KR0164282B1 - Preparation method of phb by suppression of filamentation in recombinant escherichia coli - Google Patents

Abstract

The present invention transforms Escherichia coli with a recombinant plasmid containing essential cell division protein (ftsZ) and poly-3-hydroxybutyrate (PHB) synthetic gene of Escherichia coli, and prepares a high concentration of PHB from the transformant. It is about a method. According to the PHB manufacturing method of the present invention, by overexpressing ftsZ, which can suppress filamentation, the cells are lengthened by filamentation during PHB production by recombinant E. coli, thereby slowing or stopping cell growth, Solving the problems of low productivity and low PHB yield, it is possible to economically produce a high concentration of PHB.

Description

Method for preparing high concentration of poly-3-hydroxybutyrate (PHB) from recombinant E. coli by filamentation inhibition

1 shows a genetic map of the recombinant plasmid pSYL107 prepared in the present invention.

Figure 2 (a) is a graph showing the results of measuring the concentration of each of the dry cells, PHB and surplus dry cells according to the culture time of the recombinant Escherichia coli cultured at 37 ℃.

Figure 2 (b) is a graph showing the results of measuring the concentration of each of the dry cells, PHB and surplus dry cells according to the culture time of the recombinant Escherichia coli cultured at 30 ℃.

The present invention relates to a method for producing high concentration of poly-3-hydroxybutyrate (PHB) from recombinant E. coli by inhibiting filamentation (filamentation). More specifically, the present invention transforms Escherichia coli into a recombinant plasmid containing the ftsZ gene, which is an essential cell division protein gene of Escherichia coli, thereby inhibiting the filamentation phenomenon that occurs during production of PHB by recombinant Escherichia coli, and thus, high concentration PHB from recombinant Escherichia coli. It relates to a method of manufacturing.

Recently, PHB, which has been spotlighted as a biodegradable polymer material, 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 biodegradability, so it can be completely decomposed into water and carbon dioxide by microorganisms after a certain time in nature. It is attracting attention as a raw material. In particular, 3-hydroxybutyrate, a degradation product of PHB, is a metabolite present in the body of animals as well as humans, so it is being developed for use in surgical sutures and drug delivery systems. However, due to the high production cost, it is still not used as a general packaging or container.

Therefore, for the economic production of PHB, Alkali genes 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 shown the production of PHB by recombinant microorganisms by cloning the PHB synthetic gene of Alkaligenes eutropus to E. coli. 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 devise a method for producing PHB economically by recombinant Escherichia 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 research has been carried out to establish the culture conditions for promoting PHB synthesis. Through these studies, the plasmids pSYl101 and pSYL102 with different numbers of copies, pSYL103 prepared by inserting the parB region of plasmid R1 (Gerdes, Bio / Technol., 6: 1402 (1988)) into the 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: 43-53 (1994) Lee et al., J. Biotechnol., 32: 203-211 (1994).

In addition, a stable plasmid pSYL105 with high copy number was introduced to various types of Escherichia coli such as Escherichia coli XL1-Blue, Escherichia coli DH5α, Escherichia coli JM109, Escherichia coli B, Escherichia coli W, and Escherichia coli K12. As a result, it was confirmed that PHB synthesis rate and yield vary depending on the strain characteristics (Lee et al., Biotechnol. Bioeng., 44: 1337-1347 (1994)).

In addition, through the study of the culture conditions of recombinant microorganisms for promoting PHB synthesis, it was found that PHB synthesis ability is lower in low-cost simple medium compared to the complex medium. Therefore, there was a need for a cultivation method capable of producing a high concentration of PHB economically, a method of using a simple medium with a small amount of complex nitrogen source was proposed in this way, tryptone, casamino acid (casamino acid) or It was confirmed that addition of small amounts (0.5 to 5 g / L) of complex nitrogen sources such as casein hydrolysate can enhance PHB synthesis (Lee and Chang, J. Environ. Polymer Degrad., 2: 169-176 (1994). In addition, it was confirmed that addition of amino acid or oleic acid may improve the synthesis of PHB 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 to obtain 101 g / L of dry cell concentration and 81 g / L of PHB concentration in 39 hours (Lee et al., J. Biotechnol., 32: 203). -211 (1994)). In addition, even a simple medium containing a small amount of complex nitrogen source can be obtained 80g / L PHB by fed-batch culture, it was confirmed that the production of PHB by recombinant E. coli is very effective (see Lee and Chang, J. Environ). Polymer Degrad., 2: 169-176 (1994)).

