CN110903358B - Ribosomal factor and its mutant and its use in preparing vitamin B12In (1) - Google Patents

Abstract

The invention discloses 3 ribosomal factor genes, mutant genes thereof and application thereof in preparing vitamin B₁₂The use of (1). The gene is over-expressed in Sinorhizobium meliloti, so that the vitamin B can be effectively improved₁₂Has great application and popularization value, and is suitable for research on vitamin B production₁₂The gene regulation of the strain is also of great significance.

Description

Ribosomal factor and its mutant and its use in preparing vitamin B12In (1)

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a mutant of a ribosomal factor, and application of the ribosomal factor and the mutant thereof in preparation of vitamin B₁₂The use of (1).

Background

Vitamin B₁₂(VB₁₂) The vitamin B derivative has wide application in the pharmaceutical and food industries, is called cobalamin, belongs to corrin compounds, is the only vitamin compound containing metal elements, and is a macromolecular organic compound with the latest discovery of B vitamins. 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 bacteria₁₂The research mainly focuses on the optimization of the fermentation process, and 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 the control of process conditions comprising pH and oxygen supply and the like. There are reports in the literature of increased vitamin B production by cells expressing a single copy of the Vitreoscilla vgb gene on the Pseudomonas denitrificans genome₁₂(vii) ability (Chenoporyl et al, expression of uroporphyrinogen III transmethylase from different sources in Pseudomonas denitrificans and its effect on vitamin B12 production. Industrial microorganism, 2017, Vol.47, No. 3).

A transcriptional elongation factor is a factor that enables the transcription of a gene to proceed continuously, such as a protein that activates RNA polymerase activity. The ribosome circulating factor RRF consists offrrAnd (3) coding, which plays an important role in recycling ribosome. In thatStreptomyces coelicolorOverexpression of RRF in (1) can enhance protein synthesis in the late growth phase, thereby overproducing natural products (Hosakat, Xu J, Ochi K (2006) incorporated expression of ribosome transduction factor for the enhanced protein synthesis and reduction of the substrate growth phase an anti-inflammatory-overproducing Streptomyces coelicolor ribosomal rpsL microbial 61 (883) (897)). In thatStreptomyces avermitilisIn the middle of overexpressionfrrIncrease the production of Avermectin (. Li L, Guo J, Wen Y, Chen Z, Song Y, Li J (2010) Overexpression of ribosomal transduction genes involved in the production of increased production of avermectin J Ind Microbiol Biotechnol 37(7):673 and 679)Streptomyces diastatochromogenesIn the middle of overexpressionfrrCan increase the yield of toyocamycin (Ma Z, TaoL, Bechthold A, Shentu X, Bian Y, Yu X (2014) Overexpression of ribosomal factor for improvement of nuclear antibiotic-toyocamycin 1628 Appl Microbiolbiotechnol 98(11): 5051-. In thatCorynebacterium glutamicumIn the middle of overexpressionfusA(encoding ribosomal elongation factor G) and/orfrrIncreases isoleucine production (ZHao J1, Hu X, Li Y, Wang X (2015), Overexpression of ribosomal elongation factor G and recycling factorincreases L-isoleucine production inCorynebacterium glutamicumApplMicrobiol Biotechnol. 99(11): 4795-805). However, it affects vitamin B₁₂The yield of ribosomal factors has not been reported.

Disclosure of Invention

The inventor selects a high-yield vitamin B strain in the earlier stage₁₂The Sinorhizobium meliloti CGMCC NO.9638 strain (CN 104342390A) can be fermented to produce vitamin B₁₂. Obtaining the vitamin B by mutagenizing the strain₁₂The inventor carries out mining analysis on ribosomal factors of the mutant strain with improved capability, comprises 9 different ribosomal factors, and finds that the vitamin B can be improved by over-expressing the ribosome binding factor A, the ribosome translation elongation factor Ts or the ribosome translation elongation factor NusA in the Sinorhizobium meliloti CGMCC NO.9638₁₂The yield of (2). Through further research, the invention is finally completed, and three ribosomal factors and mutants thereof are provided, and vitamin B is produced by the three ribosomal factors₁₂The use of (1).

The invention provides a mutant of ribosome binding factor A, the amino acid sequence of which is shown in SEQ ID No.2, and the 70 th amino acid is replaced by H. Specifically, the amino acid sequence is shown as SEQ ID No. 34.

The invention also provides a mutant of ribosome translation elongation factor Ts, which is characterized in that the amino acid sequence is shown in SEQ ID No.4, and the 211 th amino acid is replaced by L. Specifically, the amino acid sequence is shown as SEQ ID No. 36.

The invention also provides a mutant of ribosome translation elongation factor NusA, the amino acid sequence of which is shown in SEQ ID No.6, and the 173 th amino acid is replaced by S. Specifically, the amino acid sequence is shown as SEQ ID No. 38.

Secondly, the invention provides the coding gene of the mutant, and specifically the nucleotide sequence of the coding gene of the ribosome binding factor A is shown in SEQ ID No. 33; the nucleotide sequence of the coding gene of the ribosome translation elongation factor Ts is shown in SEQ ID No. 35; the nucleotide sequence of the ribosome transcription elongation factor NusA coding gene is shown in SEQ ID No. 37.

In a third aspect, the present invention provides a method for producing vitamin B using a gene encoding ribosome binding factor A, ribosome translation elongation factor Ts, or ribosome translation elongation factor NusA₁₂The use of (1). The ribosome binding factor A coding gene codes polypeptide with an amino acid sequence shown as SEQ ID No.2 or SEQ ID No. 34; the ribosome translation elongation factor Ts coding gene codes polypeptide with an amino acid sequence shown as SEQ ID No.4 or SEQ ID No. 36; the ribosome translation elongation factor NusA coding gene codes polypeptide with an amino acid sequence shown as SEQ ID No.4 or SEQ ID No. 36.

In one embodiment, the coding gene is introduced into Sinorhizobium meliloti for overexpression via an expression vector containing the coding gene. Further, the gene or mutant gene is located in a plasmid or chromosome.

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

Screening research on 6 ribosome factors shows that the ribosome binding factor A, the ribosome translation elongation factor Ts, the ribosome translation elongation factor NusA and mutant genes thereof are over-expressed in the sinorhizobium meliloti, so that the method not only is biosafety of genetically engineered bacteria (the over-expression in the bacteria does not influence the growth of the bacteria), but also can effectively improve the vitamin B production of the sinorhizobium meliloti₁₂The ability of the cell to perform. The experimental data show that the overexpression of the original gene in the sinorhizobium meliloti can improve the vitamin B production of the sinorhizobium meliloti by the overexpression of the ribosome binding factor A, the ribosome translation elongation factor Ts and the ribosome translation elongation factor NusA₁₂Especially their mutant gene overexpression can even further enhance vitamin B production₁₂The ability of the cell to perform. And overexpresses three other ribosomal factors (frr，ysxC，greA) Then vitamin B is produced₁₂With varying degrees of reduction in capacity.

