Acinetobacter bengal and its applications

Abstract

This invention provides Acinetobacter bengal and its applications, particularly a knockout bacterium with high screening throughput. hpxO Acinetobacter baylyi Ab-ZEN2 and its applications, accession number GDMCC No: 67727. This strain can be used to test whether a candidate gene coding region has uricase activity, and has a higher screening throughput.

Description

Technical Field

[0001] This disclosure belongs to the field of microbial technology, and in particular relates to Acinetobacter bengal and its applications. Background Technology

[0002] Acinetobacter baylyi ADP1 (Ab bacteria) is easy to culture, grows and divides rapidly, and has natural competent characteristics. It can be transformed by directly adding exogenous DNA to the culture without the need to prepare competent cells. Its 3.5 million base pairs of whole genome have been completely sequenced.

[0003] Acinetobacter baylyi hpxO The gene (NCBI GeneID: 45235716, https: / / www.ncbi.nlm.nih.gov / gene / 45235716) encodes a FAD-dependent uricase that converts uric acid to 5-hydroxyisourate (5-HIU), which can spontaneously convert to allantoin under physiological conditions. When *Ab* bacteria... hpxO Once the gene is knocked out, the organism will no longer be able to grow in uric acid-containing medium. Therefore, there is a need for a knockout gene that offers higher screening throughput, or one that reduces experimental scale and cost while maintaining the same screening throughput. hpxO The gene's chassis cells screen DNA libraries containing a large number of different coding regions. Summary of the Invention

[0004] On the one hand, this disclosure provides an Acinetobacter baylyi Ab-ZEN2, with the accession number GDMCC No: 67727. This Acinetobacter baylyi is deposited at the Guangdong Provincial Center for Microbial Culture Collection.

[0005] In some specific embodiments, the Acinetobacter Ab-ZEN2 contains the uricase gene. hpxO The absence of.

[0006] In some specific embodiments, the Acinetobacter Ab-ZEN2 contains a kanamycin resistance gene. Preferably, the IS1236 transposon in the Acinetobacter Ab-ZEN2 is replaced by a cassette expressing thymidine kinase and kanamycin resistance genes.

[0007] In some specific embodiments, the Acinetobacter Ab-ZEN2 comprises the nucleic acid sequence as described in SEQ ID NO: 25. Preferably, the uricase gene... hpxO Knockout hpxOThe gene coding region was replaced by SEQ ID NO: 25.

[0008] In some specific embodiments, the Acinetobacter Ab-ZEN2 is described with the nucleic acid sequence shown in SEQ ID NO: 26. Preferably, the nucleic acid sequence of the expression cassette expressing the thymidine kinase and kanamycin resistance genes is shown in SEQ ID NO: 26.

[0009] On the other hand, this disclosure provides an Acinetobacter bengal preparation comprising Acinetobacter bengal Ab-ZEN2 as described above, which is a solid or liquid preparation.

[0010] On the other hand, this disclosure provides the use of Acinetobacter Ab-ZEN2 as described above in screening gene coding regions with urate oxidase activity.

[0011] In some specific embodiments, the urate oxidase includes FAD-independent urate oxidase and FAD-dependent urate oxidase, wherein the FAD-dependent urate oxidase is selected from HpxO type and HpyO type, and the FAD-independent urate oxidase is selected from Pegloticase, Rasburicase and SpUricase.

[0012] In another aspect, this disclosure provides a method for screening uricase activity, wherein a gene fragment encoding the uricase is introduced into Acinetobacter benzi Ab-ZEN2 as described above, and the cells are cultured in a uric acid medium containing kanamycin, the growth of the cells representing uricase activity.

[0013] In some implementations, the culture medium is a solid culture medium, the culture temperature is 25-37°C, and the culture time is 12-96 hours.

