KR101023788B1 - Recombinant plasmid, biotin producing microorganism transformed by the recombinant plasmid and method for manufacturing biotin using the transformed biotin producing microorganism - Google Patents

Abstract

The present invention is to make a recombinant plasmid introduced with genes that play an important role in biotin biosynthesis and par locus to enhance the stability of biotin operon and plasmid consisting of one bioABFCD, a gene involved in the biosynthesis of D-biotin (D-biotin) The present invention relates to a method for transforming a biotin-producing bacterium using the plasmid and transforming it into a bacterium capable of producing a large amount of biotin, and a method for producing a biotin using the same, wherein the recombinant strain prepared according to the present invention actually has only D-biotin. It was effective in mass production of biotin by biological method, replacing the existing chemical synthesis method to selectively produce and discharge it out of cells. As such, if D-biotin is produced in a single reaction at room temperature using biological technology, it is expected to be environmentally friendly and economically competitive.

Description

Recombinant plasmid, biotin producing microorganism transformed by the recombinant plasmid and method for manufacturing biotin using the transformed biotin producing microorganism }

The present invention is one of the complexes of vitamin B and biotin (biotin, vitamin H) belonging to essential vitamins to replace the existing chemical synthesis method in order to produce a biological process by actually producing only D- biotin and biotin that is discharged out of the cell The present invention relates to a method for producing a large amount using a production bacterium. More specifically, the bioABFCD, a gene involved in biotin biosynthesis, is composed of one biotin operon and a par locus that plays an important role in biotin biosynthesis. The present invention relates to a method for producing a recombinant plasmid into which genes have been introduced, transforming a bacterium capable of producing a large amount of biotin by transducing a biotin-producing bacterium using the plasmid, and a method for producing biotin using the same.

Biotin is one of the most recently discovered B vitamins and is an essential nutrient for many microorganisms, animals and plants. The study was discovered in 1901 by Wildiers and named bios (a factor necessary for the growth of yeast). In 1935, it was first named Kotin by Koggl, and the pure form of biotin was separated and identified by Gyotgy in 1940 as the same substance as vitamin H.It is known as protective factor X, vitamin H. The structure was first revealed in 1942 and first synthesized in 1943.

Biotin is a coenzyme that helps carboxylase work when pyruvate becomes oxaloacetic acid by the action of carboxylase in the process of glucose metabolism in the cell and is transferred to tissues such as liver and converted to glucose. It is known that enzymes containing biotin work to promote cell proliferation when making nucleic acids that are important metabolism of fatty acids or amino acids and DNA components. The role in metabolism in detail is that it has the function as a complementary molecular family (an atomic group or molecular group that combines with proteins to form complex proteins) in enzymes involved in the carboxylation reaction. The function of the biotin molecules in these enzymes is to allow the enzymes to transport carbon dioxide to specific substrates to fix or retain the carbon dioxide.

Biotin is a necessary vitamin for both humans and animals, and if it is insufficient in vivo, humans have symptoms such as dermatitis, vomiting, anorexia, boredom, muscle paralysis, hypercholesterolemia, and various congenital diseases. Animals must be ingested from the outside because farm income is reduced due to diseases such as fatty liver, fatty kidneys, skin diseases, hoof rolling, and serious phenomena such as decreased growth rate, increased fat saturation, and reduced milk production. In the United States, the daily intake of biotin is defined as 100-200 µg in adults.

In microorganisms, biotin is a precursor to pimelic acid, pimelyl-CoA, 7-keto-8 amino pelargonic acid (KAPA), It is biosynthesized using 7,8-diaminoferargonic acid (DAPA) and desthiobiotin (DTB) as an intermediate, and most bacteria and fungi can self-synthesize. However, livestock and humans are vitamins that cannot be synthesized by themselves. They are very useful because they are used as feed additives to promote livestock growth or as raw materials for the synthesis and production of food additives, injections, antibiotics and other medicines. . However, no studies on biotin production by biological processes have been conducted, and the results of previous studies are not considered to be sufficient to replace organic synthesis. Therefore, research is needed to secure biological production technology of biotin at home and abroad.