Drawings

FIG. 1: plasmid vector pBBR-P21-rbfAA map of (a).

FIG. 2: plasmid vector pBBR-P21-tsfA map of (a).

FIG. 3: plasmid vector pBBR-P21-nusAA map of (a).

FIG. 4: plasmid vector pBBR-P21-frrA map of (a).

FIG. 5: plasmid vector pBBR-P21-ysxCA map of (a).

FIG. 6: plasmid vector pBBR-P21-greAA map of (a).

FIG. 7: 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)₂SO₄1，(NH₄)₂HPO₄2，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)₂SO₄2，MgSO₄1.5, 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。

Detection method

(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; after cooling to room temperature, centrifugation was carried out at 10000 rpm for 1 minute, and the supernatant was filtered through a 0.22 μm membrane (⌀ = 0.22 μm) filter into an upper flask, followed by addition of 20 μ l of 2% NaCN (w/v) 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 (20 mg/L, 50mg/L, 100mg/L, 150 mg/L).

(3) HPLC detection conditions

C18-250A column (Agilent, 4.6 mmid 9X 250 mm, 5 μm). The mobile phase is 70% organic phase (acetonitrile) and 30% inorganic phase (sodium acetate aqueous solution), the absorption wavelength is 361 nm, the column temperature is 35 ℃, the flow rate is 0.8 mL/min, and the sample amount is 20 mu 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. 7, resulting in regression equation y =19.846x-80.857, R²= 0.999, the absorbance is in a good linear relationship with the mass concentration. 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 is subjected to normal pressure and room temperature plasma (ARTP) mutagenesis. First, the lethality of CGMCCNO.9638 under the condition of plasma mutagenesis was improvedAnd (4) carrying out measurement. Culturing seed with LB culture medium to middle logarithmic phase CGMCC NO.9638 cell (OD)₆₀₀=1) wash twice with 0.85% NaCl solution, then dilute 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, 20 s and 25 s 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%, where N =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

Pick up all the plates separatelyInoculating single colony in 96 deep-well plate containing 500uL seed culture medium (each plate contains 6 control strains CGMCC NO. 9638), shake culturing at 30 deg.C and 800rpm at 80% humidity for 36h, transferring into 96 deep-well plate containing 450uL fermentation culture medium according to 10% (v/v) inoculum size, shake culturing at 30 deg.C and 800rpm at 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 80 mg/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. The point mutation of 3 ribosomal factors was found by comparing the whole genome sequences of the two strains, as shown in Table 2. To verify the mutation site to vitamin B₁₂The influence of yield, we over-express the mutated transcription regulatory factor gene in the original strain CGMCC NO. 9638. Meanwhile, other 3 kinds of ribosomal factor coding genes which are not mutated are selected according to literature reports and genome data (frr，ysxC，greA) And (6) carrying out verification.

Example 3: construction of plasmid vector

1、pBBR-P21-rbfAThe construction of (1):

respectively using the primer pair P21-XbaI-F and P21-R1, in the presence of a catalystEnsifer adhaerensCasida A (Ensifer. viscosus) genome is used as a template, amplified by PCR and introducedXbaI enzyme cutting site to obtain promoter P21-1 segment, electrophoresis verification and restriction enzyme restrictionDpnI (NEB) was treated at 37 ℃ for 30min, and the purified P21-1 fragment was obtained after recovery of the gel by electrophoresis. The P21 promoter sequence is shown in SEQ ID No.7 and is described in the patent document with the patent application number 201910929398. X.

Respectively using the primer pairs rbfA-F and rbfA-EcoRI-R in the table 3, taking the Sinorhizobium meliloti CGMCC NO.9638 genome as a template, and introducing the rSbA-F and the rbfA-EcoRI-R through PCR amplificationEcoRI, obtaining a rbfA fragment by enzyme cutting sites, carrying out electrophoresis verification,Dpnand I, treating by using an enzyme method, and recovering electrophoresis gel to obtain a purified rbfA fragment. The rbfA gene sequence is shown as SEQ ID No.1, and the coded amino acid sequence is shown as SEQ ID No. 2.

Then, using the primer pair P21-XbaI-F and rbfA-EcoRI-R, and the P21-rbfA fragment (containing the fragment) is obtained by fusion PCR using the purified P21-1 fragment and rbfA fragment as templatesXbaI andEcoRi enzyme cutting site), and obtaining a purified P21-rbfA fragment after electrophoresis verification and electrophoretic gel recovery.

The purified P21-rbfA fragment and pBBR1MCS2 plasmid were used separatelyXbaI andEcoRi, carrying out double enzyme digestion, connecting a double enzyme digestion product of a P21-rbfA fragment with a double enzyme digestion product of a pBBR1MCS2 plasmid through T4 ligase at 4 ℃ overnight, transforming the connection product into escherichia coli DH5 α, coating the escherichia coli DH5 α on an LB solid plate containing 50mg/L kanamycin, carrying out colony PCR detection after culturing for 16h, carrying out Jinzhi sequencing, and naming the obtained positive bacteria as the positive bacteria after correct sequencingE.coli/pBBR-P21-rbfA. Plasmid pBBR-P21-containing plasmid extracted by plasmid kitrbfAFor use, the plasmid map is shown in FIG. 1.

、pBBR-P21-rbfA (T208C)

The strain SM genome was used as a template to obtain an rbfA (T208C) fragment (nucleotide sequence shown in SEQ ID NO: 33, amino acid sequence shown in SEQ ID NO: 34), and the same primers were used for construction according to the construction process of pBBR-P21-rbfA to obtain plasmid pBBR-P21-rbfA (T208C).

、pBBR-P21-tsfThe construction of (1):

respectively using the primer pair P21-XbaI-F and P21-R2, using Sinorhizobium meliloti CGMCC NO.9638 genome as template, through PCR amplification and introductionXbaI enzyme cutting site to obtain promoter P21-2 fragment, electrophoresis verification and restriction enzyme restrictionDpnI (NEB) was treated at 37 ℃ for 30min to recover the nucleic acid gel, and purified P21-2 fragment was obtained.

Respectively using the primer pairs tsf-F and tsf-EcoRI-R of the table 3, taking the Sinorhizobium meliloti CGMCC NO.9638 genome as a template, and introducing the Sinorhizobium meliloti CGMCC NO.9638 genome by PCR amplificationEcoRI, obtaining a tsf fragment by enzyme cutting sites, carrying out electrophoresis verification,Dpnand I, performing enzyme treatment, and recovering electrophoresis gel to obtain a purified tsf fragment. the tsf gene sequence is shown as SEQ ID No.3, and the coded amino acid sequence is shown as SEQ ID No. 4.

Then, using the primer pair P21-XbaI-F and tsf-EcoRI-R, using purified P21-2 fragment and tsf fragment as template, and obtaining P21-tsf fragment (containingXbaI andEcoRi enzyme cutting site), and obtaining a purified P21-tsf fragment after electrophoresis verification and electrophoretic gel recovery.

