Halomonas sp. engineered strain for bio-oxidative desulfurization and method for catalyzing desulfurization by whole cell

Abstract

This invention discloses an engineered strain of *Haloxymonas* for biological oxidative desulfurization and its whole-cell catalytic desulfurization method. The engineered strain is a recombinant *Haloxymonas* containing a quinone oxidoreductase gene. The recombinant *Haloxymonas* expresses the quinone oxidoreductase. The recombinant *Haloxymonas* of this invention achieves a maximum biological hydrogen sulfide removal rate of 80.216 μmol·min under alkaline conditions. ‑1 ·g ‑1 The cell dry weight showed a highly efficient hydrogen sulfide removal rate; it can be recycled 6 times in a whole-cell catalytic desulfurization system, and the biological desulfurization rate is maintained at 40 μmol·min⁻¹. ‑1 ·g ‑1 Above the cell dry weight, it exhibited good stability, with an average rate reaching 56.843 μmol·min. ‑1 ·g ‑1 Cell dry weight. Therefore, the above-mentioned recombinant halometabolite and its whole-cell biological desulfurization process have application value in the field of biological removal of hydrogen sulfide.

Description

Technical Field

[0001] This invention relates to engineered strains of Halomonas bacteria for biological oxidative desulfurization and a whole-cell catalytic desulfurization method thereof. Background Technology

[0002] Hydrogen sulfide (H2S) is the most significant pollutant in natural gas exploration, transmission, and development. Due to its high toxicity, foul odor, and corrosiveness, it poses a threat to human health, industrial production, and the environment. Therefore, it is necessary to remove hydrogen sulfide from natural gas, i.e., natural gas desulfurization. Currently, natural gas desulfurization mainly utilizes physicochemical and biological technologies. Biological desulfurization, under normal temperature and pressure, uses the sulfur oxidation of microorganisms to convert dissolved sulfides in the absorbent into insoluble elemental sulfur, thus removing hydrogen sulfide. Compared to physicochemical methods, biological methods have advantages such as low energy consumption, low cost, no need for chemical catalysts, recyclable products, and less secondary pollution, making them more economical, efficient, and environmentally friendly, and have attracted widespread attention.

[0003] Biological desulfurization microorganisms are classified into chemoautotrophic and chemoheterotrophic bacteria based on the carbon source they utilize. Chemoautotrophic bacteria (such as *Thiobacillus denitrifyingus*) use atmospheric carbon dioxide as a carbon source and inorganic sulfur compounds such as hydrogen sulfide to provide reducing power and energy for their cells. However, the generated elemental sulfur is further oxidized to sulfate by the bacteria, which causes the reaction system to become acidic. This necessitates the addition of large amounts of alkali to maintain pH stability, increasing desulfurization costs. Alkaline conditions are beneficial for pH stability, reducing the risk of contamination by other microorganisms and improving the absorption efficiency of the absorbent for hydrogen sulfide, potentially reducing desulfurization costs. *Thioalbuminuria*, a halophilic chemoautotrophic sulfur-oxidizing bacterium, can adapt to alkaline conditions up to pH 10, offering significant advantages for hydrogen sulfide removal from alkaline absorbents.

[0004] Some chemoheterotrophic microorganisms have the ability to oxidize hydrogen sulfide, and are characterized by rapid growth rate and high biological desulfurization efficiency, showing great development potential in the field of biological desulfurization. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to enable halophilic basobacteria to have efficient biological oxidation and desulfurization capabilities.

[0006] To address the aforementioned technical problems, this invention provides a recombinant halomonas bacterium that can express sulfide quinone oxidoreductase under strongly alkaline conditions, exhibiting a highly efficient biological desulfurization rate. Furthermore, its accompanying whole-cell catalytic process allows for multiple recycling cycles.

[0007] The recombinant halomonas provided by this invention contains a quinone oxidoreductase gene.

[0008] The aforementioned recombinant Halomonas can express the quinone oxidoreductase.

[0009] In the above-mentioned recombinant Halomonas, the quinone oxidoreductase may be selected from any of the following proteins:

[0010] 1) A protein with an amino acid sequence as shown in SEQ ID No. 2;

[0011] 2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein shown in 1), which has more than 80% identity with the protein shown in 1) and has quinone oxidoreductase activity.

[0012] 3) The fusion protein is obtained by attaching a protein tag to the N-terminus and / or C-terminus of 1) or 2).

[0013] SEQ ID No.2 consists of 422 amino acid residues.

[0014] In the aforementioned recombinant Halomonas bacteria, the protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0015] In the aforementioned recombinant Halomonas, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0016] In the above-mentioned recombinant Halomonas, the quinone oxidoreductase gene may be selected from any of the following:

[0017] x1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3;

[0018] x2) The coding sequence (CDS) is the DNA molecule shown in positions 1-1329 of SEQ ID No. 3;

[0019] The DNA molecule that has more than 80% identity with the DNA molecule defined by x3) and x1) or x2) and encodes the quinone oxidoreductase.

[0020] Of these, SEQ ID No.3 consists of 1329 nucleotides.

[0021] In the aforementioned recombinant Halomonas, the "identity" or "percentage of sequence identity" of the DNA is determined by comparing two optimally aligned sequences within a comparison window. The optimal alignment provides the highest level of pairing and allows for the introduction of nucleotide additions into the test or reference sequence. The identity percentage is determined by calculating the percentage of identical nucleotides at every position throughout the entire sequence between the test and reference sequences. The optimal sequence alignment and percentage identity can be determined manually, or more preferably by computer algorithms, including but not limited to TBLASTN, FASTA, GAP, BESTFIT, and CLUSTALW (Altschul et al., 1990, J.Mol.Biol.215(3):403-10; Pearson and Lipman, 1988, Proc.Natl.Acad.Sci.USA 85(8):2444-8; Thompson, et al., 1994, Nucleic Acids Res.22(22):4673-80; Devereux et al., 1984, Nuc.Acids.Res.12:387-395; Higgins, et al., 1996, Methods Enzymol.266:383-402). Preferably, the NCBI Blast Server (http: / / www.ncbi.nlm.nih.gov) with default parameters is used to search multiple databases for homologous sequences.

[0022] In the aforementioned recombinant halomonas, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0023] In the above-mentioned recombinant halomonas, the method for constructing the recombinant halomonas includes introducing the quinone oxidoreductase gene into a recipient bacterium to obtain the recombinant halomonas, wherein the recipient bacterium is halomonas.

[0024] In the aforementioned recombinant halomonas, the recipient bacterium may be Halomonas salifodinae IM328, whose registration number at the China General Microbiological Culture Collection Center is CGMCC No. 22183.

[0025] Among the aforementioned recombinant halomonas, the recombinant halomonas may be Halomonassalifodinae IM328_sqr, whose registration number at the China General Microbiological Culture Collection Center is CGMCC No. 22179.

[0026] The present invention also provides a method for constructing the recombinant halomonas bacteria.

[0027] The method for constructing the recombinant halomonas provided by the present invention involves introducing the quinone oxidoreductase gene into a recipient bacterium to obtain the recombinant halomonas, wherein the recipient bacterium is halomonas.

[0028] In the above method, the recipient bacterium can be Halomonas salifodinae IM328, whose registration number at the China General Microbiological Culture Collection Center is CGMCC No. 22183.

