KR19990017166A - Recombinant Escherichia coli producing PHB from whey and method for producing PH B using same - Google Patents

Abstract

The present invention relates to a recombinant Escherichia coli producing PHB (poly-3-hydroxybutyrate) from whey and a method for producing PHB using the same. More specifically, the present invention is transformed into a recombinant plasmid containing a PHB synthetic gene of Alcaligenes eutrophus, so that the recombinant E. coli and Whey-containing medium to effectively produce PHB from whey It relates to a method for producing PHB to be cultured. The recombinant E. coli of the present invention effectively produces PHB from low-cost and environmentally-contaminated whey, contributing to environmental protection as well as economic production of PHB.

Description

Recombinant Escherichia coli producing PHB (poly-3-hydroxybutyrate) from whey and method for producing PHB using the same

The present invention relates to a recombinant Escherichia coli producing PHB (poly-3-hydroxybutyrate) from whey and a method for producing PHB using the same. More specifically, the present invention is transformed into a recombinant plasmid containing a PHB synthetic gene of Alcaligenes eutrophus, and cultured in a whey containing medium and recombinant E. coli which effectively produces PHB from whey It relates to a method for producing PHB.

PHB is an energy storage material synthesized and accumulated by microorganisms. PHB has similar physical, chemical, and mechanical properties to existing chemical synthetic polymers, and has excellent biocompatibility and biodegradation. When it is completely decomposed into water and carbon dioxide by microorganisms, it has been spotlighted as an environmentally friendly plastic raw material. In particular, 3-hydroxybutyrate, a decomposition product of PHB, is a metabolite present in the body of animals as well as human beings, so it is being developed for use in surgical sutures and drug delivery systems. Due to the high production cost, it has not yet been used as a general-purpose packaging material or container.

Therefore, for economic production of PHB, Algengenes eutrophus and Alcaligenes latus, known as PHB producing strains, have been intensively studied (see USP 4,433,053; Anderson and Daws). , Microbiol. Rev., 54: 450-472 (1990), recently cloned the PHB synthetase by recombinant microorganisms into E. coli by cloning the PHB synthetase of Alkaligenes eutropus to E. coli. This has also enabled the production of PHB by recombinant microorganisms (see WO 89/00202; Slater et al. J. Bacteriol., 170: 4431-4436 (1988); Schubert et al., J. Bacteriol., 170: 5837-5847 (1988); Peoples and Sinskey, J. Biol. Chem., 264: 15298-15303 (1989).

In order to find a method for producing PHB economically by recombinant E. coli, the present inventors have also developed a plasmid having a high copy number, stabilization of the plasmid, development of transformation strain, development of high concentration culture technology of recombinant microorganism, and promotion of PHB synthesis. Research has been carried out to establish the culture conditions for.

Through this study, the plasmids pSYL101 and pSYL102 having different numbers of copies, pSYL103 prepared by inserting the PARB region of plasmid R1 (Gerdes, Bio / Technol., 6: 1402 (1988)) into an electric plasmid to stabilize them, pSYL104 was introduced into Escherichia coli XL1-Blue to compare PHB synthesis ability, and it was confirmed that high copy number plasmids were used to produce high PHB (Lee et al., Ann. NY Acad. Sci. 721: 32-53 (1994); Lee et al., J. Biotechnol., 32: 203-211 (1994).

In addition, a stable plasmid pSYL105 was introduced to a variety of Escherichia coli such as Escherichia coli XL1-Blue, Escherichia coli DH5a, Escherichia coli JM109, Escherichia coli B, Escherichia coli W, and Escherichia coli K12. As a result, it was confirmed that the PHB synthesis rate and yield vary depending on the strain characteristics. Lee et al., Biotechnol. Bioeng., 44: 1337-1347 (1994).

In addition, since PHB production by recombinant Escherichia coli lengthens the cells due to filamentation, which slows down or stops the cell elongation, not only does the culture efficiency of the cells decrease, but also the productivity of the PHB has been exposed. As a result of repeated studies to inhibit or prevent the filamentation phenomenon of recombinant E. coli, the present inventors have prepared a recombinant plasmid pSYL107 including a gene of essential cell division protein (ftsZ) and a PHB gene capable of inhibiting filamentation. It has been shown that recombinant E. coli transformed therewith shows no filamentation phenomenon in culture and can produce PHB at high concentrations (S. Y Lee, Biotechnol. Lett., 16: 1247-1252 (1994); SY Lee, Kor. J. Appl. Microbiol.Biotechnol., 22: 614-620 (1994); Lee and Lee, J. Environ, Polymer Degrad., 4: 131-134 (1996)).

