Recombinant <i>Escherichia coli </i>expressing fusion protein of formamidase and phosphite dehydrogenase and construction method and use thereof

Abstract

The invention relates to a recombinant Escherichia coli expressing a fusion protein of formamidase and phosphite dehydrogenase, a construction method and use thereof. The invention includes adopting engineered E. coli DH5α as a host, amplifying a cloned formamidase gene and a cloned phosphite dehydrogenase gene into a fusion gene, ligating the fusion gene to a multiple cloning site of a vector, transforming the obtained recombinant plasmid into the E. coli DH5α, extracting the plasmid and transforming into an expression strain, and performing induction culture to obtain a recombinant E. coli. The recombinant E. coli can express a fusion protein of formamidase and phosphite dehydrogenase.

Claims

1. A method for constructing a recombinant Escherichia coli expression stain which expresses a fusion of a formamidase and a phosphite dehydrogenase, comprising the following steps: (1) amplifying a formamidase gene fragment with a linker sequence by polymerase chain reaction using a first pair of primers and a genome of Paenibacillus pasadenensis CS0611 as a template to clone a formamidase gene with a linker sequence (for-Linker) of 1041 bp, wherein the first pair of primers comprises a forward primer (primer A1) and a reverse (primer A2), wherein the nucleotide sequence of primer A1 is SEQ ID NO: 4 and the nucleotide sequence of primer A2 is SEQ ID NO: 5, wherein the Paenibacillus pasadenensis CS0611 has China Center for Type Culture Collection (CCTCC) deposit number M 2014458; (2) ligating the for-Linker into a vector to obtain a recombinant vector comprising the for-Linker (vector-for) and transforming E. coli DH5α competent cells with the recombinant vector for to obtain a recombinant E. coli DH5α containing the recombinant vector-for; (3) amplifying the for-Linker in the recombinant vector-for by PCR using a second pair of primers and the vector for as a template, wherein the second pair of primers comprises the forward primer A1 and a reverse primer (primer B1), wherein the nucleotide sequence of primer B1 is SEQ ID NO: 6; (4) amplifying a phosphite dehydrogenase gene fragment by PCR using a third pair of primers and a genome of Klebsiella pneumoniae OU7 as a template to clone a phosphite dehydrogenase gene (ptx) of 1008 bp, wherein the third pair of primers comprises a forward primer (primer B2) and a reverse primer (primer B3), wherein the nucleotide sequence of primer B2 is SEQ ID NO: 7 and the nucleotide sequence of primer B3 is SEQ ID NO: 8, wherein the Klebsiella pneumoniae OU7 has CCTCC deposit number M 2017449; (5) generating a fusion gene (for-Linker-ptx) by overlapping PCR amplification using a fourth pair of primers and the amplified for-Linker of step (3) and the amplified ptx of step (4) as templates, wherein the fourth pair of primers comprises the forward primer A1 and the reverse primer B3; (6) digesting the for-Linker-ptx fusion gene with BamHI and EcoRI and ligating the digested for-Linker-ptx fusion gene into a pGEX-2T expression plasmid digested with BamHI and EcoRI to obtain a recombinant plasmid comprising the for-Linker-ptx fusion gene (pGEX-for-Linker-ptx), and transforming E. coli DH5α competent cells with the recombinant plasmid pGEX-for-Linker-ptx to obtain a recombinant E. coli DH5α comprising the recombinant plasmid pGEX-for-Linker-ptx; (7) extracting the recombinant plasmid pGEX-for-Linker-ptx from the recombinant E. coli DH5α comprising the recombinant plasmid pGEX-for-Linker-ptx and transforming E. coli BL21(DE3) competent cells with the recombinant plasmid pGEX-for-Linker-ptx to obtain a recombinant E. coli expression strain; and (8) inoculating a LB medium with the recombinant E. coli expression strain, inducing expression of the for-Linker-ptx fusion gene, collecting the recombinant E. coli expression strain, adding the recombinant E. coli expression strain to a MOPS medium comprising formamide and phosphite, and culturing the recombinant E. coli expression strain in the MOPS medium comprising formamide and phosphite.

