TWI418631B - Method for enhancing thermotolerance of photosynthesis bacteria - Google Patents

Description

Method for improving heat tolerance of photosynthetic hydrogen producing bacteria

The present invention relates to a method for enhancing the thermal tolerance of photosynthetic hydrogen producing bacteria.

Hydrogen is a green energy source that is energy efficient, does not emit carbon dioxide, and does not contribute to the greenhouse effect. Biohydrogen production converts organic matter into hydrogen by biodegradation and bioconversion. Photosynthetic microorganisms use hydrogen to produce hydrogen by Nitrogenase, which converts hydrogen protons into hydrogen by consuming adenosine triphosphate (ATP). The role is:

2H ⁺ +2e ^- +4ATP → H ₂ +4ADP+4Pi

When cultivating photosynthetic microorganisms, the temperature of the culture tank is often increased due to illumination. When the temperature exceeds the temperature range (28-35 ° C) that the photosynthetic microorganisms can adapt to, it will affect the physiological state of the photosynthetic microorganisms and cause cell growth stagnation, resulting in hydrogen production. Reduced, thus not conducive to continuous operation.

In general, to improve the thermal tolerance of microorganisms can be achieved by two methods, one is by heterogeneous or homologous expression of multiple protein protectors (chaperone); or by natural mutation Screening microorganisms with heat tolerance traits.

One aspect of the present invention provides a method for enhancing heat tolerance of photosynthetic hydrogen producing bacteria, comprising transforming a DNA sequence comprising SEQ ID NO: 1 to a photosynthetic hydrogen producing bacterium by a vector to enable photosynthetic hydrogen production The bacteria become a transforming bacteria that can be grown and cultured in an environment of about 40 °C.

According to an embodiment of the present invention, the DNA sequence comprising SEQ ID NO: 1 is plastid-transformed so that the photosynthetic hydrogen-producing bacteria can be transformed into a growth bacterium which can be grown in a culture environment at about 40 ° C.

According to an embodiment of the present invention, the deoxyribonucleic acid sequence comprising SEQ ID NO: 1 is inserted into the chromosome of the photosynthetic hydrogen producing bacterium to make the photosynthetic hydrogen producing bacterium a transforming bacterium capable of growing in a culture environment of about 40 ° C.

According to the above, the method for enhancing the heat tolerance of photosynthetic hydrogen producing bacteria in the embodiment of the invention utilizes the expression of exogenous [FeFe]-hydrogenase in photosynthetic hydrogen producing bacteria without additional expression of chaperone protein. The host can be made to adapt to the thermal tolerance of the culture environment around 40 °C. In particular, the method of the present invention does not use only hydrogenase to enhance the hydrogen production ability of microorganisms, but produces an effect of stabilizing cell physiological activity and maintaining cell growth concentration in a high temperature environment.

According to an embodiment of the present invention, a DNA sequence comprising SEQ ID NO: 1 is transformed into a photosynthetic hydrogen producing bacterium by a vector, and the photosynthetic hydrogen producing bacterium can be transformed into a transforming bacterium which can be grown in a culture environment of about 40 ° C.

Here, the gene encoding the [FeFe]-hydrogenase can be called the hydA gene or the homologous sequence of the hydA gene, which can be obtained from the appropriate strain by means of transfection, or can be artificially synthesized. The way to get this sequence. Strains suitable for use in the present invention to obtain the hydA gene or a sequence homologous to the hydA gene include strains which can survive in the environment of 40-70 ° C, including but not limited to strains of the genus Clostridium . The strain of the genus Clostridium may be, for example, Clostridium thermocellum . Colonization can be transferred is acquired hydA gene sequences or homologous genes from the chromosomal DNA of the aforementioned strains hydA of the; hydA or synthesized gene sequence or by using the synthetic gene with homologous hydA. Those of ordinary skill in the art can use known procedures to obtain the hydA gene or a sequence homologous to the hydA gene without undue experimentation.

The above " hydA gene or a sequence homologous to the hydA gene" includes a sequence represented by SEQ ID NO: 1, a complementary sequence thereof, and a conservative analog, for example, having a degenerate codon substitution ( Degenerative codon substitutions) homologous sequences. Degenerate codon substitutions can be made via a third position in one or more selected codons in the nucleic acid sequence, according to common general knowledge in the art.