On the other hand, through the study of the culture conditions of recombinant microorganisms for promoting PHB synthesis, it was found that the PHB synthesis ability is higher in the composite medium than the low cost simple medium. Therefore, as a culture method for producing a high concentration of PHB, a method of adding a small amount of complex nitrogen source to a simple medium has been proposed, tryptone, casamino acid or casein hydrolysate, etc. By adding 0.5 to 5 g / L of the complex nitrogen source, it was confirmed that PHB synthesis could be enhanced. (See Lee and Chang, J. Environ.Polymer Degrad., 2: 169-176 (1994).) PHB synthesis could be improved by addition of amino acids or oleic acid instead of the previous complex nitrogen sources. See Lee et al., J. Ferment.Bioeng., 79: 177-180 (1995).

In addition, in order to increase the productivity of PHB, a fed-batch culture method was developed to obtain dry cell concentration 101G / L and PHB concentration 81g / L in 39 hours (Lee et al., J. Biotechnol., 32: 203). -211 (1994) In a simple medium supplemented with a small amount of nitrogen, obtaining 80 g / L of PHB by fed-batch culture, it was confirmed that the production of PHB by recombinant Escherichia coli was very effective (see Lee and Chang, J.). Environ.Polymer Degrad., 2: 169-176 (1994)).

However, since the above studies all use glucose as the main carbon source, the production cost of the resulting PHB depends a lot on the price of glucose. Therefore, if PHB is produced using a low-cost carbon source instead of glucose, the production cost can be considerably lowered.

On the other hand, whey is a liquid substance that is incidental to cheese or casein production and is rich in lactose and whey protein. In general, about 8 kg of whey is produced when 1 kg of cheese is manufactured. Such whey has a high biological oxygen demand (BOD), which greatly pollutes the environment such as rivers and is a waste of useful resources.

Alkali Kenneth Eutropus, Alkali Kenneth Rhesus, Methanol Magnetized Bacteria, which have been widely used for the production of biodegradable polymer PHB, cannot grow using whey as a carbon source, but some of E. coli can grow using it as a carbon source. .

Therefore, the present inventors have made intensive research to prepare PHB from whey. As a result, the present inventors selected six specific E. coli strains that can use whey as a carbon source, and transformed them with recombinant plasmid pSYL107 cloned with a PHB synthetic gene in a whey-containing medium. Upon culturing, it was confirmed that PHB was effectively produced, and the present invention was completed.

After all, the main object of the present invention is to provide a specific recombinant E. coli that can be transformed with a recombinant plasmid containing a PHB synthetic gene to produce PHB from whey.

Another object of the present invention is to provide a method for preparing PHB from whey using electrocombinant Escherichia coli.

1 shows a genetic map of recombinant plasmid pSYL105.

2 shows a genetic map of recombinant plasmid pSYL107.

Hereinafter, the present invention will be described in more detail.

First, in order to select E. coli with excellent growth using whey as a carbon source, simple medium (KH ₂ PO ₄ 13.5 g / L, (NH _{4) 2} HPO ₄ 4 g / L, MgSO ₄ 7H ₂ O 1.4 g / L, Citric acid 1.7 g / L, FeSO ₄ 7H ₂ O 0.1 g / L, CaCl ₂ 2H ₂ O 0.02 g / L, ZnSO ₄ 7H ₂ O, 0.022 g / L, MnSO ₄ 4H ₂ O 0.005 g / L 0.01 g / L of CuSO ₄ 5H ₂ O, (NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O 0.001 g / L Na ₂ B ₄ O ₇ 10H ₂ O 0.002 g / L and thiamine 0.01 g / L The culture medium) was cultured in a variety of Escherichia coli in whey added medium and examined for growth. As a result, six kinds of specific E. coli (Escherichia coli) That is, E. coli K12 (A wild-type strain, F ^+), E. coli HMS174 (λ ^-, recA1, IN (rrnD-rrrE), rpoB331, hsdR19, ^{F -), E. coli MG1047 (} F -, γδ (3X) :: Tn1000, λ -, recA56), E. coli B (a wild-type strain, F -, GCSC # 6572), E. coli C- Strains 1 (A wild-type strain, F ⁻ ) and E. coli B (a wild-type strain, F ⁻ , GCSC # 2507) showed relatively good growth.

