TWI429749B - A method for the polyhydroxybutyrate (phb) production by using recombinant escherichia coli - Google Patents

Description

Method for producing polyhydroxybutyl ester (PHB) by recombinant E. coli

The present invention relates to a method for producing a polyhydroxybutyl ester (PHB) strain by directly inserting a phaCAB gene into nlpB locus on the chromosome of Escherichia coli, in particular to a method for transfecting a phaCAB gene into Escherichia coli by using a gene transfer technique. E. coli was induced and produced to produce a polyhydroxybutyl ester (PHB) product.

In recent years, due to the rise of environmental awareness, the use of non-decomposable petrochemical plastic products, environmental damage and waste of resources has become a topic of concern and ambition; therefore, the search for low-cost, decomposable alternative materials is currently The goals of all parties.

In order to adapt to the changing environment of nature, microorganisms usually survive in the harsh conditions that are not conducive to their growth, and they usually survive and produce specific energy substances in the body. Among these substances, polyhydroxyalkanoates (PHAs) are used because of these. It is biocompatible, biodegradable and flexible. Its physical properties are similar to those of plastic products (such as polyethylene polyethylene). Therefore, it can be used as a substitute for petroplastic plastics. The use of petrochemical plastics can not be decomposed to cause environmental pollution problems, and can be further developed into high-value biomedical materials for bioengineering and biomedical applications.

Polyhydroxyalkyl esters (PHAs) are polyesters used to store energy when they are in excess of carbon and lack certain nutrients (eg N, P, S, O or Mg). In the environment, polyhydroxyalkyl esters (PHAs) are accumulated in cells to serve as a carbon source and store energy.

Polyhydroxyalkyl esters (PHAs) and their related derivatives are metabolized by different microorganisms, and their classification is differentiated according to the number of carbon atoms. For example, 3 to 5 carbons are polyhydroxybutyrate (PHB); short-chain-length (scl-PHAs) containing short carbon chains; and 6-10 carbons in side chains In the case of carbon, it is a medium-chain-length (mcl-PHAs) of a medium-long carbon chain.

Among the microorganisms used to produce polyhydroxybutyrate (PHB), Cupriavidus necator (formerly known as Ralstonia eutropha ) is most widely used. Ralstonia eutropha and Alcaligenes latus can produce high amounts of polyhydroxybutyl ester (PHB). Compared to other wild-type polyhydroxybutyrate (PHB) microorganisms, recombinant E. coli containing R. eutropha PHA gene is used in polyhydroxyl groups. The production of butyl ester (PHB) has advantages, including the use of a wide range of carbon sources, high accumulation of polyhydroxybutyl ester (PHB), relatively fragile cells (favorable for PHB), and during fermentation. Polyhydroxybutyl ester (PHB) is not decomposed due to the lack of intracellular polyhydroxybutyrate degrading enzymes (PHB depolymerases).

(Hemoglobin, VHb) contains a vgb gene expression in a host cell heterogeneity (heterologous bacterial hosts) of hemoglobin, particularly under conditions of oxygen limitation in this vgb gene enhances cell density, oxidative metabolism, protein production and cell repair and antibiotic activities . Other beneficial effects include increased fungal antibiotic production, increased tissue plasmin synthesis in mammalian cells, and accelerated plant growth. Some studies have pointed out that the help of VHb to the growth of bacteria is directly related to the respiratory oxidase, and this enzyme provides oxygen, thereby increasing the oxidative phosphorylation reaction (the production of adenosine triphosphate). Furthermore, we demonstrated that, in VGB phaCAB with recombinant plasmid expression help of polyhydroxybutyrate (PHB) production.

One of the strains currently used for the production of polyhydroxybutyrate (PHB) is a large-scale fermentation production using Escherichia coli. Although E. coli itself does not carry the genes related to the synthesis of polyhydroxybutyl ester (PHB), most E. coli strains produce hydroxybutyrate (PHB) by plastid carrying phaCAB , and the plastids express phaCAB . The advantage is that the number of copies is large, but the relative stability of the plastid is required to consume a large amount of antibiotic cost. Therefore, the present invention embeds the phaCAB gene into the nlpB locus of the E. coli chromosome, and successfully displays the related protein enzyme to produce poly. Hydroxybutyl ester (PHB). This recombinant strain still has its shortcomings, because the embedded chromosome makes the gene become a copy, which is much different from the number of copies of the plastid. Therefore, the ratio of the PHB inclusions in the expression is relatively better than that of the plastid. Low, this problem still needs to be further overcome.