However, filamentation is generally observed in the expression of proteins by recombinant E. coli (Schoner et al., Biotechnol., 3: 151-154 (1985); Rinas et al., J. Biotechnol., 28 : 313-320 (1993)), when the production of PHB by recombinant E. coli is prolonged by filamentation, the growth of the cells is slowed down or stopped, resulting in a decrease in the culture efficiency of the cells and a decrease in the productivity of the PHB. This has been exposed. On the other hand, the degree of filamentation varies depending on the type of E. coli. In the case of Escherichia coli XL1-Blue, which accumulates the most PHB, about 20% of the total cells were extended to 8 μm or more and 150 μm, and E. coli DH5α, E. coli JM109, etc. Other E. coli strains also exhibited filamentation during PHB accumulation (Lee et al., Biotechnol. Bioeng., 44: 1337-1347 (1994)).

Accordingly, the present inventors have conducted research to inhibit or prevent the filamentation phenomenon of recombinant E. coli, thereby producing a recombinant plasmid containing a gene of essential cell division protein (ftsZ) and a PHB gene capable of inhibiting filamentation. From this, it was found that the transformed Escherichia coli filamentation phenomenon was not exhibited, and that PHB could be produced at a high concentration, thereby completing the present invention.

After all, it is an object of the present invention to provide a method for transforming Escherichia coli with a recombinant plasmid containing ftsZ and PHB synthetic genes, and producing a high concentration of PHB from the transformant.

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

First, in order to obtain an E. coli ftsZ gene capable of inhibiting or preventing filamentation, plasmids pZAQ (Ward and Lutkenhaus, Cell, 42: 941-945 (1985)) were treated with restriction elements Hind III and Cla I. A 2.1 kb DNA fragment containing the ftsZ gene was then obtained, followed by pSYL105 (Lee et al., Biotechnol., 44: 1337), a high-copy-stable stable plasmid cloned with an alkaline gene Eutropus PHB synthetic gene. -1347 (1994)) was digested with restriction enzymes HindIII and Cla I and recombinant plasmid pSYL107 was prepared by inserting the 2.1 kb DNA fragment obtained previously.

PSYL107 prepared in advance was introduced into Escherichia coli, and then recombinant E. coli was transferred to 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, sheet Citric acid 1.7g / L, FeSO ₄ · 7H ₂ O 0.1g / L, ZnSO ₄ · 7H ₂ O 0.022g / L, CaCl ₂ · 2H ₂ O 0.02g / L, CuSO ₄ · 5H ₂ O 0.01 g / L, MnSO _{4 4} H ₂ O 0.005 g / L, Na ₂ B ₄ O ₇ 10 H ₂ O 0.002 g / L, (NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O 0.001 g / L, thiamine batch consisting of 0.01 g / L thiamine and 20 g / L glucose, combined medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L and glucose 20 g / L) Medium) or an electric simple medium containing 0.1 to 10 g / L of a complex nitrogen source, and then the degree of filamentation, cell growth, PHB production capacity and PHB yield (g PHB / g glucose) were measured. Compared to E. coli transformed with pSYL105 (Lee et. al., Biotechnol., 44: 1337-1347 (1994)) does not contain the ftsZ synthetic gene. In the above, the complex nitrogen source is tryptone, yeast extract, peptone, casamino acid, cotton seed hydrolysate, bee extract, casein hydrolysate, Collagen hydrolysate, corn steep liquor or soybean hydrolysate may be used.

