CN111057133B

CN111057133B - Response regulator and its mutant and its application in preparing vitamin B12In (1)

Info

Publication number: CN111057133B
Application number: CN201911335769.8A
Authority: CN
Inventors: 张大伟; 董会娜
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2021-09-28
Anticipated expiration: 2039-12-23
Also published as: CN111057133A

Abstract

The invention discloses a response regulatory factor gene, a mutant gene and application thereof in preparing vitamin B₁₂The use of (1). Genetically engineered bacteria of response regulator genes and mutant genes overexpressed in sinorhizobium meliloti, producing vitamin B₁₂The capability of the method is greatly improved, and the method has great application and popularization values.

Description

Response regulator and its mutant and its application in preparing vitamin B12In (1)

The technical field is as follows: the invention belongs to the technical field of biology, and particularly relates to a response regulatory factor, a mutant thereof and application thereof in preparing vitamin B₁₂The use of (1).

Background art:

vitamin B₁₂(VB₁₂) Also called cobalamin, belongs to corrin compounds, is the only vitamin compound containing metal elements, and is a macromolecular organic compound with the latest B vitamins found. Vitamin B, depending on the type of ligand (R group) above the corrin ring₁₂The method can be divided into the following steps: hydroxycobalamin, deoxyadenosylcobalamin and methylcobalamin. Vitamin B₁₂Participate in a large number of biochemical processes including DNA synthesis and regulation, fatty acid synthesis, amino acid metabolism and ability generation.

Due to vitamin B₁₂The molecular structure is complex, the artificial synthesis by a chemical method needs to consume a large amount of manpower and material resources, and the synthesis period is long. The requirements on operators during the synthesis process are too high, so that the large-scale production cannot be realized. Microbial fermentation currently produces vitamin B₁₂The method (2) can be mass-produced and popularized for use.

At present, vitamin B is targeted at home and abroad₁₂Biosynthesis of vitamin B by producing bacteria₁₂Mainly focuses on the optimization of the fermentation process, mainly relates to the optimization of a culture medium comprising a carbon nitrogen source and metal ions, the addition of betaine and the addition of rotenone, and controls the process conditions comprising pH and oxygen supplyEtc. (Chenoporyl et al, expression of uroporphyrinogen III transmethylase from different sources in Pseudomonas denitrificans and its pair for production of vitamin B₁₂Industrial microorganisms, 2017, vol 47, No. 3).

Microorganisms possess a series of mechanisms for adapting organisms to different environments by regulating the expression of their own genes. Two-component systems consist of two basic components: one is Histidine Kinase (HK) and the other is Response Regulator (RR). However, in the production of vitamin B₁₂Which response regulator in the bacterial species affects vitamin B₁₂The yield of (A) has not been reported.

The invention content is as follows:

the inventor screens high-yield vitamin B in the early stage₁₂The Sinorhizobium meliloti strain CGMCC NO.9638 (CN104342390A) is subjected to mutagenesis to obtain a strain capable of producing vitamin B₁₂A mutagenized strain with improved capacity. The present inventors have further studied to find that vitamin B is produced₁₂Genes with an effect on competence. Researches show that a response regulatory factor coding gene and a mutant gene thereof are introduced into Sinorhizobium meliloti for overexpression, so that the vitamin B production of the Sinorhizobium meliloti can be improved₁₂The ability of the cell to perform.

Firstly, the present invention provides a mutant responding to regulatory factors, which is characterized in that the polypeptide amino acid sequence has the following mutations on the basis of the original sequence shown in SEQ ID No. 2: substitution of amino acid 212 with V.

Preferably, the amino acid sequence is as set forth in SEQ ID NO: 4, respectively.

Next, the present invention provides a gene encoding the mutant responding to regulatory factors as described above.

Preferably, the nucleotide sequence is as set forth in SEQ ID NO: 3, respectively.

In a third aspect, the present invention provides a gene encoding a response regulator in the production of vitamin B₁₂The use of (1).

Specifically, the coding gene is introduced into Sinorhizobium meliloti through an expression vector containing the coding gene for overexpression, and the introduced coding gene is located in a plasmid or a chromosome.