2. The method of claim 1, wherein in step (3), the amplified for-Linker comprises the nucleotide sequence of SEQ ID NO: 1.

3. The method of claim 1, wherein in step (4), the amplified ptx comprises the nucleotide sequence of SEQ ID NO: 2.

4. The method of claim 1, wherein in step (5), the for-Linker-ptx fusion gene comprises the nucleotide sequence of SEQ ID NO: 3.

5. The method of claim 1, wherein in steps (1), (3) and (4), conditions for the PCR amplification comprise reacting at 94° C. for 5 minutes; reacting at 98° C. for 10 seconds, reacting at 55° C. for 5 seconds and reacting at 72° C. for 70 seconds, and repeating reactions for 30 times; then reacting at 72° C. for 7 minutes; and finally, cooling to 16° C.

6. The method of claim 1, wherein in step (8), inducing expression of the for-Linker-ptx fusion gene comprises culturing in a LB medium at 37° C. and 180 rpm for 12 hours to 16 hours, then inoculating in a fresh LB medium, and continuing to culture at 37° C. and 180 rpm until OD.sub.600 of the recombinant E. coli expression stain in the culture reaches 0.6, and after cooling to 20° C., adding isopropyl-3-D-thiogalactoside (IPTG) with a final concentration of 0.2 mM for induction for 16 hours.

7. The method of claim 1, wherein in step (8), collecting the recombinant E. coli expression strain comprises centrifuging the recombinant E. coli expression strain at 4° C. and 8000 rpm for 5 minutes, suspending the recombinant E. coli expression strain with physiological saline precooled to 4° C., centrifuging again at 4° C. and 8000 rpm for 5 minutes, repeating the suspending with physiological saline and centrifuging twice, and then collecting the recombinant E. coli expression strain; and wherein in the MOPS medium comprising formamide and phosphite, a final concentration of the formamide is 200 mM and a final concentration of the phosphite is 1.32 mM.

8. The method of claim 1, wherein in step (8), after adding the collected recombinant E. coli expression strain into the MOPS medium comprising formamide and phosphite, the OD.sub.600 of the recombinant E. coli expression strain in the medium is 0.1 to 0.15, inducing expression of the for-Linker-ptx fusion gene with IPTG at a final concentration of 0.2 mM, and culturing the recombinant E. coli expression strain in the MOPS medium containing formamide and phosphite at 30° C. and 180 rpm for 84 hours to 96 hours.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram illustrating a construction process of a recombinant fusion expression plasmid pGEX-for-Linker-ptx;

(2) FIG. 2 is a growth curve graph of a recombinant Escherichia coli BL21 (DE3)(pGEX-for-Linker-ptx) in a MOPS medium;

(3) FIG. 3 is a photofluorogram of the recombinant Escherichia coli BL21 (DE3)(pGEX-for-Linker-ptx) expressing a green fluorescent protein (GFP) in the MOPS medium.

DESCRIPTION OF THE EMBODIMENTS

(4) In order to better understand the present invention, the technical solution of the present invention is further described hereinafter with reference to the embodiments and the accompanying drawings, but the description is only used for illustrating the present invention, and shall not and does not limit the present invention.

(5) Paenibacillus pasadenensis. CS0611 used in the specific embodiments of the present invention was preserved in China Center for Type Culture Collection on Oct. 8, 2014 with a preservation number of CCTCC NO: M2014458, and a whole genome sequencing has been completed. A fragment of a formamidase gene containing a linker sequence obtained by amplification is shown in SEQ ID NO: 1, which has a fragment length of 1041 bp, contains the linker sequence, and encodes 347 amino acids.

(6) Klebsiella pneumonia. OU7 used in the specific embodiments of the present invention was preserved in China Center for Type Culture Collection (Wuhan University, Luojia Mountain, Wuchang Road, Wuhan City, Hubei Province, postcode: 430072) on Aug. 24, 2017 with a preservation number of CCTCC NO: M 2017449, and the Klebsiella pneumonia. OU7 is obtained by culturing and self-screening by the following method. Through the sequence analysis of phosphite dehydrogenases on the NCBI database and PCR amplification, a fragment of a phosphite dehydrogenase gene is shown in SEQ ID NO: 2, which has a fragment length of 1008 bp and encodes 336 amino acids.