The purified P21-tsf fragment and pBBR1MCS2 plasmid were used separatelyXbaI andEcoRi, carrying out double enzyme digestion, connecting a double enzyme digestion product of a P21-tsf fragment with a double enzyme digestion product of a pBBR1MCS2 plasmid through T4 ligase at 4 ℃ overnight, transforming the connection product into escherichia coli DH5 α, coating the escherichia coli DH5 α on an LB solid plate containing 50mg/L kanamycin, culturing for 16h, carrying out colony PCR detection, carrying out Jinzhi sequencing, and after the sequencing is correct, naming the obtained positive bacteria as theE.coli/pBBR-P21-tsf. Plasmid pBBR-P21-containing plasmid extracted by plasmid kittsfFor use, the plasmid map is shown in FIG. 2.

、pBBR-P21-tsf (T631C)

The strain SM genome was used as a template to obtain a tsf (T631C) fragment (nucleotide sequence shown in SEQ ID NO: 35, amino acid sequence shown in SEQ ID NO: 36), and the same primers were used for construction according to the pBBR-P21-tsf to obtain plasmid pBBR-P21-tsf (T631C).

、pBBR-P21-nusAThe construction of (1):

respectively using the primer pair P21-XbaI-F and P21-R3, in the presence of a catalystEnsifer adhaerensCasida A (Ensifer. viscosus) genome is used as a template, amplified by PCR and introducedXbaI enzyme cutting site to obtain promoter P21-3 fragment, electrophoresis verification and restriction enzyme restrictionDpnI (NEB) was treated at 37 ℃ for 30min, and the purified P21-3 fragment was obtained after recovery of the gel by electrophoresis.

The primer pairs nusA-F and nusA-HindIII-R in the table 3 are respectively utilized, the Sinorhizobium meliloti CGMCC NO.9638 genome is taken as a template, PCR amplification is carried out, and the primer pairs are introducedHindIII, obtaining a nusA fragment by enzyme cutting, verifying by electrophoresis,Dpnenzyme method I, and recovery of electrophoresis gel to obtain purified nusA fragment. The sequence of the nusA gene is shown as SEQ ID No.5, and the coded amino acid sequence is shown as SEQ ID No. 6.

Then, using the primer pair P21-XbaI-F and nusA-HindIII-R, using purified P21-3 fragment and nusA fragment as template, and performing fusion PCR to obtain P21-nusA fragment (containingXbaI andHindIII cleavage site), and obtaining a purified P21-nusA fragment after electrophoresis verification and electrophoretic gel recovery.

The purified P21-nusA fragment and pBBR1MCS2 plasmid were usedXbaI andHindIII, carrying out double digestion, connecting a double digestion product of the P21-nusA fragment with a double digestion product of a pBBR1MCS2 plasmid through T4 ligase at 4 ℃ overnight, transforming the connection product into escherichia coli DH5 α, coating the escherichia coli DH5 α on an LB solid plate containing 50mg/L kanamycin, carrying out colony PCR detection after culturing for 16h, carrying out Jinzhi sequencing, and naming the obtained positive bacteria as the bacteria after the sequencing is correctE.coli/pBBR-P21-nusA. Plasmid pBBR-P21-containing plasmid extracted by plasmid kitnusAFor use, the plasmid map is shown in FIG. 3.

、pBBR-P21-nusA (C517T)

The strain SM genome is used as a template to obtain a nusA (C517T) fragment (the nucleotide sequence is shown as SEQ ID NO: 37, and the amino acid sequence is shown as SEQ ID NO: 38), and the same primers are used for construction according to the construction process of pBBR-P21-nusA to obtain a plasmid pBBR-P21-nusA (C517T).

、pBBR-P21-frr

Respectively using the primer pair P21-XbaI-F and P21-R4, in the presence of a catalystEnsifer adhaerensCasida A (Ensifer. viscosus) genome is used as a template, amplified by PCR and introducedXbaI enzyme cutting site to obtain promoter P21-4 segment, electrophoresis verification and restriction enzyme restrictionDpnI (NEB) was treated at 37 ℃ for 30min to recover the nucleic acid gel, and purified P21-4 fragment was obtained.

Respectively using the primer pairs frr-F and frr-HindIII-R in Table 3, introducing the genome of Sinorhizobium meliloti CGMCC NO.9638 as a template through PCR amplificationHindIII, obtaining frr fragments by enzyme cutting sites, carrying out electrophoretic verification,Dpnand I, treating by an enzyme method, and recovering electrophoresis gel to obtain a purified frr fragment. The frr gene sequence is shown in SEQ ID No.8, and the coded amino acid sequence is shown in SEQ ID No. 9.

Then, using the primer pair P21-XbaI-F and frr-HindIII-R, using purified P21-4 fragment and frr fragment as template, and performing fusion PCR to obtain P21-frr fragment (containingXbaI andHindIII enzyme cutting site), and obtaining a purified P21-frr fragment after electrophoresis verification and electrophoretic gel recovery.

The purified P21-frr fragment and pBBR1MCS2 plasmid were used separatelyXbaI andHindIII, carrying out double enzyme digestion, connecting the double enzyme digestion product of the P21-frr fragment with the double enzyme digestion product of pBBR1MCS2 plasmid through T4 ligase at 4 ℃ overnight, transforming the connecting product into escherichia coli DH5 α, coating the escherichia coli DH5 α on an LB solid plate containing 50mg/L kanamycin, culturing for 16h, carrying out colony PCR detection, carrying out Jinzhi sequencing, and after the sequencing is correct, naming the obtained positive bacteria as theE.coli/pBBR-P21-frr. Plasmid pBBR-P21-containing plasmid extracted by plasmid kitfrrFor use, the plasmid map is shown in FIG. 4.

、pBBR-P21-ysxC

Respectively using the primer pair P21-XbaI-F and P21-R5, in the presence of a catalystEnsifer adhaerensCasida A (Sword bacterium adhesion)Genome as template, amplified by PCR, and introducedXbaI enzyme cutting site to obtain promoter P21-5 segment, electrophoresis verification and restriction enzyme restrictionDpnI (NEB) was treated at 37 ℃ for 30min to recover the nucleic acid gel, and purified P21-5 fragment was obtained.

Respectively utilizing the primer pair ysxC-F and ysxC-HindIII-R of the table 3, taking the Sinorhizobium meliloti CGMCC NO.9638 genome as a template, and introducing the genome by PCR amplificationHindIII, enzyme cutting sites are cut to obtain ysxC fragments, electrophoresis verification is carried out,Dpnand I, performing enzyme method treatment, and recovering electrophoresis gel to obtain a purified ysxC fragment. The ysxC gene sequence is shown as SEQ ID No.10, and the coded amino acid sequence is shown as SEQ ID No. 11.