[0014] Beneficial effects This disclosure provides an Acinetobacter bacillus strain Ab-ZEN2, with accession number GDMCC No: 67727, deposited at the Guangdong Provincial Microbial Culture Collection Center. The transformation efficiency of strain Ab-ZEN2 is approximately 10 times higher than that of strain Ab-HPXOKO1 obtained by conventional methods, supporting higher throughput screening systems, while also exhibiting a higher natural transformation rate of exogenous DNA compared to strains in the same batch. Attached Figure Description

[0015] This disclosure can be more fully understood with reference to the following figures.

[0016] Figure 1 A flowchart illustrating the construction process of the Ab-ZEN2 strain is shown.

[0017] Figure 2 It shows hpxO Electrophoresis diagrams of the left and right homologous arms upstream and downstream of the gene coding region.

[0018] Figure 3 Plates showing transformation of wild-type Ab bacteria are displayed.

[0019] Figure 4 The image shows a colony PCR electrophoresis pattern.

[0020] Figure 5 The electrophoresis diagram of the PCR products of the three DNA fragments used to construct the pZEN1.1-BB plasmid is shown.

[0021] Figure 6 The image shows a colony PCR electrophoresis pattern.

[0022] Figure 7 The plate showing the transformation of Ab-HPXOKO1 bacteria is shown.

[0023] Figure 8 The image shows a colony PCR electrophoresis pattern.

[0024] Figure 9 The PCR electrophoresis diagrams of the left and right homologous arms upstream and downstream of transposon IS1236 are shown.

[0025] Figure 10 The plate showing the transformation of Ab-HPXOKO2 bacteria is shown.

[0026] Figure 11 The image shows a colony PCR electrophoresis pattern.

[0027] Figure 12 The Sanger sequencing peak diagram shows the Ab-ZEN2 strain. hpxO The coding region of the gene was successfully deleted and replaced with a 26-base-pair sequence.

[0028] Figure 13 The Sanger sequencing peak diagram shows that the kanamycin resistance gene expression cassette replaces the original transposon IS1236 gene sequence at the transposon IS1236 locus in the expected manner.

[0029] Figure 14 The flowchart for constructing the Ab-ZEN2-cmp strain, which is complementary to Ab-ZEN2, is shown.

[0030] Figure 15 The growth of Ab-ZEN2 strain and Ab-ZEN2-cmp strain in ammonium chloride and uric acid media at different time points is shown.

[0031] Figure 16 The experimental flowchart for screening genes with urate oxidase activity using the Ab-ZEN2 strain is shown.

[0032] Figure 17 The experimental flowchart for screening genes with urate oxidase activity using the Ab-ZEN2 strain is shown.

[0033] Figure 18 A plate showing the conversion of Ab-ZEN2 using Golden Gate Assembly products is shown. Detailed Implementation

[0034] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0035] As used herein, urate oxidase (UOX), also known as uricase, refers to an enzyme that catalyzes the oxidation of uric acid to allantoin. The urate oxidase described in this invention includes naturally occurring protein sequences or their variants or truncated forms. It should be understood that the gene encoding the urate oxidase is not specifically limited, as long as it has the activity of degrading uric acid. The urate oxidase of this invention can also be from different sources, such as bacteria, fungi, or mammals. In some embodiments, the urate oxidase is derived from humans, chimpanzees, orangutans, gorillas, gibbons, baboons, macaques, cynomolgus monkeys, night monkeys, rabbits, mice, dogs, cattle, and pigs. In some embodiments, the urate oxidase is a natural urate oxidase (Uricozyme) derived from Aspergillus flavus. In some embodiments, the uricase is a recombinant enzyme, such as recombinant Aspergillus flavus uricase-rasburicase or pegloticized recombinant uricase. In some specific embodiments, the uricase comprises FAD-independent urate oxidase (EC 1.7.3.3), FAD-dependent urate oxidase (EC 1.14.13.113), and uricase integrated into the cell membrane. In some embodiments, the FAD-dependent uricase can be of the HpxO and HpyO types. Acinetobacter baylyi... hpxO The gene (NCBI GeneID: 45235716, https: / / www.ncbi.nlm.nih.gov / gene / 45235716) encodes an FAD-dependent uricase that converts uric acid into 5-hydroxyisourate (5-HIU), which can spontaneously convert into allantoin under physiological conditions.