As a result of comparing the degree of filamentation inhibition, cell growth, PHB production capacity and PHB yield, E. coli transformed with the recombinant plasmid pSYL107 containing the ftsZ gene of the present invention, filamentation phenomenon is inhibited or prevented, cell growth , It was confirmed that the recombinant PHB production capacity and the PHB yield (g PHB / g glucose) having a molecular weight of 1 million or more is significantly superior to E. coli transformed with pSYL105. In addition, the recombinant PHB produced in the present invention was a novel polymer, which is very different from the conventional PHB, which generally has a molecular weight of 50 to 800,000.

Therefore, by culturing the recombinant Escherichia coli containing the recombinant plasmid of the present invention in which filamentation is suppressed and prevented, it is possible to increase the PHB synthesis ability and the PHB yield, and in particular, a simple medium containing a low cost simple medium or a complex nitrogen source. It was confirmed that the medium can further increase the PHB synthesis ability and PHB yield.

On the other hand, when the recombinant Escherichia coli amplified ftsZ gene is incubated at 37 ℃, there is a report that the strain becomes unstable by forming a minicell (Ward and Lutkenhaus, Cell, 42: 941-947 (1985)) , When the E. coli strain transformed with the recombinant plasmid pSYL107 of the present invention was cultured at a temperature of 37 ° C. in various culture mediums as described above, only strains cultured in a complex medium formed minicells, resulting in slow growth of cells and low production of PHB. It was confirmed. Therefore, it can be seen that the E. coli strain transformed with the recombinant plasmid of the present invention is preferably cultured in a temperature range of 20 ℃ to 40 ℃, more preferably 25 ℃ to 35 ℃.

According to the PHB manufacturing method of the present invention, by overexpressing ftsZ, which can suppress filamentation, the cells are lengthened by filamentation during PHB production by recombinant E. coli, thereby slowing or stopping cell growth, Solving the problem of lowering productivity and PHB yield, it is possible to economically produce a high concentration of PHB.

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 to these examples according to the gist of the present invention. In particular, according to the purpose of the present invention, not only a method for producing PHB in high concentration using recombinant plasmids containing the ftsZ and PHB synthetic genes exemplified in the examples, but also a recombinant plasmid containing each of these genes at the same time It will be appreciated that methods of producing PHB by transformation also fall within the scope of the present invention.

Example 1 Preparation of Recombinant Plasmid pSYL107

In order to inhibit or prevent the filamentation of recombinant E. coli, plasmid pSYL107 containing the ftsZ and PHB synthetic genes was prepared as follows: First, to obtain the ftsZ gene of E. coli, plasmid pZAQ (see Ward and Lutkenhaus, Cell) , 42: 941-945 (1985)) were treated with restriction enzymes Hind III and Cla I to obtain a 2.1 kb DNA fragment containing the ftsZ gene. The PHB synthesizing gene of Alkaligenes eutropus was then inserted into the SmaI-EcoRI restriction enzyme site of plasmid pBluescript KS- (Stratagene, USA). Was obtained (see Schubert et al., J. Bacteriol., 173: 168 (1991)), pSK2665 was digested with restriction enzymes EcoRI and HindIII and inserted into the region with 0.6 kb of parB region. PSYL105 (Lee et al., Biotechnol., 44: 1337-1347 (1994)), a cloned high copy number stable plasmid, was digested with restriction enzymes HindIII and Cla I and the 2.1 kb DNA fragment obtained from the above was obtained. Recombinant plasmid pSYL107 was prepared by insertion. pSYL107 is a plasmid of 10.9 kb, and a gene map of pSYL107 is shown in FIG.

PSYL107 prepared in the above was introduced into Escherichia coli such as Escherichia coli XL1-Blue, Escherichia coli DH5α, Escherichia coli JM109, Escherichia coli B, and Escherichia coli W, and compared with the degree of filamentation inhibition and PHB production. -Blue was confirmed to be E. coli accumulating the largest amount of PHB as filamentation is suppressed. Therefore, the following example used Escherichia coli XL1-Blue transformed with pSYL107 as the recombinant Escherichia coli.

