KR100199993B1 - Process for producing phb from lactose using recombinant e. coli - Google Patents

Abstract

The present invention relates to a method of producing poly-3-hydroxybutyrate (PHB) from lactose or whey using recombinant E. coli. More specifically, the present invention is to transform the E. coli lactose available in the production plasmid containing a PHB synthetic gene of Alcaligenes eutriphus, and cultured in lactose or whey-containing medium It relates to a manufacturing method of PHB. According to the present invention, production of PHB from lactose or milking by six recombinant E. coli (DSM 499 (pSYL105), DSM 499 (pSYL107), DSM6056 (pSYL105), DSM 6056 (pSYL107), KCTC 2223 (pSYL107)) It is possible to improve the PHB production ability by adding a small amount of various complex nitrogen sources, and PHB can be produced more effectively using recombinant E. coli DSM 499 (pSYL107) and KCTCb 2223 (pSYL105). In addition, since the present invention can manufacture PHB from whey, which is a low cost and environmental pollutant, not only economic production of PHB, but also environmental protection.

Description

Method for preparing poly-3-hydrxybutyrate (PHB) from lactose or whey using recombinant E. coli

The present invention relates to a method for producing poly-3-hydroxybutyrate (PHB) from lactose or whey using recombinant E. coli. More specifically, the present invention provides a recombinant plasmid containing PHB synthetic gene of Alkaligenes eutrophus, which transforms lactose available Escherichia coli and cultures it in lactose or whey-containing medium. It relates to a method for producing PHB.

Recently, PHB, which has been spotlighted as a biodegradable polymer material, is an energy storage material synthesized and stored by microorganisms. PHB has similar physicochemical and mechanical properties of existing chemical synthetic polymers, and has excellent biocompatibility and biodegradability, so it can be completely decomposed into water and carbon dioxide by microorganisms after a certain time in nature. I am in the limelight. Especially. 3-hydroxybutyrate, a breakdown product of PHB, is a metabolite present in the body of animals as well as humans, and is being developed for use in surgical sutures and drug delivery systems. Due to the production cost, it has not been used as a general packaging material or container yet.

Thus, for the borderline production of PHB, Alkaligenes 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)), and recently, several researchers have made it possible to produce PHB by recombinant microorganisms by cloning the PHB gene into E. coli. (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 Escherichia coli, the present inventors have also developed plasmids having high copy numbers, stabilization of plasmids, development of transformation strains, development of high concentration culture technology of recombinant microorganisms, and promotion of PHB synthesis. Research has been carried out to establish the culture conditions for. Through this study, plasmids pSYl101 and pS 102 with different copy numbers were obtained. In order to stabilize these compounds, pSYL103 and pSYL104 prepared by inserting the parB region of plasmid R1 (Gerdes, Bio / Technol., 6: 1402 (1988)) into an electric plasmid were introduced into colonic T1-Blue, and thus PHB synthesis ability As a result, it was confirmed that the production of PHB is high only by using a high copy number plasmid (Lee et al., Ann. NY Acad. Sci., 721: 43-53 (1994): Lee et al. al., J. Biotechmol., 32: 203-211 (1994)).

In addition, E. coli XL1-Blue, E. coli DH5 Escherichia coli JM109. As a result of research on the rate and yield of PHB synthesis and the yield of stable plasmid pSYL105 with high copy number to E. coli B, Escherichia coli W, E. coli K12, etc. This difference was confirmed (lee et al., Biotechnol. Ioeng, 44: 1337-1347 (1994)).

together. Studies on the culture conditions of the prepared microorganisms to promote the synthesis of PHB, it was found that the low PHB short-circuit capacity in low-cost simple medium compared to the composite medium. Therefore, a re-cultivation method was required to produce high-quality PHB economically, and a method using a simple medium added with a complex nitrogen source of HOS was proposed in this way, tryptone and xasamino acid. Or it was confirmed that by adding a flavor nitrogen (0.5 to 5g / L), such as casein hydrolysate (casein hydrolysate), PHB synthesis can be enhanced (see Lee and Chang, J. Environ. Polymer Degrad., 2 : 169-176 (1994) It has also been shown that the addition of amino acids or oleic acids can improve PHB synthesis instead of the former complex nitrogen source: Lee et al., J. Ferment. Bioeng., 79 177-180 (1995).