In one embodiment of the present invention, a vector is constructed by using the obtained hydA gene or a sequence homologous to the hydA gene, and a photosynthetic hydrogen producing bacteria is transformed by the vector to convert the photosynthetic hydrogen producing bacteria into 40 ° C. Transforming bacteria grown in the environment.

A vector suitable for use in the present invention, including but not limited to a plasmid which can be replicated in a photosynthetic hydrogen producing host, is transformed into a photosynthetic hydrogen producing host by transformation; or a phage is used as a vector Or a homologous recombination plastid is a vector, and the deoxyribonucleic acid fragment is directly embedded in the chromosome of the photosynthetic hydrogen producing bacterium.

The vector may have at least one deoxyribonucleic acid fragment having a hydA gene or a sequence homologous to the hydA gene. For example, the vector may comprise a deoxyribonucleic acid fragment having a sequence as shown in SEQ ID NO: 1. In one embodiment, the plastid is pMGPHT and is transformed into photosynthetic hydrogen producing bacteria by an electroporation method. pMGPHT comprises the deoxyribonucleic acid sequence of SEQ ID NO: 3.

The promoter region may comprise a sustained phenotype promoter or an inducible promoter. Unless otherwise specified, the "promoter region" as used herein encompasses a promoter sequence recognized by an RNA polymerase, one or more regulatory sequences recognized by a regulatory gene, and a TATA-BOX sequence. , transcription start point sequence, Shine-Dalgarno (SD) sequence.

The photosynthetic hydrogen producing bacteria suitable for use in the present invention may include, but are not limited to, strains of the genus Rhodopseudomonas, the genus Rhodospirillaceae, and the genus Rhodobacter . The strain of Rhodopseudomonas can be, for example, a purple sulphur-free photosynthetic bacterium ( Rhodopseudomonas palustris ).

In addition, a DNA sequence homologous to the hydA gene can be replaced with a preferred codon of the target host according to different hosts to promote expression of a hydrogen-producing [FeFe]-hydrogenase.

The deoxyribonucleic acid fragment (SEQ ID NO: 2) containing the sustained phenotype promoter region and the hydA gene (SEQ ID NO: 1) was constructed to form plastid pMGPHT (SEQ ID NO: 3) and transformed into Rhodopseudomonas palustris CGA009 (purchased). As an example from the American Type Culture Collection (ATCC) host, the method of the present invention and the effect of the heterologous hydrogen production gene on the heat tolerance of the host in photosynthetic hydrogen producing bacteria are demonstrated.

Construction of plastid pMGPHT

The first picture shows the construction flow chart of the plastid pMGPHT. In this example, Clostridium thermocellum ATCC 27405 was used as a template (purchased from the Center for Biological Resources attached to the Food Industry Research), using the introduction of HydA-F (SEQ ID NO: 4) and the introduction of HydA-R (SEQ ID NO: 5). The hydA gene, the start codon was modified to ATG to enhance the expression of the hydA gene in the host; a promoter (Promoter) fragment was added upstream of the hydA gene (SEQ ID NO: 6), and a terminator fragment was added downstream of the hydA gene. (SEQ ID NO: 7), a deoxyribonucleic acid fragment as shown in SEQ ID NO: 2 was obtained, wherein the 5' end has an Xba I cleavage site and the 3' end has a Sac I cleavage site. In this example, the sequence of the promoter and terminator is derived from the pckA gene of R. palustris P4.

The DNA fragment of SEQ ID NO: 1 is further utilized with a pMG105 shuttle vector (Shuttle vector; Research Institute of Innovative Technology for the Earth, Kyoto, Japan; SEQ ID NO: 8), which is cleaved by the same restriction enzyme Xba I/ Sac I. T ₄ ligase (T ₄ DNA ligase) was ligated at an appropriate ratio to obtain a constructed pMGPHT plastid (SEQ ID NO: 3).

The above plastid colonization operation may include, but is not limited to, the following steps.