[0029] In the above method, the recombinant halomonas can be Halomonas salifodinae IM328_sqr, whose registration number at the China General Microbiological Culture Collection Center is CGMCCNo.22179.

[0030] The aforementioned Halomonas salifodinae IM328 also falls within the scope of protection of this invention.

[0031] The application of the aforementioned recombinant halomonas bacteria in the biological removal of hydrogen sulfide also falls within the scope of protection of this invention.

[0032] In the above applications, the recombinant halomonas bacteria can remove hydrogen sulfide under conditions of pH 7-10.

[0033] In the above applications, the biological removal of hydrogen sulfide is carried out using whole-cell catalysis by the aforementioned recombinant halomonas bacteria.

[0034] In the above application, the biological removal of hydrogen sulfide can be divided into two stages: the cultivation and growth of microorganisms and the oxidative desulfurization stage. The cultured microbial cells can be directly used for oxidative desulfurization and cycled at least six times, and no additional nutrients are needed during the oxidative desulfurization stage.

[0035] This invention introduces the quinone oxidoreductase gene (Sqr) into *Halomonas salifodinae* IM328, resulting in a recombinant engineered bacterium, IM328_sqr (recombinant *Halomonas*). This recombinant bacterium can oxidize sulfides under highly alkaline conditions (pH 10), achieving a removal rate of up to 98% within 5 minutes of whole-cell catalytic reaction. This high removal efficiency is maintained even after six consecutive cycles. The recombinant *Halomonas* of this invention achieves a maximum rate of 80.216 μmol·min⁻¹ for the biological removal of hydrogen sulfide under alkaline conditions. -1 ·g -1 The cell dry weight showed a highly efficient hydrogen sulfide removal rate; it can be recycled 6 times in a whole-cell catalytic desulfurization system, and the biological desulfurization rate is maintained at 40 μmol·min⁻¹. -1 ·g -1 Above the cell dry weight, it exhibited good stability, with an average rate reaching 56.843 μmol·min. -1 ·g -1 Cell dry weight. Therefore, the above-mentioned recombinant halometabolite and its whole-cell biological desulfurization process have application value in the field of biological removal of hydrogen sulfide.

[0036] Preservation Instructions

[0037] 1. Strain name: *Haloxylon ammodendron*

[0038] Latin name: Halomonas salifodinae

[0039] Strain ID: IM328_sqr

[0040] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0041] Collection institution abbreviation: CGMCC

[0042] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0043] Deposit date: April 14, 2021

[0044] Registered with the China National Collection Center (CGMCC) No. 22179.

[0045] 2. Strain name: *Haloxylon ammodendron*

[0046] Latin name: Halomonas salifodinae

[0047] Strain number: IM328

[0048] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0049] Collection institution abbreviation: CGMCC

[0050] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0051] Deposit date: April 14, 2021

[0052] CGMCC Registration Number: CGMCC No. 22183 Attached Figure Description

[0053] Figure 1 Evolutionary relationship diagram of 16S rRNA of Halomonas salifodinae IM328.

[0054] Figure 2 The images show the plasmid maps of pBBR-ptac-rfp, pBBR-ptac-sqr, and pBBR-ptac-ck.

[0055] Figure 3 The growth of the strains used for transformation on different solid selection media.

[0056] Figure 4 Electrophoresis gel images of engineered strain IM328-sqr and control strain IM328-ck for colony PCR validation.

[0057] Figure 5 To validate the expression of heterogeneous Sqr in IM328_sqr using SDS-PAGE.

[0058] Figure 6 This is the standard curve for hydrogen sulfide concentration.

[0059] Figure 7 The efficiency of whole-cell catalytic oxidation of sulfides by engineered bacteria

[0060] Figure 8 The optimal pH for the whole-cell catalytic oxidation of sulfides by engineered bacteria IM328_sqr is determined.

[0061] Figure 9 The efficiency of cyclic desulfurization of sulfides catalyzed by the whole-cell catalytic oxidation of engineered bacteria IM328_sqr is measured. Detailed Implementation

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0064] The pBBR1MCS plasmid used in the following examples is found on page 449, fifth line from the bottom left column of the following document. This biological material is available to the public from the applicant and is intended solely for the purpose of repeating the experiments of this invention and shall not be used for any other purpose: Mu et al., 2017, Biotechnol. Lett. 39:447-452.

[0065] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0066] Example 1: The recombinant halomonas salifodinae IM328_sqr was constructed and its registration number at the China General Microbiological Culture Collection Center is CGMCCNo.22179.

[0067] 1. Discovery and acquisition of functional genes for biological desulfurization

[0068] Based on the sequencing and functional annotation of metagenomics of microorganisms in the saline-alkali environment of Inner Mongolia, 484 genes encoding sulfide:quinone oxidoreductase (Sqr) and cytochrome c: sulfide dehydrogenase (FCSD) were extracted. Phylogenetic tree construction using maximum likelihood analysis showed that these 484 sequences were divided into Sqr types I-VI, one unclassified Sqr sequence, and FCSD. Among them, Sqr-II genes were the most abundant, totaling 307, followed by type I (10), type III (57), type IV (3), type VI (13), and type V (no sequence found). Additionally, 50 protein sequences lacked corresponding reference sequences, and their classification is currently uncertain. 44 sequences belonged to FCSD. The top four most abundant proteins in the 18 samples all belonged to type II. The total abundance of the 1st, 2nd and 4th most abundant proteins accounted for nearly 20%, and their phylogenetic relationships were very close. They all showed high similarity to the Sqr gene of Spiribacter sp.IM2438, a high-abundance bacterium isolated from the salt lake (96.15%, 98.05%, and 95.93%, respectively). Therefore, the sqr gene of Spiribacter sp.IM2438 was finally used as the target gene for heterologous expression.

[0069] 2. Establishment of methods for isolating, screening, and genetically manipulating halophilic single cells.

[0070] The *Halomonas salifodinae* IM328 provided by this invention was obtained from sampling at Yanhaizi Lake in Inner Mongolia (40°9'6"N", 108°26'14"E). Single colonies were isolated by spreading the suspension from the salt lake sediment sample onto HM3.6 solid medium at 28°C. Identification using 16S rRNA of the strain showed that *Halomonas salifodinae* IM328 shared 99.71% homology with *Halomonas salifodinae* BC7T. Figure 1 This indicates that IM328 belongs to Halomonas salifodinae, and it was eventually named Halomonas salifodinae IM328.

[0071] Halomonas salifodinae IM328 cells are short rod-shaped, 0.5-1.0 μm wide and 1.0-2.0 μm long. When cultured on compound agar at 37°C, single colonies are round, typically 1-2 mm in diameter, smooth, and orange-yellow in color.

[0072] Halomonas salifodinae IM328 was deposited on April 14, 2021, at the China General Microbiological Culture Collection Center (CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 22183. It will be referred to as Halomonas IM328.

