Recombinant Shewanella strain synthesizing poly 3-hydroxybutyrate using formic acid and its construction method and application

Abstract

The invention discloses a recombinant Shewanella strain for synthesizing poly 3-hydroxybutyrate using formic acid, and a construction method and application thereof, comprising the following steps: introducing a gene FTL containing a Saccharomyces cerevisiae S288C source into a wild-type Shewanella halifaxensis through an FTLpHG12-pBAD plasmid to obtain a recombinant strain Sh01 for utilizing formic acid. In Sh01, poly 3-hydroxybutyrate synthesis genes phaA, phaB1, and phaC1 from Ralstonia eutropha H16 are introduced through a plasmid phaABC-FtL-pHG12-pBAD to construct a Shewanella strain Sh02 for synthesizing poly 3-hydroxybutyrate (PHB) using formic acid. The method for synthesizing poly 3-hydroxybutyrate using formic acid and seawater fermentation by microorganisms is simple to operate, and poly 3-hydroxybutyrate with high yield can be obtained at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and more particularly to a recombinant Shewanella halifaxensis strain for synthesizing poly 3-hydroxybutyrate using formic acid and a construction method thereof. Background Art

[0002] Poly (3-hydroxybutyrate) (PHB) is a bioplastic synthesized by microorganisms under nutrient-restricted conditions. It is biodegradable and compatible, and therefore has great development potential in replacing petrochemical-derived plastics and has broad application prospects in the medical field, packaging industry, nanotechnology, and agriculture.

[0003] At present, the commercial application of PHB is mainly limited by its high production cost. The main reason is that expensive sugars and other substances need to be used as substrates to produce PHB, as well as the electricity cost required for steam sterilization and sterility to prevent bacterial contamination. Formic acid is a cheap and readily available chemical that can be obtained by photocatalysis or electrochemical catalysis of carbon dioxide. Therefore, using formic acid as a substrate to produce PHB is a feasible solution to reduce costs. At the same time, Shewanella halifaxensis is a marine strain, a Gram-negative bacterium, and a facultative anaerobic microorganism. Using marine bacteria for fermentation can reduce bacterial contamination, reduce fermentation costs, and utilize marine resources. Summary of the invention

[0004] The invention aims to provide a marine bacterium Shewanella halifaxensis strain which utilizes formic acid to synthesize poly 3-hydroxybutyrate.

[0005] The second object of the present invention is to provide a method for constructing a Shewanella halifaxensis strain that synthesizes poly 3-hydroxybutyrate using formic acid.

[0006] The third object of the present invention is the use of the recombinant Shewanella halifaxensis strain to produce poly 3-hydroxybutyrate using formic acid.

[0007] The technical solution of the present invention is summarized as follows:

[0008] The method for constructing a Shewanella halifaxensis strain for synthesizing poly 3-hydroxybutyrate comprises the following steps:

[0009] (1) The formate-tetrahydrofolate ligase (FTL) gene from Saccharomyces cerevisiae S288C was introduced into Shewanella halifaxensis via the FTL-pHG12-pBAD plasmid to obtain the recombinant strain Sh01 that can utilize formic acid;

[0010] (2) The poly 3-hydroxybutyrate synthesis genes phaA, phaB1, and phaC1 derived from Ralstonia eutropha H16 were introduced into the recombinant strain Sh01 via the plasmid phaABC-FtL-pHG12-pBAD to construct the Shewanella halifaxensis recombinant strain Sh02 that can synthesize poly 3-hydroxybutyrate using formic acid;

[0011] The nucleotide sequence of the FTL gene is shown in SEQ ID NO.1;

[0012] The nucleotide sequence of the phaA gene is shown in SEQ ID NO.2;

[0013] The nucleotide sequence of the phaB1 gene is shown in SEQ ID NO.3;

[0014] The nucleotide sequence of the phaC1 gene is shown in SEQ ID NO.4.

[0015] The poly 3-hydroxybutyrate-synthesizing Shewanella halifaxensis recombinant strain Sh02 constructed by the above method was obtained.

[0016] The use of the recombinant strain Sh02 in producing poly 3-hydroxybutyrate includes the following steps: culturing and fermenting the poly 3-hydroxybutyrate synthesizing Shewanella strain Sh02 in a fermentation medium to obtain poly 3-hydroxybutyrate, wherein the fermentation medium is formulated as 40 g / L sea salt, 20 g / L sodium formate, and the rest is water.