Embodiment 1

(7) Acquisition of a Formamidase Gene for (Containing a Linker Sequence)

(8) Paenibacillus pasadenensis. CS0611 was cultured in a LB medium at 37° C. and 180 rpm for one day; cultured bacteria were centrifuged at 4° C. and 8000 rpm for 5 minutes to collect the bacteria, the bacteria were washed twice with physiological saline to remove residual medium, and then a genome of the Paenibacillus pasadenensis. CS0611 was extracted according to a specific method of an OMEGA bacterial genome DNA extraction kit.

(9) The extracted genome of the Paenibacillus pasadenensis. CS0611 was used as a template, A1 (5′-CGCGGATCCGATGAACGGACTGGGCGGCTTGAAC-3′) of SEQ ID NO: 4 and A2 (5′-CGACCCACCACCGCCCGAGCCACCGCCACCTCGCGCCGCGCCTCCCTTCGC-3′) of SEQ ID NO: 5 were respectively used as a forward primer and a reverse primer, and a formamidase gene for-Linker was amplified by PCR.

(10) An enzyme reagent used for PCR was PrimeSTAR® HS DNA Polymerase with GC buffer from TaKaRa Company; and a PCR reaction system and conditions were as follows:

(11) Composition of PCR reaction liquid (25 μL)

(12) TABLE-US-00001 Volume 2 × Prime STAR buffer 12.5 μL dNTP mixture 2μ A1 primer 0.5 μL A2 primer 0.5 μL Template 1 μL PrimeSTAR HS DNA polymerase (2.5 U/μL) 0.25 μL Deionized water 8.25 μL

(13) PCR reaction conditions were as follows: reacting at 94° C. for 5 minutes; reacting at 98° C. for 10 seconds, reacting at 55° C. for 5 seconds and reacting at 72° C. for 70 seconds in sequence, and repeating the reactions for 30 times; then reacting at 72° C. for 7 minutes; and finally, cooling to 16° C.

(14) A DNA product obtained by the PCR amplification was subjected to electrophoresis with 1 wt % agarose gel, a gel extraction kit from the OMEGA Company was used to perform gel extraction purification according to steps in the instruction, then the DNA product was sent for sequencing, and the result showed that a formamidase gene sequence containing a linker sequence with a fragment length of 1041 bp was obtained, and was named as for-Linker.

Embodiment 2

(15) Acquisition of a Phosphite Dehydrogenase Gene Ptx

(16) A phosphite dehydrogenase gene was derived from self-screened Klebsiella pneumonia. OU7, and was screened by our laboratory.

(17) A genome of the screened Klebsiella pneumonia. OU7 was extracted according to a specific method of an OMEGA bacterial genome DNA extraction kit, the genome of the screened Klebsiella pneumonia. OU7 was used as a template, B2 (5′-TGGCTCGGGCGGTGGTGGGTCGATGCTGCCGAAACTCGTTATA-3′) of SEQ ID NO: 7 and B3 (5′-CCGGAATTCCGACATGCGGCAGGCTCGGCCTTGGGC-3′) of SEQ ID NO: 8 were respectively used as a forward primer and a reverse primer, and a phosphite dehydrogenase gene ptx was amplified by PCR.

(18) An enzyme reagent used for PCR was PrimeSTAR® HS DNA Polymerase with GC buffer from TaKaRa Company; and a PCR reaction system and conditions were as follows:

(19) Composition of PCR reaction liquid (25 μL)

(20) TABLE-US-00002 Volume 2 × Prime STAR buffer 12.5 μL dNTP substrate 2 μL B2 primer 0.5 μL B3 primer 0.5 μL Template 1 μL PrimeSTAR HS DNA polymerase (2.5 U/μL) 0.25 μL Deionized water 8.25 μL

(21) PCR reaction conditions were as follows: reacting at 94° C. for 5 minutes; reacting at 98° C. for 10 seconds, reacting at 55° C. for 5 seconds and reacting at 72° C. for 70 seconds in sequence, and repeating the reactions for 30 times; then reacting at 72° C. for 7 minutes; and finally, cooling to 16° C.