Preferably, the Sinorhizobium meliloti has a preservation number of CGMCC NO. 9638.

Further, the response regulator encoding gene encodes a polypeptide having the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

More preferably, the gene encoding the mutant response regulator has the sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

Proved by research, the genetic engineering bacteria of the invention responding to the coding gene and the mutant gene of the regulatory factor are biologically safe (the over-expression in the bacteria does not influence the growth of the bacteria), and can effectively improve the vitamin B production of the sinorhizobium meliloti₁₂The experimental data show that the over-expression of the original gene in Sinorhizobium meliloti can improve the vitamin B production₁₂The capacity of the mutant gene reaches 9.8 percent, and the over-expression of the mutant gene in Sinorhizobium meliloti can further improve the vitamin B production₁₂The capacity of (2) is improved by 15.9%.

Drawings

FIG. 1: map of plasmid vector pBBR-P21-RR.

FIG. 2: VB of different sinorhizobium meliloti strains fermented for 144h₁₂And (4) yield.

FIG. 3: biomass of different sinorhizobium meliloti strains after fermentation for 144 h.

FIG. 4: vitamin B₁₂Standard graph of (2).

Detailed Description

The following examples and figures of the present invention are merely illustrative of specific embodiments for carrying out the invention and these should not be construed as limiting the invention and any changes which may be made without departing from the principles and spirit of the invention are within the scope of the invention.

The experimental techniques and experimental methods used in this example are conventional techniques unless otherwise specified. The materials, reagents and the like used in the present examples are all available from normal commercial sources unless otherwise specified.

The formula of the culture medium is as follows:

LB medium (g/L): 10 parts of sodium chloride, 10 parts of tryptone, 5 parts of yeast extract and 15 parts of agar powder added into a solid culture medium.

Seed medium (g/L): sucrose 40, corn steep liquor 20, betaine 5, (NH)₄)₂SO₄ 1，(NH₄)₂HPO₄ 2，MnSO₄·H₂O 0.8，CoCl₂·6H₂O 0.02，MgO 0.3，DMBI 0.01，ZnSO₄·7H₂O 0.01，CaCO₃1.5, and controlling the pH value to be 7.0-7.4 by NaOH.

Fermentation medium (g/L): sucrose 80, corn steep liquor 30, betaine 15, (NH)₄)₂SO₄ 2，MgSO₄ 1.5,K₂HPO₄0.75， CoCl₂·6H₂O 0.14,DMBI 0.075，ZnSO₄·7H₂O 0.08，CaCO₃1, controlling the pH value to be 7.0-7.4 by NaOH.

Vitamin B₁₂Is detected by

(1) Sample pretreatment

Taking 1mL of fermentation liquor, adding 8% sodium nitrite solution and glacial acetic acid, shaking up 0.25mL each, and placing in a water bath at 95-100 ℃ for 30-40 min; cooling to room temperature, centrifuging at 10000 rpm for 1min, and passing the supernatant through a 0.22 μm membrane

The filter was filtered into the sample vial, and 20. mu.l of 2% NaCN (w/v) was added to 1mL of the supernatant. The addition amount of the sodium nitrite solution and the glacial acetic acid can be correspondingly adjusted along with the amount of the fermentation liquor.

(2) Preparation of standards

Configuring gradient vitamin B₁₂Standard substance (20mg/L, 50mg/L, 100mg/L, 150 mg/L).

(3) HPLC detection conditions

C18-250A column (Agilent, 4.6mmid 9X 250mm, 5 μm). The mobile phase comprises 70% organic phase (acetonitrile) and 30% inorganic phase (sodium acetate aqueous solution), the absorption wavelength is 361nm, the column temperature is 35 deg.C, the flow rate is 0.8mL/min, and the sample injection amount is 20 μ L.

(4) Vitamin B₁₂Drawing of standard curve

Performing HPLC detection on the standard substances with different concentrations according to the above conditions, and drawing peak area A-VB₁₂Concentration standard curve. Using the measured peak area A as the ordinate, vitamin B₁₂The mass concentration C (mg/L) is recorded as the abscissa and vitamin B is plotted₁₂A standard curve. See fig. 4, resulting in the regression equation y 19.846x-80.857, R²The absorbance is well linear with mass concentration at 0.999. After the liquid phase is finished, according to vitamin B₁₂The standard curve calculates the sample yield.