Example 2 Measurement of Filamentation Inhibition Degree by Recombinant Escherichia Coli

As a result of measuring the degree of filamentation inhibition of recombinant Escherichia coli, Escherichia coli XL1-Blue containing pSYL105 showed a filamentation phenomenon in the process of synthesizing PHB when cultured in complex medium, but containing pSYL107 cloned with ftsZ gene. One Escherichia coli XL1-Blue showed no filamentation at all when cultured in a mixed medium, and had a cell length of 4 µm or less, similar to or slightly smaller than that of E. coli.

In addition, when filamentous Escherichia coli XL1-Blue containing pSYL107 was cultured in a simple medium, some filamentation occurred, but the amount was much smaller than that of Escherichia coli XL1-Blue containing pSYL105, about 5%. Only cells below or below showed filamentation. The slight filamentation in the simple medium even after the amplification of the ftsZ gene is presumed to be due to the fact that ftsZ protein expression in the simple medium is relatively less than that in the complex medium, which does not completely inhibit the filamentation.

Therefore, E. coli XL1-Blue containing pSYL107 cloned ftsZ gene overexpressed the ftsZ protein, it can be seen that it can significantly inhibit or completely prevent filamentation.

The composition of the simple medium and the composite medium used above are shown in Table 1 and Table 2, respectively.

Example 3 Comparison of Cell Growth and PHB Production Capacity with and without Filamentation

E. coli XL1-Blue containing pSYL107, which inhibits filamentation, and E. coli XL1-Blue containing pSYL105, which filamentation occurs, respectively, in 30 ml or 37 ° C in a 250 ml flask containing 50 ml of mixed medium and simple medium. After shaking culture at temperature and 300rpm, the dry cell concentration, PHB concentration and PHB content were measured. At this time, the PHB concentration was measured by the method of Braunegg et al. (Braunegg et al., Eur. J. Appl. Microbiol. Biotechnol., 6: 29-37 (1978)). In addition, the result of incubation for 48 hours in complex medium, 60 hours in simple medium is shown in Table 3 below, PHB content was defined as the percentage (%) of PHB concentration to dry cell concentration ratio.

As shown in Table 3, Escherichia coli XL1-Blue containing pSYL107 cultured at 30 ° C. using a composite medium had better cell growth and PHB production capacity than Escherichia coli XL1-Blue containing pSYL105. , PHB content was found to be 85.5%. In addition, when cultured at 37 ° C. using a mixed medium, Escherichia coli XL1-Blue containing pSYL107 had a lower cell concentration and less PHB than Escherichia coli XL1-Blue containing pSYL105 due to the formation of minicells as described above. It was produced.

In addition, cell growth and PHB content by filamentation inhibition were more prominent in simple medium, so that XL1-Blue containing pSYL107 was higher than Escherichia coli XL1-Blue containing pSYL105 at both 30 ° C and 37 ° C. Fast growth and more than twice the production of PHB, PHB content was very high (71.7%) when incubated at 30 ℃. This means that PHB can be produced effectively even in simple medium, which is much cheaper than compound medium.

Example 4 PHB Synthesis Characteristics of Recombinant Escherichia Coli XL1-Blue According to Culture Temperature in Complex Medium

Since the recombinant Escherichia coli amplified ftsZ gene forms a minicell and the strain becomes unstable, while culturing Escherichia coli XL1-Blue transformed with pSYL107 in a complex medium, the characteristics of PHB synthesis according to the culture temperature are investigated, Decision was made (see FIGS. 2 (a) and 2 (b)). 2 (a) and 2 (b) show the concentrations of dry cells, PHB and surplus dry cells (excluding PHB in dry cells) according to the incubation time of recombinant E. coli cultured at 37 ° C. and 30 ° C., respectively. It is a graph showing the result of a measurement.

As shown in FIG. 2 (a) and FIG. 2 (b), it can be seen that the cell growth and PHB synthesis ability are significantly different due to the different incubation temperature. It can be seen that concentration and PHB concentration are more than doubled. The maximum PHB synthesis rate was 0.207g PHB / g surplus dry cell and 0.191g PHB / g surplus dry cell at 37 ° C, respectively, but it was slightly faster at 37 ° C. After about 35 hours of incubation, the PHB synthesis rate at 37 ℃ and 30 ℃ was 0.040g PHB / g surplus dry cell and 0.106g PHB / g surplus dry cell, respectively. And it was found. At this time, the PHB synthesis rate was defined as the gram PHB produced per gram excess dry cells per hour.