In this example, the reactants and reagents of the polymerase chain reaction are carried out in the following ratios: 1 μg of template DNA, 2 μL of dNTP solution (1.25 mM), 2.5 μL of 10 times concentrated polymerase buffer (10X-Taq DNA polymerase buffer), 10 pmole primer pair and 2.5 unit of Taq-polymerase (Taq DNA polymerase), adjusted to a total volume of 25 μL in sterile deionized water, placed in a polymerase chain reaction reactor (GeneAmp PCR system 2400; Perkin Elmer) was subjected to a polymerase chain reaction. The reaction conditions were 94 ° C for 5 minutes, 94 ° C for 1 minute (10 cycles; 10 cycles), 50 ° C for 1 minute, 72 ° C for 1 minute and 20 seconds, and 94 ° C for 1 minute (20 cycles; 20 cycles). The reaction was carried out at 60 ° C for 1 minute, 72 ° C for 1 minute and 20 seconds, and after the reaction was completed, the reaction was continued at 72 ° C for 10 minutes, and then the temperature was lowered to 4 ° C, and the results of the PCR were observed by agarose gel electrophoresis.

The PCR product recovery can be first separated by agarose gel electrophoresis. The colloid containing the target fragment is excised and recovered using methods or commercial kits commonly used in the art for recovering DNA. The recovered product can be subjected to subsequent shearing and joining reactions.

DNA molecular cleavage is performed according to the protocol provided by the restriction enzyme manufacturer. After appropriate DNA sample is mixed with sterile deionized water and restriction enzyme buffer, then add appropriate amount of restriction enzyme to mix evenly, and react at the optimum reaction temperature of the enzyme.

DNA joining reaction is carried cut in accordance with T ₄ ligase manufacturers to provide the process. The plastid DNA and the DNA fragment to be inserted are mixed in an appropriate ratio, mixed with the sterilized deionized water and the ligase buffer, and then mixed with an appropriate amount of ligase, and the reaction is terminated at the optimum reaction temperature of the enzyme for a while. It can be transformed.

Selection of photosynthetic hydrogen-producing strains containing plastid pMGPHT

R. palustris is a purple non-sulfur bacteria (PNSB) that produces hydrogen by nitrogenase. Purple sulfur-free bacteria are photosynthetic microorganisms with the best hydrogen production efficiency, and can produce hydrogen in both light and dark places.

The shuttle plastid pMGPHT was transformed into R. palustris electric competent cells by electric transformation, and colonies with antibiotic resistance of kanamycin were screened after culture. The selected transformed strain was named R. palustris strain pMGPHT.

The above-mentioned transformation plant selection operation may include, but is not limited to, the following steps. For example, in this example, R. palu stris was cultured in Rhodospirillaceae medium under an anaerobic environment at 32 ° C and provided with 50 W/m ² of light. Rhodospirillum medium contains K ₂ HPO ₄ (0.5 g/L), KH ₂ PO ₄ (0.5 g/L), MgSO ₄ ‧7H ₂ O (0.2 g/L), NaCl (0.4 g/L), CaCl ₂ ‧2H ₂ O (0.05 g/L), yeast extract (0.2 g/L), 1.0 g/L Iron citrate solution (5 mL/L) and trace element solution (1 mL). The trace element solution contains ZnCl ₂ (70 mg/L), MnCl ₂ ‧4H ₂ O (100 mg/L), H ₃ BO ₃ (60 mg/L), CoCl ₂ ‧6H ₂ O (200 mg/L), CuCl ₂ ‧2H ₂ O (20 mg/L), NiCl ₂ ‧6H ₂ O (20 mg/L), NaMoO ₄ ‧2H ₂ O (40 mg/L); 25% HCl (1 mg/L).

The above agent may be pre-formulated as a concentrate: (A) KH ₂ PO ₄ + K ₂ HPO ₄ (50 g / L: 50 g / L); (B) MgSO ₄ ‧7H ₂ O (20 g / L); (C) NaCl + CaCl ₂ ‧ 2H ₂ O (40 g / L: 5 g / L). When preparing 300 mL, first fill about 150 mL of deionized water in a triangular flask, then add concentrate (A) (B) (C) 3 mL, Fe-citrate solution 1.5 mL, trace element solution 0.3 mL, yeast extract (Yeast Extract) 0.06 g, glutamic acid (Glutamate) 0.12 g and CH ₃ COONa 0.3 g as a carbon source. The carbon source can be replaced with lactic acid (Lactate) as needed. The pH of the medium was adjusted to 7.1 using 0.1 M NaOH, and the top space of the flask was filled with helium to become an anaerobic state.

