introduced into a fluidized bed reactor, and react at 290-360 ℃ under the catalysis of V ₂ O ₅ to generate nicotinonitrile, then the nicotinonitrile is hydrolyzed in a sodium hydroxide aqueous solution at a high pressure at 160 ℃ to generate sodium nicotinate, and finally the nicotinic acid is acidified by hydrochloric acid to obtain the nicotinic acid, the nicotinonitrile is generated and hydrolyzed into the nicotinic acid by a chemical reaction under the conditions of high temperature, high pressure and strong acid, the requirements on equipment are met, the equipment is not corroded, and the environment is friendly, CN114288A of the Qinghua viola company reports that 3-methylpyridine is used for generating the nicotinic acid, the nicotinic acid is generated by a one-step catalytic reaction, the nicotinic acid sulfate is used as a strong alkali sulfate, the nicotinic acid sulfate is added into the alkaline sulfate, and the nicotinic acid sulfate is used as a strong alkali sulfate, and the nicotinic acid sulfate is generated by adding the alkaline sulfate and the sulfuric acid.

In the chemical synthesis process of the nicotinic acid, a specific high-temperature and high-pressure environment is required or strong acid, strong base or a chemical catalyst is adopted for treatment, the reaction selectivity is not high, a large number of byproducts are generated, the product yield is not high, and the environmental pollution is large. In contrast, the biological method for preparing nicotinic acid has the characteristics of high substrate selectivity, high catalytic efficiency, mild reaction conditions, small environmental pollution and the like. In addition, the biological method is easy to amplify, has low cost and is suitable for large-scale industrial production and application. The biocatalytic production of nicotinic acid from 3-cyanopyridine has been reported using the microorganisms Bacillus subtilis, Rhodococcus rhodochrous, Nocardia, Fusarium solani and Pseudomonas putida for fermentation. However, the biocatalytic method for preparing nicotinic acid mainly relies on microbial fermentation to produce nitrilase, and the nitrilase catalyzes the conversion of 3-cyanopyridine into nicotinic acid by the nitrilase, and the raw material 3-cyanopyridine still needs to be added.

disclosure of Invention

the invention aims to overcome the defects of the prior art and provides a construction method of super recombinant mycobacterium smegmatis for producing nicotinic acid.

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

A construction method of super recombinant mycobacterium smegmatis for producing nicotinic acid comprises the following steps:

1) construction of plasmid pMHS-NrtR _ms:

1-1) carrying out PCR amplification by using a genomic DNA of mycobacterium smegmatis mc ² 155 as a template and primers shown in SEQ ID No. 1-4, and carrying out restriction enzyme digestion by Van91I for later use after electrophoretic separation;

1-2) carrying out amplification culture on Escherichia coli DH5 alpha strain containing pMHS plasmid, then collecting thalli, extracting plasmid, carrying out electrophoretic separation after the plasmid is cut by Van91I restriction endonuclease, and recovering DNA fragments with 1600bp interval and 3600bp interval;

1-3) reacting the DNA fragments obtained in the steps 1-1 and 1-2 with T4 DNA ligase at 16 ℃ overnight, transforming the ligation product into escherichia coli DH5 alpha competent cells for expanded culture, and extracting plasmids to obtain a plasmid pMHS-NrtR _ms;

2) construction of plasmid pHAGE-NrtR _ms:

2-1) respectively carrying out amplification culture on Escherichia coli DH5 alpha strains containing plasmids pHAGE and pMHS-NrtR _ms, extracting plasmids, linearizing the two plasmids by PacI restriction endonuclease, then reacting at 16 ℃ overnight by using T4 DNA ligase, packaging a ligation product by using a packaging kit, transforming the ligation product into Escherichia coli HB101 competent cells, carrying out amplification culture, and extracting plasmids to obtain plasmid pHAGE-NrtR _ms;

3) Preparation of recombinant TM4 phage:

3-1) carrying out amplification culture on mycobacterium smegmatis mc ² 155, carrying out shaking culture until logarithmic phase, centrifuging to collect thalli, washing the thalli by using precooled 10% sterile glycerol, then adding precooled 10% glycerol to resuspend the thalli, and freezing and storing at-80 ℃ for later use;

3-2) transferring the plasmid pHAGE-NrtR _ms into a Mycobacterium smegmatis mc ² 155 electrotransformation competent cell, incubating for 10min on ice, then performing electric shock transformation, adding a 7H9 liquid culture medium after transformation, incubating overnight in an incubator at 37 ℃, adding a bacterium solution placed at 37 ℃ into an EP tube containing top agar, uniformly mixing, pouring and flatly paving on a 7H10 solid plate, and placing the plate in the incubator at 30 ℃ for culturing for 2-3 days to obtain a recombinant TM4 bacteriophage;

4) Constructing nrtR _ms gene-deleted strain mycobacterium smegmatis mc ² 100:

Culturing mycobacterium smegmatis mc ² 155 with a 7H9 liquid culture medium until logarithmic growth phase, centrifugally collecting thalli, washing the thalli obtained by centrifugation with an MP buffer, then re-suspending the thalli with the MP buffer, mixing the re-suspended mycobacterium smegmatis mc ² 155 with a recombinant TM4 phage 1 with the titer of 10 ¹⁰ PFU by volume of 1:1, incubating in an incubator, centrifugally collecting the upper clear liquid of the thalli, re-suspending with a 7H9 liquid culture medium, placing in the incubator for incubating overnight, centrifugally collecting the upper clear liquid again, coating the thalli with a 7H10 solid plate for culturing, and obtaining mutant mycobacterium smegmatis mc ² 100 secreting nicotinic acid;