[0017] Beneficial effects of the present invention:

[0018] The invention discloses a Shewanella halifaxensis strain for synthesizing poly 3-hydroxybutyrate using formic acid, which can synthesize poly 3-hydroxybutyrate using formic acid as a carbon source and seawater as a culture medium. The method for synthesizing poly 3-hydroxybutyrate using formic acid and seawater fermentation by microorganisms is simple to operate, and can obtain poly 3-hydroxybutyrate with high yield. Therefore, using Shewanella halifaxensis bacteria to produce PHB using formic acid is a cheap, readily available and environmentally friendly method. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The poly 3-hydroxybutyrate synthesis pathway in the recombinant Shewanella halifaxensis strain;

[0020] Figure 2 The yield-time graph of Shewanella halifaxensis strain Sh02 for the synthesis of poly 3-hydroxybutyrate;

[0021] Figure 3 This is a comparison chart of the production of poly 3-hydroxybutyrate synthesized by Shewanella halifaxensis strains Sh01 and Sh02. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0023] Example 1

[0024] 1. Strain construction

[0025] The construction of a fixed formic acid-producing PHB Shewanella halifaxensis strain includes the following steps: introducing the FTL enzyme to open up the reductive acetyl-CoA pathway that can assimilate formic acid in the bacteria, splicing the Ftl gene (derived from Saccharomyces cerevisiae S288C) with the pHG12-pBAD plasmid to obtain a recombinant plasmid FTL-pHG12-pBAD plasmid, and introducing it into the original Shewanella strain to assimilate formic acid to obtain the recombinant strain Sh01.

[0026] The genes phaA, phaB1, phaC1 and FTL from Ralstonia eutropha H16 were spliced ​​with the pHG12-pBAD plasmid to obtain the phaABC-FtL-pHG12-pBAD plasmid, which was introduced into the wild-type strain to obtain the recombinant strain Sh02 for producing PHB.

[0027] The nucleotide sequence of the Ftl gene is shown in SEQ ID NO.1; the nucleotide sequence of the phaA gene is shown in SEQ ID NO.2; the nucleotide sequence of the phaB1 gene is shown in SEQ ID NO.3; the nucleotide sequence of the phaC1 gene is shown in SEQ ID NO.4;

[0028] The specific steps are:

[0029] (1) Construction of FTL-pHG12-pBAD plasmid and phaABC-FtL-pHG12-pBAD plasmid.

[0030] The gene Ftl from Saccharomyces cerevisiae S288C and the pHG12-pBAD vector sequence were obtained from the NCBI database. The complete sequence of the FTL-pHG12-pBAD plasmid was synthesized by codon optimization DNA. The genes phaA, phaB1, phaC1 and FTL gene from Ralstonia eutropha H16 were obtained from the NCBI database and spliced ​​with the pHG12-pBAD vector to obtain the phaABC-FtL-pHG12-pBAD plasmid sequence by DNA synthesis.

[0031] (2) Transform the plasmid into E. coli S17-1

[0032] All operations in this experiment need to be carried out in a low temperature environment

[0033] Take out the E. coli competent cells from the -80℃ freezer, and immediately put them in an ice box to slowly thaw, then add the target plasmid (take recombinant plasmid 1 as an example. The operation of recombinant plasmid 2 is the same and will not be repeated here) into the competent cells, and gently tap the tube wall several times with your fingers to mix the various ingredients, then put them in an ice box to chill for 30 minutes, and adjust the water bath to 42℃ for use.

[0034] Put the EP tube on the float, heat it in a 42℃ water bath for 90 seconds, then take it out and put it in an ice box to chill for 5 minutes.

[0035] After the ice bath, add 1 ml of antibiotic-free LB medium to the tube, pipette gently to mix, clamp the EP tube on the float again, and then fix it in the shaker and culture it at 37°C and 220 rpm for 1 hour.

[0036] The culture medium was taken out from the shaker, and 100-150 μl was evenly spread on the LB solid plate (Kan50 resistance) according to the concentration of the bacterial solution, and then inverted and cultured overnight in a constant temperature incubator at 37°C.

[0037] LB medium ingredients: 10g / L sodium chloride, 5g / L yeast extract, 10g / peptone (for solid culture medium, add 15g / L agar powder).

[0038] (3) Combine with E. coli S17-1 to introduce the plasmid into the wild-type strain.

[0039] Take out the E. coli glycerol bacteria introduced with the target plasmid from the -80℃ refrigerator, melt it on ice, aspirate 10 μl of the glycerol bacteria solution, and transfer it into 3 mL of LB liquid culture medium, and then shake it in an incubator at 37℃ and 220 rpm for 10 hours; in addition, inoculate 10 μl of Shewanella halifaxensis glycerol bacteria into 3 mL of non-resistant LB culture medium and culture it in an incubator at 30℃ and 200 rpm for 10 hours.

[0040] Use a pipette to transfer 500 μl of each of the two bacterial solutions into a sterile EP tube, centrifuge at 500 rpm for 5 minutes, remove the supernatant in an ultra-clean bench, resuspend the cells with 1 mL of LB culture medium, and statically culture them in a 30°C incubator for 1 hour.

