KR20020075068A - Yeast strain have iron at high level and the same methode - Google Patents

Abstract

PURPOSE: A yeast strain containing a large quantity of iron and a method for producing the same are provided, therefore the yeast strain can be useful for supplementation of iron in a human or an animal. CONSTITUTION: The yeast strain containing a large quantity of iron comprises a recombinant yeast capable of expressing cDNAs of liver ferritin H chain and L chain and producing a ferritin protein, wherein the recombinant yeast is produced by: PCR amplifying CYC1 terminator of S. cerevisiae; inserting the amplified gene into a recognition site between Sac1 and Sac11 of pBluscript11 SK vector; inserting the PCR amplified Inu1 promoter gene of Kluyveromyces maxianus into a recognition site between Kpn1 and Sall of pBluscript11 SK vector; and inserting 2u origin, which is obtained by digesting pYESTrp2 with Ssp1, and a fragment containing Trp1 gene into the pBluscript11 SK vector.

Description

Yeast strain containing a large amount of iron and a method for producing the same {Yeast strain have iron at high level and the same methode}

The present invention relates to the development of a yeast (Saccharomyces cerevisiae) strain containing a large amount of organo iron to produce a large amount of feratin.

In general, iron is a trace mineral that is essential for animals as well as the human body, and is an important factor in maintaining life in the living body such as oxygen transport and electron transfer in the living body, and red blood cell reduction and hemoglobin number when the iron is deficient. Due to the reduction of anemia, anorexia, diarrhea, difficulty breathing, growth and susceptibility to stress and disease causes problems in metabolism in vivo.

In vivo, iron is contained in the body about 0.04%, of which about 70% is contained in red blood cells in the form of hemoglobin, which carries oxygen, and about 30% in the liver, spleen, bone marrow, etc. As a constituent such as cytochrome, cardalase and oxidase, which are electron transfer elements which are important for bioenergy acquisition, they exist as indispensable elements for maintaining life of a living body.

As mentioned above, iron, which plays an important role in the living body, has been pointed out as a big problem because its absorption rate is very low when absorbed into the living body in the case of a natural metal salt. In particular, it is necessary to supplement the iron deficiency of piglets so that the loss due to abnormal delivery, poor growth and development, mortality due to iron deficiency is large.

Although the baker's yeast Saccharomyces cerevisiae KCTC 1216 may be used as the iron supplement, the yeast produces ferritin-like iron storage protein, but the storage capacity is very low and most intracellular iron is wrapped in the vacuole, thereby reducing its effectiveness.

The present invention develops a yeast strain for iron supplement to solve the above problems.

The present invention introduces an expression vector capable of expressing liver ferritin H chain and L chain cDNA in vivo to strains such as baker's yeast.

The vector expressing the cDNA was amplified by PCR amplifying the CYC1 terminator of S. cerevisiae into pBlucriptll SK vector, and cut and inserted into Sacl and Sacll, amplified by Inlu1 promoter of Kluyveromycesmaxianus by PCR, and cut into pPESTESTrp2. The pYEIP vector obtained by inserting a fragment containing the 2u origin and Trp1 gene obtained by cutting the vector into Sspl is used.

Accordingly, an object of the present invention is to introduce an expression vector capable of resolving liver ferritin H chains and L chain cDNAs of living organisms in yeast to increase the iron content by overproducing ferritin protein.

1 is a view for explaining the production process of the yeast strain of the present invention.

In order to achieve the above object, the present invention is to increase the iron content in yeast by producing ferritin protein using recombinant yeast expressing liver ferritin H chain and L chain cDNA.

The recombinant yeast amplifies the S.cerevisiae CYC1 terminator by PCR and inserts it into Sacl and Sacll, amplifies the Inu1 promoter of Kluyveromyces maxianus by PCR, and inserts the pYESTrp2 vector into the pBlucriptll SK vector. Characterized by obtaining a fragment containing the 2u origin and Trp1 gene cut out by Sspl.

Hereinafter, the present invention will be described in detail with reference to FIG. 1.