In order to produce biotin, which is one of essential vitamins, in a biological process instead of the conventional chemical synthesis method, a biotin-producing bacterium which selectively produces only D-biotin and discharges it out of the cell was attempted. Development of high yielding recombinant biotin-producing strains that overproduce biotin using genetic recombination techniques that transform host microorganisms incorporating biotin operon into vectors, and also at the genetic level of biotin biosynthesis An object of the present invention is to produce biotin in high yield and high productivity by developing a biological fermentation process of a high recombinant biotin producing strain based on the results of research, genomics, proteomics and the like.

     In order to achieve the above object, the present invention provides a recombinant plasmid into which biotin operon and par locus DNA are introduced, and furthermore, a recombinant plasmid into which the biotin B gene is further introduced under PL Promotor and the recombinant plasmid. Provided is a biotin production method characterized by adding an antibiotic, in particular, ampicillin, to a bacterial culture in a converted biotin producing bacterium and a biotin production method using the transformed biotin producing bacterium.

In the present invention as described above, the biotin operon and biotin B gene is biotin producing bacteria, preferably Serratia marsense ( Serratia) marcescens ).

The recombinant strain prepared according to the present invention was effective in mass production of biotin by a biological method in place of the conventional chemical synthesis method to actually generate only D-biotin and discharge it out of cells. As such, if D-biotin is produced in a single reaction at room temperature using biological technology, it is expected to be environmentally friendly and economically competitive.

      Specific details for the implementation of the present invention as described above will be described in detail by way of examples.

In the following examples, Ceratia marsense, which selectively generates only D-biotin and discharges it out of cells, is not limited thereto, and it is known to selectively generate only D-biotin and discharge it out of cells. Any biotin-producing bacterium can be applied, and the vector and plasmid of the above-described embodiment are not limited to the description of the examples, and modifications substituted with other vectors and plasmids known to play an equivalent role are also included in the present invention.

Referring to FIG. 1, in which the entire process of the embodiments to be described below is shown in a flow chart, the embodiment is largely a gene cloning of a biotin operon, a recombinant plasmid preparation using a vector pBR322, a biotin operon gene cloning into a pEYR vector into which par locus is introduced, and bioA. Wow bioB Cloning of genes, cloning the bioB gene into a pLEX vector with a powerful PL promoter, constructing a recombinant plasmid containing the bioB gene, biotin operon and par locus under the PL promoter, transforming into Serratia marsense, using bioassay and HPLC Biotin production will be analyzed in order.

Example  1. Polymerase chain reaction Polymerase Chain Reaction ) Biotin Operon  Amplification and restriction enzyme Biotin Operon  Preparation of Recombinant Vectors Including

Biotin operon in biotin operon involved in biotin synthesis (ATCC 14764) was selectively amplified using a polymerase chain reaction (PCR) to selectively amplify the biotin operon and clone it into a vector. . The structure of the biotin operon gene is shown in FIG. 2.

The biotin operon was divided into two sections because the size of the total biotin operon was relatively large, 7.2 kb. The primers used for PCR are as follows: biotin 1F, biotin 1R and biotin 2F, biotin 2R primers 20pmole, magnesium chloride 25mM, dNTP's mix 2.5mM, designed to specifically amplify only the genes encoding the biotin operon 5 units / μl of Taq DNA polymerase was added to 10 μl of Serratia marsense DNA template to use DNA Thermal Cycler. Amplification PCR conditions were subjected to denaturation (denaturation) for 5 minutes at 94 ℃ and then 30 cycles amplification. Denaturation was performed at 95 ° C. for 1 minute, annealing at 63 ° C. for 2 minutes, and extension at 72 ° C. for 3 minutes. In the last 30 cycles, the reaction was completed by increasing the elongation time at 72 ° C for 10 minutes.

