KR100199994B1 - Process for preparing phb from xylose using recombinant e. coli - Google Patents

Abstract

The present invention relates to a method for producing a poly-3-hydroxybutyrate (PHB) from xylose using recombinant E. coli. More specifically, the present invention relates to a PHB transforming Escherichia coli of xylose available with a recombinant plasmid containing PHB synthetic gene of Alxaligenes eutrophus and culturing it in a xylose-containing ash. It relates to a manufacturing method of. According to the present invention, xylose 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 acidity can be improved by adding small amounts of various complex nitrogen sources, and PHB can be produced more effectively using recombinant E. coli DSM 499 (pSYL107) and DSM 6056 (pSYL107). In addition, the present invention can be produced PHB from hemicellulose, which is mainly composed of xylose and is widely present in nature, and is also useful for economic production of PHB.

Description

Method for producing PH-3-polybutyrate from xylose using recombinant E. coli

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

PHB, which has recently been spotlighted as a biodegradable polymer material, is an energy storage material synthesized and accumulated by microorganisms. PHB has similar physicochemical and mechanical properties to other types of chemical synthetic polymers, and has excellent biocompatibility and biodegradability. After a certain time in nature, PHB is completely decomposed into water and carbon dioxide. I am in the limelight. In particular, 3-hydroxybutyrate, a degradation product of, is a metabolic product present in the body of animals as well as humans, so it is being developed for use in surgical sutures and drug delivery systems. Due to its high production cost, it is still not used as a general packaging material or container.

Therefore, for the economic production of PHB, Algengenes eutrophus and Alcaligenes latus, which are known as PHB producing strains, have been intensively studied (see USP 4,433,053: Anderson and Daws). , Micrebiol. Rev., 54: 450-472 (1990)), and recently, several researchers have cloned the synthetic genes of Alkaligenes eutropus into E. coli to enable production of recombinant abiotic. 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 that can be economically produced 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 synthesis. Research has been conducted on establishing culture conditions. In 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, As a result of comparing pSYL104 to Escherichia coli XL1-Blue, it was confirmed that high copy number plasmid was used to produce high PHB (Lee et al., Ann, NY Acad. Sc., 721). : 43-53 (1994); Lee et al., J. Biotechnol., 32: 203-211 (1994).

, 대장균 JM109, 대장균 B, 대장균 W, 대장균 K12 등 여러 종류의 대장균에 높은 복제수를 가지면서 안정한 플라스미드 pSYL105를 도입하여, PHB 합성속도 및 수율 등에 대한 연구를 수행한 결과, 균주특성에 따라 PHB 합성속도와 수율 등이 다름을 확인하였다(참조: Lee et al., Biotechnol. Bioeng., 44:1337-1347(1994)).In addition, E. coli XL1-Blue, E. coli DH5

Introduced stable plasmid pSYL105 with high copy number to E. coli, E. coli JM109, E. coli B, E. coli K, E. coli K12, PHB synthesis rate and yield The rate and yield were confirmed to be different (Lee et al., Biotechnol. Bioeng., 44: 1337-1347 (1994)).

In addition, through the study of the recombinant microorganism culture conditions 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 culture method capable of producing a high concentration of PHB, and a method using a simple medium with a small amount of complex nitrogen source was proposed in this way, tryptone, xasamino acid or It was confirmed that addition of a small amount (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) Also, it was confirmed that the addition of amino acids or oleic acid instead of the former complex nitrogen source can improve PHB synthesis (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 in a simple medium containing a small amount of complex nitrogen source, 80g / L PHB can be obtained by fed-batch culture, and it was confirmed that the production of PHB by recombinant Escherichia coli was very effective (Lee and Chang, J. Environ. Polymer Degrad., 2: 169-176 (1994)).

In addition, the production of PHB by recombinant E. coli is prolonged by filamentation, which slows down or stops the growth of cells, thereby reducing the cultivation efficiency of the cells and lowering the productivity of PHB. 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 containing 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 exhibits no filamentation phenomenon in culture and can produce PHB at high concentrations (see SY Lee, Biotechnol. Lett., 16: 1247-1252 (1994): S. Y Lee Kor, J Appl. Microbiol.Biotechnol., 22: 614-620 (1994): Lee and Lee, J. Environ.Polymer Degrad., 4: 131-134 (1996).