[0073] The HM3.6 isolation and screening medium for Halomonas salifodinae IM328 provided by this invention consists of: sodium chloride 36 g / L, potassium chloride 2 g / L, magnesium sulfate heptahydrate 1.0 g / L, calcium chloride 0.27 g / L, sodium bromide 0.23 g / L, sodium bicarbonate 0.06 g / L, trace amounts of ferric chloride, peptone 5 g / L, yeast extract 10 g / L, pH 8.5, agar 1.5%, and water as the solvent. The LB-60 growth medium for IM328 under aerobic conditions consists of: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 60 g / L, water as the solvent, and pH 8.0. The LB-20 incubation medium used for IM328 conjugation transformation consists of: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 80 g / L, and water as the solvent. Screening medium LB-80: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 80 g / L, chloramphenicol 25 mg / L, solvent is water.

[0074] 3. Construction of the expression carrier

[0075] The pBBR1MCS plasmid was linearized by double digestion with Hind III and Nhe I. The insert fragment was amplified by three-step PCR.

[0076] 1) First, using the genome of Spiribacter sp.IM2438 as a template, based on the Sqr coding region in sequence 3 and its upstream and downstream nucleotide sequences, amplification primers sqr-F / sqr-R for the upstream and downstream exchange arms were designed. This resulted in the addition of a homologous sequence (5'-CACTGCAGGAGGAAGCTT-3') containing a Hind III restriction site that perfectly matches the plasmid pBBR1MCS to the 5' end of the amplified product sqr, and the addition of the 5' end portion of the TER terminator structure of the plasmid pBBR1MCS to the 3' end.

[0077] 2) Additionally, TER-F and TER-R primers were designed to amplify the plasmid pBBR1MCS terminator structure (TER) containing the Nhe I restriction site. Primer TER-F contains the 3' end sequence of sqr. The 3' end of the amplified product TER' is complementary to the 5' end sequence of the first amplified product sqr', serving as a template for the next overlap PCR. Amplification using sqr-F and TER-R primers resulted in the final PCR product having sequences (15bp-25bp) at both the 5' and 3' ends identical to those at the ends of the linearized vector (nucleotide sequence of this PCR product is sequence 4). A 200μL reaction system is required as follows:

[0078] 10×buffer 20μL

[0079] dNTPs 20μL

[0080] MgSO4 12μL

[0081] sqr-F (10μM) 8μL

[0082] TER-R (10μM) 8μL

[0083] 4 μL of KOD enzyme

[0084] Template 1 (sqr': 92.9 ng / μL) 2μL

[0085] Template 2 (TER': 29.6 ng / μL) 6 μL

[0086] ddH2O 120μL

[0087] The prepared system was kept at 95°C for 5 minutes, then cooled to 4°C. The PCR product with vector end sequences at both ends was then gel-recovered to obtain the target gene fragment.

[0088] 3) Mix the recovered PCR product and the linearized vector at a ratio of 4:6, and react at 50°C for 50 min under the catalysis of the Hieff Clone™ Plus OneStep Cloning Kit enzyme. Then, clone the target gene sqr (nucleotide sequence is SEQ ID No. 3) into plasmid pBBR1MCS (i.e., using recombination methods). Figure 2 The pBBR-ptac-rfp expression plasmid was obtained by using the Tac promoter to initiate the transcription of the target gene quinone oxidoreductase (Sqr) gene sqr, ultimately resulting in the expression plasmid pBBR-ptac-sqr expressing the target gene sqr. Figure 2 pBBR-ptac-sqr contains the quinone oxidoreductase (Sqr) gene sqr, whose coding sequence (CDS) is sequence 3, and whose transcription is initiated by the promoter Tac.

[0089] pBBR-ptac-rfp( Figure 2 The rfp gene in pBBR-ptac-rfp is removed, while the other nucleotide sequences of pBBR-ptac-rfp remain unchanged. The resulting recombinant vector is denoted as pBBR-ptac-ck. Figure 2 (), as a control without load.

[0090] Colony PCR was performed on DH5α cells containing the pBBR-ptac-sqr and pBBR-ptac-ck plasmids using primers sqr-F / sqr-R (amplifying sequence 3) and CX-F / CX-R (amplifying sequence 5 in plasmid pBBR1MCS), respectively. Sequencing then confirmed successful plasmid construction. The nucleotide sequences of the primers are as follows:

[0091] sqr-F:5'- CACTGCAGGAGGAAGCTT ATGCCCAACGAATCA-3' (underlined sequence indicates a homologous sequence that perfectly matches plasmid pBBR1MCS)

[0092] sqr-R:5'-TCCCAGCTCAACGCCCTAGATTCGGCCACG-3'

[0093] TER-F:

[0094] 5'-TGCTACGTGGCCGAATCTAGGGCGTTGAGCTGGGATTAACCCGGCGAGGCGGAGACCCAACAGAACGGAGCCAGGGAGATGGCGACGCAG-3'

[0095] TER-R:

[0096] 5'- ACTCGATTGACTGGGGGGCTAGCTGCGTTGAGGAGCCAGCCAGCGCCACTGGGGTCAAACCTTGT CTGTTGACCCTCGTGCCC-3' (underlined to indicate a homologous sequence that perfectly matches plasmid pBBR1MCS)

[0097] CX-F:5'-ACTGCATAATTCGTGTCGCT-3'

[0098] CX-R:5'-AAGAGGAGCAACGCGATCTA-3'

[0099] 4. Establishment of genetic manipulation methods for IM328 and construction of desulfurization engineered strains.

[0100] 4.1 Construction of recombinant Halomonas salifodinae IM328_sqr

[0101] pBBR-ptac-sqr was introduced into Escherichia coli S17 (abbreviated as S17) (Mu et al., 2017, Biotechnol. Lett. 39:447-452, page 449, left column, line 26) to obtain recombinant Escherichia coli S17 / pBBR-ptac-sqr (abbreviated as S17_sqr).

[0102] To construct the genetic operating system for IM328, a conjugation transformation method was used. Recombinant *E. coli* S17 / pBBR-ptac-sqr was used as the donor, and the plasmid pBBR-ptac-sqr, containing the tac promoter and chloramphenicol resistance gene, was transformed into *Halomonas salifodinae* IM328. The selection conditions for conjugation transformation were determined by experimenting with different culture media. Since *Halomonas salifodinae* IM328 is sensitive to chloramphenicol, and *E. coli* S17 does not grow at salinity above 8%, LB-80 medium containing chloramphenicol and with a salinity of 8% was used to select IM328 transformants. Under these conditions, neither *E. coli* S17 nor wild-type *Halomonas salifodinae* IM328 (hereinafter referred to as IM328) grew. Figure 3 ).

[0103] Recombinant *Escherichia coli* S17 / pBBR-ptac-sqr and *Halomonas salifodinae* IM328 were incubated overnight. 1 mL of each culture was centrifuged (5000 g, 4 °C, 10 min) to harvest the cells. The recombinant *Escherichia coli* S17 / pBBR-ptac-sqr and *Halomonas salifodinae* were then washed with LB medium and LB-60 medium (solutes: sodium chloride 60 g / L, yeast extract 5 g / L, and peptone 10 g / L; solvent: water), respectively. The bacteria were mixed twice with 1:1 ratio and dropped onto LB-20 agar plates (solutes: 20 g / L sodium chloride, 5 g / L yeast extract, and 10 g / L peptone, solvent: water) and incubated at 37°C for 18 hours. Finally, the conjugated bacteria were evenly spread on LB-80 agar plates (solutes: 80 g / L sodium chloride, 5 g / L yeast extract, and 10 g / L peptone, 1.5% (w / w) agar powder, 25 mg / L chloramphenicol; solvent: water) containing a final concentration of 25 mg / L chloramphenicol and cultured at 37°C for four days to obtain the transformant Halomonas salifodinae IM328_sqr, which is the recombinant Halomonas salifodinae IM328_sqr of this invention.