Then, using the primer pair P21-XbaI-F and ysxC-HindIII-R, and obtaining a P21-ysxC fragment (containing a P21-ysxC fragment) by fusion PCR with the purified P21-5 fragment and the ysxC fragment as templatesXbaI andHindIII enzyme cutting site), and obtaining a purified P21-ysxC fragment after electrophoresis verification and electrophoretic gel recovery.

The purified P21-ysxC fragment and pBBR1MCS2 plasmid were used separatelyXbaI andHindIII, carrying out double enzyme digestion, connecting a double enzyme digestion product of a P21-ysxC fragment with a double enzyme digestion product of a pBBR1MCS2 plasmid through T4 ligase at 4 ℃ overnight, transforming the connection product into escherichia coli DH5 α, coating the escherichia coli DH5 α on an LB solid plate containing 50mg/L kanamycin, carrying out colony PCR detection after culturing for 16h, carrying out Jinzhi sequencing, and naming the obtained positive bacteria as the positive bacteria after correct sequencingE.coli/pBBR-P21-ysxC. Plasmid pBBR-P21-containing plasmid extracted by plasmid kitysxCFor use, the plasmid map is shown in FIG. 5.

、pBBR-P21-greA

Respectively using the primer pair P21-XbaI-F and P21-R6, in the presence of a catalystEnsifer adhaerensCasida A (Ensifer. viscosus) genome is used as a template, amplified by PCR and introducedXbaI enzyme cutting site to obtain promoter P21-6 segment, electrophoresis verification and restriction enzyme restrictionDpnI (NEB) was treated at 37 ℃ for 30min to recover the nucleic acid gel, and purified P21-6 fragment was obtained.

Primer pairs greA-F of Table 3 were used respectivelyAnd greA-HindIII-R, using Sinorhizobium meliloti CGMCC NO.9638 genome as template, making PCR amplification, introducingHindIII, obtaining greA fragments by enzyme cutting sites, carrying out electrophoretic verification,Dpnand I, treating by an enzyme method, and recovering electrophoresis gel to obtain a purified greA fragment. The gene sequence of greA is shown in SEQ ID No.12, and the coded amino acid sequence is shown in SEQ ID No. 13.

Then, using the primer pair P21-XbaI-F and greA-HindIII-R, and obtaining a P21-greA fragment (containing the P21-greA fragment) by fusion PCR with the purified P21-6 fragment and the greA fragment as templatesXbaI andHindIII enzyme cutting site), and obtaining a purified P21-greA fragment after electrophoresis verification and electrophoretic gel recovery.

The purified P21-greA fragment and pBBR1MCS2 plasmid were used separatelyXbaI andHindIII, carrying out double enzyme digestion, connecting the double enzyme digestion product of the P21-greA fragment with the double enzyme digestion product of pBBR1MCS2 plasmid through T4 ligase at 4 ℃ overnight, transforming the connecting product into escherichia coli DH5 α, coating the escherichia coli DH5 α on an LB solid plate containing 50mg/L kanamycin, carrying out colony PCR detection after culturing for 16h, carrying out Jinzhi sequencing, and naming the obtained positive bacteria as the bacteria after correct sequencingE.coli/pBBR-P21-greA. Plasmid pBBR-P21-containing plasmid extracted by plasmid kitgreAFor use, the plasmid map is shown in FIG. 6.

Example 4: construction of plasmid vector-containing Strain

The 10 plasmids in 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 12,000 rpm 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 12,000 rpm 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. Obtaining positive sinorhizobium meliloti, SM/pBBR (control bacterium), SM/pBBR-P21-rbfA(abbreviated as SM 1), SM/pBBR-P21-rbfA(T208C) (abbreviated as SM 2), SM/pBBR-P21-tsf(abbreviated as SM 3), SM/pBBR-P21-tsf(T631C) (abbreviated as SM 4), SM/pBBR-P21-nusA(abbreviated as SM 5), SM/pBBR-P21-nusA(C517T) (abbreviated as SM 6), SM/pBBR-P21-frr (abbreviated as SM 7), SM/pBBR-P21-ysxC (abbreviated as SM 8), SM/pBBR-P21-greA (abbreviated as SM 9).

Example 5: evaluation of different strains

1. The culture conditions of the Sinorhizobium meliloti are as follows:

control bacteria, SM1, SM2, SM3, SM4, SM5, SM6, SM7, SM8 and SM9 strains are streaked on LB solid medium containing 100mg/L kanamycin with an inoculating needle under aseptic conditions, and are kept standing at a constant temperature of 30 ℃ for 48 h for culture, so that single colonies are obtained. 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 (250 mL shake flask). After shaking (220 r/min) culture at 30 ℃ for 144 h, 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 Capacity and Biomass

Strains SM1, SM2, SM3, SM4, SM5 and SM6 produce vitamin B compared to control bacteria₁₂All the capabilities are improved. The results are shown in Table 4.

From the above, it was found that SM1 was increased by 4.8%, SM2 by 8.4%, SM3 by 25.3%, SM4 by 32.5%, SM5 by 20.5%, and SM6 by 27.7%. While SM6, SM7 and SM8 produce vitamin B₁₂With varying degrees of reduction in capacity. The biomass of each strain is not changed greatly, which indicates that the over-expression of each ribosomal factor gene does not influence the growth of the thalli.

Sequence listing

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

<120> ribosomal factor, mutant thereof and application thereof in preparation of vitamin B12