Example 1: determination of mutagenesis time of Atmospheric Room Temperature Plasma (ARTP), construction of mutant library and acquisition of high-yield strain

(1) Determination of lethality

In order to obtain a wide mutant library, the chassis cell Sinorhizobium meliloti CGMCC NO.9638 was subjected to atmospheric pressure room temperature plasma (ARTP) mutagenesis. First, the lethality of cgmccno.9638 was determined under plasma mutagenesis conditions. Culturing seed with LB culture medium to middle logarithmic phase CGMCC NO.9638 cell (OD)₆₀₀1) two washes with 0.85% NaCl solution, then diluted to 10 with 0.85% NaCl solution⁸Individual cells/mL of suspension. 10uL of the suspension is uniformly coated on an iron sheet and subjected to ARTP mutagenesis for 0s, 5s, 10s, 15s, 20s and 25s respectively, 2 times of mutagenesis time points and 3 times of plate coating of each time point. After mutagenesis, the iron sheet with the cells is placed in 1mL of sterile water to wash the cells by vortex oscillation, and the cell suspension is diluted to 10 times of gradient^-3And taking 100 mu L of diluted cell suspension, coating the diluted cell suspension on an LB culture medium plate, and counting the number of colonies after culturing for 72h at 30 ℃. The lethality at different mutagenesis times was calculated according to the following formula, and a lethality curve was drawn with the mutagenesis time as abscissa and the lethality at different mutagenesis times as ordinate. The lethality rate is calculated by the formula: lethality (%) - (number of mutagenized 0s colonies-number of mutagenized Ns colonies)/number of mutagenized 0s colonies]X 100%, wherein N is 5, 10, 15, 20, 25.

As is clear from table 1, since the mortality rate at 10s was 82.2%, the mortality rate at 15s was 95% or more, and the probability of positive mutation after mutagenesis was the highest at 80% to 90%, 10s was selected as the mutagenesis time for finally constructing the mutant library.

(2) Construction and screening of mutant libraries

Taking the concentration as 10⁸10uL of each/mL cell suspension was coated on an iron plate and subjected to ARTP mutagenesis for 10s, cells on 3 iron plates were subjected to mutagenesis treatment, and after mutagenesis, the cells on the 3 iron plates were resuspended in a 1.5mL centrifuge tube containing 1mL of LB medium by vortexing. 10-fold gradient dilution of cell suspension to 10^-3And taking 100 mu L of diluted cell suspension, coating the diluted cell suspension on an LB medium plate, and culturing at 30 ℃ for 72h to grow 270 single colonies.

(3) Mutant strain 96 deep-hole plate fermentation primary screen

Respectively picking up all single colonies on the plate, inoculating the single colonies in a 96-deep-well plate containing 500uL seed culture medium (each plate contains 6 control strains CGMCC NO.9638), carrying out shake culture at 30 ℃, 800rpm and 80% humidity for 36h, transferring the single colonies into a 96-deep-well plate containing 450uL fermentation culture medium according to the inoculum size of 10% (v/v), carrying out shake culture at 30 ℃, 800rpm and 80% humidity for 120h, and detecting vitamin B₁₂And (4) yield.

(4) Mutant strain 96 deep-hole plate fermentation rescreening

Inoculating a single colony (containing a control strain CGMCC NO.9638) of the strain with the yield of 30 before in the step (3) into a 96 deep-well plate containing 500uL of seed culture medium, carrying out shake culture at 30 ℃, 800rpm and 80% humidity for 36h, then transferring the single colony into the 96 deep-well plate containing 450uL of fermentation culture medium according to the inoculation amount of 10% (v/v) (3 strains are parallel), carrying out shake culture at 30 ℃, 800rpm and 80% humidity for 120h, and detecting vitamin B₁₂And (4) yield.

Repeating the steps (3) and (4) for three times to finally obtain a strain with high vitamin B yield₁₂The yield of the strain SM in the 96-well plate is improved from 50mg/L to 80mg/L, which is 1.6 times of that of the chassis strain.