[0104] Halomonas salifodinae IM328_sqr was deposited on April 14, 2021, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 22179. It will be referred to as the engineered strain IM328_sqr.

[0105] 4.2 Construction of control strain IM328_ck

[0106] Except for replacing pBBR-ptac-sqr with pBBR-ptac-ck in 4.1, the other operations are the same as in 4.1. The plasmid pBBR-ptac-ck is transformed using the above conjugation transformation method to obtain the control strain Halomonas salifodinae IM328_ck (abbreviated as IM328_ck).

[0107] PCR was performed using pBBR-ptac-sqr and pBBR-ptac-ck as templates. The engineered strain IM328-sqr (IM328-sqr), the control strain Halomonas salifodinae IM328_ck (IM328_ck), and the recipient strain Halomonas salifodinae IM328 (IM328) were used as templates. Colony PCR was performed using two pairs of primers, sqr-F / sqr-R and CX-F / CX-R, and 2×mix Taq Master Mix enzymes to verify the engineered strain IM328_sqr and the control strain IM328_ck after multiple subcultures. The engineered strain IM328_sqr contained the quinone oxidoreductase gene sqr, while neither the recipient strain Halomonas salifodinae IM328 nor the control strain Halomonas salifodinae IM328_ck contained the quinone oxidoreductase gene sqr. Using IM328_sqr cells as a template, the PCR product length was 1362 bp. The observed cloning bands were close to the theoretical value, but no bands were observed in IM328 and IM328_ck samples (containing pBBR_ptac_ck plasmid, used as a negative control). Figure 4 This result proves that the IM328_sqr strain was successfully constructed. Furthermore, colony PCR identification of IM328_ck using primers CX-F / CX-R showed a length corresponding to the theoretical size of 551 bp. Figure 4 ).

[0108] To verify the expression of Sqr in the engineered bacterium IM328_sqr, SDS-PAGE analysis was performed. 5% polyacrylamide stacking gel (pH 6.8) and 12% polyacrylamide separating gel (pH 8.8) were used to prepare the gels, with a loading volume of 20 μL of protein extract for each gel. Electrophoresis was performed at 4°C. The molecular weight of the target protein Sqr was approximately 48.1 kDa. SDS-PAGE showed that, compared with the control bacterium, the engineered bacterium IM328_sqr contained some protein bands with different molecular weights (possibly at the theoretical molecular weight). Figure 5Therefore, gels potentially containing the target protein (numbered 1 to 5) were recovered and analyzed by mass spectrometry. The results showed that samples 1, 2, and 3 (marked with #) contained peptides with the target protein's Sqr sequence (expected values ​​all less than 0.05). Sample 2 contained 33 trypsin-digested peptide sequences that matched the Sqr protein sequence (all peptides had expected values ​​less than 0.05), achieving a protein sequence coverage of 47.4%. Samples 1 and 3 had relatively fewer matching peptides (2 and 19, respectively), and no matching peptides were observed in samples 4 and 5. Further protein-level analysis confirmed that the engineered bacterium IM328_sqr expresses the sqr gene, while the control bacterium Halomonas salifodinae IM328_ck does not.

[0109] Example 2: Determination of the biological desulfurization capacity of engineered bacteria IM328_sqr

[0110] The test reagents used in the following experiments include:

[0111] Zinc acetate-sodium acetate solution: Dissolve 10g zinc acetate and 2.5g sodium acetate in 200mL of water, shake well, and store at 4℃.

[0112] Ferric chloride solution: Dissolve 6g of ferric chloride hexahydrate in water and bring the volume up to 6mL.

[0113] Working solution for p-aminodimethylaniline: While stirring, pour 50g of concentrated sulfuric acid into 50mL of water. After cooling, add 0.4g of p-aminodimethylaniline hydrochloride and dilute with water to 100mL. Store at 4℃.

[0114] Mixed colorimetric reagent: Mix p-aminodimethylaniline working solution and ferric chloride solution in a 20:1 ratio, and prepare and use immediately.

[0115] Diammonium hydrogen phosphate solution: Weigh 40g of diammonium hydrogen phosphate and dilute to 100mL with water.

[0116] 1) Plot the standard curve for determining sodium sulfide.

[0117] The preparation method of sodium sulfide standard working solution (calculated as S, 0.312 mmol / L) is as follows: First, prepare 250 mL of alkali solution A (pH 11, adjusted with NaOH granules, solute is NaOH, solvent is water); after adding 125 mL of solution A to a volumetric flask, add 2 mL of zinc acetate-sodium acetate solution and mix well; take 20 mL of 100 mg / L sodium sulfide standard solution, add it dropwise while shaking, and make up to 200 mL with alkali solution A to obtain a zinc sulfide suspension. This zinc sulfide suspension is the sodium sulfide standard working solution, and the sulfide ion concentration of this sodium sulfide standard working solution is 10 mg / L. Shake well before use.

[0118] Take 7 centrifuge tubes with 15mL each, add the standard working solution according to Table 1, and then dilute to 10mL with distilled water.

[0119] Table 1. Reagents and their contents used in measuring the sodium sulfide standard curve

[0120]

[0121] Add 100 μL of the mixed colorimetric reagent to each tube, immediately cap, invert and slowly shake to mix, and let stand for 30 min. Add 50 μL of diammonium hydrogen phosphate solution to eliminate the color from ferric ions, and mix well. Measure the absorbance at 670 nm using a 1 cm cuvette, with water as a reference. Plot a standard curve against absorbance and hydrogen sulfide content (mM). Figure 6 ).

[0122] Finally, the equation of the standard curve was obtained as Y = 24.68X + 0.0212, R. 2 = 0.9949. (X represents the optical density value at 670 nm, and Y represents the hydrogen sulfide content (mM) in the liquid being tested)

[0123] 2) Desulfurization rate determination by engineered bacteria

[0124] The reagents used in the whole-cell catalytic reaction include:

[0125] Tricine buffer: 50g NaCl (10%), 3.0285g Tricine, adjust pH to 8 with NaOH, bring volume to 500mL with water, sterilize at 120℃ for 20min.

[0126] Sodium sulfide mother liquor (400mM Na2S·9H2O): 4.8g Na2S·9H2O is dissolved in 50mL of oxygen-free water, filtered and sterilized, and then evacuated and purged with nitrogen.

[0127] The experiment included two treatments: an experimental group and a control group.

[0128] Experimental group: The engineered strain IM328_sqr and the control strain IM328_ck were cultured in LB-60 medium containing 25 mg / L chloramphenicol until the late logarithmic growth phase (OD2). 600nm 4) Transfer 1 mL of bacterial culture to a 1.5 mL centrifuge tube, centrifuge at 6000 rpm and 4°C for 5 min, discard the supernatant, and retain the pellet (cells); wash the pellet (cells) once with 25 mM Tricine buffer (pH 8.0) (dissolve Tricine in distilled water to a concentration of 25 mM, adjust the pH to 8.0 with NaOH), and resuspend in 950 μL of 25 mM Tricine buffer (pH 8.0) to OD. 600nmThe values ​​were approximately 4 (4.51 for engineered strain IM328_sqr and 4.25 for control strain IM328_ck), and centrifuge tubes containing engineered strain IM328_sqr and control strain IM328_ck were obtained respectively.