The six E. coli strains were plasmid pSYL105 comprising a plasmid cloned with a poly-3-hydroxybutyrate (PHB) syngene, preferably beta-lactamase, a PHB syngene and the parB region of plasmid R1 (see Lee et al. , Biotechnol.Bioeng. 44: 1337-1347 (1994)) or plasmid pSYL107 (SY Lee. Biotechnol. Lett., 16: 1247-1252) prepared by inserting the gene of essential cell division protein (ftsZ) into pSYL105. (1994)), and most preferably each was transformed with the plasmid pSYL107 described above.

The transformants thus obtained were cultured in the simple medium containing whey containing medium, preferably whey at a concentration of 10 to 80 g / L, most preferably 30 to 60 g / L, and the final dry cell concentration, PHB concentration and The PHB content (% of PHB concentration per dry cell concentration) was determined. As a result, all six transformants were able to effectively produce PHB from whey.

In addition to the simple medium described above, the six transformants of the present invention will exhibit higher PHB production capacity in simple medium with a small amount of complex nitrogen source added. At this time, the complex nitrogen source is tryptone, yeast extract, peptone, casamino acid, casamino acid, cotton seed hydrolysate, bee extract, casein hydrolysate Corn steep liquor or soybean hydroysate may be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of Recombinant E. Coli That Can Synthesize PHB from Whey

In order to select E. coli with excellent growth in simple medium containing whey as a carbon source, 30 g / L of whey was added to the simple medium shown in Table 1 below, and various coliform cultures were flask cultured. As a result, a relatively high growth of six kinds of E. coli as the primary, that is E. coli K12 (a wild-type strain, F +), E. coli HMS174 (λ -, recA1, IN (rrnD-rrrE), rpoB331, hsdR19 ^{, F -), E. coli MG1047} (F -, γδ (3X) :: Tn1000, λ -, recA56), E. coli B (a wild-type strain, F -, GCSC # 6572), E. coli C -1 (a wild-type strain, F -) , and E. coli B (a wild-type strain, F -, GCSC # 2507): (see Table 2) were selected as the strains of the present invention.

Subsequently, recombinant plasmid pSYL107 prepared by inserting a beta-lactamase, a PHE syngene, the parB region of plasmid R1, and the essential cell division protein (fetZ) (see FIG. 1; SY Lee, Biotechnol. Lett., 16). : 1247-1252 (1994)) were transformed into each of six strains of E. coli (E. coli) selected above, to obtain six transformants as shown in Table 2 below. At this time, transformation was performed by an electroporation method (Dower et al., Nucleic Acids Res., 16: 6127-6145 (1988)).

The plasmids were isolated from the transformants thus obtained and digested with restriction enzymes to confirm that they were identical to the transformed plasmids.

Example 2: Preparation of PHB from Whey Using Recombinant E. Coli

To determine the PHB production capacity from whey, the six recombinant E. coli listed in Table 2 were incubated for 60 hours at 30 ° C. and 250 rpm in the simple medium of Table 1 added with 35 g / L or 70 g / L of whey. Then, dry cell concentration, PHB concentration and PHB content were determined, and the results are shown in Table 3 below.

As shown in Table 3, the PHB production amount is slightly different depending on the strain, it was confirmed that all six recombinant strains can be produced PHB from whey. In addition, doubling the amount of whey did not increase the production of PHB. Actually, all strains except recombinant E. coli 2507 (pSYL107) showed a decrease in PHB concentration when the whey concentration increased.

Example 3 Comparison of Different PHB Production Capacity to Whey Concentration Change

In order to determine how the concentration change of whey affects PHB production ability, E. coli 4401 (pSYL107) and E. coli 6576 (pSYL107) having the highest PHB production capacity in Example 2 were 10, 20, 30, 40, 50, After 60 hours incubation at 30 ℃, 250rpm condition in the simple medium of Table 1 added at a concentration of 60, 70g / L, the dry cell concentration, PHB concentration and PHB content was determined, and the results are shown in Table 4 below. Indicated.