Meanwhile. In order to increase the productivity of PHB, a fed-batch culture method was developed, and the dry cell concentration 101g / L in 39 hours. A PHB concentration of 81 g / L was obtained. See Lee et al. J. Biotechnol., 32: 203-211 (1994)). In addition, 80g / L PHB can be obtained by fed-batch culture in simple medium containing small amounts of complex nitrogen sources, indicating that PHB production by recombinant E. coli is very effective (see Lee and Chang, J. Environ. Polymer Degrad). , 2: 169-176 (1994)).

In addition, since the growth of the cells is slowed or stopped by filamentation during the production of PHB by recombinant E. coli, the growth rate of the cells is reduced, as well as the productivity of PHB has been exposed. Accordingly, the present inventors have repeatedly studied to inhibit or prevent the filamentation phenomenon of recombinant E. coli, thereby producing a recombinant plasmid pSYL107 containing a gene of essential cell division protein (ftsZ) and a PHB gene that can inhibit filamentation. The recombinant Escherichia coli transformed therewith showed no filamentation in culture and was able to produce PHB at a high concentration (see SY Lee. Biotechnol., Lett, 16: 1247-1252 (1994); SY Lee.Kor. J. Appl.Microbiol.Biotechnol ,. 22: 614-620 (1994); Lee and Lee.Je nviron, Polymer Degrad., 4: 131-134 (1996)).

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

Whey is a liquid substance that is a by-product of cheese or casein production and is rich in lactose and whey protein. In general, about 8 kg of whey is produced for 1 kg of cheese. Such whey has a high biological oxygen demand (BOD) and greatly pollutes the environment such as rivers, and wastewater of useful resources is required for its effective use.

Alkali genes Eutropus, Alkogen Generus, Methanol magnetization bacteria, which have been used early for the production of biodegradable polymer PHB, cannot grow using Judas as a carbon source, but some of E. coli can grow it as a carbon source.

Therefore, the present inventors introduced the recombinant plasmid pSYL105 or pSYL107 cloned with the PHB synthetic gene into seven types of E. coli, which can use lactose as a carbon source. Recombinant Escherichia coli, which produces PHB in a lactose or whey containing medium, was selected and completed the present invention.

After all, an object of the present invention is to provide a method for producing PHB by transforming E. coli of lactose available with a recombinant plasmid containing a PHB synthetic gene, and culturing the transformant in lactose or whey-containing medium.

1 shows a genetic map of recombinant plasmid pSYL105.

2 shows a genetic map of recombinant plasmid pS 107.

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

First, in order to screen E. coli with excellent growth using lactose as a carbon source, simple medium (KH ₂ PO ₄ 13.5 g / L, (KH ₂ ) ₄ 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 THIAMINE 0.01 gL) 7 known Escherichia coli strains (Escherichia coli), E. coli (ATCC 9492), E. coli W (ATCC 9637), E. coli K12 (ATCC 23716), E. coli B (DSM 499), E. coli TG1 (DSM 6056), E. coli K12 (KCTC 1116) and E. coli W3110 (KCTC 2223) were cultured and examined for growth and extent. As a result, the stocks of E. coli B (DSM 499), E. coli TG1 (DSM 6056), and E. coli W3110 (KCTC 2223) grew relatively.

The three E. coli strains were recombinant plasmid pSYL105 comprising a plasmid with a poly-3-hydroxybutyrate (PHB) synthetic gene, preferably beta-lactamase, a PHB synthetic user, and the paeB region of plasmid R1 (Lee et al. al., Biotechnol., Bioeng ,. 44: 1337-1347 (1994)) or recombinant plasmid pSYL107 (SY Lee, Biotechnol., Lett.) prepared by inserting the gene of essential cell division protein (ftsZ) into pSYL105. , 16: 1247-1252 (1994), respectively.