However, since the above studies all use glucose as the main carbon source, the resulting production cost of PHB depends a lot 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.

Xylose has become a major component of hemicellulose, which accounts for the largest portion of the available resources in the natural world after cellulose. However, since strains using xylose efficiently are relatively smaller than glucose-using strains, there is a demand for effective use of xylose.

Alkali genes or methanol magnetization bacteria, which have been widely used for the production of biodegradable polymer PHB, cannot grow using xylose as a carbon source, but some of E. coli can grow as a carbon source.

Therefore, the present inventors introduced the recombinant plasmid pSYL105 or pSYL107, in which the PHB synthetic gene was cloned, into three types of E. coli, which can use xylose as a carbon source, and then, recombinant E. coli efficiently producing PHB in xylose-containing medium. Selection and completion of the present invention.

After all, an object of the present invention is to provide a method for producing PHB, wherein the recombinant plasmid containing PHB synthetic gene is transformed into E. coli xylose, and the transformant is cultured in xylose-containing medium.

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, to determine whether E. coli, which is known to be able to grow with xylose as a carbon source, grows in PHB production medium containing xylose, it is 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, 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 Xylose was added to a medium consisting of 0.002 g / L and thiamine 0.01 g / L, and three xylose available Escherichia coli, E. coli B ( DSM 499), E. coli TG1 (DSM 6056) and E. coli W3110 (KCTC 2223) were screened and then grown in PHB production medium. As a result, the above-mentioned strains were confirmed to grow in the PHB production medium to which xylose was added.

The first three E. coli plasmids cloned with a poly-3-hydroxybutyrate (PHB) -containing mutant, preferably beta-lactamase, Alkogen genes Eutropus is a recombinant plasmid containing the PHB syngene and the parB region of plasmid R1. Each was transformed with the synthetic plasmid pSYL107 (SY Lee Biotechnol. Lett., 16: 1247-1252 (1994)) prepared by inserting the gene of essential cell division protein (ftsZ) into pSYL105. In this case, the PHB synthetic gene may be used not only PHB synthetic gene derived from Alkagenes eutropus, but also PHB synthetic gene derived from other bacteria.

내지 40

, 보다 바람직하게는 25

내지 38

, 가장 바람직하게는 37

에서 배양하였고, pSYL107의 형질전환체는 미니셀(minicell)의 형성을 방지하기 위하여 20

내지 40

, 보다 바람직하게는 25

내지 35

, 가장 바람직하게는 30

에서 배양하였다(참조: Lee, Biotechnol. Lett., 16:1247-1252(1994))The simple medium comprising the transformants thus obtained contains a xylose-containing ash ratio, preferably xylose at a degree of 10 to 100 g / L. Or cultured in the simple medium containing xylose at a concentration of 10 to 100 g / L and adding 0.1 to 10 g / L of the complex nitrogen source, and final dry cell concentration, PHB concentration and PHB content (PHB concentration per dry cell concentration). Cents). 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 30

(Lee, Biotechnol. Lett., 16: 1247-1252 (1994))

As a result, all six transformants were able to produce PHB from xylose, and PHB production ability was better in simple medium containing complex nitrogen source than simple medium.

In the complex nitrogen source, tryptone, yeast extract, peptone, casamino acid, cotton seed hydrolysate, juicy liquor or soybean hydrolyzate ( soyvean hydrelysate) and the like can be used. In addition, in this invention, the hydrolyzate of hemicellulose can be used instead of xylose.

According to the present invention, a recombinant plasmid containing a PHB synthetic gene can be transformed into E. coli xylose, and the transformant can be cultured in a xylose-containing medium to effectively produce PHB. PHB synthetic concentrations could be improved. In addition, the present invention is useful for economical production of PHB, since the main component of xylose can be produced PHB from hemicellulose, which is widely present in nature.