Example 2: comparative genomic analysis of mutant strains with the original strains

The strain SM and the original strain CGMCC NO.9638 are sent to Jinzhi biotechnology limited to carry out whole genome sequencing. By comparing the whole genome sequences of the two strains, it was found that a point mutation occurred in response to the regulator-encoding gene RR, which contained a mutation at nucleotide 635, wherein the mutation was a substitution of C with T. To verify the mutation site to vitamin B₁₂Influence of yield, we over-express the response regulator coding gene RR before and after mutation in the original strain CGMCC NO. 9638.

Example 3: construction of plasmid vector

Construction of pBBR-P21-RR:

the primer pairs P21-XbaI-F and P21-R in Table 2 are respectively utilized, the genome of Ensifer adhaerens Casida A (Excellent Zygomyces agglomerans) is used as a template, an XbaI restriction site is introduced through PCR amplification to obtain a promoter P21 fragment, the promoter P21 fragment is treated for 30min at 37 ℃ by using restriction enzyme DpnI (NEB company), and the purified P21 fragment is obtained after nucleic acid electrophoresis gel recovery. The P21 promoter sequence is shown in SEQ ID No.5 and is described in the patent document with the patent application number 201910929398. X.

Respectively utilizing the primer pairs RR-F and RR-EcoRI-R of the table 2, taking the genome of Ensifer adhaerens Casida A (Ensifer encephalus) as a template, carrying out PCR amplification, introducing an EcoRI enzyme cutting site to obtain an RR fragment, carrying out electrophoresis verification, carrying out DpnI enzyme method treatment, and recycling electrophoresis gel to obtain a purified RR fragment. The RR gene sequence is shown in SEQ ID No.1, and the coded amino acid sequence is shown in SEQ ID No. 2.

Then, by using a primer pair P21-XbaI-F and RR-EcoRI-R, the purified P21 fragment and RR fragment are used as templates, a P21-RR fragment (containing XbaI and EcoRI cleavage sites) is obtained through fusion PCR, and the purified P21-RR fragment is obtained after electrophoretic verification and electrophoretic gel recovery.

The purified P21-RR fragment and pBBR1MCS2 plasmid were digested simultaneously with XbaI and EcoRI, and the digested product of P21-RR fragment and pBBR1MCS2 plasmid were ligated with T4 ligase overnight at 4 ℃. The ligation products were transformed into E.coli DH5 α, spread on LB solid plate containing 50mg/L kanamycin, cultured for 16h, and then colony PCR was performed, and after sequencing was performed by Kingo-wisely, the obtained positive bacteria were named E.coli/pBBR-P21-RR after correct sequencing. The plasmid pBBR-P21-RR was extracted with a plasmid kit for use, and the plasmid map is shown in FIG. 1.

pBBR-P21-RR(C635T)

Plasmid pBBR-P21-RR as template and primer pair C635T-F/C635T-R are used for reverse PCR amplification to obtain fragment DpnI with about 6.9kb in size, and the purified product is obtained after recovery of electrophoresis gel. 30ng of the purified product was added to 2. mu.l of 10 × T4 ligase buffer (NEB), 1. mu. l T4 Polynucleotide kinase (NEB), distilled water was added thereto to 20. mu.l, and the mixture was reacted at 37 ℃ for 30min, 1. mu. l T4 ligase (NEB) was added thereto, and the reaction mixture was reacted at room temperature for 2 hours to obtain a ligated product. The ligation products were transformed into E.coli DH 5. alpha. and spread on LB solid plates containing 50mg/L kanamycin, and after 16h of culture, colony PCR was performed, and then subjected to Kingo-only sequencing, and after the sequencing was correct, the resulting positive bacteria were named E.coli/pBBR-P21-RR (C635T). Plasmid pBBR-P21-RR (C635T) was extracted using a plasmid kit for further use. Wherein, the mutated RR gene sequence is shown as SEQ ID No.3, and the coded amino acid sequence is shown as SEQ ID No. 4.