The transformants thus obtained contain lactose or whey containing medium, preferably the simple medium containing lactose or whey at a concentration of 1 to 80 g / L, or lactose or whey at a concentration of 10 to 80 g / L Incubated in the simple medium with 0.1 to 10 g / L nitrogen source, final dry cell concentration, PHB concentration and PHB content (percent of PHB concentration per crude cell concentration) were determined. At this time, the transformant of pSYL105 is 20 To 40 , More preferably 25 To 38 , Most preferably 37 And pSYL107 transformants were cultured at 20 to prevent minicell formation. To 40 , More preferably 25 To 35 , Most preferably 37 And pSYL107 transformants were cultured at 20 to prevent minicell formation. To 40 , More preferably 25 To 35 , Most preferably 30 (Lee, Biotechnol., Lett., 16: 1247-1252 (1994))

As a result, all six transformants were able to produce PHB from lactose or whey, and PHB production ability was better in simple medium supplemented with complex nitrogen source than simple medium.

In the above, as a source of nitrogen, tryptone, yeast extract, peptone, casamino acid, cotton seed hydrolysate, juicy extract, casein hydrolysate (xasein) hydrolysate). Corn steep liquor or soybean hydreoysate may be used.

According to the present invention, a recombinant plasmid containing a PHB synthetic gene is transformed into Escherichia coli, which is lactose available, and the transformant can be cultured in a lactose or milk-containing medium to effectively produce PHB, and by adding a small amount of a complex nitrogen source. PHB synthesis ability could be improved. In particular, since the present invention can produce PHB from low-cost whey, not only economic production of PHB, but also environmental protection.

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, 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. D

Example 1

Comparison of Escherichia Coli Growth in Lactose-Based Medium

In order to select an E. coli strain having excellent growth in a simple medium using lactose as a carbon source, 20 g / L of lactose was added to the simple medium shown in Table 1 below, and flask cultures of 7 known E. coli strains known as lactose available strains Growth capacity was compared (see Table 2)

As shown in Table 2, the growth of strains E. coli B (DSM 499), E. coli TG1 (DSM 6056) and E. coli W3110 (KCTC 2223) was found to be relatively good. Strain was selected as the strain of the present invention.

Example 2

Preparation of a transformant capable of synthesizing PHB from lactose

Recombinant plasmid pSYL105 (see Figure 1; Lee et al., Biotechnol., Bioeng ,. 44: 1337-1347 (1994)) and electric pSYL105 comprising the beta-lactamase, PHB syngene and the parB region of plasmid R1 A recombinant plasmid pSYL107 (see FIG. 2; SY Lee, Biotechnol., Lett, 16: 1247-1252 (1994)) prepared by inserting the gene of essential cell division protein (ftsZ) into Each of the three E. coli strains was transformed. Transformation was performed by the electroporation (elextroporation) method. (Dower et al., Nucleic Acids Res., 16: 6127-6145 (198)).

The plasmids were isolated from the six transformants thus prepared, and digested with restriction enzymes, followed by electrophoresis, thereby confirming that the electric transformants contained pSYL105 or pSYL107, and they were dsm 499 (pSYL105), respectively. DSM 499 (pSYL107), DSM 6056 (pSYL105). DSM 0656 (pSYL107), KCTC 2223 (pSYL105) and KCTC 2223 (pSYL107).

Example 3

Preparation of PHB from Lactose

PHB was efficiently produced by culturing the recombinant E. coli prepared above in a medium containing lactose as the main carbon source.

Example 3-1

Determination of Optimal Lactose Concentration

Before examining the PHB production capacity from lactose, to determine the optimal lactose concentration, participate in the simple medium of Table 1 so that lactose concentration of 10, 20, 30, 45, 65 g / L After preparation, the six transformants obtained in Example 2 were inoculated and incubated for 60 hours, and the absorbance (OD) was measured at 600 nm (see Table 3). At this time, the pSYL105 transformant is 37 , pSYL107 transformants were 30 Incubated at.