In the solid medium, 1.5% of bacterial agar (Bacto Agar; Difco) was added to the liquid medium, and after sterilization and dissolution in the sterilization pot, antibiotics were added as needed, and the culture dishes were placed (90×15 mm Petri dishes, Alpha). In plus), it can be used after it has been coagulated and dried. In this example, the preparation and electroporation transformation of R. palustris electrical competent cells was carried out in accordance with the method published by Donohue and Kaplan in 1991. R. palustris was cultured in the above culture solution for 8-24 hours, repeatedly diluted with sterile secondary water, centrifuged 7-8 times, and then 10% glycerol was added to prepare an electric competent cell. After mixing 40 μL of polyethylene glycol 6000 (PEG 6000) (50%), 20 μL of plastids and 40 μL of electric competent cells, the shock wave was shocked by 8.5 m/sec and placed on ice for 1 minute. (BIO-RAD GENE PULSER II), electrical transformation conditions are resistance 400Ω, 2.5kV, capacitance 25μF, electric field strength 12.5kV/cm. The transformed cells were cultured in a medium containing Kanamycin (200 μg/mL), and a transformed strain having antibiotic resistance was screened.

Fig. 2 is a result of gel electrophoresis after screening the transformed transformant R. palustris strain pMGPHT plastid and digesting with restriction enzyme Bgl II. Among them, the lane M is a molecular weight marker, and the lane 1 is the result of the extracted plastid by Bgl II. According to the pMGPHT restriction enzyme map shown in Fig. 1, after cleavage with Bgl II, a fragment of 5704 base pairs (bp), 1460 bp, and 471 bp was theoretically obtained. The gel electrophoresis results in Figure 2 showed fragments of about 5.7 kb, about 1.5 kb, and about 0.47 kb, which were consistent with the estimated fragment size and confirmed to be pMGPHT.

In addition, all colonies of the completed R. palustris strain pMGPHT retained kanamycin antibiotic resistance after the second passage, indicating that the plastid pMGPHT is stable in R. palustris . Figure 3 shows the results of detecting the hydA gene of pMGPHT in the host by reverse transcription polymerase chain reaction (RT-PCR).

The total RNA of the transformed strain R. palustris strain pMGPHT was extracted and extracted with the specificity of the hydA sequence PHT-45F(R) (SEQ ID NO: 9) in combination with PHT-45F(L) ( SEQ ID NO: 10) RT-PCR was performed to detect whether the selected hydA gene was successfully expressed in the host. M is the molecular weight marker (100 bp DNA Ladder), RT (+) is the experimental group (adding reverse transcriptase and reaction buffer, primer PHT-45F (R), total RNA), RT (-) is the control group (not Add reverse transcriptase). Shown by the results of FIG. 3, RT (+) is complementary to the experimental group an increase of success of the DNA hydA (Complementary DNA; cDNA) fragments, indeed represents hydA gene expression in the host.

Effect of exogenous [FeFe]-hydrogenase protein expression on enhancing host heat tolerance

Figure 4 is a comparison of the cell growth rates of the R. palustris strain pMGPHT transformed strain and the control group containing the empty vector (pMG105; no hydA gene) at different temperature conditions.

During the temperature test, the strains to be tested were cultured in 38 ° C, 40 ° C, 42 ° C, and 45 ° C water baths in an anaerobic sealed tube. The cell growth condition (concentration) was detected by a spectrophotometer. Each set of data is expressed as the mean and standard deviation of at least three independent experiments.

The results in Fig. 4 show that the R. palustris strain pMGPHT transformed strain has a significantly shorter cell doubling time than the control group containing only empty vector when cultured at about 40 °C.