[0129] Dilute the sodium sulfide stock solution with distilled water to obtain a 20 mM sodium sulfide solution. Add 50 μL of the 20 mM sodium sulfide solution (prepared by mixing 225 μL of 400 mM sodium sulfide stock solution with 4275 μL of distilled water) to each centrifuge tube containing the engineered bacteria IM328_sqr and the control bacteria IM328_ck in a specific order to achieve a final Na2S concentration of 1 mM to initiate the reaction. At time 0, after adding the sodium sulfide solution to the experimental group, centrifuge rapidly at 4°C and 12000 rpm for 30 s. Take 33.3 μL of the supernatant from each of the engineered bacteria IM328_sqr and control bacteria IM328_ck reaction solutions, and dilute them 15-fold with 466.7 μL of sterile water respectively to obtain the reaction dilutions for the engineered bacteria IM328_sqr tube and the control bacteria IM328_ck tube at time 0. Centrifuge tubes containing the engineered bacteria IM328_sqr and the control bacteria IM328_ck were quickly capped and incubated at 37°C and 200 rpm for 5 minutes on a shaker. Then, they were centrifuged at 4°C and 12000 rpm for 30 seconds. 500 μL of the supernatant from the engineered bacteria IM328_sqr reaction solution was collected to obtain the reaction solution for the engineered bacteria IM328_sqr tube after 5 minutes of reaction. 100 μL of the supernatant from the control bacteria IM328_ck reaction solution was collected, and 400 μL of sterile water was added to dilute the test system 5-fold to obtain the diluted reaction solution for the control bacteria IM328_ck tube after 5 minutes of reaction. The sulfide solution to be tested was diluted to a final volume of 500 μL.

[0130] Control group: The engineered strain IM328_sqr and the control strain IM328_ck were cultured in LB-60 medium containing 25 mg / L chloramphenicol until the late logarithmic growth phase (OD2). 600nm 4) Transfer 1 mL of bacterial culture to a 1.5 mL centrifuge tube, centrifuge at 6000 rpm and 4°C for 5 min, discard the supernatant, and retain the pellet (cells); wash the pellet (cells) once with 25 mM Tricine buffer (pH 8.0) (dissolve Tricine in distilled water to a concentration of 25 mM, adjust the pH to 8.0 with NaOH), and resuspend in 950 μL of 25 mM Tricine buffer (pH 8.0) to OD. 600nmThe concentration was approximately 4 (4.51 for engineered strain IM328_sqr and 4.25 for control strain IM328_ck), resulting in centrifuge tubes containing engineered strain IM328_sqr and control strain IM328_ck, respectively. These centrifuge tubes were then incubated in a 100°C water bath for 10 minutes to obtain heat-killed cell-contaminated IM328_sqr and IM328_ck centrifuge tubes, respectively.

[0131] Dilute the sodium sulfide stock solution to 20 mM with distilled water to obtain a sodium sulfide solution. Add 50 μL of sodium sulfide solution (20 mM concentration) to each heat-killed cell IM328_sqr and heat-killed cell IM328_ck centrifuge tube in a specific order to achieve a final Na2S concentration of 1 mM to initiate the reaction. At time 0, after adding the sodium sulfide solution to the heat-killed cell IM328_sqr and heat-killed cell IM328_ck centrifuge tubes, centrifuge rapidly at 4°C, 12000 rpm for 30 s. Take 33.3 μL of the supernatant from each of the engineered bacteria IM328_sqr and control bacteria IM328_ck reaction solutions, and add 466.7 μL of sterile water to dilute the test system 15-fold, obtaining the reaction dilutions for the heat-killed cell IM328_sqr and heat-killed cell IM328_ck centrifuge tubes at reaction time 0. The heat-killed cell IM328_sqr and heat-killed cell IM328_ck centrifuge tubes were quickly capped after 5 minutes of reaction and incubated at 37°C and 200 rpm for 5 minutes on a shaker. Then, they were centrifuged at 4°C and 12000 rpm for 30 seconds. 33.3 μL of the supernatant from each of the engineered bacterial IM328_sqr and control bacterial IM328_ck reaction solutions were added, and 466.7 μL of sterile water was added to each, diluting the test system 15-fold. This yielded the reaction dilutions for the heat-killed cell IM328_sqr and IM328_ck centrifuge tubes, respectively, without the addition of cells compared to the experimental group. The sulfide solution to be tested was diluted to a final volume of 500 μL for both.

[0132] The sulfide content was determined using the diphenylamine colorimetric method described below. Add 10 μL of zinc acetate-sodium acetate solution to each of the above tubes (500 μL of reaction (diluted) solution, shake, centrifuge for 5 min, discard the supernatant, add 1 mL of deionized water, then add 100 μL of the mixed colorimetric reagent to each tube, allow to stand for 30 min, add 50 μL of diammonium hydrogen phosphate solution, and measure the OD. 670nmCalculate the sulfide concentration in the diluted solution at time 0 and after 5 minutes of reaction based on the standard curve above. The concentration of the sulfide solution before dilution is equal to the concentration of the corresponding diluted solution × the dilution factor × 2 (the dilution factor is the number of times the reaction solution is diluted with deionized water, and 2 is the number of times the volume differs before and after color development, i.e., 2 times). The difference represents the concentration of oxidized sulfide within 5 minutes of reaction.

[0133] After the reaction, the cells were harvested, washed with buffer, freeze-dried for 24 hours, and the dry weight of the cells was measured.

[0134] q=(C0-C t )×V / (m×T)

[0135] Quantitative analysis was performed on the sulfide consumption rate (desulfurization rate) q, where C0 and C t is the sulfide concentration (μM (as hydrogen sulfide)) at reaction time 0 and after reaction time t minutes; V is the volume of the test solution (mL), in this experiment the volume of the reaction solution when sodium sulfide was added was 1 mL; m is the dry weight of the cells (g); T is the incubation time (min), in this experiment it was 5 min.

[0136] The results showed that the dry weight of IM328_ck cells in the experimental group was 0.0325 g, and the dry weight of IM328_sqr cells was 0.0345 g. Within 5 minutes of whole-cell catalytic reaction, the sulfide oxidation amount in the centrifuge tube of engineered bacteria IM328_sqr was 643.067 μM (calculated as hydrogen sulfide), while the sulfide oxidation amount in the centrifuge tube of control bacteria IM328_ck was 470.216 μM (calculated as hydrogen sulfide). In the control group, the sulfide oxidation amount in the centrifuge tube of heat-killed cells IM328_sqr was 44.977 μM, and the sulfide oxidation amount in the centrifuge tube of heat-killed cells IM328_ck was 49.836 μM. The hydrogen sulfide consumption rate of engineered bacteria IM328_sqr in the whole-cell catalytic reaction was approximately 35 μmol·min⁻¹. -1 ·g -1 The cell dry weight was higher than that of the control bacterium IM328_ck, which was 26 μmol·min⁻¹. -1 ·g -1 Cell dry weight ( Figure 7 ).