5) amplification of the nucd gene and construction of plasmid pMV361-NudC _ms:

5-1) nudC gene amplification, namely performing PCR amplification on a nudC gene ORF sequence by using a primer pair shown in SEQ ID No. 9-10 and using Mycobacterium smegmatis mc ² 155 genome DNA as a template, performing electrophoretic separation and recovery on a PCR product, performing enzyme digestion by using EcoRI and HindIII restriction enzymes, and recovering the DNA subjected to enzyme digestion for later use;

5-2) enzyme cutting: LB culture contains pMV361 plasmid Escherichia coli DH5 alpha strain, extract plasmid, use EcoR I and Hind III restriction enzyme digestion, then electrophoretic separation, reclaim the linearized plasmid;

5-3) connecting, namely reacting the DNA fragment recovered by enzyme digestion and the linearized plasmid pMV361 recovered by the same enzyme digestion at 16 ℃ with T4 DNA ligase overnight, transforming escherichia coli DH5 alpha competent cells by the connecting product and coating an LB solid plate, and culturing the plate in a constant temperature incubator at 37 ℃ overnight to obtain a plasmid pMV361-NudC _ms, wherein the LB solid plate contains 100 mu g/ml kanamycin sulfate;

6) Constructing the super recombinant mycobacterium smegmatis with nrtR gene knockout/nucDC gene overexpression:

6-1) preparing competent cells of the mutant mycobacterium smegmatis mc ² 100, namely culturing the mutant mycobacterium smegmatis mc ² 100 in a 7H9 liquid culture medium until logarithmic phase, centrifugally collecting thalli, washing the thalli at least twice by using precooled 10% sterile glycerol, finally adding a proper amount of precooled 10% glycerol, uniformly blowing the thalli, and separately storing the thalli at-80 ℃ for later use;

6-2) transformation of mutant Mycobacterium smegmatis mc ² 100 plasmid pMV361-NudC _ms is transferred into mutant Mycobacterium smegmatis mc ² 100 electrotransfer competent cells, the cells are incubated on ice for 10min, then the transformation is performed by electric shock, then 7H9 liquid culture medium is added, the cells are incubated overnight in an incubator at 37 ℃, then a proper amount of culture solution is taken to coat a 7H10 solid plate, the plate is placed in the incubator at 37 ℃ for 3-5 days, and the super recombinant Mycobacterium smegmatis producing nicotinic acid is obtained, wherein the 7H10 solid plate contains 50 mu g/mL kanamycin sulfate.

wherein the scale-up culture in step 1-2 is to inoculate the strain to LB liquid medium containing 150. mu.g/mL hygromycin B, and to culture the strain overnight at 37 ℃.

the amplification culture in the step 1-3 is to inoculate the strain to LB solid plate culture medium containing 150 mug/mL hygromycin B, to culture the strain overnight at 37 ℃, then to pick up the monoclonal colony growing on the plate to inoculate to LB liquid culture medium containing 150 mug/mL hygromycin B, to culture the strain overnight at 37 ℃ with shaking at 200 rpm.

in step 2-1, plasmid pHAGE was expanded in LB medium containing ampicillin resistance, and plasmid pMHS-NrtR _ms was expanded in LB medium containing hygromycin B resistance.

and 2-1, performing amplification culture on the escherichia coli HB101 competent strain transformed by the ligation product, and then performing plasmid extraction, wherein the amplification culture is to inoculate the strain to an LB solid plate culture medium containing 150 mu g/mL hygromycin B, performing overnight culture at 37 ℃, then picking a monoclonal colony growing on the plate to inoculate to an LB liquid culture medium containing 150 mu g/mL hygromycin B, and performing shaking culture on a shaking table at 200rpm at 37 ℃ for overnight culture.

The method for the expanded culture of the mycobacterium smegmatis mc ² 155 in the step 3-1 comprises the steps of inoculating mycobacterium smegmatis mc ² 155 in a 7H9 liquid culture medium, carrying out shaking culture at the temperature of 37 ℃ and 200rpm until the logarithmic growth phase, wherein OD600 is 0.5-1.0, inoculating a culture in a fresh 7H9 liquid culture medium in a ratio of 1:100, and carrying out overnight culture at the temperature of 37 ℃ until OD600 is 0.6.

centrifuging the collected thalli in the step 3 for 10min at the temperature of 4 ℃ and at the rpm of 5000; centrifuging the collected thalli in the step 4 at 6000rpm for 10 min; the centrifugation conditions for collecting the bacteria in the step 6 are 4 ℃ and centrifugation is carried out for 10min at 5000 rpm.

the method for culturing the mycobacterium smegmatis mc ² 100 to the logarithmic growth phase in the 7H9 liquid medium in the step 6-1 is to inoculate a colony of the mycobacterium smegmatis mc ² 100 in 5mL of 7H9 liquid medium, culture is performed at 37 ℃ and 200rpm in a shaking mode until the OD600 of the logarithmic growth phase is 0.5-1.0, the culture is inoculated in fresh 100mL of 7H9 liquid medium in a ratio of 1:100, and culture is performed at 37 ℃ overnight until the OD600 is 0.6.

the electric shock conversion parameters in the step 3-2 and the step 6-2 are both voltage 2.5kV, resistance 1000 omega and capacitance 25 muF.