It can be seen that there are still many shortcomings in the above methods for producing PHB by means of plastid carrying phaCAB , which is not a good designer and needs to be improved.

In view of the above shortcomings derived from the above-mentioned method for producing PHB by means of plastid carrying pha CAB, the inventor of the present invention has improved and innovated, and after years of painstaking research, finally succeeded in researching and developing this piece of cis - expressing pha CAB- vgb Knock nlpB locus region (locus) of the E. coli chromosome to generate a method of PHB.

I.e., that the object of the present invention to provide a process for producing by recombinant Escherichia coli in a poly-hydroxybutyrate (PHB), the use of gene transfer methods colonization, will produce polyhydroxybutyrate (PHB) of phaCAB - vgb into transgenic E. In the bacillus.

The object of the present invention is a method for producing polyhydroxybutyl ester (PHB) by recombinant E. coli, the method comprising:

(1) providing an Escherichia coli microorganism;

(2) providing a having phaCAB - DNA structures vgb gene;

(3) transferring the DNA construct obtained in (2) to the microorganism of Escherichia coli of (1);

(4) screening the recombinant Escherichia coli obtained in (3);

(5) cultivating the recombinant Escherichia coli obtained in (4);

(6) The (5) obtained E. coli cells were collected and purified to obtain polyhydroxybutyl ester (PHB).

The phaCAB - vgb Mount Adapter gene (Cassette) inserted into E. coli K12 is nlpB region (locus), for screening and analysis of recombinant E. coli, the gentamicin-resistant section (gentamicin, GM) Mount Adapter gene (Cassette) connected to a downstream nlpB :: phaCAB - vgb gene vector fragment, and further transferred into E. coli colonization. Engaged by germ (Conjugation) will nlpB :: phaCAB - vgb transfer gene fragment from E. coli S17-1 (λpir) to E. coli K12, and then via homologous recombination method for inserting genes into the chromosome, thereby completing comprising phaCAB A recombinant strain of -vgb , called Escherichia coli YH100 ( E. coli YH100).

First, the experimental materials: Strain and plastid

The strains and plastid types used in this experiment are summarized in Table 1. The medium was adjusted to pH 6.8 before sterilization. E. coli was cultured in Luria-Bertani medium (composed of 10 g/liter tryptone, 5 g/liter yeast extract 10 g/liter sodium chloride (NaCl) and 1% glucose) in, 1.5% Bacto ^TM agar (agar) medium was added to form a solid.

Enzymes and drugs

DNA restriction enzymes and modification enzymes were purchased from Roche. Tag polymerase (Tag Polymerase) cable and a polymerization chain reaction enzyme (polymerase chain reaction, PCR) Related products purchased from Perkin-Elmer or Takara Biomedicals. Other experimental grade chemicals were purchased from Sigma Chemical Company.

Second, the experimental method: Polyhydroxybutyl ester (PHB) quantitative method

The bacterial strain was placed in the strain culture solution at 37 ° C, and after 48 hours of shaking culture (200 rpm), the dry weight of the strain and the weight of polyhydroxybutyl ester (PHB) were measured. 0.5 g of dried cells were added to 5 mL of sodium dodecyl sulfate (SDS) for 5 minutes at 50 ° C, centrifuged with water and dried to obtain polyhydroxybutyl ester (PHB). . The polyhydroxybutyl ester (PHB) was dissolved in chloroform at 60 ° C for 24 hours, and then quantified by gas chromatography (GC), and the content was expressed by dividing the weight of polyhydroxybutyl ester (PHB) by the dry cell weight (wt%). The concentration is calculated from the volume of the bacterial suspension and the content of polyhydroxybutyl ester (PHB).