[0137] Example 3: Optimization and Improvement of Biological Desulfurization Efficiency

[0138] Hydrogen sulfide is a weakly acidic gaseous pollutant, and a high pH system is more conducive to its absorption. The acidity / alkalinity of the whole-cell catalytic desulfurization system was optimized.

[0139] The engineered strain IM328_sqr was cultured in LB-60 medium containing 25 mg / L chloramphenicol until the late logarithmic growth phase (OD2). 600nm4) Take 500 μL of bacterial culture, transfer it to a 1.5 mL centrifuge tube, centrifuge (6000×g, 5 min) to collect cells, and then resuspend it with 950 μL of 25 mM different buffer to obtain the engineered bacteria IM328_sqr centrifuge tube. Four pH gradients (7.0, 8.0, 9.0, and 10.0) were set up using 25mM different buffer solutions. The pH 7.0 buffer was prepared using 25mM MOPS buffer solution (MOPS dissolved in distilled water to a concentration of 25mM, pH adjusted to 7.0 with NaOH) and 6% NaCl as the solute. The pH 8.0 buffer solution was prepared using 6% NaCl as the solute and 25mM Tricine buffer solution as the solvent (Tricine dissolved in distilled water to a concentration of 25mM, pH adjusted to 8.0 with NaOH). The pH 9.0 buffer solution was prepared using 6% NaCl as the solute and 25mM CHES buffer solution as the solvent (CHES dissolved in distilled water to a concentration of 25mM, pH adjusted to 9.0 with NaOH). The solute of the 10.0 buffer is 6% NaCl, and the solvent is 25mM CAPS buffer solution (dissolve CAPS in distilled water until the CAPS content is 25mM, and adjust the pH to 10.0 with NaOH).

[0140] Add 50 μL of freshly prepared 20 mM Na2S solution (prepared by mixing 225 μL of 400 mM sodium sulfide stock solution with 4275 μL of distilled water) to each of the above-mentioned engineered bacteria IM328_sqr centrifuge tubes to bring the final Na2S concentration in the system to 1 mM to initiate the reaction. For the experimental group at time 0, after adding the sodium sulfide solution, centrifuge rapidly at 4°C and 12000 rpm for 30 s. Take 25 μL of the supernatant from each of the engineered bacteria IM328_sqr reaction solutions under different pH conditions, and dilute 20 times with 475 μL of sterile water to obtain the reaction dilution solution for the engineered bacteria IM328_sqr tubes at reaction time 0. For the experimental group reacting for 5 minutes, quickly cap the tubes and place them on a shaker at 37°C and 200 rpm. Then, the cells were centrifuged at 12000 rpm for 30 seconds at 4℃. 50 μL of the supernatant from the reaction solution of the engineered bacteria IM328_sqr under different pH conditions was collected, and 450 μL of sterile water was added to dilute the test system 10-fold, obtaining the reaction dilution of the engineered bacteria IM328_sqr tube after 5 minutes of reaction. The sulfide concentration in the dilution at time 0 and after 5 minutes of reaction was analyzed using the diphenylamine colorimetric method described above. The concentration of the sulfide solution before dilution was equal to the concentration of the corresponding dilution × the dilution factor × 2 (the dilution factor is the number of times the reaction solution is diluted with deionized water, and 2 is the volume difference before and after colorimetric development, i.e., 2 times). The difference represents the concentration of oxidized sulfide within 5 minutes of reaction. After the reaction, the cells were harvested, washed with buffer, lyophilized for 24 hours, and the dry weight of the cells was measured.

[0141] q=(C0-C t)×V / (m×T)

[0142] Quantitative analysis was performed on the desulfurization rate q, where C0 and C t is the sulfide concentration (μM) at the start and start of sampling; V is the volume of the solution being tested (mL), in this experiment the reaction solution volume when sodium sulfide was added was 1 mL; m is the dry weight of the cells (g); T is the incubation time (min), in this experiment it was 5 min.

[0143] The results showed that the dry weight of IM328_sqr cells was 0.0153 g. Within 5 minutes of whole-cell catalytic reaction, the sulfide oxidation rate in the centrifuge tubes of engineered bacteria IM328_sqr was 135.203 μM (calculated as hydrogen sulfide) at pH 7, 405.293 μM at pH 8, 589.520 μM at pH 9, and 571.690 μM at pH 10. This indicates that the desulfurization rate of IM328_sqr was higher at pH 9, reaching 77.061 μmol·min⁻¹. -1 ·g -1 ( Figure 8 ).

[0144] Example 4: Whole-cell catalytic process for biological desulfurization

[0145] To assess the industrial applicability of the whole-cell catalytic desulfurization process and test the desulfurization effect of continuous reaction of the strain, six desulfurization experiments were conducted in cycles. This experiment was performed at 37℃ and 200 rpm in 15 mL centrifuge tubes containing 10 mL of 25 mM CHES buffer (pH 9.0, solute 6% NaCl, solvent 25 mM CHES buffer solution (CHES dissolved in distilled water to a CHES concentration of 25 mM, pH adjusted to 9.0 with NaOH)). The concentration of the engineered strain IM328_sqr was OD. 600nm The design involved six cycles of desulfurization reaction. In each cycle, Na₂S was added to a final concentration of 2 mM to initiate the reaction, and after 40 minutes, it was allowed to completely oxidize before being added again. The remaining sodium sulfide content in the system was measured 10 minutes after the start of each reaction (experimental group, denoted as "IM328_sqr"). A sterile reaction buffer was used as the control group (CK). Figure 9 The average sulfide oxidation amount in the IM328_sqr centrifuge tube of the engineered bacteria in the first whole-cell catalytic reaction (10 minutes) was 1227.310 μM (calculated as hydrogen sulfide), the second was 1039.600 μM, the third was 887.250 μM, the fourth was 634.198 μM, the fifth was 775.337 μM, and the sixth was 654.484 μM.

[0146] q=(C0-C t )×V / (m×T)

[0147] The dry weight m of IM328_sqr cells was 1.53 g / L, and the incubation time T (min) was 10 minutes in this experiment. The reaction volume V was 0.01 L.

[0148] Calculations showed that the desulfurization rates for six consecutive cycles were 80.216, 67.984, 57.990, 41.451, 50.676, and 42.777 μmol·min, respectively. -1 ·g -1 Cell dry weight. The results showed that the sulfide oxidation rate of heterotrophic cells IM328 expressing heterologous Sqr could be maintained between 41.451 and 80.216 μmol·min⁻¹. -1 ·g -1 Between cell dry weights, a high sulfide removal rate was observed, and the sulfide oxidation rate was very close to that of previously reported chemoautotrophic desulfurization bacteria. Therefore, this engineered bacterium can be recycled at least six times in a whole-cell catalytic desulfurization system.