[0036] Acinetobacter baylyi can grow using uric acid as the sole nitrogen source, and its uric acid culture medium is shown in Table 2, with the formulation of the 1000X SL-10 solution shown in Table 1. The ammonium chloride culture medium for Acinetobacter baylyi using ammonium chloride as the sole nitrogen source is shown in Table 3. The primer sequences used in specific embodiments of this invention are shown in Table 4, and the gene or plasmid sequence information is shown in Table 5.

[0037] Table 1. Formulation of 1000X SL-10 solution

[0038] Table 2. Uric acid medium (medium using uric acid as the sole nitrogen source)

[0039] Table 3. Ammonium chloride medium (medium using ammonium chloride as the sole nitrogen source)

[0040] Table 4. Primer sequence information

[0041] Table 5. Sequence Information

[0042] Example To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0043] Example 1: Construction of Ab-ZEN2 strain 1.1 Construction of Ab-HPXOKO1 strain (1) Wild-type Ab→Ab-HPXOKO1 Genomic DNA was extracted from Acinetobacter baylyi ADP1 (ATCC No.: ATCC33305; https: / / www.atcc.org / products / 33305) and amplified using KAPA HiFiHotStart DNA polymerase (Kapa Biosystems, catalog number: KK2502) and primers OG-HpxO-u1177-fwd / OG-HpxO-KO-R1 and OG-HpxO-KO-F2 / OG-HpxO-d1261-rev. hpxO The left and right homologous arms upstream and downstream of the gene coding region; electrophoresis images of the left and right homologous arms are shown below. Figure 2 Using the left homologous arm, right homologous arm, and pBTK622 plasmid ( Suárez GA, Dugan KR, Renda BA, Leonard SP, Gangavarapu LS, Barrick JE. Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 Genome streamlining. Nucleic Acids Res. 2020 May 7;48(8):4585-4600. doi: 10.1093 / nar / gkaa204. PMID: 32232367; PMCID: PMC7192602.The Golden Gate Assembly reaction was carried out using BsaI Type IIS restriction endonuclease (New England Biolabs, catalog number R3733) according to the reaction system in Table 6 and the reaction conditions in Table 7.

[0044] Table 6. Golden Gate Assembly Reaction System

[0045] Table 7. Golden Gate Assembly Reaction Conditions

[0046] a. Transformation of wild-type Ab bacteria: Take 70 µL of wild-type Ab bacteria cultured overnight in LB medium and add it to a shaker tube containing 1 mL of fresh LB medium. Incubate at 30°C and 200 rpm for 2 hours with shaking. Add the entire 20 µL Golden Gate Assembly reaction mixture to the culture and continue incubating at 30°C and 200 rpm for 2 hours with shaking. Collect the bacterial cells and spread them at an appropriate ratio (e.g., 1:1000) onto LB agar plates containing 30 µg / mL kanamycin. Figure 3 As shown. After incubating the plates overnight at 30°C, single colonies were picked and colony PCR was performed to identify whether the expected gene editing had successfully occurred at the genomic location.

[0047] b. Colony PCR identification of successful DNA fragment insertion: Pick a single colony from the plate and resuspend it in 10 µL of water. Take 1 µL of the bacterial suspension as a PCR template and perform colony PCR using primers OG_HpxO-KO_Colony_fwd / OG_HpxO-KO_Colony_rev and KAPA 2G Fast Genotyping Mix (Kapa Biosystems, catalog number KK5103). The corresponding electrophoresis pattern is shown below. Figure 4 As shown. The suspension of strain No. 2 with the correct PCR band was inoculated into LB medium and cultured overnight at 30°C with shaking at 200 rpm. Then, the experiment was continued or glycerol was added to a final concentration of 25% and stored at -80°C.