As shown in Table 3, the optimum lactose concentration was different depending on the transformant bar DSM 499 (pSYL105) is 30g / L; DSM 499 (pSYL107) is 60 g / L; DSM 6056 (pSYL105) at 20 g / L; DSM 6056 (pSYL107) is 10 g / L; KCTC 2223 (pSYL105) at 30 g / L; KCTC 2223 (pSYL107) showed maximum growth at a lactose concentration of 20 g / L.

Example 3-2

Preparation of PHB in Lactose-Containing Simple Medium

5 mL of simple medium containing lactose at the optimal concentration determined in Example 3-1 was placed in a 250 mL flask, and the transformants which showed the maximum growth at the corresponding lactose concentration were inoculated and shaken for 60 hours to determine the PHB production capacity. . Ewha. pSYL105 transformant is 37 , pSYL107 transformants were 30 Incubated at. PHB production capacity is the dry cell concentration measured by a known method. PHB concentration. Determined by the PHB content (Lee et sl., Biotechnol., Bioeng., 44: 1337-1347 (1994)). rm results are shown in Table 4.

As shown in Table 4, all six transformants were able to produce PHB from lactose, and among them, two types of E. coli DSM 499 (pSYL107) and KCTC 2223 (pSYL105) produced PHB more efficiently. Could know.

Example 3-3

Preparation of PHB in Lactose-Containing Simple Medium Added Complex Nitrogen

Two prepared strains DSM 499 (pSYL107) and KCTC 2223 (pSYL105), which showed more efficient PHB production in Table 4, were used in Example 3-2 and Lactose-containing simple medium containing a small amount of the complex nitrogen source of Table 5 below. The same culture was incubated. That is, DSM 499 (pSYL107) was cultured in a medium containing 60 g / L lactose and KCTC 2223 (pSYL105) was incubated in a medium containing 30 g / L lactose, respectively, and cell growth and PHB concentrations were measured (see Table: 6). At this time, complex nitrogen sources were added at concentrations already reported to provide high yields of PHB production (Lee and Chang, J. Environ. Polyver Degrad., 2: 169-176) 1994).

As shown in Table 6, the PHB synthesis ability of the recombinant strain was improved by the addition of a small amount of the complex nitrogen source, the improvement was found to be greater in the case of DSM 499 (pSYL107). In particular, the PHB concentration of DSM 499 (pSYL107) was highest at 4.82 g / L with the addition of cornspiker, and the pH of KCTC 2223 (pSYL105) was slightly higher at 3.6 g / L with the addition of peptone.

Example 4

PHB manufactured from whey

PHB was efficiently prepared by culturing the recombinant E. coli prepared in Example 2 in a medium containing whey as the main carbon source.

Example 4-1

Determination of Optimal Whey Concentration

It confirmed that PHB can be produced from lactose by using recombinant E. coli, and that whey released into the rivers as a by-product of cheese or casein contains a large amount of lactose, thus making it possible to produce PHB using low-cost whey as a carbon source. Percussion. First, in order to determine the whey concentration of sediment for each recombinant strain, four kinds of media were prepared by adding whey to the simple medium of Table 1 to 10,20,40,80 g / LDMLSHD, and then in Example 2 Six transformants obtained were inoculated and incubated for 60 hours, and the absorbance (OD) was measured at 600 nm (see Table 7). At this time, whey is added to the powdery whey (sigma. USA) to distilled water at 200 g / L and stirred for 10 minutes while slightly heating to completely dissolve 121 Autoclave for 15 minutes at. The supernatant obtained by centrifugation at 6000 rpm for 15 minutes was used.

As shown in Table 7, all six recombinant E. coli can be grown using whey as the main carbon source, and among them, four recombinant E. coli DSM 499 (pSYL105), DSM 499 (pSYL107), KCTC 2223 (pSYL105), and KCTC. The growth of 2223 (pSYL107) was excellent. Optimal whey concentrations were different for different transformants. DSM 499 (pSYL105) at 20 g / L; DSM 499 (pSYL107) is 40f / L; DSM 6056 (pSYL105) is 80 g / L; DSM 6056 (pSYL107) is 80 g./L; KCTC 2223 (pSYL105) was 40 g / L; KCTC (pSYL107) showed maximum growth at a concentration of 10 g / L whey.