Among them, as shown in the following Table 1, when cultured at 38 ° C, 40 ° C, 42 ° C and 45 ° C, R. palustris strain pMGPHT transformed strain can shorten the ratio of cell doubling time (control group / transformation strain) compared with the control group The values of 1.04, 1.46, 1.32 and 1.34, respectively, indicate that the R. palustris strain pMGPHT transformant does have the ability to adapt to higher temperature culture environments. Table 1. Cell growth rate of transformed strains and control groups under different temperature conditions

Figure 5 is a schematic diagram of the mechanism of exogenous [FeFe]-hydrogenase for enhancing the heat tolerance of photosynthetic hydrogen-producing bacteria.

Photosynthetic microorganisms use Nitrogenase to produce hydrogen to balance the reducing energy in cells. By consuming adenosine triphosphate (ATP), hydrogen protons are converted to hydrogen, which requires higher activation energy. Its chemical action is:

2H ⁺ +2e ^- +4ATP → H ₂ +4ADP+4Pi

Hydrogenase can decompose organic matter in an anaerobic fermentation environment, and some electrons generated by the process of metabolizing organic matter are generated by hydrogen transfer to hydrogen protons in water. Its chemical action is:

2H ⁺ +2e ^- → H ₂

As shown in the route (A) of Figure 5, when photosynthetic hydrogen-producing bacteria use hydrogenase to produce hydrogen, it is necessary to consume ATP to convert hydrogen protons into hydrogen. When the cells additionally express exogenous [FeFe]-hydrogenase, it is speculated to provide another energy-saving intracellular reducing energy balance pathway (B).

When the cells are cultured in a harsh environment, they may affect cell metabolism and slow down the growth rate. When the extracellular [FeFe]-hydrogenase is additionally expressed by the cells, hydrogen can be produced partially via the route (B), and the energy consumed by the route (A) is relatively saved, thereby saving the energy by the machine to promote the bacteria. Body growth helps to adapt to a higher temperature culture environment.

It can be seen from the above embodiments of the present invention that the present invention differs from the traditional meristem method in that a plurality of protein protectors are expressed to protect the host protein to enhance the heat tolerance of the microorganism, but only in the photosynthetic hydrogen producing bacteria. Heterologous expression of [FeFe]-hydrogenase can effectively improve the heat tolerance of photosynthetic hydrogen-producing bacteria, so that the photosynthetic hydrogen-producing bacteria originally adapted to growth under the environment of 28-35 °C can adapt to the temperature of about 40 °C. .

Therefore, the embodiment of the present invention solves the problem that the photosynthetic bacteria cannot adapt to the excessive temperature of the culture tank after the sunlight is irradiated. The invention can achieve the goal of using sunlight as a source of light and heat energy for photosynthetic bacteria to save the operating cost of the culture tank. Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be modified and modified without departing from the spirit and scope of the present invention. The scope is subject to the definition of the scope of the patent application attached.

<110>National Chung Hsing University

<120>Heat-tolerant photosynthetic hydrogen producing strain

<160>9

<210>SEQ ID NO: 1

<211>1739

<212>DNA

<213>Artificial sequence

<220>CDS

<223> hydA gene sequence

<400>2

<210>SEQ ID NO: 2

<211>1894

<212>DNA

<213>Artificial sequence

<220>CDS

<223> DNA fragment capable of transcribed [FeFe]-Hydrogenase

<400>2

<210>SEQ ID NO: 3

<211>7635

<212>DNA

<213>Artificial sequence

<220>cyclic nucleic acid sequence

<223> plastid pMGPHT sequence which expresses HydA protein in a photosynthetic bacteria host

<400>3

<210>SEQ ID NO: 4

<211>28

<212>DNA

<213>Artificial sequence

<220>primer

<223> Increment of the hydA gene (HydA-F)

<400>4

<210>SEQ ID NO: 5

<211>28

<212>DNA

<213>Artificial sequence

<220>primer

<223> Increment of the hydA gene (HydA-R)