[0048] 1.2 Construction of Ab-HPXOKO1→Ab-HPXOKO2 strain (1) Construction of pZEN1.1-BB plasmid The pUC57-Kan plasmid was obtained from GeneWiz. The pZEN1.1 plasmid backbone fragment (2666 bp) was amplified by PCR using primers OG-pZEN-make-F1-2 / OG-pZEN-make-R1-2 and KAPA HiFi HotStart DNA polymerase.

[0049] Genomic DNA was extracted from wild-type Absculinus and amplified using KAPA HiFi HotStart DNA polymerase and primers OG-pZEN-make-F2-2 / OG-pZEN-make-R2 and OG-pZEN-make-F3 / OG-pZEN-make-R3-2. hpxO The left and right homologous arms upstream and downstream of the gene coding region.

[0050] Electrophoresis diagram of PCR products as shown below Figure 5 As shown, the PCR products were purified using AmPure Xp magnetic beads to obtain three fragments: pZen1.1-Frag1, pZen1.1-Frag2, and pZen1.1-Frag3. Gibson Assembly reactions were performed using NEB Gibson Assembly MasterMix (New England Biolabs, catalog number E2611S), followed by transformation into DH5α chemocompetent cells. The cells were then plated onto LB agar plates containing 30 µg / mL kanamycin. After overnight incubation at 37°C, single colonies were picked and colony PCR was performed to identify the presence of the expected plasmid pZEN1.1-BB.

[0051] Pick a single colony from the plate and resuspend it in 10 µL of water. Take 1 µL of the bacterial suspension as a PCR template and perform colony PCR using primers pZEN1.1-F1-2 / pZEN1.1-R1-2 and KAPA 2G Fast Genotyping Mix. The colony PCR electrophoresis image is shown below. Figure 6 As shown, colonies 2 and 4, carrying the correct plasmid, were cultured overnight in LB medium, followed by plasmid mini-prep. The sequences were verified as correct by Sanger sequencing, and colony 2 was ultimately selected for subsequent experiments. The pZEN1.1-BB plasmid sequence is shown in SEQ ID NO: 33.

[0052] (2) Construction of Ab-HPXOKO2 strain Transformation of Ab-HPXOKO1 bacteria: 1000 ng of pZEN1.1-BB plasmid was added to a 50 μL reaction system, followed by PmeI restriction endonuclease (New England Biolabs, catalog number R0560S) and the corresponding digestion buffer. The mixture was linearized at 37°C for 2 hours, yielding a product with both ends... hpxO DNA fragments of homologous arms of the gene. Take 70 µL of Ab-HPXOKO1 bacteria cultured overnight in LB medium. Figure 6 Add the bacteria (strain #2) to a 1 mL fresh LB medium shake tube and incubate at 30°C and 200 rpm for 2 hours. Add the entire 20 µL PmeI digestion reaction mixture to the culture and continue incubation at 30°C and 200 rpm for 2 hours. Collect the bacterial cells and spread them at an appropriate ratio (e.g., 1:1000) onto LB agar plates containing 25 mg / mL Lazidothymidine. Figure 7 As shown. After incubating the plates overnight at 30°C, single colonies were picked and colony PCR was performed to identify whether the expected gene editing had successfully occurred at the genomic location.

[0053] Colony PCR identification hpxO Correct gene editing at the locus: Pick a single colony from the plate and resuspend it in 10 µL of water. Take 1 µL of the bacterial suspension as a PCR template and perform colony PCR using primers forward primer 1 / reverse primer 1 and KAPA 2G FastGenotyping Mix. PCR electrophoresis image as shown. Figure 8 As shown. The suspension of bacteria No. 6 with the correct PCR band was inoculated into LB medium and cultured overnight at 30°C with shaking at 200 rpm. Then, the experiment was continued or glycerol was added to a final concentration of 25% and stored at -80°C.