[0149] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Institute of Microbiology, Chinese Academy of Sciences <120> Engineered strains of Halomonas bacteria for biological oxidative desulfurization and their whole-cell catalytic desulfurization method <130> 1 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 1387 <212> DNA <213> Halomonas salifodinae <400> 1 taagctaacc acttctggtg cagtccactc ccatggtgtg acgggcggtg tgtacaaggc 60 ccgggaacgt attcaccgtg acattctgat tcacgattac tagcgattcc gacttcacgg 120 agtcgagttg cagactccga tccggactga gaccggcttt atgggattag ctccacctcg 180 cggtattgca accctttgta ccgaccattg tagcacgtgt gtagccctac ccgtaagggc 240 catgatgact tgacgtcgtc cccaccttcc tccggtttgt caccggcagt ctccctagag 300 ttcccgaccg aatcgctggc aaatagggac aagggttgcg ctcgttacgg gacttaaccc 360 aacatttcac aacacgagct gacgacagcc atgcagcacc tgtctgtgcg ttcccgaagg 420 caccaatcca tctctggaaa gttcgcacga tgtcaagggt aggtaaggtt cttcgcgttg 480 catcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc atttgagttt 540 taaccttgcg gccgtactcc ccaggcggtc gacttatcgc gttaactgcg ccacaaagtt 600 ctcaaggaac ccaacggcta gtcgacatcg tttacggcgt ggactaccag ggtatctaat 660 cctgtttgct acccacgctt tcgcacctca gtgtcagtgt cagtccagaa ggccgccttc 720 gccactggta ttcctcccga tctctacgca tttcaccgct acaccgggaa ttctaccttc 780 ctctcctgca ctctagcctg acagttccgg atgccgttcc caggttgagc ccggggcttt 840 cacaaccggc ttatcaagcc acctacgcgc gctttacgcc cagtaattcc gattaacgct 900 cgcaccctcc gtattaccgc ggctgctggc acggagttag ccggtgcttc ttctgcgagt 960 gatgtccttc ctgaagggta ttagccctca ggccttcttc ctcgctgaaa gtgctttaca 1020 acccgaaggc cttcttcaca cacgcggcat ggctggatca gggtttcccc cattgtccaa 1080 tattccccac tgctgcctcc cgtaggagtc tgggccgtgt ctcagtccca gtgtggctga 1140 tcatcctctc agaccagcta cggatcgtcg ccttggtgag ccgttacctc accaactagc 1200 taatccgaca taggctcatc cgatagcgca aggtccgaag atcccctgct ttctccccgta 1260 ggacgtatgc ggtattagcg tgagtttccc cacgttatcc cccactatcg ggcagattcc 1320 tatgcattac tcaccccgtcc gccgctcgac gcctggaagc aagcttccat cgttccgctc 1380 gactgca 1387 <210> 2 <211> 442 <212> PRT <213> Spiribacter sp <400> 2 Met Pro Asn Glu Ser Ala Ile Asn Thr Ser Arg Arg Arg Phe Leu Ala 1 5 10 15 Gly Ser Ala Ala Val Ala Gly Ala Ala Thr Val Gly Thr Gly Ser Leu 20 25 30 Leu Thr Ser Gly Pro Ala Arg Ala Val Ser Thr Asp Ala Arg Ile Val 35 40 45 Ile Ala Gly Ala Gly Ala Ala Gly Ile Ser Ile Ala Thr Arg Leu Ser 50 55 60 Arg Gln Leu Asp Gly Ala Thr Ile Ile Val Ile Asp Ser Arg Glu Thr 65 70 75 80 His Phe Tyr Gln Pro Gly Leu Thr Leu Val Ala Thr Gly Leu Trp Thr 85 90 95 Pro Ser Arg Val Glu Asp Ala Asn Thr Arg Phe Met Pro Ala Gly Val 100 105 110 Asp Trp Met Lys Asp Asp Val Val Glu Ile Asn Ala Asp Asn Asn Gln 115 120 125 Val Met Thr Ala Gly Gly Asp Thr Val Asp Tyr Asp Tyr Leu Val Val 130 135 140 Ser Thr Gly Leu Gln Ile Asn Phe Asp Glu Ile Asp Gly Met Ser Pro 145 150 155 160 Asp Leu Ile Gly His Asn Gly Val Gly Cys Val Tyr Ala Asn Pro Asp 165 170 175 His Ala Ala Arg Thr Trp Gln Ala Ala Glu Arg Tyr Ile Glu Glu Gly 180 185 190 Gly Ile Gly Leu Phe Thr Arg Pro Arg Gly Pro Ile Lys Cys Ala Gly 195 200 205 Ala Pro Leu Lys Val Thr Met Ile Ile Glu Asp Ala Leu Gln Glu Arg 210 215 220 Gly Asn Arg Glu Arg Ala Glu Met His Tyr Leu Pro Pro Gly Lys Gly 225 230 235 240 Leu Phe Ser Gln Pro Asp Ile Asp Thr Phe Leu Lys Asp Tyr Phe Pro 245 250 255 Ser Gln Arg Asp Ile Ala Ile Asp Tyr Asn His Pro Leu Ser Ala Ile 260 265 270 Asp Pro Asp Arg Arg Glu Ala Thr Phe Ala Thr Pro Glu Gly Pro His 275 280 285 Thr Met Glu Tyr Asp Phe Ile His Val Val Pro Pro Met Ser Ala Pro 290 295 300 Asp Met Ile Arg His Gly Glu Leu Gly Trp Gln Asn Gly Ser Phe Glu 305 310 315 320 Gly Trp Met Glu Val Asp Gln Tyr Ser Met Gln His Arg Arg Tyr Pro 325 330 335 Asn Val Phe Gly Ala Gly Asp Val Val Gly Thr Pro Ile Gly Lys Thr 340 345 350 Ala Ala Ser Val Lys Ala Gln Ala Pro Val Val Ala Asp Asn Leu Val 355 360 365 Ala Thr Ile Ala Gly Gln Glu Met Pro Met Ala Trp Asn Gly Tyr Thr 370 375 380 Ser Cys Pro Leu Ile Thr Ala Arg Gly Glu Ala Met Leu Val Glu Phe 385 390 395 400 Asp Phe Ser Leu Ala Met Lys Pro Ser Phe Ser Phe Ile Asp Pro Met 405 410 415 Lys Gln Gln Trp Ala Pro Trp Phe Leu Lys Asp Gln Met Leu His Ala 420 425 430 Ala Tyr Asn Ala Met Leu Arg Gly Arg Ile 435 440 <210> 3 <211> 1329 <212> DNA <213> Spiribacter sp <400> 3 atgcccaacg aatcagccat caacacgtcc cggcggcggt ttctcgccgg ctcggccgcg 60 gttgccgggg ccgcgacagt gggcaccggc agcctgctga ccagtgggcc cgcccgcgcc 120 gtatccacgg acgcgcgaat cgtcatcgcg ggagccggtg ccgcggggat atccattgcc 180 acccgtctct ctcgtcagct ggacggtgcc acgatcattg tgatcgacag ccgcgaaacc 240 catttctacc agcccggctt aacgctggtg gcaaccggac tgtggacgcc ctcccgggtc 300 gaggacgcca atactcggtt catgcccgct ggcgtggact ggatgaagga cgacgtggtc 360 gagataaacg ccgacaacaa ccaggtcatg accgccggtg gcgataccgt cgactatgac 420 tacctggtgg tcagtaccgg actgcagatc aactttgacg aaatcgacgg catgtcgccg 480 gacctcatcg gccataacgg cgtcggctgc gtgtacgcga atccggatca tgccgcccgc 540 acctggcagg ccgcggagcg gtatattgaa gaaggcggta tcgggctatt cactcgtcca 600 cgggggccga tcaaatgcgc tggggcgccg ctcaaggtca ccatgatcat tgaagatgcg 660 ctgcaggagc gcggcaaccg cgaacgggcg gaaatgcatt atctcccgcc tggcaaagga 720 ctgttctcgc agcccgacat cgataccttc ctgaaagact atttcccgtc ccagcgggac 780 attgccattg actacaacca cccgctgtcg gctattgacc ctgaccgccg cgaagcgacc 840 ttcgccacgc cggaggggcc acataccatg gagtatgact tcatccacgt ggtgcctccg 900 atgtccgcgc cggacatgat tcggcatggc gagctgggct ggcagaacgg cagcttcgag 960 ggctggatgg aagtcgacca gtacagcatg cagcatcgcc ggtatcccaa cgtgtttggc 1020 gccggggatg tggtgggtac gcccatcggc aagaccgccg ccagcgtgaa ggcgcaggcg 1080 ccggtggtgg cggacaacct ggtggcaacc attgccgggc aggaaatgcc catggcctgg 1140 aacggataca cctcctgtcc gctgatcacg gcccggggcg aggcaatgct ggtggagttc 1200 gacttcagcc ttgccatgaa gccatcattc agcttcattg acccaatgaa acagcagtgg 1260 gcgccctggt tcctcaagga ccagatgctg catgcggcct ataacgccat gctacgtggc 1320 cgaatctag 1329 <210> 4 <211> 1670 <212> DNA <213> Artificial sequence <400> 4 cactgcagga ggaagcttat gcccaacgaa tcagccatca acacgtcccg gcggcggttt 60 ctcgccggct cggccgcggt tgccggggcc gcgacagtgg gcaccggcag cctgctgacc 120 agtgggcccg cccgcgccgt atccacggac gcgcgaatcg tcatcgcggg agccggtgcc 180 gcggggatat ccattgccac ccgtctctct cgtcagctgg acggtgccac gatcattgtg 240 atcgacagcc gcgaaaccca tttctaccag cccggcttaa cgctggtggc aaccggactg 300 tggacgccct cccgggtcga ggacgccaat actcggttca tgcccgctgg cgtggactgg 360 atgaaggacg acgtggtcga gataaacgcc gacaacaacc aggtcatgac cgccggtggc 420 gataccgtcg actatgacta cctggtggtc agtaccggac tgcagatcaa ctttgacgaa 480 atcgacggca tgtcgccgga cctcatcggc cataacggcg tcggctgcgt gtacgcgaat 540 ccggatcatg ccgcccgcac ctggcaggcc gcggagcggt atattgaaga aggcggtatc 600 gggctattca ctcgtccacg ggggccgatc aaatgcgctg gggcgccgct caaggtcacc 660 atgatcattg aagatgcgct gcaggagcgc ggcaaccgcg aacgggcgga aatgcattat 720 ctcccgcctg gcaaaggact gttctcgcag cccgacatcg ataccttcct gaaagactat 780 ttcccgtccc agcgggacat tgccattgac tacaaccacc cgctgtcggc tattgaccct 840 gaccgccgcg aagcgacctt cgccacgccg gaggggccac ataccatgga gtatgacttc 900 atccacgtgg tgcctccgat gtccgcgccg gacatgattc ggcatggcga gctgggctgg 960 cagaacggca gcttcgaggg ctggatggaa gtcgaccagt acagcatgca gcatcgccgg 1020 tatcccaacg tgtttggcgc cggggatgtg gtgggtacgc ccatcggcaa gaccgccgcc 1080 agcgtgaagg cgcaggcgcc ggtggtggcg gacaacctgg tggcaaccat tgccgggcag 1140 gaaatgccca tggcctggaa cggatacacc tcctgtccgc tgatcacggc ccggggcgag 1200 gcaatgctgg tggagttcga cttcagcctt gccatgaagc catcattcag cttcattgac 1260 ccaatgaaac agcagtgggc gccctggttc ctcaaggacc agatgctgca tgcggcctat 1320 aacgccatgc tacgtggccg aatctagggc gttgagctgg gattaacccg gcgaggcgga 1380 gacccaacag aacggagcca gggagatggc gacgcagcaa cagaagaggg cgccgcagga 1440 cggcgccctt ttttgtgcgg gcgcttcggg cacgagggtc aacagacaag gtttgacccc 1500 agtggcgctg gctggctcct caacgcagct agccccccag tcaatcgagt caagtggttt 1560 aagagcgaaa aacctgagga aacaacccgt ggcatggagt gtacggatcc actagtgtgt 1620 tcccagggga taggagaagt cgcttgatat ctagtatgac gtctgtcgca 1670 <210> 5 <211> 551 <212> DNA <213> Artificial sequence <400> 5 actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga 60 catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg 120 tataatgtgt ggaattgtga gcggataaca atttcacaca ggaaacactg caggaggaag 180 cttggcgttg agctgggatt aacccggcga ggcggagacc caacagaacg gagccaggga 240 gatggcgacg cagcaacaga agagggcgcc gcaggacggc gccctttttt gtgcgggcgc 300 ttcgggcacg agggtcaaca gacaaggttt gaccccagtg gcgctggctg gctcctcaac 360 gcagctagcc ccccagtcaa tcgagtcaag tggtttaaga gcgaaaaacc tgaggaaaca 420 acccgtggca tggagtgtac ggatccacta gtgtgttccc aggggatagg agaagtcgct 480 tgatatctag tatgacgtct gtcgcacctg cttgatcgcg gccgcgatag ctagatcgcg 540 ttgctcctct t 551