The method also comprises the following steps after the step 5-3:

5-4) selecting a monoclonal colony growing on the plate, inoculating the monoclonal colony into an LB liquid culture medium, shaking by a shaker at 37 ℃ at 200rpm for overnight culture, extracting a plasmid, carrying out restriction enzyme digestion on the obtained plasmid by using EcoRI and Hind III restriction enzymes, detecting and verifying by nucleic acid gel electrophoresis, verifying that a nucd gene ORF sequence is connected to a pMV361 vector, and simultaneously verifying that the plasmid sequence has no mutation.

the method also comprises the following steps after the step 6-2:

6-3) selecting monoclones growing on a plate, inoculating the monoclones into 5mL of 7H9 liquid culture medium, culturing for 2-3 days until the logarithmic phase, centrifugally collecting thalli, extracting genomic DNA as a template, and verifying the transformation of pMV361-NudC _ms plasmid into mutant mycobacterium smegmatis mc ² 100 by using a primer pair shown in SEQ ID No. 13-14 through PCR.

The invention has the beneficial effects that: the invention prepares the super recombinant mycobacterium smegmatis by constructing nrtR gene knockout and ncdC gene overexpression, the engineering bacteria prepared by the invention can directly obtain nicotinic acid in one step without depending on adding 3-cyanopyridine, not only avoids various defects in the chemical synthesis process of the nicotinic acid, but also has simple production condition, no pollution, simple and easy operation, easy amplification and low cost, is suitable for large-scale industrial production and application and has great popularization and application values.

drawings

FIG. 1 is an electrophoretogram of mc ² 155 gene of Mycobacterium smegmatis amplified by PCR, wherein, in FIG. 1, a Lane M is a DNA marker, a Lane 1 is a DNA fragment shown in SEQ ID No.6, and a Lane 2 is a DNA fragment shown in SEQ ID No. 5;

FIG. 2 is a diagram of the electrophoresis of pMHS plasmid after digestion; lane M in fig. 2: DNA marker; lane 1: the arrows indicate 1600bp and 3600bp DNA fragments after pMHS plasmid enzyme digestion;

FIG. 3 shows the nucleic acid gel electrophoresis of pMV361-NudC _ms plasmid after restriction enzyme digestion with EcoRI and Hind III restriction enzymes, M is DNA marker, 1-3 is positive plasmid;

FIG. 4 is a schematic diagram of a successfully constructed plasmid map of pMV361-NudC _ms;

FIG. 5 is a diagram showing a genome of a super recombinant Mycobacterium smegmatis and a verification result, wherein in FIG. 5A, the genome of the super recombinant Mycobacterium smegmatis is shown schematically, in FIG. 5B, 1, a PCR verification diagram of a knockout strain, 2, a wild type control strain and in FIG. 5C, a verification diagram of PCR verification Pmv361-NudC _ms plasmid transformation into mutant Mycobacterium smegmatis mc ² 100 are shown;

FIG. 6 shows HPLC separation chromatograms of culture solution filtrate of wild bacteria and super recombinant strain and nicotinic acid standard substance, wherein A is culture solution filtrate of super recombinant strain, B is culture solution filtrate of control strain Mycobacterium smegmatis mc ² 155, and C is nicotinic acid standard substance.

Detailed Description

the concept and technical effects of the present invention will be clearly and completely described in the following embodiments to fully understand the objects, aspects and effects of the present invention. It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.

EXAMPLE 1 construction of plasmid pMHS-NrtR _ms

1) Performing PCR amplification by using a genomic DNA of mycobacterium smegmatis mc ² 155 as a template and primers shown in SEQ ID Nos. 1-4, wherein as shown in FIG. 1, a target DNA fragment is separated by performing gel electrophoresis on a PCR product nucleic acid, the target DNA fragment shown in SEQ ID Nos. 5-6 is recovered by using a colloidal recovery kit of Omega company, the recovered DNA is subjected to enzyme digestion by Van91I restriction enzyme, and the enzyme-digested DNA is recovered by using a Cycle-pure recovery kit of Omega company for later use;

2) inoculating Escherichia coli DH5 alpha strain containing pMHS plasmid into LB liquid medium (adding 150. mu.g/mL hygromycin B), culturing overnight at 37 ℃, then collecting thalli, extracting plasmid by using Omega plasmid extraction kit, performing nucleic acid gel electrophoresis after the obtained pMHS plasmid is cut by Van91I restriction enzyme, and recovering about 1600bp and 3600bp two DNA fragments (as shown by arrows in figure 2) by using Omega gel recovery kit;

3) reacting the DNA fragments obtained in the steps 1 and 2 with T4 DNA ligase at 16 ℃ overnight, transforming the ligation product into escherichia coli DH5 alpha competent cells, coating an LB solid plate (containing 150 mu g/mL hygromycin B), placing the plate in a 37 ℃ constant temperature incubator for overnight culture, then picking out a single colony growing on the plate, inoculating an LB liquid culture medium (containing 150 mu g/mL hygromycin B) on a shaking table at 200rpm for overnight culture at 37 ℃, then using an Omega plasmid extraction kit to extract a plasmid, digesting the Van91I restriction enzyme with the obtained plasmid, connecting the DNA fragment to a pMHS vector through nucleic acid gel electrophoresis detection and verification, and obtaining a positive plasmid pMHS-NrtR _ms.

Meanwhile, the correct plasmid pMHS-NrtR _ms is verified to have no mutation in sequence by the sequencing verification of Onck corporation.