[0054] 1.3 Construction of Ab-HPXOKO2→Ab-ZEN2 strain Genomic DNA was extracted from wild-type Ab bacteria. Using KAPA HiFi HotStart DNA polymerase and primers OG-IS1KO-F1 / OG-IS1KO-R1 and OG-IS1KO-F2 / OG-IS1KO-R2, the left and right homologous arms upstream and downstream of transposon IS1236 were amplified. The PCR results are as follows: Figure 9 As shown. Using the left and right homologous arms, and the pBTK622 plasmid, along with the BsaI Type II restriction endonuclease (New England Biolabs, catalog number R3733), the Golden Gate Assembly reaction was performed according to the reaction system in Table 8 and the reaction conditions in Table 7.

[0055] Table 8. Golden Gate Assembly Reaction System

[0056] Ab-HPXOKO2 transformation: Take 70 µL of Ab-HPXOKO2 bacteria that have been cultured overnight in LB medium ( Figure 8 Add the bacterial strain (strain #6) to a 1 mL fresh LB medium shake tube and incubate at 30°C and 200 rpm for 2 hours. Add the entire 20 µL Golden Gate Assembly reaction mixture to the culture and continue incubation at 30°C and 200 rpm for 2 hours. Collect the bacterial cells and spread them at an appropriate ratio (e.g., 1:1000) onto LB agar plates containing 30 µg / mL kanamycin. Figure 10 As shown. After incubating the plates overnight at 30°C, single colonies were picked and colony PCR was performed to identify whether the expected gene editing had successfully occurred at the genomic location.

[0057] Colony PCR to identify successful DNA fragment insertion: Pick a single colony from the plate and resuspend it in 10 µL of water. Take 1 µL of the bacterial suspension as a PCR template and perform colony PCR using forward primer 2 / reverse primer 2 and KAPA 2G FastGenotyping Mix. The corresponding electrophoresis image is shown below. Figure 11 As shown. Bacterial suspensions with the correct PCR bands were inoculated into LB medium and cultured overnight at 30°C with shaking at 200 rpm. Experiments were then continued, or glycerol was added to a final concentration of 25% and stored at -80°C. Genomic DNA was extracted from the overnight cultured cells, and Sanger sequencing was used to determine if the DNA sequence at the target location matched expectations. Colony 1 was ultimately selected as strain Ab-ZEN2.

[0058] Sanger sequencing peaks show that in the Ab-ZEN2 strain hpxO The coding region of the gene was successfully deleted and replaced with a 26-base-pair sequence. Figure 12 The kanamycin resistance gene expression cassette replaced the original transposon IS1236 gene sequence at the IS1236 locus in the expected manner. Figure 13 ).

[0059] In summary, among the Ab-ZEN2 strains, hpxOThe entire coding region of the gene, from the start codon to the stop codon, was deleted and replaced with an irrelevant 26-base-pair sequence. IS1236 was replaced by an expression cassette that simultaneously expresses thymidine kinase and the kanamycin resistance marker (Tdk-Kan). The Tdk-Kan expression cassette was derived from the pBTK622 plasmid ( ref: Suárez GA, Dugan KR, Renda BA, Leonard SP, Gangavarapu LS, Barrick JE. Rapid and Assured genetic engineering methods applied to Acinetobacter baylyi ADP1 Genome streamlining. Nucleic Acids Res. 2020 May 7;48(8):4585-4600. doi: 10.1093 / nar / gkaa204. PMID: 32232367; PMCID: PMC7192602. Its construction process is as follows: Figure 1 As shown.

[0060] Example 2: Phenotypic analysis of Ab-ZEN2 strain 2.1 Different Ab-ZEN2 clones exhibited differences in the spontaneous transformation rate of exogenous DNA. Genomic DNA was extracted from wild-type Ab bacteria, and a DNA fragment containing hpxO CDS and a left homologous arm of approximately 1000 base pairs upstream and a right homologous arm of approximately 1000 base pairs downstream was amplified using KAPA HiFi HotStart DNA polymerase and primers OG-HpxO-u1177-fwd / OG-HpxO-d1261-rev.