Claims

1. A recombinant Halomonas bacterium, characterized in that: The recombinant Halomonas contains a quinone oxidoreductase gene, and the recombinant Halomonas expresses the quinone oxidoreductase; the quinone oxidoreductase is selected from any one of the following proteins: 1) a protein with an amino acid sequence as shown in SEQ ID No. 2; 2) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of 1).

2. The recombinant Halomonas sp. of claim 1, wherein: The quinone oxidoreductase gene is selected from any one of the following: x1) a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 3; x2) a DNA molecule with a coding sequence (CDS) as shown in positions 1-1329 of SEQ ID No.

3.

3. The recombinant Halomonas sp. of claim 1 or 2, wherein: The construction method of the recombinant Halomonas comprises introducing the quinone oxidoreductase gene into a recipient bacterium to obtain the recombinant Halomonas, and the recipient bacterium is Halomonas.

4. The recombinant Halomonas sp. of claim 3, wherein: The acceptor bacteria is Halomonas sp. (HJ-1) Halomonas salifodinae ) IM328 ， It is registered in the China General Microbiological Culture Collection Center with the registration number of CGMCC No. 22183.

5. The recombinant Halomonas sp. of claim 1 or 2, wherein: The recombinant Halomonas is an engineered strain of Halomonas salina Halomonassalifodinae IM328_sqr, having the accession number CGMCC No. 22179 at the China General Microbiological Culture Collection Center.

6. The construction method of the recombinant Halomonas according to claim 3.

7. The recombinant Halomonas according to claim 1, used in biological removal of hydrogen sulfide.

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