biotin 1 F: 5 'AAG CTT GCC GCC GAA CAG CGC G 3'

biotin 1 R: 5 'CAG ATC GAT CGG TGA GAT CGC 3'

biotin 1 F: 5 'TGC TGT TCG AAG CGC AAA CC 3'

biotin 2 R: 5 'CGC CCC GGG GGC ACC GRC GGC G 3'

Since the biotin operon prepared by the above method contains adenine (A) at both ends of the 3 ', the ligation is performed on a pGEM-T easy vector (Promega, USA) having thymine (T) at both ends. The recombinant plasmids pGEM-bio1 and pGEM-bio2 were produced. The recombinant plasmids were transformed into Escherichia coli Top10F 'to prepare a transformed strain. Thus inserted gene was PCR using the same primers and conditions as described above, it was confirmed that the biotin operon gene was correctly inserted (see Figure 3).

Example  2. Recombinant plasmid pBR322 - biotin operon ) Production and transformation strain Development

A recombinant vector was prepared to carry the biotin operon gene into the Serratia marsense strain and to insert and express it in the genome. I.e., the recombinant plasmid in Example 2 pGEM-bio1, of the pGEM-bio2 restriction enzymes Spe l, BstB After each cleavage into l-site, bio2 fragments were ligated to pGEM-bio1 to complete the whole biotin operon (FIG. 4). Completely transformed plasmid pBR322 from pGEM-biotin operon and Serratia marsense, the restriction enzyme EcoR l and Hind After cutting with lll, the biotin operon fragment was ligated to pBR322 to prepare pbioBR322 (FIG. 5).

Biotin Operon Restriction Enzyme Hind lll, Bst B l, Spe l, Bam H l, Sca After cutting using l, the analysis was carried out to confirm the orientation and ligation of the biotin operon (Fig. 6). In order to determine whether the finished biotin operon is active, a plasmid containing biotin operon was transformed into E. coli χ1776, a mutant strain lacking the bioH gene and biotin operon. Escherichia coli χ 1776 was incubated overnight in LB medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride), and then re-inoculated to incubate from OD600 to 0.6. The cells were centrifuged (4 ° C., 4000 rpm, 10 minutes), and then, 1/2 volume of cold 100 mM calcium chloride was added thereto and left at 0 ° C. for 15 minutes, followed by centrifugation again to harvest the cells. The cells were again suspended in 1/10 volume of cold 100 mM calcium chloride, left for 5 minutes on ice, and then put into a recombinant plasmid and left at 0 ° C. for 20 minutes and subjected to heat shock at 42 ° C. for 90 seconds. It was left for 5 minutes and 1 ml of LB medium was added thereto, followed by incubation at 37 ° C. This was plated in agar medium to which 50 μg / ml of ampicillin was added and cultured at 37 ° C. After transformation, the experiment was carried out by observing colony generation by smearing on a selection medium (MM) lacking biotin, and confirmed that the cloned biotin operon was properly expressed (FIG. 7). PbioBR322, which was found to have proper activity, was transformed into a Seratia marsense strain using the transformation method of calcium chloride as described below. The Serratia marsense strain was incubated overnight in LB medium, then cultured at 0.4 in OD600 and the cells were left at 0 ° C. for 10 minutes and centrifuged (4 ° C., 4000 rpm, 10 minutes). The cells are again suspended in 10 mM sodium chloride and left at 0 ° C. for 10 minutes. Thereafter, the cells were subjected to heat shock at 65 ° C. for 1 minute to inactivate nuclease, and then left at 0 ° C. for 10 minutes. The cells are centrifuged (4 ° C., 4000 rpm, 15 minutes), suspended in 30 mM calcium chloride and left at 0 ° C. for 15 minutes. After harvesting the cells by centrifugation, the cells were resuspended in 30 mM calcium chloride and 15% glycerol and left for 5 minutes on ice. Then, the recombinant plasmid was added and left at 0 ° C. for 20 minutes and subjected to thermal shock at 42 ° C. for 75 seconds. It was left on ice for 5 minutes and 1 ml of LB medium was added thereto, followed by incubation at 37 ° C. This was plated in agar medium containing 500 μg / ml of ampicillin and incubated at 37 ° C. Through this, a recombinant strain in which the recombinant plasmid pbioBR322 was transformed into a Serratia marsense strain was developed.