[0061] The above DNA fragment was used to transform Ab-ZEN2 or Ab-HPXOKO1 to complement their hpxO knockout phenotype. 70 µL of Ab-ZEN2 bacteria cultured overnight in LB medium (containing the finally selected...) Figure 11 Colony 1 (and colonies 2-7 for comparison) or Ab-HPXOKO1 were added to a 1 mL LB medium culture tube and incubated at 30°C with shaking at 200 rpm for 2 hours. 100 ng of PCR product was added to the culture, and incubation continued at 30°C with shaking at 200 rpm for another 2 hours. The bacterial cells were collected and plated 1:100,000 onto ammonium chloride and 1:10,000 onto uric acid agar plates. Incubation was continued at 30°C for 144 hours until full colony growth was observed and colonies were counted. The colony count on the ammonium chloride plate reflects the number of viable cells, while the colony count on the uric acid plate reflects the number of phenotypically complementary cells obtained through successful transformation. This allows calculation of the transformation ratio per unit of viable cells and the transformation efficiency (cfu per µg DNA) per unit of exogenous DNA.

[0062] The Ab-HPXOKO1 strain used in this experiment was the same as the knockout strain reported in the literature. hpxO Methods for constructing Ab bacteria with genes ( Suárez GA, Dugan KR, Renda BA, Leonard SP, Gangavarapu LS, Barrick JE. Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining. Nucleic Acids Res. 2020 May 7;48(8):4585-4600. doi: 10.1093 / nar / gkaa204. PMID: 32232367; PMCID: PMC7192602.The results are comparable. Experimental data show that the selected Ab-ZEN2 strain clone ( Figure 11 The transformation efficiency of colony 1 in the sample was as high as 2.93 × 10⁻⁶. 8 The cfu per µg DNA was significantly higher than that of the Ab-HPXOKO1 strain used for comparison, which was 1.27 × 10⁻⁶. 7 The CFU per µg DNA was more than 10 times higher, and also significantly higher than some other clones obtained in the same batch, such as colonies 2-7 (transformation efficiency comparable to Ab-HPXOKO1 strain, at 1.45 × 10⁻⁶). 7 (cfu per µg DNA) (Table 9). This allows for higher screening throughput when using selected Ab-ZEN2 strains as chassis cells to screen DNA libraries containing a large number of different coding regions, or reduces experimental scale and cost at the same screening throughput.

[0063] Therefore, when transformed using DNA with a left and right homologous arm of 1000 base pairs, the Ab-ZEN2 strain retained the natural transformation ability of wild-type Ab bacteria. Unexpectedly, different Ab-ZEN2 strains exhibited different transformation efficiencies, with the ultimately selected Ab-ZEN2 strain demonstrating approximately 10 times higher transformation efficiency than the Ab-HPXOKO1 strain obtained using conventional methods, thus supporting higher-throughput screening systems.

[0064] Different clones of the Ab-ZEN2 strain obtained in the same batch exhibited different spontaneous transformation rates. The selected Ab-ZEN2 clone had an unexpectedly high spontaneous transformation rate of exogenous DNA, ranking among the highest in the same batch of Ab-ZEN2 clones, and showing a higher spontaneous transformation rate of exogenous DNA compared to the Ab-HPXOKO1 strain obtained by conventional methods.

[0065] Table 9. Transformation ratio per unit viable bacterial count and transformation efficiency per unit amount of exogenous DNA

[0066] 2.2 Preservation of microbial strains Ab-ZEN2 strain ( Figure 11 Colony 1) has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. This center is a national patent culture collection institution and an international depositary unit under the Budapest Treaty. The accession number of strain Ab-ZEN2 is GDMCC No: 67727, the deposit date is January 22, 2026, and the taxonomic name is Acinetobacter baylyi.