Qualitative analysis of polyhydroxybutyl ester (PHB)

The sample (approximately 5-10 mg) was sealed in an aluminum bottle, and the thermogram temperature was scanned from -40 ° C to 200 ° C at a rate of 10 ° C per minute (first scan), and the mobile phase was nitrogen (flow rate was 40 ml/min); the sample was stored at 200 ° C for 5 minutes to eliminate thermal history, then cooled to 40 ° C at a cooling rate of 10 ° C / min, then heated to 200 ° C at a scan rate of 10 ° C / min (Second scan) and performed under the same nitrogen conditions; thermal spectrum measurements were made with blank aluminum bottles for baseline correction. The sample was further analyzed for weight loss by detecting the polyhydroxybutyl ester (PHB) in a dynamic scanning mode using a PerkinElmer Pyris-1 thermogravimetric analyzer (TGA). A 10 to 15 mg sample was subjected to scanning analysis at 30 to 400 ° C at a rate of 10 ° C per minute and a nitrogen flow rate of 40 ml / min.

Nile red stain

Polyhydroxybutyrate (PHB) particles were cultured in recombinant strains containing 1% fluorescent dye and Nile red dye on LB agar plates. The ultraviolet light conditions of the bacteria were observed under the particles of PHB.

Western blotting

The bacterial cells were washed with phosphate-buffered saline (PBS, pH 7.5) and dissolved in cell lysis buffer (pH 7.2) (20 mM piperazine-N, N'-bis (2-ethanesulfonic acid, PIPES). , 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride). The sample was ice bathed for 30 minutes and then centrifuged at 14,000 rpm for 30 minutes at 4 °C. After centrifugation, the supernatant was analyzed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein was transferred to a polyvinylidene difluoride membrane, and the polyvinylidene fluoride membrane was immersed in a blocking buffer (containing 5% milk, 0.1% Tween 20) for one hour. Further, the polyvinylidene fluoride membrane was immersed in an anti-VHb polyclonal antibody, and then the polyvinylidene fluoride membrane was immersed in the blocking solution at room temperature for 1 hour, followed by horseradish. The substrate was treated with a horseradish peroxidase-conjugated anti-rabbit second antibody for 1 hour and then exposed to X-ray film.

Transmission electron microscopy (TEM)

The polyhydroxybutyl ester (PHB) encapsulated in the cells was checked for negative staining by a transmission electron microscope (TEM). A drop of bacterial solution (containing 10 ¹² PFU/ml) was applied to a formvar surface coating grid (200 mesh copper grid), negatively stained with 2% uranyl-acetateteandrup, and then in Hitachi H-7500 transmissive electrons. The microscope was operated at 80 kV (kV).

Third, the experimental results: Embed the nlpB::phaCAB-vgb gene into E. coli

In order to introduce foreign DNA into the E. coli chromosome, an unnecessary gene or site is first selected as a target gene for homologous recombination. Previous experiments have found that the presence or absence of the nlpB gene does not affect the viability of E. coli. Therefore, the phaCAB-vgb gene cassette was inserted into the nlpB region (locus) of Escherichia coli K12. In order to screen and analyze recombinant E. coli, a 850-bp gentamicin (GM) gene cassette was ligated to the downstream nlpB::phaCAB-vgb gene fragment vector and then transferred into the large intestine. Bacillus.

Briefly, the 3' region of the nlpB gene amplifies this region by polymerase chain reaction (PCR) and using primer pair nlpB- FF/ nlpB- FR. The amplified product was transferred to pCR2.1, and the nlpB fragment was excised with SalI/ Sma I. Another DNA fragment, phaCAB-vgb , was excised from the plastid pSY02 with Sma I/ Hind III enzyme. These two fragments, nlp B and phaCAB-vgb , together with the Gm ^R gene cassette (Cassette) were cut out by Hind III/ Sma I and ligated together to the pUTmini-Tn5-km1 suicide vector (suicide) cut out by Sma I Vector) to form pUT- nlpB::phaCAB-vgb:: Gm ^R , called pYH-1 (as shown in Figure 1A).

In order to successfully insert the gene into the chromosome via homologous recombination, pYH-1 is transferred from Escherichia coli S17-1 (λpir) to E. coli K12 via conjugation, and the transconjugants are distributed in the mold. In gentamicin (15 μg/ml) LB culture dishes, the target of the transfection is screened by the use of polymerase chain reaction (PCR) and pha-F/phaC-R primer pairs (eg As shown in Fig. 1B, the resulting recombinant strain containing phaCAB-vgb is called Escherichia coli YH100 ( E. coli YH100).