<160>38

<170>PatentIn Version 3.1

<210>1

<211>372

<212>DNA

<213>Sinorhizobium meliloti

<400>1

atgctgcgcg tcggcgaaca ggtgcgcgct gccatcaccc aggttctgca acgcggcgag 60

gtccgcgatc cggtgatcga gaagaccgtc atcgcgattt cggaagtgcg catgtcaccg 120

gatctgaaga tcgccacggc ctatgtcacg ccgcttggcg tgcccaacca tgccgaggtc 180

atcgacgcgc tcaaccgcaa tgccaagtac attcgcggcc gcctcggccc gcagctgcga 240

cagatgaaat acatgcccga cgttcgtttc cgcgacgata cgagcttcga caattacaag 300

aagatcgacg agctcttgcg ttcgccggaa gttagccgcg atctcgacca gaccagcgaa 360

gacgaagaat ag 372

<210>2

<211>123

<212>PRT

<213>Sinorhizobium meliloti

<400>2

MLRVGEQVRA AITQVLQRGE VRDPVIEKTV IAISEVRMSP DLKIATAYVT PLGVPNHAEV 60

IDALNRNAKY IRGRLGPQLR QMKYMPDVRF RDDTSFDNYK KIDELLRSPE VSRDLDQTSE 120

DEE 123

<210>3

<211>906

<212>DNA

<213>Sinorhizobium meliloti

<400>3

atggtgaagg aactgcgcga aaagaccggc gcaggcatga tggactgcaa gaaggcgctc 60

gccgaaacca acggcgacat ggaagctgcg atcgactggc tgcgcgccaa gggcatcgcc 120

aaggccgaca agaagtcggg ccgcacggct gctgaaggcc tcatcggtat tgtcagcggt 180

ggcgtcaagg ctgtcgtcgt cgaaatcaac tccgagaccg atttcgttgc ccgcaacgaa 240

gccttccagg aactcgtccg cggcgttgcc aacgtagcgc tgaccaccga cggctcggtt 300

gaagccatcg cggctgcaaa ctacccggca accggcaagt cggtcgacga gaccatcaag 360

gacgcgatcg ccaccatcgg cgagaacatg acgcttcgcc gcgcagccct gctcaaggtc 420

gaagacggtg ttgtcgccac ctacatccac aacgctgccg gcgacggcat cggcaagctc 480

ggtgttctcg tcgccctgaa gtcgatcggc gacaaggaag ttctcttctc ggttggccgt 540

cagatcgcca tgcacatcgc tgcgaccaac ccgctcgcga tccgtgcgga cgaagtcgac 600

gccgctgttg ccgagcgcga acgcaacgtc ttcatcgaac agtcgcgcgc ttcgggcaag 660

ccggacaaca tcatcgaaaa gatggtcgaa ggccgcatgc gcaagttctt cgaagaagtc 720

gctctgctgt cgcaggcctt cgtcatgaac ccggaaatca ccgtcggtca ggccgtcaag 780

gacgctgaaa agctcgctgg cgcaccgatc gaagtcaccg gcatggctcg cctgctgctc 840

ggcgaaggtg tcgagaagga agagtccgac ttcgctgcag aagttgcagc cgtcgccaag 900

ggctga 906

<210>4

<211>301

<212>PRT

<213>Sinorhizobium meliloti

<400>4

MVKELREKTG AGMMDCKKAL AETNGDMEAA IDWLRAKGIA KADKKSGRTA AEGLIGIVSG 60

GVKAVVVEIN SETDFVARNE AFQELVRGVA NVALTTDGSV EAIAAANYPA TGKSVDETIK 120

DAIATIGENM TLRRAALLKV EDGVVATYIH NAAGDGIGKL GVLVALKSIG DKEVLFSVGR 180

QIAMHIAATN PLAIRADEVD AAVAERERNV FIEQSRASGK PDNIIEKMVE GRMRKFFEEV 240

ALLSQAFVMN PEITVGQAVK DAEKLAGAPI EVTGMARLLL GEGVEKEESD FAAEVAAVAK 300

G 301

<210>5

<211>1620

<212>PRT

<213>Sinorhizobium meliloti

<400>5

atggcagtca gtgctaaccg gctcgagctt ctgcagatcg cggatgctgt cgcgcgtgaa 60

aaggtgatcg accgcgagat cgttctggcc gcgatggccg acgcgatcca gaaggccgcc 120

cgctcgcgct atgggtcgga atcgaacatc cgcgccgaca tcaactccaa gactggcgaa 180

atccgcctgc agcgtcttct ggaagtggtc gagaaggctg acgactactc cacgcagatc 240

ccgctcgagc ttgctcgcga tcgcaatccg gacgccaagc tcggtgactt catcgccgac 300

ccgctgccgc cgatggattt tggccgcatc gccgcccagt cggccaagca ggtcatagtg 360

cagaaggtgc gtgaagccga gcgcgaccgt cagttcgacg agttcaagga tcgcgtcggc 420

gagatcgtca acggcacggt caagcgcgtc gaatacggca acgtcatcgt cgacctcggc 480

cgtggcgaag gcatcatccg tcgcgacgag atgatcccgc gcgaaaacat gcgctacggc 540

gatcgcgtcc gctccttcgt ctacgacgtt cgccgcgaac agcgcggccc gcagatcttc 600

ctgtcgcgta cccatccgca gttcatggtg aagctcttca ccatggaagt gcctgaaatt 660

tacgacggca tcatccagat caagtcggtt gcccgcgacc cgggttcgcg cgccaagatc 720

gcggtcgtct ccaacgatag ctcgatcgat ccggtcggcg cctgcgtcgg tatgcgcggt 780

tcgcgcgttc aggccgtcgt tggcgagttg cagggcgaaa agatcgacat cattccgtgg 840

tcgcaggatc cggcatcgtt catcgtcaac gcgctgcagc cggccgaagt cgccaaggtc 900

gttctcgacg aagacgccga gcgcatcgaa gtggtcgttc ccgacgagca gctgtcgctc 960

gccatcggcc gacgcggcca gaacgttcgc ctcgcttcgc agctgaccgg ctgggatatc 1020

gacatcctga ccgagcagga agagagcgaa cgccgccaga aggaattcac cgagcgcacc 1080

aacctcttca tggatgcgct cgacgtcgac gaaatggtcg gtcaggtgct ggcttcggaa 1140

ggctttgccg ccgtcgagga gcttgcctat gtcgatctcg acgaaattgc gtcgatcgac 1200

ggtttcgacg acgagaccgc caacgaaatc cagacccgcg ctcgcgagta cctggaccgg 1260

atcgaggcgg aaatggatgc caagcgccgc gaactcggcg ttgccgacga actgcgctca 1320

atcgacggcc tgacgagcca gatgctcgtt gcgctgggcg aagaaggcat caagacggtc 1380

gaggactttg ctggttgcgc cgctgacgat ctcgtcggct ggagcgaacg caaggacggc 1440

gagaccaagc gtttcgaagg caccttctcg aagttcgacg tctcgcgcgc cgaagccgaa 1500

gccatgatcg ttcaggcccg cctcgcagcc ggctggatca cgcaagagga cctggcaagc 1560

gaagaggtcg aaggtgaaga gccgatcgac gttgccgaag gcgccgagca ggacgcttaa 1620

<210>6

<211>539

<212>PRT

<213>Sinorhizobium meliloti

<400>6

MAVSANRLEL LQIADAVARE KVIDREIVLA AMADAIQKAA RSRYGSESNI RADINSKTGE 60

IRLQRLLEVV EKADDYSTQI PLELARDRNP DAKLGDFIAD PLPPMDFGRI AAQSAKQVIV 120

QKVREAERDR QFDEFKDRVG EIVNGTVKRV EYGNVIVDLG RGEGIIRRDE MIPRENMRYG 180