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 of ordinary skill 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

Confirmation of Escherichia Coli Growth in PHB Production Medium Using Xylose as Carbon Source

In order to determine whether the known xylose available Escherichia coli grow in a simple medium of PHB production using xylose as a carbon source, 20 g / L of xylose was added to the simple medium of Table 1 below, and the known xylose available strain Three Escherichia coli strains, namely E. coli B (DSM 499), E. coli TG1 (DSM 6056), and E. coli W3110 (KCTC 2223), were cultured in flask flasks. Was found to grow as a carbon source, therefore, in the following examples, these three strains were used as strains for transformation.

Example 2

Recombinant plasmid pSYL105 (see FIG. 1: Lee et al., Biotechnol. Bioeng., 44: 1337-1347 (1994)) comprising the PHB syngene from xylose and the parB region of plasmid R1, and cells essential for pSYL105 Three species confirmed growth in Example 1 by recombinant plasmid pSYL107 (see FIG. 2: SY Lee, Biotechnol. Lett., 16: 1247-1252 (1994)) prepared by inserting the gene of the cleavage protein (ftsZ). Of E. coli were transformed. At this time, transformation was performed by an electroporation method (Dower et al., Nucleic Acids Res., 16: 6127-6145 (1988)).

By separating the plasmids from the six transformants thus prepared, and digesting them with restriction enzymes, electrophoresis was confirmed that the electrotransformants contained exactly pSYL105 or pSYL107, and they were respectively identified as DSM 499 (6) and DSM 499. (pSYL107), DSM 6056 (pSYL105), DSM 6056 (pSYL107), KCTC 2223 (pSYL105) and KCTC 2223 (pSYL107).

Example 3

Preparation of PHB from Xylhose

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

Example 3-1

Determination of Optimal Xylose Concentration

, pSYL107 형질전환체는 30

에서 배양하였다.Prior to examining the PHB production capacity from xylose, to determine the optimal SZMTLF loss concentration for the growth of the strain, by adding xylose to the concentration of 20,50,80,120 g / L to the simple medium of Table 1 After preparing four kinds of medium, six kinds of transformants obtained in Example 2 were inoculated and incubated for 60 hours to measure absorbance (OD) at 600 NM (see Table 2). 37

, pSYL107 transformants were 30

Incubated at.

In addition, in Table 2, the optimum xylose concentration providing the best strain growth compared to carbon source consumption was different depending on the transformant, and DSM 499 (pSYL105) was 50 g / L: DSM 499 (pSYL107). Silver 80 g / L DSM 6065 (pSYL105) at 20 g / L; DSM 6056 (pSYL107) is 20 g / L; KCTC 2223 (pSYL105) at 20 g / L; KCTC 2223 (pSYL107) showed optimal growth at a xylose concentration of 20 g / L.

Example 3-2

Preparation of PHB in Xylose-Containing Simple Medium

, pSYL107 형질전환체는 30

에서 배양하였다. PHB 생산농은 공지의 방법으로 측정하는 건조균체농도. PHB농도, PHB 함유량으로 결정하였으며 (참조: Lee at al., Biotechnol. Bioeng,. 44: 1337-1347(1994)), 그 결과를 표 3에 나타내었다.50 mL of simple medium containing xylose was added to a 250 mL flask at the optimum concentration determined in Example 3-1, and each strain was inoculated with the transformant showing the optimal growth at the corresponding xylose concentration and shaken for 60 hours to induce PHB production capacity. Decided. Wherein the pSYL105 transformant is 37

, pSYL107 transformants were 30

Incubated at. PHB production concentration is the dry cell concentration measured by a known method. PHB concentration was determined by the PHB content (Lee at al., Biotechnol. Bioeng ,. 44: 1337-1347 (1994)), and the results are shown in Table 3.

As shown in Table 3, all six transformants were able to produce PHB from xylose, among which two recombinant E. coli DSM 499 (pSYL107) and DSM 6056 (pSYL107) produced PHB more efficiently. I could see.