Example  3. Biotin Operon  Recombinant strains Biotin  Check generation

Recombinant strain with pbioBR322 developed above was produced at 30 ° C. in a medium (15% sucrose, 1.5% urea, 0.1% corn steep liquor, 0.1% K2HPO4, 0.01% FeSO4, 1.5% CaCO3, 0.2% MgSO4). Incubation with a flask at 200 rpm for 1 day. After the culture was recovered, only the supernatant was obtained by using a centrifuge, and the amount of biotin produced was quantified by HPLC analysis as well as by bioassay using Lactobacillus plantarum , a biotin-identifying strain. This bioassay was added to Biotin assay media, 1.75% agar powder, 0.002% bromocresol purple, sterilized at 121 ° C for 15 minutes, cooled to 50 ° C, and then put into Lactobacillus plantarum. Dragon made agar badges. Thereafter, a hole of 0.5 cm was placed in the center of the agar medium and 10 µl of the culture solution was added thereto. The amount of biotin was determined by comparing the yellow zone generated after incubating the agar medium at 37 ° C. for 20 hours to the standard zone. HPLC analysis was quantified by HPLC (High Pressure Liquid Chromatography waters 5300) using a C18 reverse phase column (waters, USA). As a developing reagent, potassium phosphate (25 mM) / acetonitrile was used as 90:10 (v / v).

As a result, the recombinant strain with pbioBR322 showed a biotin production amount of 834 ㎍ / ㎖ by HPLC analysis, and also confirmed the activity of the approximate value in the bioassay method.

Example  4. Biotin Operon  Confirmation of stability of plasmid of recombinant strain

The effect of increased plasmid stability of biotin operon recombinant strain on biotin production was examined. Plasmid stability was determined using a medium containing antibiotics. Strains containing recombinant plasmids were cultured in a medium containing antibiotics ampicillin, then reinoculated in a medium without ampicillin, and then cultured at 30 ° C. for a period of time. The culture medium was taken every time, and the culture medium was diluted appropriately and smeared on the medium containing no ampicillin and the agar medium contained therein to compare the number of colonies to confirm the plasmid stability. As a result, the recombinant strain was observed to lose most of the plasmid after 72 hours.

Example  5. Biotin Operon  To the recombinant plasmid par locus  Increased plasmid stability through introduction and Biotin  Check generation

Recombinant plasmid pbioEYR was prepared by digesting and ligating biotin operon with restriction enzymes EcoRl and Hindlll of pEYR, a vector into which par locus DNA fragments involved in stability of plasmid were introduced in order to increase plasmid stability. ). This was transformed into a Serratia marsense strain and plasmid stability experiments were performed as indicated above. As a result, even after 144 hours, the plasmid stability of the pbioEYR-expressing strain was maintained at 60% or more.

In order to confirm the change in the amount of biotin produced as the plasmid stability was increased, it was observed in the production medium of Example 3 and then observed by HPLC analysis. As a result, the amount of biotin produced was increased by 50% or more. In this regard, the biotin production of recombinant strains is affected by the stability of the plasmid, and biotin production is increased as the plasmid is stabilized.

Example  6. Polymerase chain reaction (PCR)  Used bioA Wow bioB  Gene Amplification and Recombinant Plasmid Construction

Among the genes that make up biotin operon, bioA and bioB To clone the gene and to determine how its expression affects biotin production, bioA and bioB Genes were selectively amplified and cloned into vectors to amplify each using polymerase chain reaction (PCR) to produce recombinant vectors. with bioA bioB The structure of the gene is shown in FIG.

bioA and bioB The part including the open reading frame and the promoter of the gene was amplified by PCR. Primers used for PCR are as follows.