DRVRSFVYDV RREQRGPQIF LSRTHPQFMV KLFTMEVPEI YDGIIQIKSV ARDPGSRAKI 240

AVVSNDSSID PVGACVGMRG SRVQAVVGEL QGEKIDIIPW SQDPASFIVN ALQPAEVAKV 300

VLDEDAERIE VVVPDEQLSL AIGRRGQNVR LASQLTGWDI DILTEQEESE RRQKEFTERT 360

NLFMDALDVD EMVGQVLASE GFAAVEELAY VDLDEIASID GFDDETANEI QTRAREYLDR 420

IEAEMDAKRR ELGVADELRS IDGLTSQMLV ALGEEGIKTV EDFAGCAADD LVGWSERKDG 480

ETKRFEGTFS KFDVSRAEAE AMIVQARLAA GWITQEDLAS EEVEGEEPID VAEGAEQDA 539

<210>7

<211>1000

<212>DNA

<213>Ensifer adhaerens

<400>7

caaacagacc gggatatgcg ggtattcttc cgccgcgccg aggatgaggt ggcgcaggaa 60

cgcgtcaccg gcataggagc gggcgccacg gcttgcctga aggatgaccg ggctgtcggt 120

cgcatcggcg gcgcgcatga cggcctgaat gtattccaga ttgttcacat tgaacgccgg 180

cagcgcgtaa tcgttctccg ccgcatggtc gagcagttgc cgcaatgtga tcaatgccat 240

tcgctatctc cctttggata ctcggtgcaa cctatgcggc gcaccacaaa aacaatccgg 300

ccgttgaacc gcacgaaatg catcgatggc aaagtcgatg gccggctttt tcgtgcggcg 360

tgacggcgcg cgcgaattgg tcgcgcccac cgaagtcagg cgcacaatag ttcatcgaag 420

tggtttgaca accgggcaaa aggcaggttg ccagaggtcg aaactcgctt caatcgattt 480

tactgtggac tggatgcaac accttcagtg tgaagtgttt tcactttctg gtggtgcctg 540

agaggagggg gagtcgaggg cagtggatgc aaccattggg cgctgatttt gtctgttaca 600

ccatcgtggt ggatgccctg tcggaaacag tctgtcgaca ggaggtgaac gtcccgcaag 660

aagattgcgg caacgcccct ctttctttgc gcagattacg taaactgccg ctaaaattca 720

caaactttgc atcgcggatg attcgaggct caatccggcc gacaaaaagc gcggacctaa 780

aacgttgcag tagatttcgc aaaaatgccc tgttcacgtc atatgcccgt cgcaaaggcg 840

acgaaaagaa tcgcaaacaa aatacaacct atgggatagg ccgattcccc tcctatagat 900

aaagatgcag acagccgcag aatccgcctt gcgttcgcga acgatttgcg cttctctcct 960

gcgatcacaa acccaaaaca aggggaagga gagaaacaaa 1000

<210>8

<211>561

<212>DNA

<213>Sinorhizobium meliloti

<400>8

atgagtgaag gtgttgacct gaaggaactg aagcgtcgca tggatggcgc catttccgca 60

ttcaagagcg atatcgcgtc gctccgtacc ggccgcgcgt cggcgaacgt tctcgatccg 120

gtgaccgtcg aagcctatgg ctcacgcgtg ccgctcaacc aggtggcgaa catcacggtt 180

cccgagccgc gcatgctgtc cgtctcggtc tgggacaagc agatggtcgg cgctgtggat240

cgcggcattc gcgaatcgaa cctcggcctc aatccgatca tcgacggcca gaacctgcgc 300

attccgctgc cggaactcaa cgaagagcgc cgcaagtcgc tcgtcaaagt ggcacatgac 360

tacgccgaaa aggcgaaggt cgccgtgcgc cacgtgcgcc gtgacggcat ggacgacctg 420

aaaaaagccg aaaaggatgg cgacatcggt caggatatca gccgttctca gtccgagcgc 480

gtgcaaaaga tgaccgacga aacgatttcc gatatcgacc gcttgctcgt cgagaaggaa 540

aaggaaatca tgcaggtctg a 561

<210>9

<211>186

<212>PRT

<213>Sinorhizobium meliloti

<400>9

MSEGVDLKEL KRRMDGAISA FKSDIASLRT GRASANVLDP VTVEAYGSRV PLNQVANITV 60

PEPRMLSVSV WDKQMVGAVD RGIRESNLGL NPIIDGQNLR IPLPELNEER RKSLVKVAHD 120

YAEKAKVAVR HVRRDGMDDL KKAEKDGDIG QDISRSQSER VQKMTDETIS DIDRLLVEKE 180

KEIMQV 186

<210>10

<211>672

<212>DNA

<213>Sinorhizobium meliloti

<400>10

atgaaggacc gaccgccgat ggccgatacc agacccaaag acgacaagcc gttgttcggc 60

cggccgtgga ttttcattcg cggcgtcccc gccatgaaat tcctgccgcc ggaaggaccg 120

gccgagattg cctttgccgg tcgctcgaac gtcggcaagt cgtcgttgat caacgccctg 180

gtcggccaca agggtctggc aagaacctcc aacaccccgg gccgcacgca ggaactcaac 240

tacttcgtac cggatggatt ttccggcgag gccgacgacc tgccgccgat ggcgctcgtc 300

gacatgcccg gttacggcta tgcccaggcg ccgaaggaac aggtcgatgc ctggacaaaa 360

ctggtcttcg actacctgcg cggccgctcg acgctgaagc gcgtctatgt gctgatcgac 420

agccgccacg gcatcaagaa gaacgacgac gaggtgctcg acctgctcga caaggcagcc 480

gtctcctatc agctcgtgct gaccaagacc gacaagatca aggccgccgg cgtgccgcgg 540

ctgatcgccg agacgctcga aaagatcaag aagcatcccg ccgccttccc ggaagtcctt 600

gcgacttcct cggagaaggg cgaagggctc gacgagctgc gggcggcaat cggcatcgcg 660

atcgcgcgct aa 672

<210>11

<211>223

<212>PRT

<213>Sinorhizobium meliloti

<400>11

MKDRPPMADT RPKDDKPLFG RPWIFIRGVP AMKFLPPEGP AEIAFAGRSN VGKSSLINAL 60

VGHKGLARTS NTPGRTQELN YFVPDGFSGE ADDLPPMALV DMPGYGYAQA PKEQVDAWTK 120

LVFDYLRGRS TLKRVYVLID SRHGIKKNDD EVLDLLDKAA VSYQLVLTKT DKIKAAGVPR 180

LIAETLEKIK KHPAAFPEVL ATSSEKGEGL DELRAAIGIA IAR 223

<210>12

<211>492

<212>DNA

<213>Sinorhizobium meliloti

<400>12

atgaaggaca ttgaaatggt cgaaaaagtg ccgatgacac agggcggatt cgtcaacctg 60

caggaggagc tgcgctggcg ccagcaggaa gaacgaccgc gaatcatcga ggcgattgcc 120

gaagcccgtg cacatggcga cctgtcggaa aatgccgagt accacgcggc caaggaagcg 180

cagagccaca acgagggccg gatcaccgag ctcgaagact tgatcgcccg cgcggaagtc 240

attgatctct cgaagatgtc cggctccaag atcaagttcg gcgccaaggt caaactcgtc 300

gacgaagaca ctgaagaaga aaagacctac cagatcgttg gcgaccagga agccgacgtg 360

aaggctggcc gcatttcgat ctcgtcgccg atcgcgcgcg cgctcatcgg caaggaagtc 420

ggcgattcca tcgaagtgaa cgcgcctggc ggttccaagg cctacgaaat tctcgccgtc 480

aactggggct ga 492

<210>13

<211>163

<212>PRT

<213>Sinorhizobium meliloti

<400>13