<400>5

<210>SEQ ID NO: 6

<211>124

<212>DNA

<213>Artificial sequence

<220>promoter

<223> The pckA gene derived from Rhodopseudomonas palustris P4

<400>6

<210>SEQ ID NO: 7

<211>52

<212>DNA

<213>Artificial sequence

<220>terminater

<223> The pckA gene derived from Rhodopseudomonas palustris P4

<400>7

<210>SEQ ID NO: 8

<211>7635

<212>DNA

<213>Artificial sequence

<220>cyclic nucleic acid sequence

<223> Shuttle carrier pMG105

<400>8

<210>SEQ ID NO: 9

<211>27

<212>DNA

<213>Artificial sequence

<220>primer

<223>Introduction to the hydA sequence PHT-45F(R)

<400>

<210>SEQ ID No: 10

<211>28

<212>DNA

<213>Artificial sequence

<220>primer

<223>Specific primer for the hydA sequence PHT-45F(L)

<400>

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

The first figure is a flow chart of the construction of the plastid pMGPHT.

Fig. 2 Transformation strain R. palustris strain pMGPHT plastids were confirmed by gel electrophoresis after restriction enzyme cleavage.

Figure 3 is a graph showing the results of the detection of the fragment of SEQ ID NO: 1 of pMGPHT in a host by a reverse transcription polymerase chain reaction.

Figure 4 is a comparison of cell growth rates of R. palustris strain pMGPHT transformed strains and control groups at different temperature conditions.

Figure 5 is a schematic diagram of the mechanism of exogenous [FeFe]-hydrogenase for enhancing the heat tolerance of photosynthetic hydrogen-producing bacteria.

Claims (11)

A method for enhancing the heat tolerance of photosynthetic hydrogen producing bacteria Rhodopseudomonas palustris , comprising: transforming a photosynthetic hydrogen producing bacterium Rhodopseudomonas palustris with a vector comprising a SEQ ID NO: 1 deoxyribonucleic acid sequence, thereby making the photosynthetic hydrogen producing bacterium available One of the transformed bacteria is grown in a culture environment of 40 ° C to 45 ° C, wherein the DNA of the sequence number: 1 can encode a [FeFe]-hydrogenase protein product. A method for enhancing the heat tolerance of the photosynthetic hydrogen producing bacteria Rhodopseudomonas palustris according to claim 1, wherein the DNA sequence comprising SEQ ID NO: 1 is the sequence of SEQ ID NO: 2. A method for enhancing the heat tolerance of the photosynthetic hydrogen producing Rhodopseudomonas palustris according to claim 1, wherein the DNA sequence comprising SEQ ID NO: 1 is the sequence of SEQ ID NO: 3. A method for enhancing the heat tolerance of the photosynthetic hydrogen producing bacteria Rhodopseudomonas palustris according to claim 1, wherein the promoter sequence of the SEQ ID NO: 1 upstream comprises a promoter region. A method for enhancing the heat tolerance of the photosynthetic hydrogen producing bacteria Rhodopseudomonas palustris according to claim 4, wherein the promoter region is an inducible promoter region. A method for enhancing the heat tolerance of the photosynthetic hydrogen producing bacteria Rhodopseudomonas palustris according to claim 1, wherein the transformed bacteria is deposited in the Food Industry Development Institute (FIRDI), number BCRC 910506. As the lifting of a requested item of Rhodopseudomonas palustris tolerance to heat the hydrogen producing photosynthetic bacterium, wherein the chromosomal deoxyribonucleic acid sequence is embedded in the hydrogen producing photosynthetic bacteria. A method for enhancing the heat tolerance of the photosynthetic hydrogen producing Rhodopseudomonas palustris according to claim 1, wherein the DNA sequence comprising SEQ ID NO: 1 is derived from a chromosomal DNA of a strain which is viable above 40-70 ° C. . A method for enhancing the heat tolerance of the photosynthetic hydrogen producing bacteria Rhodopseudomonas palustris as claimed in claim 8 wherein the strain is a Clostridium genus. A method for enhancing the heat tolerance of the photosynthetic hydrogen producing bacteria Rhodopseudomonas palustris as claimed in claim 8, wherein the strain is Clostridium thermocellum . A method for enhancing the heat tolerance of the photosynthetic hydrogen producing Rhodopseudomonas palustris according to claim 1, wherein the DNA sequence comprising SEQ ID NO: 1 can be artificially synthesized.