bioA F: 5 'CAC CGA TCG ATC TGA TGG CGG 3'

bioA R: 5 'CAG ATC GAT CGG TGA GAT CGC 3'

bioB F: 5 'CAT GAT GGC CGA CCG CAT 3'

bioB R: 5 'CAT TCA GCA CCT CCA GCA G 3'

with bioA bioB To each primer 20pmole, magnesium chloride 25mM, dNTP's mix 2.5mM, 10μl of Serratia marsense DNA template was added 5unit / μl Taq DNA polymerase was used DNA Thermal Cycler. Amplification PCR conditions were performed for 5 minutes denaturation at 94 ℃ and then 30 cycles amplification. Denaturation was performed at 95 ° C for 1 minute, annealing at 57 ° C for 2 minutes, and extension at 72 ° C for 3 minutes. In the last 30 cycles, the reaction was completed by increasing the elongation time at 72 ° C for 10 minutes.

BioA prepared by the above method and bioB Since the gene contains adenine (A) at both ends of the 3 ', it is ligated to the pGEM-T easy vector (Promega, USA) which has thymine (T) at both ends, so that the recombinant plasmids pbioAS and pbioBS Was produced. The recombinant plasmid was introduced into E. coli Top10F 'to prepare a transformed strain. So a gene inserted after performing the PCR using the same primers and conditions as in the above, and bioA bioB It was confirmed that the gene was correctly inserted (see FIG. 10).

Example  7. bioA Wow bioB Confirming the activity of the recombinant plasmid and bioA Wow bioB Through development of genetically transformed strains Biotin  Check generation

with bioA bioB Recombinant vectors were prepared for delivery of the gene into the Serratia marsense strain and for insertion and expression in the genome (FIG. 11 a and b). That is, the recombinant plasmid in Example 6 pbioAS, pbioBS each bioA and bioB to see if the activity of the BioA gene using a mutant strain of MEC1, MEC2 and bioB After the gene has been spread on the plasmid (pbioAS, pbioBS) transformed culture medium (MM) selected biotin deficient after switching on the cloned observing whether the colonies generated, cloned and bioA bioB It was confirmed that the gene is properly expressed (Fig. 12).

PbioAS and pbioBS plasmids, which were confirmed to have proper activity, were transformed into Serratia marsense strains, respectively, to develop recombinant strains.

with bioA bioB The change in biotin production according to the introduction of the gene was incubated in the production medium of Example 3 and observed through HPLC analysis. As a result, more biotin production was observed in the strain transformed with the bioB gene, especially bioB rather than the bioA gene. The genes were found to have a significant effect on increasing biotin production by about 30%. Biotin synthesis inde step of the bioB gene coding process is converted to biotinylated di arylthio biotin (Destiobiotin) the most important step of this by introducing the bioB gene was increased the activity to be converted into biotin in di arylthio biotin biotin due to this The production seems to have increased.

Example  8. Piel  Promoter PL promoter With) bioB  Gene overexpression of recombinant plasmid and SDS - PAGE  Expression confirmation using electrophoresis

bioB Since the gene is thought to increase biotin production, it was expressed under a PL promoter which is stronger than the promoter of bioB gene. The pLEX vector containing the PEL promoter and pbioBS , a bioB recombinant plasmid prepared in Example 6, were digested with restriction enzyme Ndel and then ligated to prepare pbioBL (FIG. 13).

In order to determine whether pbioBL, the recombinant plasmid prepared above, has activity, E. coli JM109 was used under the Pel promoter. bioB The plasmid in which the gene was cloned was transformed. E. coli JM109 was cultured in aerobic conditions at 37 ℃ using LB medium, E. coli JM109 transformed with the recombinant plasmid pbioBL was cultured in aerobic conditions at 37 ℃ by adding 50 ㎍ / ㎖ ampicillin to the LB medium. After incubation for 24 hours, the cells were collected and crushed, and the lysed cells were suspended in physiological saline, and the cell lysate was centrifuged, and only the supernatant was obtained as a sample. This prepared sample was loaded with a loading buffer (2.5% sodium dodecyl sulfite, 5% (M / V) β-mercaptoethanol, 0.002% bromophenol blue, 6M urea, 0.01M tris-hydrochloric acid (pH6.8), 10 % (W / V) glycerol) and the same amount and then stirred in boiling water for 5 minutes, followed by electrophoresis. The gels were electrophoresed using a dye solution (0.005% Coomassie Brilliant Blue R-250) and bleached using a bleach solution (50% methanol, 10% acetic acid). As a result of analysis by SDS-PAGE electrophoresis, a band of the biotin synthase enzyme was identified at a position of about 39 KDa (FIG. 14).