A vector capable of expressing ferritin was amplified by PCR amplifying S.cerevisiae's CYC1 terminator into pBlucriptll SK vector, cut into Sacl and Sacll, and amplified Kluyveromyces maxianus' Inu1 promoter by PCR and cut into Kpnl and Sall. The pYEIP vector obtained by inserting a fragment containing the 2u origin and Trp1 genes obtained by cutting the pYESTrp2 vector with Sspl is used. From the RNA obtained from living liver cDNA, ferritin H chain 548bp and L chain 527bp primers were prepared and amplified by RT PCR, respectively, and the product of the H chain was cut into Sall and EcoRl, and the L chain was cut into EcoR1 and Sacll and pBlucriptll SK. Inserted into the vector to prepare a pBHFeHL vector, and then again amplified fragments containing the ferritin H chain and L chain using both the H and L chain primers, cut into Sall and Sacll and inserted into the pYEIP vector pYEHL vector To prepare.

The iron content of the yeast into which the expression vector prepared as described above was introduced was 1 vvm and 150 rpm in a medium containing 1% Glucose, 0.3% Yeast extract, 0.3% Malt extract, 0.5% Peptone, and 2000 ppm iron sulfate. After incubating the yeast containing KCTC1216) and the expression vector for 32 hours, cells were obtained by using a 500 rpm centrifuge, and the inorganic iron powder was washed three times with distilled water to remove distilled water by centrifugation. In order to confirm whether or not the inorganic iron adsorbed on the cell wall of the yeast cell of inorganic iron in yeast, Tween 80, which is a detergent component capable of removing the inorganic iron attached to the cell, was dissolved in 0.1% saline solution and washed.

Subsequently, the supernatant was obtained by centrifugation, and the iron content in the supernatant and the cell wall of the yeast were completely removed using hydrochloric acid, and then the iron content was examined by the pennansroline method.

As a result of the investigation, it can be seen that the iron content of the yeast into which the expression vector is introduced is significantly increased as compared to general yeast as shown in Table 1 below.

collection General Yeast Expression vector introduction yeast Iron content (ppm) Iron content (ppm) One 240 1700 2 280 1650 3 270 1720 Average 263 1690

1 liter of the yeast culture solution of the present invention prepared as described above is adsorbed on 1 kg of skim soybean meal and dried to use it as an iron-containing feed additive.

As a result of measuring the hemoglobin and hematocrit after feeding the feed additive to piglets, it can be seen that the measured value is improved as shown in Table 2 compared with the piglets not fed the feed additive.

Piglets without additives Pigment Application first 5 days later After 20 first 5 days later After 20 days Haemoglobin (g / dl) 10.4 8.8 10.2 10.8 10.4 11.2 Hematocrit (%) 37.8 31.0 37.0 41.6 38.6 42.3

In the present invention, a vector capable of expressing liver ferritin H-chain and L-chain cDNA in vivo to strains such as baker's yeast is amplified by PCR to amplify the CYC1 terminator of S. cerevisiae in pBlucriptll SK vector by Sacl and Sacll. By cutting and inserting, amplifying the Inu1 promoter of Kluyveromyces maxianus by PCR, cutting and inserting into Kpnl and Sall, and inserting a fragment containing 2u origin and Trp1 gene obtained by cutting pYESTrp2 vector with Sspl, thereby overproducing ferritin protein. Iron content can be obtained.

Claims (3)

A yeast strain containing a large amount of iron, characterized by comprising a recombinant yeast expressing the liver ferritin H chain and L chain cDNA of a living body, and producing a ferritin protein. The method of claim 1, The recombinant yeast amplifies the S.cerevisiae CYC1 terminator by PCR and inserts it into Sacl and Sacll, amplifies the Inu1 promoter of Kluyveromyces maxianus by PCR, and inserts the pYESTrp2 vector into the pBlucriptll SK vector. Yeast strain containing a large amount of iron, characterized in that obtained by inserting a fragment containing a 2u origin and Trp1 gene cut by Sspl. PCR amplification of SY. cerevisiae CYC1 terminator by pBlucriptll SK vector, cut and insert into Sacl and Sacll, amplify Inu1 promoter of Kluyveromyces maxianus by PCR, cut into Kpnl and Sall, and cut pYESTrp2 vector with Sspl Method for producing a yeast strain containing a large amount of iron, characterized in that inserting a fragment containing the 2u origin and Trp1 gene.