MKDIEMVEKV PMTQGGFVNL QEELRWRQQE ERPRIIEAIA EARAHGDLSE NAEYHAAKEA 60

QSHNEGRITE LEDLIARAEV IDLSKMSGSK IKFGAKVKLV DEDTEEEKTY QIVGDQEADV 120

KAGRISISSP IARALIGKEV GDSIEVNAPG GSKAYEILAV NWG 163

<210>14

<211>32

<212>DNA

<213> Artificial sequence

<400>14

ctagtctaga caaacagacc gggatatgcg gg 32

<210>15

<211>43

<212>DNA

<213> Artificial sequence

<400>15

ctgttcgccg acgcgcagca ttttgtttct ctccttcccc ttg 43

<210>16

<211>43

<212>DNA

<213> Artificial sequence

<400>16

caaggggaag gagagaaaca aaatgctgcg cgtcggcgaa cag 43

<210>17

<211>32

<212>DNA

<213> Artificial sequence

<400>17

ccggaattcc tattcttcgt cttcgctggt ct 32

<210>18

<211>41

<212>DNA

<213> Artificial sequence

<400>18

ttcgcgcagt tccttcacca ttttgtttct ctccttcccc t 41

<210>19

<211>41

<212>DNA

<213> Artificial sequence

<400>19

aggggaagga gagaaacaaa atggtgaagg aactgcgcga a 41

<210>20

<211>30

<212>DNA

<213> Artificial sequence

<400>20

ccggaattct cagcccttgg cgacggctgc 30

<210>21

<211>40

<212>DNA

<213> Artificial sequence

<400>21

cggttagcac tgactgccat tttgtttctc tccttcccct 40

<210>22

<211>40

<212>DNA

<213> Artificial sequence

<400>22

aggggaagga gagaaacaaa atggcagtca gtgctaaccg 40

<210>23

<211>30

<212>DNA

<213> Artificial sequence

<400>23

cccaagcttt taagcgtcct gctcggcgcc 30

<210>24

<211>492

<212>DNA

<213> Artificial sequence

<400>24

aggtcaacac cttcactcat tttgtttctc tccttcccct 40

<210>25

<211>40

<212>DNA

<213> Artificial sequence

<400>25

aggggaagga gagaaacaaa atgagtgaag gtgttgacct 40

<210>26

<211>30

<212>DNA

<213> Artificial sequence

<400>26

cccaagcttt cagacctgca tgatttcctt 30

<210>27

<211>44

<212>DNA

<213> Artificial sequence

<400>27

ccatcggcgg tcggtccttc attttgtttc tctccttccc cttg 44

<210>28

<211>44

<212>DNA

<213> Artificial sequence

<400>28

caaggggaag gagagaaaca aaatgaagga ccgaccgccg atgg 44

<210>29

<211>40

<212>DNA

<213> Artificial sequence

<400>29

aggggaagga gagaaacaaa atgagtgaag gtgttgacct 40

<210>30

<211>30

<212>DNA

<213> Artificial sequence

<400>30

cccaagcttt tagcgcgcga tcgcgatgcc 30

<210>31

<211>40

<212>DNA

<213> Artificial sequence

<400>31

aggggaagga gagaaacaaa atgaaggaca ttgaaatggt 40

<210>32

<211>31

<212>DNA

<213> Artificial sequence

<400>32

cccaagcttt cagccccagt tgacggcgag a 31

<210>33

<211>372

<212>DNA

<213>Sinorhizobium meliloti

<400>33

atgctgcgcg tcggcgaaca ggtgcgcgct gccatcaccc aggttctgca acgcggcgag 60

gtccgcgatc cggtgatcga gaagaccgtc atcgcgattt cggaagtgcg catgtcaccg 120

gatctgaaga tcgccacggc ctatgtcacg ccgcttggcg tgcccaacca tgccgaggtc 180

atcgacgcgc tcaaccgcaa tgccaagcac attcgcggcc gcctcggccc gcagctgcga 240

cagatgaaat acatgcccga cgttcgtttc cgcgacgata cgagcttcga caattacaag 300

aagatcgacg agctcttgcg ttcgccggaa gttagccgcg atctcgacca gaccagcgaa 360

gacgaagaat ag 372

<210>34

<211>123

<212>PRT

<213>Sinorhizobium meliloti

<400>34

MLRVGEQVRA AITQVLQRGE VRDPVIEKTV IAISEVRMSP DLKIATAYVT PLGVPNHAEV 60

IDALNRNAKH IRGRLGPQLR QMKYMPDVRF RDDTSFDNYK KIDELLRSPE VSRDLDQTSE120

DEE 123

<210>35

<211>906

<212>DNA

<213>Sinorhizobium meliloti

<400>35

atggtgaagg aactgcgcga aaagaccggc gcaggcatga tggactgcaa gaaggcgctc 60

gccgaaacca acggcgacat ggaagctgcg atcgactggc tgcgcgccaa gggcatcgcc 120

aaggccgaca agaagtcggg ccgcacggct gctgaaggcc tcatcggtat tgtcagcggt 180

ggcgtcaagg ctgtcgtcgt cgaaatcaac tccgagaccg atttcgttgc ccgcaacgaa 240

gccttccagg aactcgtccg cggcgttgcc aacgtagcgc tgaccaccga cggctcggtt 300

gaagccatcg cggctgcaaa ctacccggca accggcaagt cggtcgacga gaccatcaag 360

gacgcgatcg ccaccatcgg cgagaacatg acgcttcgcc gcgcagccct gctcaaggtc 420

gaagacggtg ttgtcgccac ctacatccac aacgctgccg gcgacggcat cggcaagctc 480

ggtgttctcg tcgccctgaa gtcgatcggc gacaaggaag ttctcttctc ggttggccgt 540

cagatcgcca tgcacatcgc tgcgaccaac ccgctcgcga tccgtgcgga cgaagtcgac 600

gccgctgttg ccgagcgcga acgcaacgtc ctcatcgaac agtcgcgcgc ttcgggcaag 660

ccggacaaca tcatcgaaaa gatggtcgaa ggccgcatgc gcaagttctt cgaagaagtc 720

gctctgctgt cgcaggcctt cgtcatgaac ccggaaatca ccgtcggtca ggccgtcaag 780

gacgctgaaa agctcgctgg cgcaccgatc gaagtcaccg gcatggctcg cctgctgctc 840

ggcgaaggtg tcgagaagga agagtccgac ttcgctgcag aagttgcagc cgtcgccaag 900

ggctga 906

<210>36

<211>301

<212>PRT

<213>Sinorhizobium meliloti

<400>36

MVKELREKTG AGMMDCKKAL AETNGDMEAA IDWLRAKGIA KADKKSGRTA AEGLIGIVSG 60

GVKAVVVEIN SETDFVARNE AFQELVRGVA NVALTTDGSV EAIAAANYPA TGKSVDETIK 120

DAIATIGENM TLRRAALLKV EDGVVATYIH NAAGDGIGKL GVLVALKSIG DKEVLFSVGR 180

QIAMHIAATN PLAIRADEVD AAVAERERNV LIEQSRASGK PDNIIEKMVE GRMRKFFEEV 240

ALLSQAFVMN PEITVGQAVK DAEKLAGAPI EVTGMARLLL GEGVEKEESD FAAEVAAVAK 300

G 301

<210>37

<211>1620

<212>DNA