Example 2 construction of plasmid pHAGE-NrtR _ms

Respectively culturing Escherichia coli DH5 alpha strain containing plasmid pHAGE (ampicillin resistance) and plasmid pMHS-NrtR _ms (hygromycin B resistance) by LB, extracting plasmids, linearizing the two plasmids by PacI restriction endonuclease, reacting at 16 ℃ overnight by using T4 DNA ligase, packaging a ligation product by using a packaging kit, transforming to Escherichia coli HB101 competent cells, then coating on an LB solid plate (containing 150 mu g/mL hygromycin B), placing the plate in a 37 ℃ constant temperature incubator for overnight culture, picking up monoclones growing on the plate, inoculating an LB liquid culture medium (containing 150 mu g/mL hygromycin B) for overnight shaking culture by a shaker at 37 ℃ and shaking at 200rpm, and extracting the plasmids to obtain the plasmid pHAGE-NrtR _ms.

Example 3: preparation of recombinant TM4 phage

1) inoculating mycobacterium smegmatis mc ² 155 in 5mL 7H9 liquid culture medium, performing shaking culture at 37 ℃ and 200rpm until OD600 is 0.5-1.0 in logarithmic growth phase, inoculating the culture in a fresh 100mL 7H9 liquid culture medium at a ratio of 1:100, performing overnight culture at 37 ℃ until OD600 is about 0.6, centrifuging at 4 ℃ and 5000rpm for 10min to collect thalli, washing the thalli twice with precooled 10% sterile glycerol, finally adding 10mL (proper amount) of precooled 10% glycerol, resuspending the thalli, and performing split freezing at-80 ℃ for later use;

2) The plasmid pHAGE-NrtR _ms is transferred into a Mycobacterium smegmatis mc ² 155 electrotransferase competent cell, the cell is incubated for 10min on ice, then the cell is transferred into a BTX electrotransferase cup with the diameter of 2mm, the water on the outer wall of the electrotransferase cup is wiped off, then a BTX ECM630 electrotransferase instrument is used for electric shock, the electric shock parameters are that the voltage is 2.5kV, the resistance is 1000 omega, the capacitance is 25 muF, after the electric shock is finished, 1mL of 7H9 liquid culture medium is immediately added, the cell is incubated overnight in an incubator at the temperature of 37 ℃, the bacteria liquid placed at the temperature of 37 ℃ is added into a 5mL of EP tube containing 4mL of Top Agar (Top Agar 100 mL: Middlebrook 7H9br 0.47g and Agar 0.75g) to be mixed evenly and poured and laid flat on a 7H10 solid plate, the cell is placed for 2-3 days, and the recombinant TM4 bacteriophage is obtained.

Example 4 construction of nrtR _ms Gene-deleted Strain Mycobacterium smegmatis mc ² 100

7H9 liquid medium culture of M.smegmatis mc ² 155 to logarithmic growth phase (OD 0.5-1.0), 6000rpm, 10min centrifugation collection of thallus, MP buffer washing the thallus obtained by centrifugation, then MP buffer heavy suspension of thallus, heavy suspension of Mycobacterium smegmatis mc ² 155 with the titer of 10 ¹⁰ PFU recombinant TM4 phage 1:1 volume after mixing incubator incubation, then centrifugation collection of thallus abandoning supernatant and then heavy suspension with 7H9 liquid medium, left incubator overnight, then 6000rpm, 10min centrifugation collection of thallus abandoning supernatant, thallus coating 7H10 solid plate culture (containing 150. mu.g/mL hygromycin B), obtaining nicotinic acid secreting mutant M.smegmatis mc ² 100. using microbial genome extraction kit (Omega) to extract genomic DNA, using the extracted genomic DNA as template, using primers shown in SEQ ID No. 7-8 to PCR, the verification results are shown in the verification result B in FIG. 5.

example 5 amplification and construction of the NudC Gene plasmid pMV361-NudC _ms

1) amplifying the nucc gene, namely taking the genomic DNA of mycobacterium smegmatis mc ² 155 as a template, carrying out PCR amplification on a nucd gene ORF sequence by using a primer pair shown in SEQ ID No. 9-10, separating a target DNA fragment by carrying out nucleic acid gel electrophoresis on a PCR product, recovering the target DNA fragment by using a glue-back kit of Omega company, sequencing to obtain a nucd gene ORF sequence shown in SEQ ID NO.11, wherein the amino acid sequence of the encoded protein is shown in SEQ ID NO.12, carrying out enzyme digestion on the recovered DNA by using EcoRI and HindIII restriction endonucleases, and recovering the enzyme-digested DNA for later use by using a Cycle-pure recovery kit of Omega company;

2) Enzyme digestion: LB culture containing pMV361 plasmid Escherichia coli DH5 alpha strain, use Omega company plasmid extraction kit to extract the plasmid, the pMV361 plasmid obtained uses EcoR I and Hind III restriction enzyme digestion, then carry on the nucleic acid gel electrophoresis, use Omega company glue to reclaim the linearized plasmid finally;

3) Connecting, namely reacting the DNA fragment recovered by enzyme digestion and the linearized plasmid pMV361 recovered by the same enzyme digestion at 16 ℃ with T4 DNA ligase for overnight, transforming escherichia coli DH5 alpha competent cells by using a connecting product, coating an LB solid plate (containing 100 mu g/mL kanamycin sulfate), and culturing the plate in a constant-temperature incubator at 37 ℃ for overnight to obtain a plasmid pMV361-NudC _ms;

4) Screening positive plasmids, namely selecting a monoclonal colony growing on a plate, inoculating an LB liquid culture medium, shaking at 37 ℃ and shaking at 200rpm for overnight culture, extracting the plasmids by using an Omega plasmid extraction kit, carrying out restriction enzyme digestion on the obtained plasmids by using EcoRI and Hind III, detecting and verifying by nucleic acid gel electrophoresis, connecting a nucd gene ORF sequence to a pMV361 vector, and verifying the result as shown in figure 3, simultaneously verifying that the correct plasmids pMV361-NudC _ms have no mutation through sequencing verification sequences of the promoter company, and constructing a successful pMV361-NudC _ms plasmid map as shown in figure 4.