[0067] 2.3 The Ab-ZEN2 culture strain can grow in ammonium chloride medium, but cannot grow in uric acid medium using uric acid as the sole nitrogen source. (1) Ab-ZEN2 complementary strain: Construction of Ab-ZEN2-cmp strain The construction process of the Ab-ZEN2 complementary strain Ab-ZEN2-cmp is as follows: Figure 14 As shown, the specific operation is as follows: Genomic DNA was extracted from wild-type Absculinus and amplified with homologous arms using KAPA HiFi HotStart DNA polymerase and primers OG-HpxO-u1177-fwd / OG-HpxO-d1261-rev. hpxO DNA fragments containing gene coding regions.

[0068] Take 70 µL of Ab-ZEN2 cultured overnight in LB medium and add it to a shaker tube containing 1 mL of fresh LB medium. Incubate at 30°C and 200 rpm for 2 hours. Add 100 ng of PCR product to the culture and continue incubation at 30°C and 200 rpm for 2 hours. Collect the bacterial cells and spread them at an appropriate ratio (e.g., 1:1000) onto uric acid solid medium containing 30 µg / mL kanamycin. Incubate the plates at 30°C for 48–96 hours, then pick single colonies as the complementary Ab-ZEN2-cmp strain for further experiments.

[0069] (2) Determination of growth curves of Ab-ZEN2 and Ab-ZEN2-cmp Three single colonies of Ab-ZEN2 and Ab-ZEN2-cmp were picked separately and incubated overnight at 30°C with shaking at 300 rpm in ammonium chloride medium containing 30 µg / mL kanamycin until the plateau phase. The next morning, 500 µL of each of the six cultures was taken, washed twice with 1×PBS to prevent ammonium chloride from entering the subsequent uric acid medium, and then resuspended in 500 µL of 1×PBS. Two µL of the resuspension was inoculated into 10 mL culture tubes containing either 3 mL of ammonium chloride medium or 3 mL of uric acid medium containing 30 µg / mL kanamycin, and incubated at 30°C with shaking at 300 rpm. The OD values ​​of the cultures in each tube were measured at 0, 12, 14, 16, 18, 23, 36, 44, and 60 hours. 600 Measurements are taken to determine the growth status.

[0070] Table 10. OD values ​​of Ab-ZEN2 strain and Ab-ZEN2-cmp strain in ammonium chloride and uric acid media at different time points. 600 value

[0071] The results are shown in Table 10 and Figure 15 As shown, Ab-ZEN2 bacteria can grow in ammonium chloride medium containing 30 µg / mL kanamycin, but cannot grow in uric acid medium containing 30 µg / mL kanamycin. Wild-type bacteria with uricase activity were then... hpxO When the coding region DNA of the gene is transformed into Ab-ZEN2 bacteria, it can complement the phenotype that prevents growth in uric acid medium. The complemented Ab-ZEN2-cmp strain can grow in both ammonium chloride medium containing 30 µg / mL kanamycin and uric acid medium containing 30 µg / mL kanamycin.

[0072] 2.4 Screening for gene coding regions with uricase activity using Ab-ZEN2 strain FAD-dependent urate oxidases, such as HpxO-type urate oxidases, and FAD-independent urate oxidases (such as Pegloticase, Rasburicase, and SpUricase from fission yeast) have different reaction mechanisms with uric acid. FAD-independent urate oxidases use oxygen molecules to directly oxidize uric acid, producing 5-HIU and hydrogen peroxide (H₂O₂). HpxO-type urate oxidases, in addition to using oxygen molecules, also use NADH or NADPH as a co-substrate, producing 5-HIU and NADPH as products. + and NADP + However, it does not produce hydrogen peroxide (H2O2).