<213>Sinorhizobium meliloti

<400>37

atggcagtca gtgctaaccg gctcgagctt ctgcagatcg cggatgctgt cgcgcgtgaa 60

aaggtgatcg accgcgagat cgttctggcc gcgatggccg acgcgatcca gaaggccgcc 120

cgctcgcgct atgggtcgga atcgaacatc cgcgccgaca tcaactccaa gactggcgaa 180

atccgcctgc agcgtcttct ggaagtggtc gagaaggctg acgactactc cacgcagatc 240

ccgctcgagc ttgctcgcga tcgcaatccg gacgccaagc tcggtgactt catcgccgac 300

ccgctgccgc cgatggattt tggccgcatc gccgcccagt cggccaagca ggtcatagtg 360

cagaaggtgc gtgaagccga gcgcgaccgt cagttcgacg agttcaagga tcgcgtcggc 420

gagatcgtca acggcacggt caagcgcgtc gaatacggca acgtcatcgt cgacctcggc 480

cgtggcgaag gcatcatccg tcgcgacgag atgatctcgc gcgaaaacat gcgctacggc 540

gatcgcgtcc gctccttcgt ctacgacgtt cgccgcgaac agcgcggccc gcagatcttc 600

ctgtcgcgta cccatccgca gttcatggtg aagctcttca ccatggaagt gcctgaaatt 660

tacgacggca tcatccagat caagtcggtt gcccgcgacc cgggttcgcg cgccaagatc 720

gcggtcgtct ccaacgatag ctcgatcgat ccggtcggcg cctgcgtcgg tatgcgcggt 780

tcgcgcgttc aggccgtcgt tggcgagttg cagggcgaaa agatcgacat cattccgtgg 840

tcgcaggatc cggcatcgtt catcgtcaac gcgctgcagc cggccgaagt cgccaaggtc 900

gttctcgacg aagacgccga gcgcatcgaa gtggtcgttc ccgacgagca gctgtcgctc 960

gccatcggcc gacgcggcca gaacgttcgc ctcgcttcgc agctgaccgg ctgggatatc 1020

gacatcctga ccgagcagga agagagcgaa cgccgccaga aggaattcac cgagcgcacc 1080

aacctcttca tggatgcgct cgacgtcgac gaaatggtcg gtcaggtgct ggcttcggaa 1140

ggctttgccg ccgtcgagga gcttgcctat gtcgatctcg acgaaattgc gtcgatcgac 1200

ggtttcgacg acgagaccgc caacgaaatc cagacccgcg ctcgcgagta cctggaccgg 1260

atcgaggcgg aaatggatgc caagcgccgc gaactcggcg ttgccgacga actgcgctca 1320

atcgacggcc tgacgagcca gatgctcgtt gcgctgggcg aagaaggcat caagacggtc 1380

gaggactttg ctggttgcgc cgctgacgat ctcgtcggct ggagcgaacg caaggacggc 1440

gagaccaagc gtttcgaagg caccttctcg aagttcgacg tctcgcgcgc cgaagccgaa 1500

gccatgatcg ttcaggcccg cctcgcagcc ggctggatca cgcaagagga cctggcaagc 1560

gaagaggtcg aaggtgaaga gccgatcgac gttgccgaag gcgccgagca ggacgcttaa 1620

<210>38

<211>539

<212>PRT

<213>Sinorhizobium meliloti

<400>38

MAVSANRLEL LQIADAVARE KVIDREIVLA AMADAIQKAA RSRYGSESNI RADINSKTGE 60

IRLQRLLEVV EKADDYSTQI PLELARDRNP DAKLGDFIAD PLPPMDFGRI AAQSAKQVIV 120

QKVREAERDR QFDEFKDRVG EIVNGTVKRV EYGNVIVDLG RGEGIIRRDE MISRENMRYG 180

DRVRSFVYDV RREQRGPQIF LSRTHPQFMV KLFTMEVPEI YDGIIQIKSV ARDPGSRAKI 240

AVVSNDSSID PVGACVGMRG SRVQAVVGEL QGEKIDIIPW SQDPASFIVN ALQPAEVAKV 300

VLDEDAERIE VVVPDEQLSL AIGRRGQNVR LASQLTGWDI DILTEQEESE RRQKEFTERT 360

NLFMDALDVD EMVGQVLASE GFAAVEELAY VDLDEIASID GFDDETANEI QTRAREYLDR 420

IEAEMDAKRR ELGVADELRS IDGLTSQMLV ALGEEGIKTV EDFAGCAADD LVGWSERKDG 480

ETKRFEGTFS KFDVSRAEAE AMIVQARLAA GWITQEDLAS EEVEGEEPID VAEGAEQDA 539

Claims (8)

1. A mutant of ribosome binding factor A, characterized in that the amino acid sequence is based on that shown in SEQ ID No.2, and the 70 th amino acid is replaced by H.

2. A mutant of ribosome translation elongation factor Ts, characterized in that the amino acid sequence is based on that shown in SEQ ID No.4, and the 211 th amino acid is replaced by L.

3. A mutant of ribosome translation elongation factor NusA is characterized in that the amino acid sequence is shown in SEQ ID No.6, and the 173 th amino acid is replaced by S.

4. A gene encoding the mutant as claimed in any one of claims 1 to 3.

5. Application of ribosome binding factor A, ribosome translation elongation factor Ts or ribosome translation elongation factor NusA encoding gene in preparing vitamin B12, wherein the ribosome binding factor A encoding gene encodes polypeptide with an amino acid sequence shown as SEQ ID No.2 or SEQ ID No. 34; the ribosome translation elongation factor Ts coding gene encodes a polypeptide with an amino acid sequence shown as SEQ ID No.4 or SEQ ID No. 36; the ribosome translation elongation factor NusA coding gene codes polypeptide with an amino acid sequence shown as SEQ ID No.6 or SEQ ID No. 38.

6. The use of claim 5, wherein: the nucleotide sequence of the ribosome binding factor A coding gene is shown as SEQ ID No.1 or SEQ ID No.33, the nucleotide sequence of the ribosome translation elongation factor Ts is shown as SEQ ID No.3 or SEQ ID No.35, and the nucleotide sequence of the ribosome translation elongation factor NusA coding gene is shown as SEQ ID No.5 or SEQ ID No. 37.

7. The use of claim 5 or 6, wherein the overexpression is performed by introducing the expression vector comprising the coding gene into Sinorhizobium meliloti.

8. The use of claim 7, wherein the preservation number of Sinorhizobium meliloti is CGMCC NO. 9638.

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