Example 4-2

Preparation of PHB in Whey-Containing Simple Medium

Six recombinant Escherichia coli were cultured in the same medium containing whey at the optimum concentration determined in Example 4-1, and the PHB production capacity was examined. (See Table 8)

As shown in Table 8, all six transformants were able to produce PHB from whey. Among them, DSM 499 (pSYL107) produced PHB most efficiently.

Example 4-3

Preparation of PHB in Whey-Containing Simple Medium Added Complex Nitrogen

PHB production in Table 8 is more effective, three recombinant strains DSM 499 (pSYL107), DSM6056 (pSYL107) and KCTC 2223 (pSYL105) shown in the whey-containing simple medium containing a small amount of the complex nitrogen source of Table 5 Incubated for 60 hours at, cell growth and PHB concentration was measured (see Table 9). At this time, whey was added at an optimal concentration for each recombinant strain as shown in Table 7.

As shown in Table 9, the PHB synthesis ability of the recombinant strain was improved by the addition of a small amount of the complex nitrogen source, especially in the case of DSM 499 (pSYL107) shows a PHB content of more than 60% in most medium, the PHB synthesis in the complex nitrogen source This proved to be very effective.

As described in detail above, the present invention provides a method for producing PHB from lactose or whey using recombinant E. coli transformed with a plasmid in which the PHB synthetic gene of alkali genes eutropus is flanded. According to the present invention, lactose or whey by six recombinant E. coli (DSM 499 (pSYL105), DSM 499 (pSYL107), DSM 6056 (pSYL105), DSM 6056 (pSYL107), KCTC 2223 (pSYL105) and KCTC 2223 (pSYL107) PHB can be produced, and PHB can be produced more effectively using recombinant E. coli DSM 499 (pSYL107) and KCTC 2223 (pSYL105), and PHB production ability can be improved by adding small amounts of various complex nitrogen sources. In addition, since the present invention can manufacture PHB from whey, which is a low cost and environmental pollutant, it contributes not only to economic production of PHB but also to environmental protection.

Claims (6)

Escherichia coli, which uses lactose as a carbon source, is transformed into a plasmid cloned with a poly-3-hydroxybutyrate (PHB) synthetic gene, and the transformant is a medium containing lactose or whey as a carbon source. PHB production method comprising the step of culturing in. The method of claim 1, wherein the Escherichia coli using lactose as a carbon source is E. coli TG1 (DSM 6056) or E. coli W3110 (KCTC 2223). The method of claim 1, wherein the plasmid is pSYL105 comprising beta-lactamase, the PHB synthetic gene and the parB region of plasmid R1, or beta-lactamase, the PHB synthetic gene, the parB region of plasmid R1 and essential cell division protein (ftsZ). Method for producing PHB, characterized in that pSYL107 containing the gene of. The method of claim 1, wherein the lactose- or whey-containing medium is KH ₂ PO ₄ 13.5 g / L, (NH ₄ ) ₂ HPO ₄ 4 g / L, NgSO ₄ 7H ₂ O 1.4 g / L, citric acid ) 1.7 g / L, FeSO ₄ 7H ₂ O 0.1g / 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 gL, Na ₂ B ₄ O ₇ 10H ₂ O 0.002 g / L and 0.011 g / L thiamine A method for producing PHB, characterized in that the wooner is a medium in which lactose or whey are added to a simple medium of electricity added at 0.1 to 10 g / L. The method of claim 4, wherein the complex nitrogen source is tryptone, yeast extract, peptone, casamino acid, cotton seed hydrolysate, bee extract, casein hydrolyzate (casein hydrolysate), a consteam liquor (taegu steep liquor) and soybean hydrolysate (soybean hydrolysate) is a method for producing a PHB, characterized in that at least one selected from the group consisting of. The method of claim 1 or 4, wherein the lactose or whey is added at a concentration of 10 to 80 f / L.