Example 6: construction of nrtR gene knockout/nudC gene over-expression super recombinant Mycobacterium smegmatis

1) preparing competent cells of mycobacterium smegmatis mc ² 100, namely selecting fresh mycobacterium smegmatis mc ² 100 bacterial colonies to inoculate in 5mL of 7H9 liquid culture medium, carrying out shaking culture at 37 ℃ and 200rpm until logarithmic growth phase (OD 0.5-1.0), inoculating the culture in a fresh 100mL of 7H9 liquid culture medium according to the proportion of 1:100, carrying out overnight culture at 37 ℃ until OD600 is about 0.6, centrifuging at 4 ℃ and 5000rpm for 10min to collect thalli, washing the thalli at least twice by using precooled 10% sterile glycerol, finally adding 10mL (proper amount) of precooled 10% glycerol, blowing the thalli evenly, and carrying out split charging and freezing storage at-80 ℃ for later use;

2) transformation of Mycobacterium smegmatis mc ² 100, adding correctly constructed positive plasmid pMV361-NudC _ms DNA into 200 mu L of mutant Mycobacterium smegmatis mc ² 100 electroconceptive cells, incubating on ice for 10min, transferring into a 2mm BTX electric rotor, wiping off water on the outer wall of the electric rotor, then using a BTX ECM630 electric converter to shock, wherein the shock parameters are voltage of 2.5kV, resistance of 1000 omega, capacitance of 25 mu F, immediately adding 1mL of 7H9 liquid culture medium after shock is finished, taking a proper amount of culture solution to coat a 7H10 solid plate (containing 50 mu g/mL kanamycin sulfate) after incubation in an incubator at 37 ℃ overnight, and placing the plate in the incubator at 37 ℃ for culturing for 3-5 days;

3) screening positive recombinant bacteria, namely picking single clones growing on a plate, inoculating the single clones into 5mL of 7H9 liquid culture medium, culturing for 2-3 days to a logarithmic growth phase, centrifugally collecting thalli, extracting genomic DNA by using a microbial genome extraction kit (Omega), taking the extracted genomic DNA as a template, and using primers shown in SEQ ID NO. 13-14 to carry out PCR verification on the pMV361-NudC _ms plasmid to transfer into the mutant M.smegmatis mc ² 100, wherein the verification result is shown as C in figure 5, the PCR result verifies that the strains have been successfully introduced, the strains which verify correct NudC _ms overexpression are named as super recombinant Mycobacterium smegmatis, and the genome schematic diagram of the super recombinant M.smegmatis is shown as A in figure 5.

Example 7: detection of super recombinant mycobacterium smegmatis secretion nicotinic acid

And (3) comparing and analyzing the nicotinic acid secretion levels of wild type, nrtR gene knockout/nudC gene over-expressed super recombinant Mycobacterium smegmatis 2 bacteria.

the super recombinant Mycobacterium smegmatis in which nrtR gene knockout/nudC gene overexpression is inoculated with 7H9 liquid medium, cultured at 37 ℃ until after logarithmic growth phase, the culture solution is filtered by using a Millipore 0.22 μm sterile filter and the filtered filtrate is collected, then macromolecules such as protein in the filtrate are removed by using a Millipore 3-kDa ultrafiltration concentration tube and the filtered filtrate is collected. The obtained filtrate was separated and analyzed for nicotinic acid using High Performance Liquid Chromatography (HPLC).

specific conditions of HPLC:

the equipment model is as follows: thermo Fisher Scientific UltiMate 3000 ultra-high-performance liquid chromatography;

a chromatographic column: thermo Fisher, Hypersil Gold aQ, 150X 4.6mm, 3 μm, column temperature: 30 ℃;

mobile phase: water phase: deionized water (containing 0.1% formic acid);

organic phase: acetonitrile;

Gradient elution: at 0min, 95% aqueous phase + 5% organic phase;

at 30min, 5% aqueous phase + 95% organic phase;

Flow rate: 0.3 mL/min;

sample introduction amount: 10 mu L of the solution;

Wavelength: 284 nm;

The HPLC results are shown in FIG. 6, which shows that nicotinic acid is contained in the super recombinant Mycobacterium smegmatis culture fluid with NrtR gene knockout/nucDC gene overexpression.