[0073] The reaction mechanisms of FAD-independent uricases (such as Pegloticase and Rasburicase) and HpxO-type uricases are compared as follows:

[0074] The CDS of the SpUricase gene from *Schizosaccharomyces pombe* was selectively modified by adding left and right homologous arms. This DNA was then used to transform the Ab-ZEN2 culture strain, resulting in complementary DNA. hpxOThe gene knockout phenotype resulted in the transformation of the Ab-ZEN2 strain, which was able to grow in uric acid medium.

[0075] An example of using Ab-ZEN2 chassis cells to test whether a candidate gene coding region has uricase activity is shown below (procedure as follows). Figure 16 and Figure 17 (as shown) Genomic DNA was extracted from *Schizosaccharomyces pombe*, and the DNA fragment of the uric acid oxidase gene SpUricase (SEQ ID NO: 34) was amplified using KAPA HiFiHotStart DNA polymerase and primers SpUO-U0-2 / SpUO-D888-2. The SpUricase DNA fragment was then subjected to a Golden Gate Assembly reaction with pZEN1.1-BB plasmid according to the reaction system described in Table 11 and the reaction conditions in Table 7, to... hpxO Homologous arms of the locus are added upstream and downstream of the SpUricase coding region.

[0076] Table 11. Golden Gate Assembly Reaction System

[0077] Transformation of Ab-ZEN2 using Golden Gate Assembly product: Take 70 µL of Ab-ZEN2 bacteria cultured overnight in LB medium and add it to a shaker tube containing 1 mL of fresh LB medium. Incubate at 30°C and 200 rpm for 2 hours with shaking. Add the entire 25 μL of Golden Gate Assembly product to the culture and continue incubation at 30°C and 200 rpm for 2 hours with shaking. Collect the bacterial cells, dilute 1 / 1000 with sterile water, and spread onto agar plates containing 30 µg / mL kanamycin in uric acid. Figure 18 As shown. Observe colony growth after incubation at 30°C for 48-72 hours. The presence of colonies indicates that the SpUricase gene from fission yeast has uric acid oxidase activity.

[0078] The results are as follows Figure 18 As shown: The results demonstrate that the SpUricase gene from fission yeast (S. Pomb) can complement the gene in Ab-ZEN2 strain. hpxO The -KO phenotype demonstrates that the Ab-ZEN2 strain can be used to screen for gene coding regions with uricase oxidase activity.

[0079] By incorporating references The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0080] Equivalence This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. An Acinetobacter baylyi Ab-ZEN2, with accession number GDMCC No: 67727.

2. The Acinetobacter behnkeni Ab-ZEN2 as described in claim 1, which contains the uricase gene. hpxO The absence of.

3. Acinetobacter benzi Ab-ZEN2 as described in claim 1 or 2, wherein it contains a kanamycin resistance gene.

4. Acinetobacter behnkeni Ab-ZEN2 as claimed in claim 1, comprising the nucleic acid sequence shown in SEQ ID NO:

25.

5. Acinetobacter behnkeni Ab-ZEN2 as claimed in claim 1, comprising the nucleic acid sequence shown in SEQ ID NO:

26.

6. An Acinetobacter benzi preparation comprising Acinetobacter benzi Ab-ZEN2 as described in any one of claims 1 to 5, wherein the preparation is in solid or liquid form.

7. The use of Acinetobacter benzi Ab-ZEN2 as described in any one of claims 1 to 5 in screening gene coding regions with urate oxidase activity.

8. The application as described in claim 7, wherein the urate oxidase comprises FAD-independent urate oxidase and FAD-dependent urate oxidase.

9. A method for screening uricase activity, comprising introducing a gene fragment encoding the uricase into Acinetobacter benzi Ab-ZEN2 as described in any one of claims 1 to 5, culturing the cells in a uric acid medium containing kanamycin, wherein the growth of the cells represents uricase activity.

10. The method of claim 9, wherein the culture medium is a solid culture medium, the culture temperature is 25-37°C, and the culture time is 12-96 hours.

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