[0041] Take 100 μl of the cultured bacterial solution and spread it on the corresponding LB solid plate, and culture it at 30°C until the bacteria grow into single colonies.

[0042] This operation was repeated to obtain recombinant strains Sh01 and Sh02.

[0043] 2. Fermentation method

[0044] Liquid culture medium: 16 g / L bacterial peptone, 4 g / L yeast extract, 40 g / L sea salt, and the rest is water.

[0045] Fermentation medium: 40g / L sea salt, 20g / L sodium formate, and the rest water.

[0046] Methods: The recombinant strains Sh01 and Sh02 were inoculated into 15 mL of liquid culture medium at 10°C and 150 rpm, respectively, and cultured for 24 h. The bacterial solution was then transferred to 30 mL of fresh fermentation medium and the bacterial density was adjusted to a final bacterial density of OD 600 The temperature was 2, 10°C, 150 rpm, and the culture was carried out for 120 h. The culture medium was taken for product analysis.

[0047] 3. Product analysis

[0048] PHB extraction and analysis: 2mL culture medium of Sh01 and Sh02 was centrifuged at 4℃8000rpm / min for 10 minutes to obtain cell pellets, and washed twice with double distilled water (ddH2O) to remove residual culture medium. The bacteria were placed in a vacuum freeze dryer, freeze-dried, and drained. The centrifuge tube with bacteria was weighed to calculate the dry weight of the bacteria. The bacteria were then transferred to a clean vial, and the mass of the bacteria in the vial was weighed. 500μL of esterification solution and 500μL of chloroform were quickly added to the vial, and the cap was quickly pressed and sealed. Then it was placed in ultrasonic vibration to fully disperse the bacteria, and then sealed with glue. The bottle was placed in an oven and heated at 90℃ for 4h. During this period, it can be shaken appropriately to fully lyse PHB. The vial was cooled to room temperature, the liquid was transferred to a 2mL centrifuge tube, 250μL of distilled water was added, mixed and kept away from light for 2h, and the organic phase was separated. Use a syringe to draw 200 μL of the lower layer liquid and add it to the gas phase vial to prepare for sample loading. The gas phase chromatographic column used is a RB-lonWax capillary column, and the solvent is acetone. Set the GC parameters according to the operating instructions (AuxI temperature 250°C, column temperature 250°C, split ratio 8:1, Det temperature 250°C, injection volume 1 μL, column pre-pressure 0.06 MPa, detection time 27 min)

[0049] 4. Results

[0050] In this study, the essential gene (Ftl) and plasmid were first synthesized into recombinant plasmid 1 and integrated into the starting strain Shewanella halifaxensis to obtain strain Sh01. However, no PHB was detected. In order to increase the yield of PHB, we synthesized recombinant plasmid 2 by using phaABC and Ftl genes and vectors to obtain recombinant strain Sh02. The recombinant strain Sh02 was cultured in culture medium and finally obtained 1.1 g / L PHB (see Figure 2 ), the recombinant strain Sh02 has a much greater ability to produce PHB than the Sh01 strain (see Figure 3 ).

Claims

1. A method for constructing a Shewanella halifaxensis strain for synthesizing poly 3-hydroxybutyrate, characterized in that: The steps include: (1) The formate-tetrahydrofolateligase (FTL) gene from Saccharomyces cerevisiae S288C was introduced into Shewanella halifaxensis via the FTL-pHG12-pBAD plasmid to obtain the recombinant strain Sh01 that can utilize formic acid; (2) The poly 3-hydroxybutyrate synthesis genes phaA, phaB1, and phaC1 derived from Ralstonia eutropha H16 were introduced into the recombinant strain Sh01 via the plasmid phaABC-FtL-pHG12-pBAD to construct the Shewanella halifaxensis recombinant strain Sh02 that can synthesize poly 3-hydroxybutyrate using formic acid; The nucleotide sequence of the FTL gene is shown in SEQ ID NO.1; The nucleotide sequence of the phaA gene is shown in SEQ ID NO.2; The nucleotide sequence of the phaB1 gene is shown in SEQ ID NO.3; The nucleotide sequence of the phaC1 gene is shown in SEQ ID NO.

4.

2. The synthetic poly 3-hydroxybutyrate Shewanella halifaxensis recombinant strain Sh02 constructed by the method of claim 1.

3. Use of the recombinant strain of claim 2 in producing poly 3-hydroxybutyrate.

4. The use according to claim 3, characterized in that: The method comprises the following steps: culturing and fermenting the poly 3-hydroxybutyrate synthesizing Shewanella strain Sh02 of claim 2 in a fermentation medium to obtain poly 3-hydroxybutyrate, wherein the fermentation medium formula is 40 g / L sea salt, 20 g / L sodium formate, and the rest is water.

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