SEQUENCE LISTING

<110> institute of Buddha science and technology

<120> super recombinant mycobacterium smegmatis for producing nicotinic acid and construction method thereof

<130> 2019

<160> 14

<170> PatentIn version 3.3

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cagctgctgt accgcaccac cgatcgcaac ggcgctcccg aggccgcggt caccacggtg 360

atcatgccgc ccgacgcgcg cgcgggctgc ccgatcgtgt cctaccagtg cgcgatcgac 420

gcgatctcgg ccacgtgctt cccgtcgtac gcgctgcgcc gtcacgccaa ggtcaccggt 480

ggcctcgccc agttcgagct cctgctgatc accgcggcgc tggccgaggg ctgggccgtc 540

tcggtgcccg accacgaagg cctcgacggc atgtggggcg cgccgtacga acccgggtac 600

cacgtgctcg acgggctgcg tgccgcgctg aactccgagc gcctgcccct gaccgacgcg 660

tcgcccatcg gcctgtgggg ctactcgggc ggcggtctgg caagtggctg ggccgcggag 720

atgagcggct cctacgcccc cgagctcaac atcgtcggcg ccgtactcgg ttcgccggtc 780

ggcgatctgg gccacacgtt ccggcggctc aacggcacgg tgttctcggg actgcccgca 840

ctcgtcgtcg ccgccctcgc cgacatctac ccgggcctca atcgggtcat cgccgagcac 900

gcgaccaccg agggccgcaa gctgctgcag cgcctgcaca agatgagcac catcgaggcc 960

gtgctgcggc tggcgcgcaa ggacatggac gacctggtcg acctgccgct cgaacagatc 1020

ctcgacagcc ccgaggtcac cgaggtcttc gacgacatca aactcggtgt ggcgacaccg 1080

aatccacccg tgctgctga 1099

<210> 7

<211> 18

<212> DNA

<213> Artificial Synthesis

<400> 7

gacgaggcac cgcagcaa 18

<210> 8

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 8

tcgaggagac cgagatcagc 20

<210> 9

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 9

atcgggatcc atgagcgaac accgcacgt 29

<210> 10

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 10

tgcaaagctt tcagtcgagt gcggcccagg 30

<210> 11

<211> 936

<212> DNA

<213> Artificial Synthesis

<400> 11

atgagcgaac accgcacgtt cgggctccgt aacgtcccgc tgctgtcccg ggtcggcgcc 60

gatcgcgccg ataccttgcg caccgacgtc gacgccgccc tggcgggctg gcccgacgcg 120

ctggtgctac gcgtggaccg ccgcaaccag gtgctcatcg ccaacggtca ggtggtgctc 180

ggtgaggccg gcgcactcgg agaccggccg cccgagcacg cggtgttcct gggacgtctg 240

caggacggca ggcacgtatg gggtatccgg gcggatctgg aggcgcccga ggatgccgac 300

ctggggaccg aggtgctcga cctgcgccgg gccgggcaga tcttcgacga caccagcgcc 360

cagttggtgg cgaccgccac ggcgctgctc aactggcatg acaacgcgcg gcacagcgcg 420

atcgacgggg cgccgacccg gcccgccaag ggcggctggt cgcgcgtcaa cccgctgacc 480

ggccacgagg agttcccgcg gatcgacccc gccatcatct gcctggtgca cgacgggcat 540

gaccgggcgg tgctggcccg tcagacgctg tggccggagc ggttgttctc gatcctggcc 600

ggcttcgtcg aggccggcga gtcgttcgag acatgcgtgc agcgcgagat cgccgaggag 660

atcgggctca cggtcaccga cgtgcagtac ctcggcagtc agccgtggcc gttcccgcgc 720

tcgctcatgg tgggattcca cgcgatcggc gacccggagc agccgttctc ctacaacgac 780

ggcgagatcg ccgaggccgc gtggttcacc cgcgatgaga tccgcgcggc actcgatcag 840

ggggactgga acagcgactc gccgtcacgg ctcctgctgc caggctccat ctcgatcgcc 900

cgcgagatca tcgaatcctg ggccgcactc gactga 936

<210> 12

<211> 311

<212> PRT

<213> Artificial Synthesis

<400> 12

Met Ser Glu His Arg Thr Phe Gly Leu Arg Asn Val Pro Leu Leu Ser

1 5 10 15

Arg Val Gly Ala Asp Arg Ala Asp Thr Leu Arg Thr Asp Val Asp Ala

20 25 30

Ala Leu Ala Gly Trp Pro Asp Ala Leu Val Leu Arg Val Asp Arg Arg

35 40 45

Asn Gln Val Leu Ile Ala Asn Gly Gln Val Val Leu Gly Glu Ala Gly

50 55 60

Ala Leu Gly Asp Arg Pro Pro Glu His Ala Val Phe Leu Gly Arg Leu

65 70 75 80

Gln Asp Gly Arg His Val Trp Gly Ile Arg Ala Asp Leu Glu Ala Pro

85 90 95

Glu Asp Ala Asp Leu Gly Thr Glu Val Leu Asp Leu Arg Arg Ala Gly

100 105 110

Gln Ile Phe Asp Asp Thr Ser Ala Gln Leu Val Ala Thr Ala Thr Ala

115 120 125

Leu Leu Asn Trp His Asp Asn Ala Arg His Ser Ala Ile Asp Gly Ala

130 135 140

Pro Thr Arg Pro Ala Lys Gly Gly Trp Ser Arg Val Asn Pro Leu Thr

145 150 155 160

Gly His Glu Glu Phe Pro Arg Ile Asp Pro Ala Ile Ile Cys Leu Val

165 170 175

His Asp Gly His Asp Arg Ala Val Leu Ala Arg Gln Thr Leu Trp Pro

180 185 190

Glu Arg Leu Phe Ser Ile Leu Ala Gly Phe Val Glu Ala Gly Glu Ser

195 200 205

Phe Glu Thr Cys Val Gln Arg Glu Ile Ala Glu Glu Ile Gly Leu Thr

210 215 220

Val Thr Asp Val Gln Tyr Leu Gly Ser Gln Pro Trp Pro Phe Pro Arg

225 230 235 240

Ser Leu Met Val Gly Phe His Ala Ile Gly Asp Pro Glu Gln Pro Phe

245 250 255

Ser Tyr Asn Asp Gly Glu Ile Ala Glu Ala Ala Trp Phe Thr Arg Asp

260 265 270

Glu Ile Arg Ala Ala Leu Asp Gln Gly Asp Trp Asn Ser Asp Ser Pro

275 280 285

Ser Arg Leu Leu Leu Pro Gly Ser Ile Ser Ile Ala Arg Glu Ile Ile

290 295 300

Glu Ser Trp Ala Ala Leu Asp

305 310

<210> 13

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 13

gtggcagcga ggacaacttg 20

<210> 14

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 14

gatgcctggc agtcgatcgt ac 22

Claims (10)