Example  9. Expression Confirmed Recombinant Plasmid pbioBL Transformation strain development and Biotin  Production quantity check bioB ( biotin synthase Enzyme activity assay

BioB under the Piel promoter cloned in Example 8 above A recombinant strain was developed by transforming pbioBL, a recombinant plasmid confirming that the gene is properly expressed, with a Serratia marsense strain.

Subsequently, the recombinant plasmid, pbioBL, was transformed to induce the expression of Seratia marsense strain in the production medium, and biotin production was quantified by bioassay and HPLC, and the bioB gene was expressed under a self promoter. Expression under the promoter increased biotin production by about 30% (885 µg / ml). This is bioB Enzyme analysis of biotin synthase encoded by the bioB gene was performed because it is thought to be a change in biotin production due to gene activity. 25 mg / ml cell extract, 1 mM disthiobiotin, 1 mM NADPH, 0.1 mM SAM, 1 mM Fe (ll) -gluconate, 2 mM DTT, 0.2 mM PMSF were added in 500 μl and finally 100 mM tris-hydrochloric acid (pH7. 5). After the enzyme reaction was carried out for 1 to 3 hours at 37 ℃. After centrifugation, the bioassay was performed using the supernatant, and the enzyme activity was increased. This suggests that overexpression of the bioB gene may have a significant effect on increasing biotin production.

Example  10. Piel  Under the promoter bioB  Gene and Biotin Operon , par locus Recombinant plasmid production comprising

In the recombinant plasmid pbioEYR prepared in Example 5, a recombinant plasmid in which the overexpressed bioB gene was introduced under the PEL promoters identified in Examples 8 and 9 was prepared to increase biotin production. PbioEYR the recombinant plasmid and a recombinant plasmid with restriction enzymes Sal pbioBL After cleavage with l, the bioB gene fragment including the PI promoter was ligated to the recombinant plasmid pbioEYR to prepare a recombinant plasmid pJEYR having the following characteristics (FIG. 15).

pJEYR: bioB + biotin operon + par locus + pBR322 under PI promoter

Example  11.recombinant plasmid pJEYR Transformation Strain Development and Plasmid Stability Biotin  Check generation

PJEYR, a recombinant plasmid prepared in Example 10, was transformed into a Serratia marcesense strain (Serratia marcescens ctd 40 (pJEYR), Accession No .: KCTC 11307BP, Deposit date: April 2008 Day 1: see attached depositary).

And then the transformed strain was cultured in a production medium results of the plasmid stability tests, since it contains the par locus maintained stably for more than 60% after the plasmid stability over a 144 times compared to a recombinant plasmid which does not include the par locus It could be observed. As a result of observing the amount of biotin produced using HPLC, it was confirmed that the amount of biotin produced was very high at 1988 µg / ml.

Example  12. Improvement of Plasmid Stability with Antibiotic Addition and Biotin  Increase in production

The transformed strain prepared in Example 11 was continuously cultured in a production medium, separated, stabilized, and purified to isolate a highly productive recombinant strain. The recombinant plasmid pJEYR contains an ampicillin resistance gene as an antibiotics marker.When inoculating a culture medium into the production medium from the initial seed culture, the addition of ampicillin increases plasmid stability, which affects biotin production. After the injection of ampicillin at the time of inoculation, the stability of the plasmid was examined. After culturing the strain, the biotin production was observed by HPLC analysis. As a result, the biotin production was high at 2350 µg / ml.