Example 4: construction of plasmid vector-containing Strain

The 3 plasmids pBBR1MCS2, pBBR-P21-RR and pBBR-P21-RR (C635T) from example 3 were transferred into Sinorhizobium meliloti CGMCC NO.9638 as follows:

(1) inoculating newly activated Sinorhizobium meliloti CGMCC NO.9638, Escherichia coli (containing corresponding plasmids) and auxiliary vector MT616, and performing shake culture in culture boxes at 30 deg.C and 37 deg.C respectively until OD value is about 1.0;

(2) separately transferring 500. mu.L of the bacterial liquid of Sinorhizobium meliloti CGMCC NO.9638, MT616 and the bacterial liquid of Escherichia coli to a 1.5mL sterile EP tube under aseptic condition, and centrifuging at 4 ℃ and 12,000rpm for 1 min.

(3) The supernatant was discarded under sterile conditions, and the pellet was suspended with 1mL of 0.85% sterile physiological saline.

(4) Centrifugation was again carried out at 12,000rpm for 1min at 4 ℃ and the supernatant was removed under aseptic conditions.

(5) The recipient cells, E.coli and MT616 pellet were suspended with 500. mu.L of fresh LB liquid medium, respectively.

(6) Three kinds of the bacterial solutions, each 2. mu.L, were dropped on the same position of LB solid medium to which no resistance was added, and carefully mixed. The bacterial liquids of single components and the bacterial liquids mixed between every two components are respectively sampled and used as test control groups.

(7) After the bacterial liquid is naturally air-dried, the bacterial liquid is inversely cultured in an incubator at 37 ℃ for about 1 day until a single bacterial colony grows out.

(8) Different single colonies were picked and streaked onto plates containing the corresponding antibiotics, and the plates were inverted and incubated in an incubator at 30 ℃ until colonies grew out. Meanwhile, different single colonies in the control group are selected and streaked on the plate containing the corresponding antibiotics.

(9) Colonies were picked from the resistant plates and verified by colony PCR. The positive S.meliloti was obtained as SM/pBBR (control), SM/pBBR-P21-RR (abbreviated as SM1), and SM/pBBR-P21-RR (C635T) (abbreviated as SM 2).

Example 5: evaluation of different strains

The culture conditions of the Sinorhizobium meliloti are as follows:

the control bacteria, SM1 and SM2 strains were streaked on LB solid medium containing 100mg/L kanamycin with an inoculating needle under aseptic conditions, and were allowed to stand at a constant temperature of 30 ℃ for 48 hours for culture to obtain single colonies. A single colony was picked up with an inoculating needle in a test tube containing 5mL of LB liquid medium containing 100mg/L kanamycin, and cultured at 30 ℃ and 200rpm for 36 hours. The seed medium was inoculated at a rate of 10% into 30mL of a fermentation medium containing 100mg/L kanamycin (250mL shake flask). After shaking (220 r/min) culture at 30 ℃ for 144h, the thalli are collected and the yield is detected. Shake flask fermentations were performed in 3 replicates per experiment.

VB produced by different sinorhizobium meliloti strains₁₂Comparison of capabilities

Two strains of SM1 and SM2 produced vitamin B compared to control₁₂All improved (see table 3 and figure 2). Among them, SM2 was improved by 15.9%, and SM1 was improved by only 9.8%. The results demonstrate that Sinorhizobium meliloti overexpressing response regulators of the invention produces vitamin B₁₂The ability of (a) is enhanced. The biomass of the three strains changed little, which indicates that the over-expression of the response regulator gene has no influence on the growth of the strains (see table 3 and figure 3).

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> response regulator, mutant thereof and application thereof in preparation of vitamin B12

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Claims

1. Response regulatory factor coding gene in preparation of vitamin B₁₂Wherein the response regulator encoding gene encodes a polypeptide having the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

2. The use of claim 1, wherein the response modifier-encoding gene has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

3. The use of claim 1, wherein overexpression is achieved by introducing the coding gene into Sinorhizobium meliloti via an expression vector comprising the coding gene.

4. The use according to claim 3, wherein Sinorhizobium meliloti has a preservation number of CGMCC No. 9638.

5. The use of claim 3 or 4, wherein the introduced coding gene is located on a plasmid or chromosome.