Recombinant Escherichia coli ( E. coli ) YH100 phenotype

The cell growth rate of phaCAB-vgb recombinant Escherichia coli YH100 and wild-type E. coli K12 was compared and analyzed (as shown in Figure 2). The cell growth curve was obtained by measuring the optical density value at 600 nm. In the absence of antibiotics, YH100 strain was obtained. The growth rate (grown over 48 hours) was significantly higher than that of the K12 strain (as shown in Figure 2A).

To confirm phaCAB - vgb gene expression, Nile red staining (Nile red) and Western printing method (Western blotting) were used to assess the functionality and PhaCAB VHb expression, recombinant strain YH100 exposed to the ultraviolet light is red ( Positive results); in the Nile red staining method, different from the performance of the K12 strain (as shown in Figure 2B).

VHb performance through coding vgb through Western printing method (Western blotting) and anti VHb expression polyclonal antibody (anti-VHb polyclonal antibodies) confirmed; VDb showed a molecular weight of 14kDa, the performance of the recombinant strain YH100, but does not exhibit On the wild-type K12 strain (as shown in Figure 2C). These results indicate the success of the way through this phaCAB - vgb in gene expression in E. coli.

Recombinant Escherichia coli ( E. coli ) YH100 produces polyhydroxybutyl ester (PHB)

Recombinant Escherichia coli YH100 was cultured in LB medium and cultured with glucose as the sole carbon source for 48 hours, and polyhydroxybutyl ester (PHB) was detected to accumulate in recombinant Escherichia coli YH100. Polyhydroxybutyrate (PHB) was extracted with excess chloroform at 60 ° C for 24 hours and analyzed by Gas Chromatography (GC). Finally, the dry cell weight (CDW), polyhydroxybutyl ester (PHB) concentration and polyhydroxybutyl ester (PHB) content were determined to be 1.2 g / liter, 0.25 g / liter, weight and 20.83%. Wild type E. coli K12 does not produce polyhydroxybutyl ester (PHB).

Polyhydroxybutyl acrylate (PHB) properties produced by recombinant Escherichia coli ( E. coli ) YH100

The polyhydroxybutyl ester (PHB) produced by recombinant Escherichia coli ( E. coli ) YH100 was confirmed by Transmission electron microscopy (TEM). Polyhydroxybutyrate (PHB) accumulates in the recombinant Escherichia coli YH100 strain (as shown in Figure 3A), but wild-type E. coli K12 does not produce polyhydroxybutyl ester (PHB) (as shown in Figure 3B). Characterization of the produced polyhydroxybutyl ester (PHB), polyhydroxybutyl ester (PHB) purified by recombinant Escherichia coli YH100 and commercial polyhydroxybutyl ester (PHB) (Sigma-Aldrich) The molecular weight was evaluated (as shown in Table 2). The polyhydroxybutyl ester (PHB) produced by recombinant Escherichia coli YH100 has a mean molecular weight (Mw) and a number average molecular weight that are two times higher than the commercially available polyhydroxybutyl ester (PHB).

The polydispersity (PDI) of polyhydroxybutyl ester (PHB) produced by recombinant Escherichia coli YH100 is closer to unity than the commercially available polyhydroxybutyl ester (PHB), which means that recombinant Escherichia coli YH100 The product of the produced polyhydroxybutyl ester (PHB) conforms to the characteristics of a commercially available polymer. The physical properties of polyhydroxybutyl ester (PHB) produced by recombinant Escherichia coli YH100 were analyzed by differential scanning calorimetry (DSC) and thermogravimetric analyzer (TGA). The results are presented in Table 3. And Table IV. The thermal stability of polyhydroxybutyl ester (PHB) produced by recombinant Escherichia coli YH100 is similar to that of commercially available polyhydroxybutyl ester (PHB).

The invention provides a method for directly producing the polyhydroxybutyl ester (PHB) strain by directly inserting the phaCAB gene into the nlpB locus on the chromosome of Escherichia coli, and has the following advantages when compared with other conventional techniques:

1. Develop strains capable of stable production of strains capable of stably producing polyhydroxybutyl ester (PHB).

2. Solve the problem of instability of polyhydroxybutyl ester (PHB) using plastids.

3. Solve the problem of using expensive antibiotics.

4. Improve the production and quality of polyhydroxybutyl ester (PHB).

The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case.