On the other hand, if ampicillin is added at 48 hours, when plasmid stability is rapidly decreased during culture, it is thought that plasmid stability will be increased to affect biotin production. Therefore, ampicillin was added at 48 hours after cultivation rather than at the time of initial inoculation. After the biotin production was investigated, high biotin production was shown at 2200 ~ 2500㎍ / ㎖.

1 is a flow chart showing a production method according to the present invention.

Figure 2 shows the structure and primer binding sites of the biotin operon gene.

Figure 3 is an electrophoresis picture confirmed by PCR the biotin operon cloned in the pGEM vector.

lane 1: Size marker

lane 2: Biotin operon

Figure 4 shows the structure of the biotin operon gene cloned into the pGEM vector.

Figure 5 shows the structure of the biotin operon gene cloned into the pBR322 vector.

Figure 6 is an electrophoresis picture confirming the direction and ligation of the cloned biotin operon using restriction enzymes.

lane 1: Size marker

lane 2: pbioBR322

lane 3: pbioBR322 digested with Hind lll

lane 4: pbioBR322 digested with Hind lll and Bst Bl

lane 5: pbioBR322 digested with Hind lll and Spe l

lane 6: pbioBR322 digested with Bam Hl

lane 7: pbioBR322 digested with Sca l

7 is a plate photograph confirmed by E. coli χ 1776 whether the finished biotin operon is active.

A: E. coli χ1776 in Minimum media-biotin

B: E. coli χ1776 in Minimum media + 100ng / ml biotin

C: pbioBR322 + E. coli χ1776 in Minimum media

Figure 8 shows the structure of the biotin operon gene cloned into the pEYR vector introduced par locus.

Figure 9 shows the structure and primer binding sites of the bioA and bioB genes.

Figure 10 is an electrophoresis picture confirmed by PCR the bioA and bioB gene cloned into the pGEM vector.

Left photo (A): PCR product of bioA

lane1: size marker

lane2: pbioAS

Right photo (B): PCR product of bioB

lane1: size marker

lane2: pbioBS

FIG. 11 shows the structures of bioA (FIG. 11A) and bioB gene (FIG. 11B) cloned into the pGEM vector.

12 is a plate photograph confirmed by E. coli MEC1, MEC2 whether the cloned bioA and bioB gene is active.

A: MEC1 is bioA deletion mutant

B: pGEMT in MEC1

C : pGEMT- bioA in MEC1

D: MEC2 is bioB deletion mutant

E: pGEMT in MEC2

F: pGEMT- bioB in MEC2

Figure 13 shows the bioB cloned into the pLEX vector into which the PI promoter was introduced. The structure of the gene is shown.

Figure 14 is an SDS-PAGE electrophoresis picture overexpressing the bioB gene under the PI promoter.

Lane1: Size marker

Lane2: JM109

Lane3: JM109 harboring pLEX

Lane4: JM109 harboring pLEX- bioB (pbioBL)

Figure 15 shows the structure of a vector (pJEYR) comprising the bioB gene, biotin operon and par locus under the PI promoter.

Claims (13)

Biotin operon and par locus DNA from Serratia marcescens are introduced, and Biotin B gene from Serratia marcescens under PL Promotor is additionally introduced. Introduced recombinant plasmid. delete delete delete Biotin operon and par locus DNA from Serratia marcescens are introduced, and Biotin B gene from Serratia marcescens under PL Promotor is additionally introduced. Biotin-producing bacteria transformed by the introduced recombinant plasmid. delete delete delete Biotin operon and par locus DNA from Serratia marcescens are introduced, and Biotin B gene from Serratia marcescens under PL Promotor is additionally introduced. Biotin production method characterized in that the antibiotic is administered to the culture medium for culturing the biotin-producing bacteria transformed by the introduced recombinant plasmid. delete delete delete 10. The biotin production method according to claim 9, wherein the antibiotic is ampicillin.

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