(22) A DNA product obtained by the PCR amplification was subjected to electrophoresis with 1 wt % agarose gel, a gel extraction kit from the OMEGA Company was used to perform gel extraction purification according to steps in the instruction, then the DNA product was sent for detection and sequencing, and the result showed that a phosphite dehydrogenase gene sequence with a fragment length of 1008 bp was obtained, and was named as ptx.

Embodiment 3

(23) Acquisition of a Fusion Gene for-Linker-Ptx by Overlapping PCR Amplification

(24) The for-Linker and ptx fragments obtained by amplification were used as templates, A1 (5′-CGCGGATCCGATGAACGGACTGGGCGGCTTGAAC-3′) of SEQ ID NO: 4 and B3 (5′-CCGGAATTCCGACATGCGGCAGGCTCGGCCTTGGGC-3′) of SEQ ID NO: 8 were respectively used as a forward primer and a reverse primer, and a fusion gene for-Linker-ptx was obtained by amplification.

(25) Amplification conditions were as follows: reacting at 94° C. for 5 minutes; reacting at 98° C. for 10 seconds, reacting at 55° C. for 5 seconds and reacting at 72° C. for 70 seconds in sequence, and repeating the reactions for 30 times; then reacting at 72° C. for 7 minutes; and finally, cooling to 16° C.

Embodiment 4

(26) Construction of a Recombinant E. coli BL21(DE3) (pGEX-for-Linker-Ptx)

(27) Double digestion was performed on the fusion gene for-Linker-ptx obtained in the embodiment 3 and a plasmid pGEX-2T respectively with BamH I and EcoR I, an underlined and italic part of a forward primer was a restriction enzyme cutting site of BamH I, an underlined and italic part of a reverse primer was a restriction enzyme cutting site of EcoR I, and digestion conditions were as follows: digesting at 37° C. for 120 minutes;

(28) Digestion system:

(29) TABLE-US-00003 Fusion gene (μL) Plasmid (μL) ddH.sub.2O 5 0 10 × buffer 3 2 Fragment/plasmid 20 16 BamH I + EcoR I 1 + 1 1 + 1 Total volume 30 20

(30) Gel extraction purification was performed on a digested product respectively, and a extraction method and steps referred to a gel extraction kit from the OMEGA Company; the digested product was subjected to electrophoresis with 1 wt % agarose gel after extraction, and a extraction rate was detected; and then a extracted target fragment was ligated to a plasmid, T4 DNA Ligase from Thermo Fisher SCIENTIFIC Company was used as a ligation kit, and a ligation system was as follows:

(31) TABLE-US-00004 DNA fragment 6 μL Plasmid 9 μL 10 × T4 buffer 2 μL T4 DNA ligase 1 Weiss U Deionized water 2 μL Total volume 20 μL

(32) A molar ratio of the fusion gene to the pGEX-2T plasmid was 5:1.

(33) A ligation product was transformed into an E. coli DH5α, and transformation steps were as follows: 10 μL of the ligation product was mixed with 100 μL of competent cells of the E. coli DH5α, the mixture was placed into ice bath for 30 minutes, heat shock at 42° C. for 90 seconds, and then ice bath for 2 minutes, then 890 μL of LB medium was added, and after shaking culture at 37° C. and 180 rpm for 1 hour, the mixture was centrifuged at 4000 rpm for 5 minutes to collect bacteria; 890 μL of supernatant medium was taken, the bacteria at a bottom of a tube were resuspended, evenly coated on a LB solid plate containing ampicillin (containing 100 μg/mL ampicillin sodium), and cultured at 37° C. for 16 hours; and after transformants grew on the plate, the transformants were selected for PCR verification and sent for detection and sequencing, and ORF search was performed on the sequencing result using DNAssist software.

(34) The result shows that the obtained fusion gene sequence (for-Linker-ptx) has been correctly inserted into a multiple cloning site of pGEX-2T, and the pGEX-for-Linker-ptx plasmid has been successfully obtained and transformed into E. coli BL21(DE3) competent cell to obtain a recombinant E. coli BL21(DE3)(pGEX-for-Linker-ptx).