In summary, this case is not only innovative in terms of method, but also the microbes produced can enhance the above-mentioned multiple functions compared with the conventional microorganisms. It should fully comply with the statutory invention patent requirements of novelty and progress, and apply for it according to law. You have approved this invention patent application, in order to invent invention, to the sense of virtue.

Figure 1. (A) Construction of a recombinant strain (YH) in which the phaCAB gene is inserted into the nlpB region of the Escherichia coli chromosome. Using the gene homology interchange technique, the production of PHB-related genes was placed behind the nlpB gene, allowing phaCAB to express PhaCAB- associated protein enzymes in the nlpB region. (B) The phaC gene of E. coli YH100 was confirmed by polymerase chain reaction (PCR). The PCR templates were E. coli K12 and E. coli YH100, respectively, showing the molecular weight marker (M) and the positive control group.

Figure 2. Comparison of characteristics of wild-type Escherichia coli ( E. coli K12) and recombinant Escherichia coli YH100 ( E. coli YH100). (A) The microbial growth curve of the culture in LB medium was measured at an absorbance of 600 nm. (B) When the phaCAB gene exhibits an enzyme protein, it can synthesize PHB biopolyester , and the resulting (PHB) can be observed by Nile Red staining (iile Red). Visible light (ii) ultraviolet light (300 nm). (C) Western blotting was used to verify the production of VHb; (i) was a crude extract of cells stained with 12% SDS-PAGE and stained with Coomassie Brilliant Blue; (ii) polyclonal antibody with anti-VHb expression The expression of VHb was confirmed by anti-VHb polyclonal antibodies, wherein the control group was wild type Escherichia coli ( E. coli K12) and the experimental group was recombinant Escherichia coli YH100 ( E. coli YH100).

Figure 3. Transmission electron microscopy (TEM) observation method can directly observe whether the strain has PHB force. The TEM results in Figure 3 show that YH100 strain produces white PHB biopolyester ( A) No PHB particles (B) were produced compared to the wild strain K12.

Claims (8)

A method for producing polyhydroxybutyrate (PHB) by recombinant Escherichia coli, the method comprising: (1) providing an Escherichia coli microorganism; (2) providing a DNA construct having a phaCAB-vgb gene; 3) The DNA construct obtained in (2) is transferred to the Escherichia coli microorganism of (1), wherein the phaCAB-vgb gene is embedded in the position of nlpB locus of Escherichia coli; (4) the result obtained by (3) The recombinant Escherichia coli is screened; (5) the recombinant Escherichia coli obtained in (4) is cultured; (6) the obtained E. coli cells are collected and purified to obtain polyhydroxybutyl ester (PHB). A method for producing polyhydroxybutyl ester (PHB) by recombinant Escherichia coli according to claim 1, wherein the Escherichia coli strain is Escherichia coli K12. A method for producing polyhydroxybutyrate (PHB) by recombinant Escherichia coli according to claim 1, wherein the phaCAB-vgb gene is a gene for synthesizing poly-3-hydroxybutyl ester (PHB), The sequence of SEQ ID NO: 1. A method for producing polyhydroxybutyrate (PHB) by recombinant Escherichia coli according to the first and third aspects of the patent application, wherein the phaCAB-vgb gene is linked to a gentamicin (GM) Gene cassettes (cassette). A method for producing polyhydroxybutyrate (PHB) by recombinant Escherichia coli according to the first and third aspects of the patent application, wherein the recombinant Escherichia coli is cultured in a gentamicin-containing (GM) Screening of recombinant strains is performed in the medium. A method for producing polyhydroxybutyrate (PHB) by recombinant Escherichia coli according to claim 1, wherein the phaCAB-vgb gene is conjugated by a method of homologous recombination E. coli S17-1 was transferred to E. coli K12 ( Escherichia coli K12). A method for producing polyhydroxybutyrate (PHB) by recombinant Escherichia coli according to claim 1, wherein the recombinant Escherichia coli strain is Escherichia coli YH100. A method for producing polyhydroxybutyl acrylate (PHB) by recombinant Escherichia coli according to claim 1, wherein the medium is LB medium.