Therefore, when producing PHB using the recombinant filamentation suppressed Escherichia coli, 30 ° C is more suitable than the culture temperature 37 ° C, which is usually used, 20 ° C to 40 ° C, more preferably 25 ° C to 35 ° C It was confirmed that the culture in the temperature range is appropriate.

Example 5 Determination of PHB Production Capacity in Simple Medium Added Complex Nitrogen

From Table 3, E. coli XL1-Blue containing pSYL107 without filamentation, containing pSYL105 with filamentation, regardless of the type of the batch and the culture temperature, except for the complex medium and the culture conditions at 37 ° C It was confirmed that it can produce a higher concentration of PHB than E. coli XL1-Blue.

In this regard, if Escherichia coli XL1-Blue containing pSYL107 can be cultured in a low-cost simple medium to increase the PHB concentration, the PHB production cost can be further lowered. E. coli XL1-Blue containing pSYL107 at First, the optimal concentration of the composite nitrogen source showing the highest PHB concentration was determined, followed by culturing Escherichia coli XL1-Blue containing pSYL107 in a simple medium containing the compound nitrogen source of this concentration to measure the cell growth and PHB production capacity.

Example 5-1 Determination of Optimal Concentration of Complex Nitrogen Sources

First, the ten complex nitrogen sources shown in Table 4 were added to simple medium while changing to 0.5 to 5 g / L, and then inoculated with Escherichia coli XL1-Blue containing pSYL107 and incubated at 30 ° C. for 60 hours, and dried cells and PHB concentration was measured. Then, the concentration of the composite nitrogen source showing the highest PHB concentration for each composite nitrogen source was the optimal addition concentration, and the results are shown in Table 5 below.

Example 5-2 Measurement of PHB Production Capacity by Addition of Complex Nitrogen Source

The complex nitrogen source was added to the simple medium at the optimum concentration determined in Example 5-1, and then inoculated with E. coli XL1-Blue containing pSYL107 and incubated at 30 ° C. for 48 hours, followed by dry cell concentration, PHB concentration, and excess drying. Cell concentration, PHB content and PHB yield were measured. The result of culturing in a simple medium without a complex nitrogen source was used as a control, and the measurement results are shown in Table 6 below.

As shown in Table 6, it was found that the addition of a small amount of complex nitrogen source can increase the cell growth, PHB production capacity and PHB content. In particular, when added to the juice extract (abbreviation 'bar') 5g / L, the dry cell and PHB concentration was 8.68g / L and 7.46g / L, respectively, the PHB content is 86%, which is obtained in the mixed medium It is similar to the result (see Table 3). In addition, the concentration of the excess dry cells did not change significantly with the addition of the composite nitrogen source, from which it can be seen that the addition of the composite nitrogen source slightly increased the growth of the cells, while PHB production capacity greatly increased. It was confirmed that the addition of the complex nitrogen source not only increased PHB production capacity, but also increased the PHB yield from glucose, which means effectively converting the carbon source glucose into PHB.

As described and demonstrated in detail above, the present invention provides a method for transforming Escherichia coli with a recombinant plasmid containing ftsZ and PHB synthetic gene, and to prepare a high concentration of PHB from the transformant. According to the PHB manufacturing method of the present invention, by overexpressing ftsZ, which can suppress filamentation, the cells are lengthened by filamentation during PHB production by recombinant E. coli, thereby slowing or stopping cell growth, Solving the problems of low productivity and low PHB yield, it is possible to economically produce a high concentration of PHB.

Claims (1)

Escherichia coli transformed with a recombinant plasmid containing both E. coli's essential cell division protein (ftsZ) and PHB (poly-3-hydroxybutyrate) synthetic genes were mixed with 10 g / L tryptone and yeast extract. A method for producing PHB from recombinant E. coli comprising the step of culturing at a temperature of 28 to 33 ℃ in a complex medium consisting of 5g / L, 10g / L NaCl and 20g / L glucose.