(35) A construction process of the recombinant E. coli BL21(DE3)(pGEX-for-Linker-ptx) assimilating and metabolizing formamide and phosphite to become dominant engineered bacteria in a medium is shown in FIG. 1. Due to insertion of a fusion gene (a formamidase gene+Linker+a phosphite dehydrogenase gene) into the E. coli BL21(DE3)(pGEX-for-Linker-ptx), the modified E. coli BL21(DE3)(pGEX-for-Linker-ptx) not only can decompose formamide into NH.sub.4.sup.+ but also can oxidize phosphite to phosphate, thus providing a nitrogen source and a phosphorus source for the growth of the E. coli BL21(DE3)(pGEX-for-Linker-ptx), while other miscellaneous microorganisms cannot grow due to the lack of the two pathways.

Embodiment 5

(36) Growth of an E. coli BL21(DE3)(pGEX-for-Linker-Ptx) in a Specific MOPS Medium

(37) Induction culture was performed on the obtained recombinant expression strain E. coli BL21(DE3)(pGEX-for-Linker-ptx), and a specific process was as follows:

(38) E. coli BL21(DE3)(pGEX-for-Linker-ptx) was inoculated to 30 mL of LB medium according to a volume ratio of 1:100, and cultured at 37° C. and 180 rpm overnight for 16 hours; induction culture was performed, 1 mL of recombinant E. coli cultured overnight was inoculated to 100 mL of fresh LB medium, and cultured at 37° C. and 180 rpm until a concentration of recombinant bacteria reached OD.sub.600=0.6, and after cooling to 20° C., IPTG with a final concentration of 0.2 mM was added for induction for 16 hours, and bacteria were collected by centrifuging at 4° C. and 8000 rpm for 5 minutes.

(39) The collected bacteria were suspended with physiological saline (precooled at 4° C.), and centrifuged at 4° C. and 8000 rpm for 5 minutes to collect bacteria, and this step was repeated twice to remove residual LB medium; the bacteria were added to a basic MOPS medium containing formamide (200 mM) and phosphite (1.32 mM) to make OD.sub.600 of the bacteria be 0.1, IPTG with a final concentration of 0.2 mM was added, and the recombinant E. coli was continued to be cultured at 30° C. and 180 rpm.

(40) The growth of the recombinant E. coli in the MOPS medium is observed, a growth curve graph of the recombinant E. coli BL21(DE3)(pGEX-for-Linker-ptx) in the MOPS medium containing formamide (200 mM) and phosphite (1.32 mM) is shown in FIG. 2. It can be seen from FIG. 2 that the recombinant E. coli BL21(DE3)(pGEX-for-Linker-ptx) can grow in this medium, a concentration of bacteria reaches a maximum value after the third day, and A600 is 2.223, while a control strain E. coli BL21(DE3) basically does not grow in the MOPS medium.

(41) In order to verify an express ability of the recombinant E. coli BL21(DE3)(pGEX-for-Linker-ptx) to synthesize exogenous gene in the MOPS medium, a green fluorescent protein (GFP) gene was used to verify an ability of the recombinant E. coli to express an exogenous gene. The pET-28a-GFP plasmid was transformed into E. coli BL21(DE3)(pGEX-for-Linker-ptx) to form the recombinant E. coli, which was named as E. coli BL21(DE3)(pGEX-for-Linker-ptx+pET-28a-GFP), and the obtained recombinant E. coli was induced to express GFP in the MOPS medium containing formamide and phosphite. Then, the cultured and fermented recombinant E. coli was observed with a fluorescence inversion microscope with an excitation wavelength of 488 nm and an emission wavelength of 507 nm. It can be seen from FIG. 3 that the recombinant E. coli can grow normally in the MOPS medium and can express an exogenous green fluorescent protein gene.

(42) The instant application contains a Sequencing Listing which has been submitted electronically in ASCII text file and is hereby incorporated by reference in its entirety. Said ASCII text file, created on Jan. 7, 2023, is named 096868-US-sequence listing ST25 and is 7,681 bytes in size.