1. A construction method of super recombinant mycobacterium smegmatis for producing nicotinic acid is characterized by comprising the following steps:

1) construction of plasmid pMHS-NrtR _ms:

1-1) carrying out PCR amplification by using a genomic DNA of mycobacterium smegmatis mc ² 155 as a template and primers shown in SEQ ID No. 1-4, and carrying out restriction enzyme digestion by Van91I for later use after electrophoretic separation;

1-2) carrying out amplification culture on Escherichia coli DH5 alpha strain containing pMHS plasmid, then collecting thalli, extracting plasmid, carrying out electrophoretic separation after the plasmid is cut by Van91I restriction endonuclease, and recovering DNA fragments with 1600bp interval and 3600bp interval;

1-3) reacting the DNA fragments obtained in the steps 1-1 and 1-2 with T4 DNA ligase at 16 ℃ overnight, transforming the ligation product into escherichia coli DH5 alpha competent cells for expanded culture, and extracting plasmids to obtain a plasmid pMHS-NrtR _ms;

2) Construction of plasmid pHAGE-NrtR _ms:

2-1) respectively carrying out amplification culture on Escherichia coli DH5 alpha strains containing plasmids pHAGE and pMHS-NrtR _ms, extracting plasmids, linearizing the two plasmids by PacI restriction endonuclease, then reacting at 16 ℃ overnight by using T4 DNA ligase, packaging a ligation product by using a packaging kit, transforming the ligation product into Escherichia coli HB101 competent cells, carrying out amplification culture, and extracting plasmids to obtain plasmid pHAGE-NrtR _ms;

3) preparation of recombinant TM4 phage:

3-1) carrying out amplification culture on mycobacterium smegmatis mc ² 155, carrying out shaking culture until logarithmic phase, centrifuging to collect thalli, washing the thalli by using precooled 10% sterile glycerol, then adding precooled 10% glycerol to resuspend the thalli, and freezing and storing at-80 ℃ for later use;

3-2) transferring the plasmid pHAGE-NrtR _ms into a Mycobacterium smegmatis mc ² 155 electrotransformation competent cell, incubating for 10min on ice, then performing electric shock transformation, adding a 7H9 liquid culture medium after transformation, incubating overnight in an incubator at 37 ℃, adding a bacterium solution placed at 37 ℃ into an EP tube containing top agar, uniformly mixing, pouring and flatly paving on a 7H10 solid plate, and placing the plate in the incubator at 30 ℃ for culturing for 2-3 days to obtain a recombinant TM4 bacteriophage;

4) constructing nrtR _ms gene-deleted strain mycobacterium smegmatis mc ² 100:

culturing mycobacterium smegmatis mc ² 155 with a 7H9 liquid culture medium until logarithmic growth phase, centrifugally collecting thalli, washing the thalli obtained by centrifugation with an MP buffer, then re-suspending the thalli with the MP buffer, mixing the re-suspended mycobacterium smegmatis mc ² 155 with a recombinant TM4 phage 1 with the titer of 10 ¹⁰ PFU by volume of 1:1, incubating in an incubator, centrifugally collecting the upper clear liquid of the thalli, re-suspending with a 7H9 liquid culture medium, placing in the incubator for incubating overnight, centrifugally collecting the upper clear liquid again, coating the thalli with a 7H10 solid plate for culturing, and obtaining mutant mycobacterium smegmatis mc ² 100 secreting nicotinic acid;

5) amplification of the nucd gene and construction of plasmid pMV361-NudC _ms:

5-1) nudC gene amplification, namely performing PCR amplification on a nudC gene ORF sequence by using a primer pair shown in SEQ ID No. 9-10 and using Mycobacterium smegmatis mc ² 155 genome DNA as a template, performing electrophoretic separation and recovery on a PCR product, performing enzyme digestion by using EcoRI and HindIII restriction enzymes, and recovering the DNA subjected to enzyme digestion for later use;

5-2) enzyme cutting: LB culture contains pMV361 plasmid Escherichia coli DH5 alpha strain, extract plasmid, use EcoR I and Hind III restriction enzyme digestion, then electrophoretic separation, reclaim the linearized plasmid;

5-3) connecting, namely reacting the DNA fragment recovered by enzyme digestion and the linearized plasmid pMV361 recovered by the same enzyme digestion at 16 ℃ with T4 DNA ligase overnight, transforming escherichia coli DH5 alpha competent cells by using a connecting product and coating an LB solid plate, and culturing the plate in a constant-temperature incubator at 37 ℃ overnight to obtain a plasmid pMV361-NudC _ms, wherein the LB solid plate contains 100 mu g/mL kanamycin sulfate;