As shown in Table 4, both recombinant strains showed the highest PHB concentration when the whey concentration was 30g / L, the lowest PHB concentration when 10g / L. In the case of E. coli 4401 (pSYL107), when the concentration of whey was more than 30g / L, PHB concentration was always higher than 3g / L, PHB content was also very high, 78% or more. In addition, for E. coli 6576 (pSYL107), when the concentration of whey was 30 g / L ~ 60 g / L, the PHB concentration was 3g / L or more, the PHB content was also very high as 79% or more; When the whey concentration was 20g / L, the PHB concentration was relatively high at 3.56g / L, but the PHB content was 56.5%.

Therefore, it was confirmed that the recombinant E. coli 4401 (pSYL107) and E. coli (pSYL107) can be prepared in a large amount of PHB when cultured in simple medium containing whey.

Recombinant Escherichia coli 4401 (pSYL107) and Escherichia coli 6576 (pSYL107) were named Escherichia coli / pSYL107 GCSC 4401 and Escherichia coli / pSYL107 GCSC 6576, respectively. It was deposited in the KCTC, the Institute for Biotechnology Research, affiliated with the Institute of Technology, with the access numbers KTCT 0350BP and KTCT 0351BP, respectively.

As described and demonstrated in detail above, six recombinant E. coli transformed with plasmid pSYL107, that is, E. coli 4401 (pSYL107), 6576 (pSYL107), 6197 (pSYL107), 6572 (pSYL107), 3221 (pSYL107) and 2507 (pSYL107). ) Can effectively produce PHB from whey, especially recombinant E. coli 4401 (pSYL107) and 6576 (pSYL107) produce higher levels of PHB. In addition, since the present invention can produce PHB from the low cost and environmental pollution whey, not only economic production of PHB, but also contribute to environmental protection.

Claims (9)

Recombinant E. coli, wherein the poly-3-hydroxybutyrate (PHB) synthetic gene is introduced into Escherichia coli where the plasmid uses whey as a carbon source to produce PHB from whey. The method of claim 1, wherein the E. coli using whey as a carbon source is E. coli K12 (a wild-type strain, F +), E. coli HMS174 (λ -, recA1, IN (rrnD-rrrE), rpoB331, hsdR19, F ^{-), E. coli MG1047 (F} -, γδ (3X) :: Tn1000, λ -, recA56), E. coli B (a wild-type strain, F -, GCSC # 6572), E. coli C-1 (A wild-type strain, F ⁻ ) and E. coli B (a wild-type strain, F ⁻ , GCSC # 2507). The recombinant Escherichia coli according to claim 1, wherein the plasmid is plasmid pSYL105 or pSYL107 having a genetic map as follows: Recombinant Escherichia coli / pSYL107 GCSC 4401 (KCTC 0350BP) transformed with plasmid pSYL107 with the following genetic map to produce poly-3-hydroxybutyrate (PHB) from whey: Recombinant Escherichia coli / pSYL107 GCSC 6576 (KCTC 0351BP) transformed with plasmid pSYL107 with the following genetic map to produce poly-3-hydroxybutyrate (PHB) from whey: A method for producing poly-3-hydroxybutyrate (PHB) comprising the step of culturing the recombinant E. coli of claim 1 in a whey-containing medium. The method of claim 6, wherein the whey-containing medium is KH ₂ PO ₄ 13.5 g / L, (NH _{4) 2} HPO ₄ 4 g / L, MgSO ₄ 7H ₂ O 1.4 g / L, citric acid 1.7 g / L, FeSO ₄ 7H ₂ O 0.1 g / L, CaCl ₂ 2H ₂ O 0.02 g / L, ZnSO ₄ 7H ₂ O 0.022 g / L, MnSO ₄ 4H ₂ O 0.005 g / L, CuSO ₄ 5H ₂ O 0.01 g / L, (NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O 0.001 g / L, Na ₂ B ₄ O ₇ 10H ₂ O 0.002 g / L and 0.01 g / L thiamine Method for producing PHB, characterized in that the medium to which whey is added to the added simple medium. 8. The complex nitrogen source according to claim 7, wherein the complex nitrogen source is tryptone, yeast extract, peptone, casamino acid, cotton seed hydrolysate, bee extract, casein hydrolyzate (casein hydrolysate), corn steep liquor (corn steep liquor) and soybean hydrolysate (soybean hydrolysate) is a method for producing PHB, characterized in that at least one selected from the group consisting of. 8. The method of claim 6 or 7, wherein whey is added at a concentration of 10 to 80 g / L.