Example 3-3

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

In the production of PHB from glucose using preparative E. coli, small amounts of complex nitrogen sources may increase PHB synthesis (Lee and Chang, J. Environ. Polymer degrad., 2: 169-176 (1994)) In order to determine whether addition of a composite nitrogen source may increase PHB synthesis even when synthesizing PHB from xylose on the basis of the above, except that a small amount of the composite nitrogen source of Table 4 is added to the xylose-containing simple medium, After incubating each recombinant E. coli in the same manner as in Example 3-2, cell growth and PHB concentration were measured (see Table 5). At this time, the complex nitrogen source was added at a concentration already reported to provide high yield of PHB production (Lee and chang, J. Environ. Polymer degrad., 2: 169-176 (1994)).

As shown in Table 5, the PHB synthesis ability of the recombinant E. coli strain was improved by the addition of a small amount of a composite nitrogen source, the improvement effect was found to be greater in the case of 499 (pSYL107) and dsm 6056 (pSYL107). Especially, in case of DSM 400 (pSYL107), PHB concentration was 3.91 g / L and PHB content was 71.4% when soybean hydrolyzate was added, and PHB concentration was 3.17 g / L even at low cost cornspiker. The PHB content was found to be very effective as 62.2%. In addition, even in the case of DSM 6056 (pSYL107), the concentration of the yeast extract, casamino acid, cotton seed hydrolyzate or soybean hydrolyzate is more than 3 g / L and the PHB content is 60% or more. It can be seen that the addition of the very effective PHB production. The addition of soybean hydrolyzate among the above composite nitrogen sources showed that the PHB concentration produced by DSM 6056 (pSYL107) was the most effective, reaching 4.40 g / L PHB content of 74%, and 20 g / L xylose. Based on 4.4 g / L of PHB produced from it, the yield was found to be very high, 0.22 g of PHB per g of xylose.

As described in detail above, the present invention provides a method for producing PHB from xylose using a recombinant E. coli transformed with a plasmid cloned with an alkali genes eutropus cloned PHB synthetic gene. According to the present invention, xylose by six recombinant E. coli (DSM 499 (pSYL105), DSM499 (pSYL107), DSM 6056 (pSYL105), DSM 6056 (pSYL107), KCTC 2223 (pSYL105), and KCTC 2223 (pSYL107) PHB production is possible, and in particular, high concentration of PHB can be produced more effectively by using recombinant E. coli DSM 499 (pSYL107) and DSM 6056 (pSYL107), and improve the PHB production ability by adding small amount of various complex nitrogen sources. In addition, the present invention is also useful for economical production of PHB, since PHB can be prepared from hemicellulose, which is mainly composed of xylose and is widely present in nature.

Claims (6)

Escherichia coli using xylose as a carbon source was transformed into a plasmid cloned with a poly-3-hydroxybutyrate (PHB) synthetic gene, and the transformant was cultured in a medium containing xylose as a carbon source. Method for producing a PHB comprising the step. Escherichia coli using xylose as a carbon source is E. coli B (DSM 499), E. coli TG1 (DSM 6056) or E. coli W3110 (KCTC 2223). The plasmid is pSYL105 comprising the beta-lactamase, the PHB syngene and the parB region of plasmid R1, or pSYL107 comprising the beta-lactamase PHB synthetic gene, the parB region of plasmid R1 and the gene of essential cell division protein (ftsZ). PHB manufacturing method characterized by rut. The method of claim 1, wherein the xylose-containing medium is KH ₂ PO ₄ 13.5 g / L, (NH ₄ ) ₂ HOP 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 G / l) of simple medium or complex nitrogen source 0.1 to Method for producing H ₂ O, characterized in that the medium to which the xylose or hemicellulosic hydrolyzate is added to a simple medium of electricity added at 10 G / l. The method of claim 4, wherein the complex nitrogen source is tryptone, yeast extract, peptone, casamino acid, cotton seed hydrolysate, juicy extract, casein hydrolyzate A method for producing PHB, characterized in that it is at least one nitrogen source selected from the group consisting of casein hydrolysate, corn steep liquor and soybean hydrolysate. The method for producing PHB according to claim 1 or 4, wherein xylose is added in a concentration of 10 to 100 g / L.

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