6) Constructing the super recombinant mycobacterium smegmatis with nrtR gene knockout/nucDC gene overexpression:

6-1) preparing competent cells of the mutant mycobacterium smegmatis mc ² 100, namely culturing the mutant mycobacterium smegmatis mc ² 100 in a 7H9 liquid culture medium until logarithmic phase, centrifugally collecting thalli, washing the thalli at least twice by using precooled 10% sterile glycerol, finally adding a proper amount of precooled 10% glycerol, uniformly blowing the thalli, and separately storing the thalli at-80 ℃ for later use;

6-2) transformation of mutant Mycobacterium smegmatis mc ² 100 plasmid pMV361-NudC _ms is transferred into mutant Mycobacterium smegmatis mc ² 100 electrotransfer competent cells, the cells are incubated on ice for 10min, then the transformation is performed by electric shock, then 7H9 liquid culture medium is added, the cells are incubated overnight in an incubator at 37 ℃, then a proper amount of culture solution is taken to coat a 7H10 solid plate, the plate is placed in the incubator at 37 ℃ for 3-5 days, and the super recombinant Mycobacterium smegmatis producing nicotinic acid is obtained, wherein the 7H10 solid plate contains 50 mu g/mL kanamycin sulfate.

2. The method of claim 1, wherein the bacteria collected in step 3 are centrifuged at 5000rpm for 10min at 4 ℃; centrifuging the collected thalli in the step 4 at 6000rpm for 10 min; the centrifugation conditions for collecting the bacteria in the step 6 are 4 ℃ and centrifugation is carried out for 10min at 5000 rpm.

3. The method of claim 1, wherein the scale-up culture in step 1-2 is performed by inoculating the strain to LB liquid medium containing 150. mu.g/mL hygromycin B and culturing overnight at 37 ℃.

4. the method according to claim 1, wherein the expansion culture in step 1-3 is performed by inoculating the strain to LB solid plate medium containing 150. mu.g/mL hygromycin B, culturing overnight at 37 ℃, then picking the single colony growing on the plate to inoculate to LB liquid medium containing 150. mu.g/mL hygromycin B, and culturing overnight at 37 ℃ with shaking at 200 rpm.

5. The method according to claim 1, wherein plasmid pHAGE is expanded in LB medium containing ampicillin resistance in step 2-1, and plasmid pMHS-NrtR _ms is expanded in LB medium containing hygromycin B resistance.

6. The construction method according to claim 1, wherein the strain HB101 competent strain of Escherichia coli transformed with the ligation product in step 2-1 is subjected to amplification culture and then plasmid extraction, wherein the amplification culture comprises inoculating the strain to LB solid plate medium containing 150. mu.g/mL hygromycin B, overnight culture at 37 ℃, then picking the monoclonal colony growing on the plate and inoculating to LB liquid medium containing 150. mu.g/mL hygromycin B, and shaking and culturing at 37 ℃ with shaking at 200rpm for overnight culture.

7. The construction method according to claim 1, wherein the Mycobacterium smegmatis mc ² 155 in the expanded culture in step 3-1 is prepared by inoculating Mycobacterium smegmatis mc ² 155 in 7H9 liquid medium, culturing at 37 ℃ and 200rpm with shaking until logarithmic growth phase, OD600 is 0.5-1.0, inoculating the culture in fresh 7H9 liquid medium at a ratio of 1:100, culturing at 37 ℃ and overnight until OD600 is 0.6, culturing the mutant Mycobacterium smegmatis mc ² 100 in 7H9 liquid medium in step 6-1 until logarithmic growth phase is prepared by inoculating the mutant Mycobacterium smegmatis mc ² 100 colony in 5mL 7H9 liquid medium, culturing at 37 ℃ and 200rpm with shaking until logarithmic growth phase OD600 is 0.5-1.0, and inoculating the culture in fresh 100mL 7H9 liquid medium at a ratio of 1:100 and culturing at 37 ℃ and overnight until OD600 is 0.6.

8. The method of claim 1, wherein the shock transformation parameters in steps 3-2 and 6-2 are voltage 2.5kV, resistance 1000 Ω, and capacitance 25 μ F.

9. The construction method according to claim 1, further comprising the steps after step 5-3 of:

5-4) selecting a monoclonal colony growing on the plate, inoculating the monoclonal colony into an LB liquid culture medium, shaking and overnight culturing at the speed of 200rpm of a shaking table at 37 ℃, extracting a plasmid, carrying out restriction enzyme digestion on the obtained plasmid by using EcoRI and Hind III restriction enzymes, detecting and verifying through nucleic acid gel electrophoresis, verifying that a nucd gene ORF sequence is connected to a pMV361 vector, and simultaneously verifying that the plasmid sequence has no mutation;

the method also comprises the following steps after the step 6-2:

6-3) selecting monoclones growing on a plate, inoculating the monoclones into 5mL of 7H9 liquid culture medium, culturing for 2-3 days until the logarithmic phase, centrifugally collecting thalli, extracting genomic DNA as a template, and verifying the transformation of pMV361-NudC _ms plasmid into mutant mycobacterium smegmatis mc ² 100 by using a primer pair shown in SEQ ID No. 13-14 through PCR.

10. A super recombinant Mycobacterium smegmatis producing nicotinic acid, characterized in that it is produced by the construction method of any one of claims 1 to 9.